Adjustable Thermo-Responsive Cell Carrier and Implants From Three Armed Macromers_KETPAT_R6

Universität Leipzig
DISSERTATION
Adjustable Thermo-Responsive Cell Carrier and Implants from

Three Armed Macromers
KETPAT VEJJASILPA
2023
Adjustable Thermo-Responsive cell carrier and implants
from three armed macromers
Dissertation
zur Erlangung des akademischen Grades
Dr. rer. nat.
an der Medizinischen Fakultät

der Universität Leipzig
eingereicht von:
KETPAT VEJJASILPA
Geburtsdatum / Geburtsort:
05.05.1989 / PETCHABURI
angefertigt an / in:
Pharmazeutische Technologie, Institut für Pharmazie, Medizinische Fakultät, Universität
Leipzig
Betreuer:
Prof. Dr. Michaela Schulz-Siegmund
Dr. rer. nat. habil. Michael C. Hacker
Beschluss über die Verleihung des Doktorgrades vom:

>> Science is not only a disciple of reason but, also, one of romance
and passion<<
Stephen Hawking
Table of Contents
CHAPTER 1……………………..……………...…………………………..…4
Introduction
CHAPTER 2……………………..…………………………..……………….29
Material and Methods
CHAPTER 3……………………………………..…..……………………….52
Thermo-Responsive Polymer from Thermal Synthesis Studies
CHAPTER 4…………………………………..……………………………...70
An Adjustable Thermo-Responsive Polymer from Photo Synthesis
CHAPTER 5……………………………………………………………....….88
Fabrication of Thermo-Responsive Scaffolds from DLP Printing
CHAPTER 6…………………...…………………………………………....107
3D Scaffold Biocompatibility Studies
CHAPTER 7…………………...……………………………………………139
Discussions
CHAPTER 8…………………...……………………………………………161
Summery
APPENDIX…………………...………………………………………….…166
Bibliography, List of Publications, CV, Declaration of Authorship, Acknowledgements,
Related publication
LIST OF ABBREVIATIONS
1
H-NMR…………………. proton nuclear magnetic resonance
2D…………………………two-dimensional
3D…………………………three-dimensional
4D………………………… four-dimensional
AIBN…………….………...azobisisobutyronitrile
AMO………………………4-Acryloylmorpholine
BAPO…………………….. UV initiator Irgacure 819
C2C12 cells………………..myoblast cell line
CDCl3………..……………deuterated chloroform
CL………………………… ε-caprolactone
Col-HA…………………….collagen hydroxyapatite composite
CIJ………………………… continuous Inkjet printing
DAPI………………………cell nucleic staining solution (4',6-diamidino-2-phenylindole)
DDS………………………. drug delivery systems
DFG……………………….German Research Institute (Deutsche Forschungsgemeinschaft)
DI water…………………...deionized water
DLP………………………..Digital Light Processing (3D printing technique)
DMAEA…………………..dimethylaminoethyl acrylate
DMEM………………….... Dulbecco’s Modified Eagle Medium
DNA………………………deoxyribonucleic acid
DoD………………………..Drop on Demand
DSC……………………….differential scanning calorimetry
e.g…………………………example given
ECM (EZM)…… …………extracellular matrix
EDTA…………………….. ethylenediaminetetraacetic acid
et al. ………………………et alii = and others
PAGE 1
ES…………………………electrical stimulation
EUR……………………….Euro
FBS………………………. fetal bovine serum
FDA……………………….Food and Drug administration
FTIR………………………fourier-transform infrared spectroscopy
G'………………………….storage modulus
G"…………………………loss modulus
GF…………………………glycofurol, tetraglycol, BioXtra
GPC……………………….gel permeation chromatography
HA……………………….. hydroxyapatite
hASCs………………….....human adipose tissue derived stem cells
hvFb……………,,,……… human vascular fibroblast
HPLC……………………..high-performance liquid chromatography
IR…………………………infrared
Irgacure2959……………...photo initiator, α-Hydroxy-4-(2-hydroxyethoxy)-α-methylpropiophenone
L929 cells…………………fibroblast cell line
LA………………………...lactic acid = D,L-lactide acid
LCST…………………….. lower critical solution temperature
LIFT………………………laser-induced forward transfer
MA………………………..methacrylic acid
MNP……………,,……… magnetic nanoparticles
MMA……………………..methyl methacrylate
Mn…………………...........number average molecular weight
MS…………………..........mechanical stimulation
MSCs……………………..mesenchymal stem cells
MW……………………….molecular weight
NFI…………………..…....net fluorescence intensity
PAGE 2
NMR……………….……... nuclear magnetic resonance
NiPAAm…………….……. N-isopropyl acrylamide
OH…………….….………...hydroxyl group
PBS…………..…………….phosphate buffer saline
PCL…………..…………… poly(ε-caprolactone)
PDS………….……………..polydiooxanone
PE………….……………....polyethylene
PEG………….…………….poly(ethylene glycol)
PHB………….……………. poly(3-hydroxybutyrate)
PI……….…………………..photo initiator
pKa………….….…………..acid dissociation constant
PLA…………..…………….polylactic acid
PGA…………..………...…. polyglycolic acid
PLGA……………….…….. poly(lactic acid-co-glycolic acid)
PNVCL……………..………Poly(N–Vinylcaprolactam)
PMMA…………….………. poly(methyl methacrylate)
SEM……………….……….scanning electron microscope
SLA………….……………. stereolithography
TCP……………………...…thin Conducting Polymer
THF………….……………. tetrahydrofuran
TMPTA………..……………trimethylolpropane triacrylate
TMS…………..…………….tetramethylsilane
TPO…………..…………..... diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide
Ttrans…………..…………......Transition temperature
w/o…………..……….….......without
PAGE 3
CHAPTER 1. INTRODUCTION
CHAPTER I
Introduction
PAGE 4
When the tissues or organs have been severely damaged by either disease or trauma, the
ability of the human body to self-regenerate is limited, and this poses a tremendous burden on
and challenge to medical systems in the short and long term.1 As has been described above,
conservative treatment is still facing major hindrances such as lack of donors and
biocompatibility issues.2 On the other hand, a development of biomaterials could play a crucial
role as a scaffold or even total substitution of lacking native donor tissues by allowing the cells
to proliferate and differentiate inside the materials into fully functional tissues. In this context,
an establishment of stimulus-responsive materials could provide the essential missing piece
such as mechanical stimulation during cultivation to establish new tissue. 3
1.1 Mechanical Cell Stimulation
Lacking dynamicity in static scaffolds has been one of the major drawbacks for in vitro
experiments.4 Mechanical stimulation has been long investigated as one of the stimulating force
factors. Cells in everyday life are exposed to various forces such as shear stress, stretching, and
compression on daily basis. For example, the gravitational compressive force affects bone cells
such as osteoblast-like cells and chondrocytes.5 In another example, endothelial cells in the
vascular system are constantly exposed to the shear stress from blood flow. The earliest
mechano-stimulus experiment was reported in 1939 by Gluckmann, stated the impact of
mechanical stress to the endosteal cells from embryonic chick tibiae.6 The majority of the
experiments investigating the influence of mechanical stimulation on tissue formation were
focused on external stimuli to the cell through such mechanisms as compressive loading
systems, longitudinal stretch systems (uniaxial tension), systems utilizing substrate bending, bi-
axial traction systems, and shear stress input systems.7 In recent works, cell manipulation
methods using external mechanical stimulation (MS) and electrical stimulation (ES) have been
proven to have an effect on cell differentiation via the release of biosignals and growth factors.
For example, the living cells such as murine myocytes respond to mechanical stimuli and
PAGE 5
convert them into electro-biological signals by changing intracellular Ca2+ influx.8 Dan P. and
his colleagues have used Mesenchymal stem cells (MCSs) that are capable of differentiating
into endothelial cells and smooth muscle cells (SMCs). They stated that both shear stress and
cyclic strain can contribute to MSCs’ differentiation into endothelial cells.9 The review suggests
that a combination of mechanical and biochemical stimuli could synergize the expression of
endothelial cell functionality and endothelial cell markers.
1.1.1 Stimuli-Responsive Materials
In order to study how cells behave in a living organism, researchers often use in vitro
experiments, which are conducted in a controlled environment outside of the body. These
experiments often involve applying mechanical inputs, such as hydrostatic pressure or shear
stress, to the cells using specialized laboratory equipment, such as a compressive loading
system or stretch system. These controlled delivery mechanisms help researchers to better
understand how cells respond to different mechanical inputs and how they might behave in
different parts of the body where they are subjected to different mechanical forces.7 Those
methods, however, depend heavily on direct physical force and require complex equipment set-
up, which may increas the possibility of contamination.10
To circumvent these complexities, the term “smart material” was created to define a
material or scaffold that can respond to the surrounding environment and change accordingly.
In a physiological environment, the tissue has been exposed to periodic temperature conditions
such as changes in temperature or pH, which makes the substitute material not only adaptive to
the environment but also promotes cell proliferation and tissue formation. Stimuli-responsive
polymers offer an appealing blueprint in such demand and could be the key to developing new
PAGE 6
smart scaffolds and modulating cell-directing cues, enabling in-depth studies to evaluate cell
elements without in vivo experimentation.
Currently, there are various types of thermo-responsive polymers which have promising
applications as they have controllable properties.11,12 This approach could provide tremendous
advantages over traditional biomaterials and traditional systems. In general, smart materials
should be able to respond to surrounding external or internal stimuli and have controllable
functions for tissue regeneration in a valuable way.13
Three-dimensional Smart Three-dimensional

printing scaffold
Materials Polymers, metals, ceramics, Responsive materials that
biomaterials, and composite could change property of
materials that unable to function in response to
response to stimuli stimuli
Materials programmability Not possible Material properties and
functions could change
depending on material’s
composition and its
arrangement in response to
stimuli
Object shape Stable over time Configuration changes over
a specific stimulus
Applications Medical, engineering, Same as three-dimensional
dentistry, automotive, etc. printing applications, where
constant dynamic change in
response to stimuli is
required.
Table 1-1: Comparison between normal three-dimensional scaffold and smart biomaterials.14
PAGE 7
1.1.2 Thermal-Responsive Scaffolds
Thermo-responsive scaffolds have been shown to be useful tools to investigate effects
of mechanical stimulation in attaching cells.15 Thermo-responsive polymers such as NiPAAm
have been well studied and have shown potential in experiments for drug delivery and tissue
engineering.16-18 The Lower critical solution temperature (LCST) of the thermo-responsive
polymer can be controlled by introducing hydrophilic or hydrophobic co-monomers into the
polymer.19 Monomeric NiPAAm is notoriously known for cytotoxicity, yet it is one of the most
popular options for synthesis of stimulus-responsive polymers. The thermo-responsive
PNiPAAm could be used for a variety of applications such as copolymers and coating. For
example, Akiyama Y. et al. reported PNiPAAm coated on the substrates by plasma
polymerization on polystyrene Surfaces enables production of a cell monolayer sheet from
cardiac or hepatic cells.20,21
Poly(N-isopropylacrylamide) (PNiPAAm) is a type of polymer that has been shown to
be useful in creating three-dimensional platforms that mimic the conditions of living tissue.
These platforms can be made dynamic by adding thermo-responsive polymers, which allow
researchers to control the structure and alignment of cells within the platform. The cells can
then be harvested when they have reached a desired structure or alignment. While three-
dimensional scaffolds can be used to create complex structures, some of them may not be
biocompatible, meaning they may not be suitable for use in living tissue. This can be because
the changing temperature that is used to control the cells may be too extreme, leading to
excessive mechanical stimulation that could harm the cells.22,23
1.1.2.1 Poly(N-isopropyl acrylamide)
Poly(N-isopropyl acrylamide) or PNiPAAm is one of the most well-known thermo-
responsive polymers with a lower critical solution temperature (LCST) of around 32℃, which
PAGE 8
is close to the human physiological temperature, and has proven biocompatibility with a variety
of cells such as mesenchymal stem cells derived from rat bone marrow (BM-MSCs), human
adipose tissue (AT-MSCs), and smooth muscle cell (SMC).23,24 PNiPAAm has been used as a
building block for biomaterial structures and has potential for clinical applications such as drug
delivery systems (DDS) and tissue regeneration.
PNiPAAm, is a type of polymer that can be dissolved in water (an aqueous state) at a
temperature of around 32℃. PNiPAAm is known as an amphiphilic polymer because it has
both hydrophilic and hydrophobic parts. The amide groups of PNiPAAm are hydrophilic, while
the isopropyl groups are hydrophobic. This combination of hydrophilic and hydrophobic
properties allows PNiPAAm to show with both hydrophilicity and hydrophobicity. At
temperatures above the LCST, the polymer becomes hydrophobic and the network contracts.
The polymer shows a coil-to-globule transition, as the globule conformation hides hydrophilic
groups and displays the hydrophobic chains, thus expelling water molecules. This process is
reversible as the temperature goes below LCST. At lower LCST, the enthalpy from the
hydrogen bond becomes negative, causing the PNiPAAm to absorb water molecules and
become hydrophilic again.25-28
PAGE 9
Figure 1-1: PNiPAAm polymer network chain in the coil-to-globule transition. Below the
LCST, the polymer chain is bound with water molecules. At temperatures above its lower
critical solution temperature (LCST), a polymer will shrink and become hydrophobic, as it loses
bond with water molecules. 29
Authors (Year) Descriptions Ref.

Mizutani et al. (2008) PIPAAm brush surfaces were found to influence adhesion 30
and detachment of bovine carotid artery endothelial cells
(ECs)
Klaikherd et al. (2009) Multi-sensitive block copolymer for control of drug delivery 31
Chen et al. (2009) Surface modification strategy for fabricating blood- 32

compatible materials
Yoshida (2010) Self-oscillating gels without any on–off switching of external 33
stimuli
Lin et al. (2012) Inexpensive, biocompatible, oxygen permeable 34
Microtextured PNIPAAm-poly(dimethylsiloxane) (PDMS) for
vascular smooth muscle cell (VSMC) sheets
Bakarich et al. (2015) Mechanical and thermal actuating 4D printed smart valve 10
Li et al. (2016) Minimally invasive hydrogels that solidify at the pH of an 35

infarcted heart site
Shida et al. (2016) 4D printed smart scaffolds from soybean oil-epoxidized 36
acrylate that can support hMSCs
Liu et al. (2018) Photosensitizer from a novel comb-shaped porphyrin end- 37
functionalized toward HeLa cancer cells therapy
Table 1-2: Current PNiPAAm platforms that have been researched in the biomedical field.
PAGE 10
1.1.2.2 Poly-N-Vinylcaprolactam
Poly-N-Vinylcaprolactam (PNVCL) presents many similarities with PNiPAAm in many
aspects (i.e., LCST is variable between 25-50 °C). It has shown some potential to be used in
biomedical applications.38 PNVCL is known to be biocompatible and to show hydrolysis
resistance. PNVCL consists of both hydrophobic and hydrophilic parts, which in turn can be
manipulated for many applications. It also has good solubility in organic solvents and dissolves
in water at around 14–20℃ with high concentration (≥40% w/w).
1.2 Four Dimensional (4D) Scaffolds
In recent decades, stimuli- responsive scaffolds processed with three-dimensional
printing does referred to as fourth-dimensional scaffolds, have received attention in polymer
research. The 4-dimensional printing technique may provide next-generation scaffold for
regenerative medicine as it allows the scaffolds to change its properties in response to a defined
stimulation, such as temperature. Smart materials integrate additive components and are able to
self-transform into pre-programmed functions according to the surrounding environment, and
they are able to display smart properties such as shape memory and self-actuation.39
There is a large number of publications of environmentally responsive materials that
have been utilized in 4D scaffold printing.39,40 In these publications, the key component of
thermo-responsive polymers is PNiPAAm, which has a transition temperature of around 32℃
and it can be controlled by adjusting ratios by copolymerization with adjuvant monomer, thus
affecting the polymer volume change.41,42 For example, Zhibing Hu and his colleagues have
developed moldable polymer gels by combining two layers of poly(acrylamide), or PAAM, and
poly(acrylamide)-co-poly(N-isopropyl acrylamide) interpenetrating networks, or PAAM-NIPA
IPN. The combination of two materials creates a shape-memory gel.43 Stile et al. have reported
the development of injectable three-dimensional polymeric scaffolds from the cross-linked
PAGE 11
network of poly(N-isopropyl acrylamide) and acrylic acid (AAc), or P(NiPAAm-co-AAc). The
gel was placed inside a tube-like structure and then forced through a small, needle-like aperture
at a temperature of 22℃. The network has shown to be solidified at physiological temperature.
The biocompatibility was tested with bovine chondrocyte. The study found that the scaffold
promoted the proliferation of chondrocyte in vitro.44
1.2.1 Scaffold Modification Strategy
Scaffolds have been used in various therapeutic applications such as smart-surface,
where a tissue layer with extracellular matrix (ECM) can be collected without chemical
intervention (trypsinization) via PNiPAAm surface coating as lowering the temperature
reconstitutes water molecules into the polymer networks and causes detachment of all thin
tissue layers. On the other hand, to use thermo-responsive materials such PNiPAAm as cell
stimulation platform, it is crucial that targeted cells adhered on the material surface both above
and below the transition temperature. In order to achieve this, charged monomers have been
introduced to the polymer network. Heydarifard et al. focused on creating chitosan-based
hydrogels using two cationic monomers. The resulting hydrogels were analyzed using various
techniques to determine the impact of the monomers' counter ions on the hydrogels'
characteristics and performance. The results showed that the counter ions had a significant
impact on the hydrogels' properties, including water uptake and swelling, as well as their ability
to adsorb an anionic dye.45
Another example that emphasizes the benefit of three-dimensional system with
modified surface chemistry has been reported by Kwon and Matsuda on a copolymer block of
PNiPAAm and PEG as thermoresponsive support for rabbit chondrocytes in three-dimensional
gel, which LCSTs ranged from 31.3℃ to 34℃. The results demonstrated minimal decrease in
PAGE 12
cell number with excellent cell viability during a 7-day culture.46 Nihan Ozturk et al. used a
tissue engineering scaffold made of poly(N-isopropylacrylamide) (pNIPAM) to examine the
effect of strain on cell proliferation and differentiation, and the influence of surface chemistry
and topography on bone marrow mesenchymal stem cells. The pNIPAM films were modified
with a temperature-sensitive elastin-like protein (ELP) and subjected to periodic temperature
changes. The results showed that the ELP enhanced initial cell attachment to the films and
helped maintain cell attachment during dynamic culturing, but had no effect on long-term cell
proliferation. Dynamic culturing improved cell proliferation, but decreased differentiation. The
modified pNIPAM scaffold had a positive influence on cell populations under dynamic culture
conditions..47
1.3 Macromers in Biomedical Applications
A macromer is a large molecule that can be chemically modified in order to impart
specific properties. In the biomedical field, macromers are often used to synthesize polymeric
materials for a variety of applications, including drug delivery, tissue engineering, and wound
healing.48 One example of a macromer used in the biomedical field is a polyethylene glycol
(PEG) macromer. PEG is a biocompatible and biodegradable polymer that can be easily
modified with a variety of functional groups.49 PEG macromers can be used to synthesize
hydrogels, which are hydrophilic networks of polymers that can be used for drug delivery and
tissue engineering.50 Other examples of macromers used in the biomedical field include
polysaccharides51, polypeptides52, and dendrimers.53 These macromers can be used to
synthesize a wide range of polymeric materials with specific properties, such as
biocompatibility, biodegradability, and drug delivery capabilities.
PAGE 13
Trimethylolpropane triacrylate, or TMPTA, is a chemical compound that is commonly
used as a cross-linker in the synthesis of polymeric materials for biomedical applications.54-56
It is a macromer, which means that it is a large molecule that can be chemically modified in
order to impart specific properties. TMPTA has a unique chemical structure, with three "arms"
that can react with other molecules and form chemical bonds.57 These arms make TMPTA a
multifunctional cross-linker, which means that it can react with multiple other molecules at the
same time and form a network of interconnected polymers. TMPTA is known to have good
biocompatibility.58 This makes it a useful material for constructing biomaterial platforms, such
as scaffolds and hydrogels, which are used in tissue engineering and drug delivery applications.
1.4 Photoinitiator for Three-Dimensional Printing
One of the crucial components of photo-polymerization is the photoinitiator, which
could have a decisive impact on the feasibility of the photosensitive three-dimensional printing
technique. When the photoinitiator is exposed to UV light, the photo-initiator creates reactive
molecules, i.e., free radicals, cations, or anions. There are numerous commercialized
photoinitiators that have been widely used in 3D printing among them Irgacure819 or BAPO,
which is a free radical high reactivity photoinitiator. When the molecules are exposed to UV
light, homolytic cleavage of the excited α-carbon bond takes place, which leads to two radical
fragments. Then, the free radicals initiate polymerization.59 One advantage of Irgacure 819 is
that it has a low absorption in the visible light range, which makes it suitable for use with
transparent or translucent systems. This means that it can be used to initiate polymerization
reactions in materials that are not fully opaque, and it can be used in applications where it is
important to maintain the transparency or translucency of the final product. Another advantage
PAGE 14
of Irgacure 819 is that it has a relatively low molecular weight, which makes it easy to
incorporate into polymer systems. It is also relatively stable, and it has a low tendency to
migrate or leach out of polymer systems over time.
Irgacure 2959, on the other hand, has a higher absorption in the visible light range,
which makes it more suitable for use in opaque systems. It is also more efficient at initiating
polymerization reactions than Irgacure 819, which makes it a good choice for applications that
require a faster cure time. However, it is more sensitive to moisture and oxygen, which can
affect its stability and performance in certain applications. 60,61
Photoinitiators Light absorption (λmax)
~380 nm62
TPO
~274 and ~330 nm63
Irgacure 2959
~370 nm64
BAPO or Irgacure819
Table 1-3: Commercialized photoinitiators for three-dimensional printing.
PAGE 15
1.5 Three-Dimensional Scaffold Fabrication Methods
The ability to fabricate complex structures is the main advantage of the three-
dimensional printing technique. As the technology advances, there are numerous fabrication
techniques that have been proven to generate biocompatible scaffolds in the biomedical
research field. The material can be created by either bottom-up (the light source is positioned
below the photoresist mixture) or top-down (the light source is positioned above the photoresist
mixture) approaches.65 Furthermore, the printing strategies can be used alone or in combination
to achieve desired applications.
Figure 1-2: Diagram of SLA (Stereolithography) and DLP (Digital Light Processing) three-
dimensional printing. SLA uses a laser as the light source. DLP is generally faster than SLA
because it uses a digital projector to cure the resin, which allows it to cover a larger surface area
at once compared to a laser.
PAGE 16
1.5.1 SLA and DLP
Stereolithography (SLA) and digital light processing (DLP) printing techniques have
been described as a suitable micro-nano-structural approach. The main advantage of these
techniques is controllable porosity and mechanical properties, which are the main factors of cell
proliferation and differentiation in biomaterials.66,67 Both methods use photo-curable resin and
exposure with UV light to produce highly accurate prototypes. While both methods have
similarity in principal, SLA uses a laser and DLP a projector.68,69 The first commercialized
three-dimensional stereolithography printing technique was developed by Chuck Hull in
1986.70 An electromagnetic light, mainly a UV laser, is directed to photocurable resin and
induces polymerization and cross-linking. The printing system allows the laser to move in three
directions (x, y, and z axis). A photocurable resin is polymerized in a planar pattern with a
spatially controlled laser beam. After the layer is cured, the platform that contains the cured
structure, is ascended or descended (in the bottom-up approach) and another layer of uncured
resin spreads over the bottom. In detail, the predefined depth of photon-irradiating resin is
greater than the step height of fabrication; thus, the first layer of photo-polymerization consists
of unreacted functional groups and is polymerized with a subsequent resin layer. Usually, post-
treatment often requires washing off the incomplete conversion of reactive groups and
improving the mechanical property. In recent years, the progression of SLA technology allows
cells to be incorporated into the resin. This technique has been reported for use with cell carrier
ink, resulting in high cell viability (>90%) and high resolution (as low as 100 µm) with printing
time less than 30 minutes.71 Though the SLA method holds many advantages over other
techniques, it is often expensive, the photocurable solution must be specially handled, and it
sometimes requires washing or curing after the printing process.
3D-DLP (digital light processing) is a technology that is used to create three-
dimensional objects by curing layers of photopolymer resin with a digital projector. In the
PAGE 17
biomedical field, 3D DLP technology has been used to create a variety of medical devices and
implants, such as scaffolds for tissue engineering and custom-made prostheses. One example
of a biomedical application for DLP technology is the production of scaffolds for tissue
engineering to create β-tricalcium phosphate (β-TCP) scaffolds for bone regeneration. A low
viscosity ceramic slurry was developed and the resulting scaffolds had a maximum compressive
strength of 9.89 MPa with 40% porosity. The biocompatibility of the scaffolds was also
confirmed through cell proliferation. These porous scaffolds have potential for use in bone
regeneration and could expand the use of DLP technology in the biomedical field.72
1.6 Aim of the Thesis
In this thesis we aim to set-up a thermo-responsive material platform. Scaffolds from
this motivated platform not only harbor and support adhered cells but also provide them with
mechanical stimulation in response to periodic temperature shifts, thereby stimulating and
encouraging the cells to proliferate and eventually to differentiate. In order to achieve our goals,
we have developed a three-pronged approach to determine the feasibility of the thermo-
responsive platform (Figure 1-3). In stage I, we will investigate a broad spectrum of monomer
ratios for cross-polymerization, as described in Figure 1-4, unless stated otherwise. NiPAAm
is used as the thermo-responsive, whereas TMPTA, or trimethylolpropane triacrylate, is used
as the core cross-linker for the scaffold. We hypothesize that the lower critical solution
temperature (LCST) can be adjusted by incorporation of additive components such as DMAEA
(Dimethylaminoethyl Acrylate) and AMO (4-Acryloylmorpholine). This hypothesis will also
be used in stages II and III, where given the concentration of photoinitiator and cross-linking
time for three-dimensional printers, the change in compositions will influence scaffold swelling
ratio, transition temperature, and biocompatibility. This is crucial step in order to understand
the effect of monomer and macromer interaction on transition temperature in soluble state.
PAGE 18
The III stage utilizes the second stage results to fabricate complex scaffolds in various
configurations by the cDLP technique. Thermo-responsive biomaterials with transition
temperatures at physiological level and adjustable properties for mechanical cell stimulation
will be proposed. Various photo-initiator ratios, printing medium solvents, and times of
exposure per layer will be determined. The scaffold’s Ttrans will be analyzed by DSC to ensure
that optimized transition temperatures are obtained. Thermo-mechanical properties under
changing temperature conditions will be determined by rheology. Platform biocompatibility
will be investigated with various cell types in periodic temperature conditions.
PAGE 19
Figure 1-3: Schematic illustration of first, second, and third stages. Starting from studies of the
monomer and macromer relationship in first stage (top) to photo cross-linking in the second
stage (middle). Afterward, the results from the first and second stages have been used in the
third stage for three-dimensional printing (bottom).
Figure 1-4: General chemical schematic structure of three-armed macromer. The scaffold
composition consists of a three-armed functional core (TMPTA, red), positive charge (DMAEA,
pink), and heterocycle amine (AMO, blue).
PAGE 20
Overall, the proposed material-platform should be able to adjust properties through
composition and on-demand printing. Furthermore, the method should allow for surface
modification. The main objectives are listed below:
• To create a new cell carrier platform made of thermo-responsive polymers,
including TMPTA, NiPAAm, and a cationic monomer. The thermo-responsive
polymer properties, such as transition temperature and hydrophilicity are
adjustable through additive monomer compositions.
• To develop an easy, on-demand complex three-dimensional biomaterial
structure, in particular with controllable porosity. Porosity is one of the
important factors of biomaterials. Three-dimensional printing technology
allows controllable porosity from the programming.
To explore biocompatibility from the scaffolds using the three-dimensional continuous
digital light processing (cDLP) method with live/dead staining with both animal and human
cells in constant and periodic temperature conditions. It is assumed that the potential toxicity
from unreacted monomers would be extracted from the scaffold by step diffusion after
fabrication. Structural morphology and porosity are also expected to affect the cell distribution
on the scaffold, and the cationic charge from DMAEA in the polymer chain is also expected to
provide cell adhesion in temperature-shifted episodes.
In summary, the experiment development will be systematically separated into three
stages: thermally induced solution polymerization, photo-induced bulk polymerization, and
three-dimensional printing. In the first stage, three-armed macromers, different ratios, and
various additive monomers will be utilized to create a thermo-responsive copolymer that
characterizes the potential for usage in the thermo-responsive platform. The influence of
macromer and additive monomers will be characterized by H1-NMR and FTIR for composition
analysis, GPC for molecular weight, and DSC for Ttrans.
PAGE 21
A mechanical cell platform stimulation via stimulus-responsive polymer-based scaffold
printing could be used to address needs in clinical applications. A proof-of-concept mechanical
stimulation scaffold by three-dimensional printing technique will be studied. The technique
should be able to stimulate cells using periodic temperature changes around the material’s phase
transition temperature, whereas the cationic molecule that is copolymerized will provide
covalent immobilization for cell attachment and proliferation as illustrated in Figure 1-5.
Figure 1-5: Thermo-responsive scaffold scheme. The swelling from the scaffold mechanically
gives stress to the cell. The reactive surface of the scaffold provides covalent immobilization
force (positive charge) for cells to attach in event of scaffold expansion.
PAGE 22
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PAGE 28
CHAPTER 2. MATERIALS & METHODS
CHAPTER II
Materials and Methods
PAGE 29
2.1 Materials
All materials and chemicals were used as received unless stated otherwise.
2.1.1 Monomers and macromer
NiPAAm monomers (TCI Chemical, Tokyo, Japan) were dissolved in warm dried hexane
under constant magnetic stirring. The solution was placed at -20℃ to encourage recrystallization
of the NiPAAm. Recrystallized NiPAAm flasks were separated and kept with a cylindrical funnel,
then they were washed again with cold hexane and dried under reduced atmospheric pressure for
72 hours. Dimethylaminoethyl acrylate (DMAEA), 4-acryloylmorpholine (AMO), and
trimethylolpropane triacrylate (TMPTA) were purchased from Sigma-Aldrich, Germany.
2.1.2 Solvents
2.1.2.1 Thermally induced solution polymerization
Tetrahydrofuran (THF, gradient grade) and diethyl ether were purchased from VWR
(Darmstadt, Germany). 2,2-Azobis(2-methylpropionitrile) or AIBN was purchased from Sigma-
Aldrich and used as received.
2.1.2.2 Photo-induced bulk polymerization
Photo-initiators 2-Hydroxy-4 ′ -(2-hydroxyethoxy)-2-methylpropiophenone
(Irgacure2959, Sigma-Aldrich, Germany) and Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide
(BAPO or Irgacure819, Sigma-Aldrich, Germany) were purchased from Sigma-Aldrich, Germany.
The photoresist solution contains various ratios of monomer, cross-linker, and photoinitiator ratios
PAGE 30
which dissolve in absolute ethanol (absolute, VWR, Germany) or tetrahydrofurfuryl alcohol
polyethylene glycol ether (glycofurol or GF, Sigma-Aldrich, Germany).
2.2 Methods
Nomenclature. Scaffold formulations are represented in x, y, and z% w/w as Nx-Dy-Az, where N
is NiPAAm, D is DMAEA, and A is AMO. TMPTA was introduced into the polymeric network
as shown in the following example: 10% TMPTA N80-D15-A5 (where the scaffold composes 80%
w/w NiPAAm), 15% w/w DMAEA, 5% w/w AMO, and 10% w/w TMPTA were added with
respect to the total mass of the monomer solution.
TMPTA (added extra)

Nx-Dy-Az x y z Total
Percentage (%) 80 15 5 100 3
Amount (g) 2 0.39 0.11 2.5 0.075

Table 2-1: Amount and percentage of N80-D15-A5. Additional 3% w/w TMPTA was added to the
total mass of monomers.
2.2.1 Stage I (Thermally Induced Solution Polymerization of Monomer Mixtures)
The synthesis of the polymers began when recrystallized NiPAAm was added to a round-
bottomed flask and dissolved with anhydrous THF in a nitrogen atmosphere, and the temperature
was then raised to 60℃. The monomer (DMAEA), additive monomer (AMO), and cross-linker
(TMPTA) were introduced along with AIBN as a catalyst, and the reaction was carried out for 24
hours. After that, the copolymer was twice precipitated in a 10:1 ratio of diethyl ether. The
supernatants were separated and discarded by a centrifuge at 7,600 RPM for 3 minutes, then dried
PAGE 31
under nitrogen flow for three hours and vacuum-dried for 24 hours. The ratios of polymers are
shown in Table 2-2. The polymers were characterized with NMR and FT-IR.
Figure 2-1: Schematic of thermo-responsive polymers in the first phase, where the TMPTAs were
polymerized either with DMAEA and AMO. All synthesizes were carried out with 4%w/w AIBN
at 60℃, 24 hours.
PAGE 32
%TMPTA %NiPAAm %DMAEA

(w/w)
100 0
98 2
95 5
0, 5, 10, 15
90 10
85 15
80 20
Table 2-2: Synthesized chemical compositions of poly(NiPAAm-co-DMAEA)
PAGE 33
2.3. Stage II (Photo-induced bulk polymerization)
The optimized polymeric thermo-responsive polymer formulations were further utilized in
photo-induced polymerization in order to obtain a higher cross-linked ratio for scaffold
development. The method has been chosen as a potential method as it offers on-demand
polymerization and tunable properties through various combinations of factors (for example,
monomers, photo-initiator, concentration, and intensity). In short, the monomer mixture was
dissolved in ethanol at ratio of 10 % (w/v). It should be noted that 10 % (w/v) comprised 100 mg
of monomer mixture in 1 mL of ethanol. Then, photo-initiator or PI (Irgacure2959 or Irgacure819)
was added into the mixture solution at a 4% w/w ratio. The solution was pipetted into an Eppendorf
cap (200 µL) and exposed in a commercial UV chamber 36 Watt (Melody Susie Violet II, Model
DR-301C, 36w) for two minutes. The hydrogels were subsequently transferred into 70% ethanol
to wash unreacted monomers for 48 hours and in distilled water for 48 hours for further
measurement.
PAGE 34
Figure 2-2: Fabrication of hydrogel in UV chamber and its product after UV irradiation.
PAGE 35
CODE NiPAAm (%w/w) AMO(%w/w) DMAEA (%w/w)

N100-D0-A0 100 0 0
N90-D0-A10 90 10 0
N80-D0-A20 80 20 0
N70-D0-A30 70 30 0
N60-D0-A40 60 40 0
N50-D0-A50 50 50 0
N90-D10-A0 90 0 10
N80-D20-A0 80 0 20
N70-D30-A0 70 0 30
N60-D40-A0 60 0 40
N50-D50-A0 50 0 50
N90-D50-A5 90 5 5
N80-D10-A10 80 10 10
N70-D15-A15 70 15 15
N60-D20-A20 60 20 20
N50-D25-A25 50 25 25
Table 2-3: Synthesized photo-induced polymerization lists.
2.3.1. Polymeric Film Silanization
The circular disks were obtained from Henneberg & Co. (Martinroda, Germany) and
cleaned with low-linting cloth (Ko-Ton tissue by Chicopee, Katwijk, the Netherlands) to wipe out
macroscopic dust. First, the disks were sonicated for five minutes to clean remaining dust particles.
Then, the glass disks were immersed and washed in acetone, 2-propanol, and distilled water,
respectively. The activation step of the glass was performed with 35% aqueous hydrogen peroxide
with magnetic stirring (1,200 rpm, 60 min) and rinsed with distilled water and 2-propanol. The
activated glasses were silanized with 3% (v/v) of 3-(Trimethoxysilyl)propyl methacrylate (Sigma-
Aldrich, USA) and rinsed again with 2-propanol and water. The glasses were dried in the lamina
PAGE 36
fume hood to prevent particle contamination. The disks were stored with Ko-Ton tissue wrap at
4℃ for further usage.
Figure 2-3: Fabrication of copolymerized film on the silane surface modified glass slide.
Figure 2-3: Fabrication of copolymerized-film on the silane surface modified glass slide.
2.4. Stage III (Scaffold by Three-Dimensional Printing)
The use of 3D printing allowed for the creation of intricate scaffold shapes. A variety of
combinations of TMPTA, DMAEA, and AMO macromers and monomers were mixed with various
ratios of PIs in either ethanol or glycofurol. The Asiga Freeform Pico 2 HD UV385, a commercial
digital light processing 3D printer with a small printing head, was purchased from Asiga to execute
this process. The photoresist was transferred into the building tray with a pipette in a customized
Teflon container at room temperature, and the scaffolds were printed with 36.0 mW·cm−2 of light
intensity along the z-plan (thickness of 50 µm/layer) at room temperature. The scaffold fabrication
was constructed as preprogrammed by the Composer program (Version 1.2.5, Asiga). The
exposure time per layer was varied along with the different ratios of photoinitiators. The designated
scaffolds were placed in the UV chamber for three, six, and 12 minutes after treatment. The printed
PAGE 37
scaffolds were leached out of unreacted monomers by step diffusion at 48 hours using 70% ethanol
and distilled water or phosphate buffer saline (PBS) for further use, respectively. The printed
scaffolds were then separated into two experimental groups: the swelling ratio study group and the
biocompatibility study group.
Figure 2-4: Monomers and cross-linker were mixed and printed according to the preprogrammed
3D object, then the scaffolds were soaked in 70% EtOH for 48 hours to leach out unreacted
monomers.
PAGE 38
NAME %TMPTA %DMAEA %AMO %PI Printing time per layer

(w/w) (w/w) (w/w) (w/w) (second)
N95-D0-A5 3 0 5 4 10
N93-D2-A5 3 2 5 4 10
N95-D5-A5 3 5 5 4 10
N85-D10-A5 3 10 5 4 10
N80-D15-A5 1.5, 3 15 5 1, 2, 4 10, 20, 30
N75-D15-A5 3 15 10 4 10, 20, 30
N75-D20-A5 3 20 5 4 10
Table 2-4: The chemical composition list and conditions of the 3D scaffold printing.
Figure 2-5: The 3D objects were preprogrammed and uploaded into a 3D printer. The polymer
resin was transferred into a building tray with customized Teflon block to reduce needed
photoresist volume.
PAGE 39
(A) (C) (E)
(B) (D)
Figure 2-6: The visualization of 3D configurations in the COMPOSER program, the
preprogrammed models are designed as follow: 2D planar configuration (7.6 × 7.6 × 1.8 mm) (A,
and B), 3D lattices scaffold (7.6 × 7.7 × 2 mm) (C), Dumbbell (3 × 8 × 2.7 mm) (D), and Cylindrical
lattices scaffold (7.6 × 7.7 × 2 mm) (E).
PAGE 40
Figure 2-7: 3D example in dumbbell configuration after printing.
2.5. Characterization
2.5.1. 1H-NMR
Polymeric thermo-responsive compositions have been determined with 1H-NMR (Varian
Mercury, Varian, Darmstadt, Germany). Briefly, the polymers were dissolved in deuterated
chloroform containing 0.03% (w/v) tetramethylsilane (TMS) (Armar GmBH, Leipzig, Germany)
at 20 mg/mL concentration. The spectra were recorded at 300 MHz. The data was processed by the
MestreNOVA LITE 11.0 NMR software package (Mestrelab Research S.L., Spain). The signals
were calculated relative to the three protons from the TMPTA core and used to determine the unit
of DMAEA or AMO molecules.
2.5.2. Fourier-Transform Infrared Spectroscopy (FT-IR)
The samples were further characterized with FT-IR. The polymeric powders were placed on
an infrared device, the FTIR-ATR (Perkin Elmer UATR Spectrum TM Two). In brief, the dried
polymer was crushed in the motor into powder form, then it was placed at the analyzing chamber.
PAGE 41
The samples were scanned from 400 to 4,000 Hz. The data were processed and exported in ASCII
form for further analysis.
2.5.3. Gel Permeation Chromatography (GPC)
Average molecular weight distribution of the copolymers was determined with GPC (Agilent
Technologies, Boeblingen, Germany). The samples were taken by auto-sampler and passed
through a differential refractometer equipped with series analytical columns (PSS DEV (5 µm),
PSS SDV 1000 (5 µm), and PSS SDV 105 (5 µm), PSS Polymer Standard service GmbH). The
samples were dissolved by THF at 10 mg/mL concentration. The dissolved solution samples were
filtered through a 0.45 µm PTFE filter before determination. Molecular weights were calculated
using the WinGPC Unity Standard program (PSS). The average molecule number (M n), average
molecular weight (Mw), and polydispersity index (PDI) were investigated. All experiments were
triplicated to confirm the results.
2.5.4. Ttrans Measurement by DSC
The transition temperatures of the copolymer, discs, and scaffolds were determined using
differential scanning calorimetry (DSC) measurement (Polymer DSC R with TS0801 R0 Sample
Robot, Mettler Toledo) from a temperature range of 10 °C to 60 °C with a heating rate of 3 °C per
minute under constant nitrogen flow. To prepare the samples for DSC measurement, they were first
immersed in distilled water for 48 hours. A sample of 10–15 mg was placed into a 40 µL aluminum
crucible along with 15 mg of water as the reference. The temperature at which the first onset of an
endothermic phase occurred was designated as the Ttrans. The data were exported in ASCII format
and plotted for further analysis.
PAGE 42
Figure 2-8: DSC device used for determination of the copolymer T trans. The samples were placed
in an aluminum crucible and sealed.
2.5.5. Swelling Ratios
Water uptake by the materials were evaluated, the thermo-responsive discs and scaffolds
were determined after a period of incubation at 25 ℃ and 37 ℃ (n = 4) for 24 hours in water with
incubation interval at 1 day. At both time points wet weights of the samples were measured after
excess water was absorbed on cellulose filter paper. The materials were thereafter lyophilized for
48 hours before recording the dry weight. The swelling was calculated using equation below:
Weight of swollen construct(g)

Swelling ratio (g/g) =
Dry weight of construct (g)
2.5.6. Delta Swelling Ratio
To demonstrate changes in swelling ratio in the desired temperature range, we measured
the difference in swelling ratios (g/g) at 37 °C and 25 °C by subtracting the swelling ratio
PAGE 43
determined at 25 °C from the value determined after equilibration at 37 °C, and this value is
reported as ΔSR (37/25). When the material exhibits thermo-responsiveness, a negative value is
indicated for this parameter, as water molecules are pushed out of the polymeric matrix at the phase
transition temperature. A positive value indicates that the swelling increases with incubation
temperature and no phase transition affecting the swelling ratio occurs between 25 °C and 37°C.
2.5.7. Rheological Properties of Scaffolds
After the samples 3D printed scaffolds were cleansed of unreacted monomers and solvent.
The samples were immersed in distilled water for 48 hours at room temperature in an orbital
shaking machine at 100 rpm. The samples were then placed on tissue paper to remove excess water
before being placed under the rheometer (MCR301, Anton Paar, Austria) to determine the property
at changing temperature with analytical protocol: Equilibrium swollen samples were kept and
measured under initiate normal force at 0.2 N using an 8 mm parallel plate geometry in a
temperature sweep experiment with an oscillation frequency of 1 Hz and an amplitude gamma of
1% strain. After an equilibration time of 10 minutes, the temperature was ramped from 20 ℃ to
50℃ and vice versa with a heating/cooling rate of 3 ℃/min. Storage and loss moduli were recorded
by RHEOPLUS program (Version 3.4).
PAGE 44
2.5.8. In vitro Experiments
2.5.7.1. Cell Cultivation
Mouse fibroblast cell lines (L929, Cell Lines Service, Eppelheim, Germany), myoblast cell
lines (C2C12, Cell Lines Service, Eppelheim, Germany), human mesenchymal stem cells (hMSC
8F3434, Lonza, Germany), human adipose tissue-derived stem cells (hASCs, ID: F3C, Passage 2,
Lonza, Germany), and primary human fibroblasts (hvFb, Passage 4, self-isolated, kindly provided
by Prof. Dr. Tilo Pompe, Institute of Biochemistry, Universität Leipzig, Germany) were used to
test cytocompatibility of the 3D scaffold.
The cells were cultivated in a cell culture flask (Sarstedt, Germany) in low-glucose or high-
glucose Dulbecco’s modified Eagle's medium (DMEM, Sigma-Aldrich) or otherwise, fetal bovine
serum (FBS, Sigma-Aldrich) and penicillin-streptomycin (P/S, Sigma-Aldrich). All cells were
cultivated at 37 °C and 5% carbon dioxide until usage. The culture medium compositions for each
cell type are described below:
PAGE 45
Culture Additional
Cell FBS (%) P/S (%)
medium chemical(s)
Low-glucose
L929 10 1
DMEM
High-glucose
C2C12 10 1
DMEM
Low-glucose 1%Non-essential
hMSC 10 1
DMEM amino acids
Low-glucose
hvFb 10 1
DMEM
Low-glucose
DMEM/
hASC 10 1
DMEM/F-12,
GlutaMAX™
Table2-5: The culture medium composition for each cell type.
The cells were grown to a certain degree (70–80% confluence) before being harvested and
washed with DPBS. Trypsin with EDTA (Sigma-Aldrich, Germany) was added to separate the
cells for five minutes at 37 ℃. The trypsinization process was stopped by adding 10% FBS/PSB
to the suspension. The cell suspension was then transferred into a 50 mL centrifuge tube and
centrifuged at 340 rcf at room temperature for five minutes. The supernatant was removed and
remixed with cultured medium for cell seeding.
The cells were counted using the Neubauer hemocytometer. In short, after remixing with
cultured medium, 100 µL were pipetted out and mixed with 900 µL of culture medium to dilute in
1 mL Eppendorf tubes. Then, trypan blue staining color was mixed with 20 µL of the mixture from
the Eppendorf tubes in 96 culture well plates. The cell numbers were calculated using the equation
as follows:
PAGE 46
𝑇𝑜𝑡𝑎𝑙𝑙 𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑝𝑒𝑟 𝑚𝐿 = 𝑁 × 𝐷𝐹 × 𝐷 × 10
where: N = average counted cells from hemacytometer
DFtrypanblue = factor of dilution with trypan blue
D = dilution factor in Eppendorf tubes
103 = factor of Neubauer hemacytometer
2.5.9. Polymeric Film Biocompatibility
The preliminary biocompatibility test was carried out by seeded L929 cells on top of the
cross-linked polymer film (1×104 cells). Afterward, the polymeric films were cleansed of unreacted
monomer and ethanol as previously described. The cell supernatant was seeded on top of the
polymeric film and covered with culture medium. The experiments ended at 24 hours. The films
were stained with DAPI/Alexa-Fluor® 488 staining and observed under fluorescence microscopy.
The samples were removed from the culture medium, washed three times with DPBS, and
fixed with 4% formaldehyde for 10 minutes at room temperature. The samples were washed before
and after with DPBS before adding 0.5% Triton X-100 in DPBS for five minutes. Subsequently,
cell-seeded polymeric films were incubated with staining solutions (DAPI, Thermo Fisher
Scientific) for 10 minutes, then washed with DPBS and counterstained with Alexa-Flour TM 568
Phalloidin (Thermo Fisher Scientific) for 15 minutes.
PAGE 47
2.5.10. Scaffold Biocompatibility in Constant and Periodic Temperature Cultivations
After elimination of unreacted monomers and solvent by step diffusion, the cell suspensions
(5 × 104 cells) were added to each scaffold construct. The cell-seeded scaffolds were placed into
an incubator for cells to settle for two minutes before the designated cell culture medium was
toppled until the solution covered the scaffold (2 mL). The mediums were replaced every 48 hours
and taken out at one, three, and seven days to stain for cytocompatibility evaluation. The dynamic
condition of the scaffolds was generated by reduced environmental temperature of the scaffold
down to 30℃ for one hour each day after 48 hours.
Cytocompatibility tests were performed by seeded cells on the material surface, and the
scaffolds were then stained with Molecular Probes™ Calcein-AM (Invitrogent TM) (λex = 488 nm,
λem = 520 nm) and ethidium homodimer-1 (Invitrogent TM) (λex = 528 nm, λem = 617 nm). The
samples were rinsed with PBS and stained with staining solution for 45 minutes. The samples were
washed with PBS to remove excess dye and placed under a fluorescence microscope (Nikon) to
evaluate.
Figure 2-9: A purposed study of swelling ratio via temperature manipulation. The swelling ratios
should not exceedingly swell or shrink, thereby allowing the cells for mechanical stimulation while
PAGE 48
attaching. In the figure shown cultivation timeline between day 0 to 7 day where the cells were
seeded at 37℃ and cultivated with periodic temperature 1 hour per day at 30℃ after day 1. R T=
room temperature (25 ℃)
2.5.11. Poly-l-Lysine Surface Coating
Poly-L-lysine solution (0.01%, Lot: 3399368, EMD Millipore Corp, USA) was used to
improve biocompatibility of the scaffolds. The 3D scaffolds were immersed in the solution for 24
hours. After that, the scaffolds were washed with PBS three times and dried again under an
incubator (37℃, 5% CO2) for 24 hours. To ensure and maximize biocompatibility, the scaffolds
were soaked with PBS and cultured medium for 24 hours each before use.
Figure 2-10: Scaffold surfaces are coated again with 0.1% poly-L-lysine solution to improve cell
adhesiveness.
PAGE 49
2.5.12. Quantitative Analysis
The scaffolds were stained, and images were obtained in ND2 format, then analyzed by Fiji
software and 3D reconstruction were performed by Fiji software (ImageJ).1 Live cell staining was
quantified by analyzing the micrographs (TIFF format, recorded with an exposure time of 600 ms)
(n=3). The normalized fluorescent intensity (NFI) was calculated by averaging the fluorescent
signal (Ia) and the average background intensity (Ib) from the cell-depleted scaffold, and again
dividing by average background intensity. The equation is shown below:
𝐼 −𝐼
NFI =
𝐼
PAGE 50
REFERRENCES
1. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat
Methods 9, 676-682 (2012).
PAGE 51
CHAPTER 3. THERMO-RESPONSIVE POLYMER FROM THERMAL SYNTHESIS
CHAPTER III
Thermally Induced Solution Polymerization of Monomer
Mixtures
PAGE 52
3.1 Introduction
Thermo-responsive biomaterials have gained attention for their ability in an adhesiveness
in response to temperature shift. This allows for stress and lift them without systematic stress. In
this thesis, we aimed to take a next step and added adhesive uses to allow the cells to maintain on
the and experience mechanical stimulation upon provided temperature shifts the crucial factor is
material chemical modification that allows thermo-responsive material properties to be adjusted
such as transition temperature and cell harboring ability. One heavily investigated monomer is N-
isopropylacrylamide, or NiPAAm. For example, after polymerization, PNiPAAm-grafted collagen
scaffolds were developed for noninvasive delivery of cells to the back of the eye for retinal
degenerative diseases. The cells remained viable when cultured with the scaffolds, and expulsion
was prevented by incubating the scaffolds at room temperature before phase transition through
“coil-globule effect”.1 At temperature below the LSCT point the polymer becomes hydrophilic,
absorbs water molecules and swells and in consequence detaches the cells from the surface. In this
thesis we aimed to combine with adhesiveness this “stimuli-responsive” property can be also
utilized into mechanical stimulative platform, for adherent cell as it becomes clear that the
mechanical force is known to play an important role in cell function and differentiation. 2-4
Another material that need to be considered is TMPTA, a biocompatible trifunctional
macromer that is used as cross-linker, TMPTA offers a high degree of site reactivity and is able to
copolymerize with NiPAAm to achieve desired properties. The TMPTA’s hydroxyl groups can be
used in ring-opening polymerization and has knowingly been used for biocompatible polymeric
biomaterials.5,6 Dimethylaminoethyl acrylate (DMAEA) is cationic comonomer is used as
important building block to adjust transition temperature (Ttrans) of the copolymer network is
adjusted by incorporation of 4-acryloylmorpholine (AMO).7 The concept of this work is to create
PAGE 53
a thermo-responsive platform that allows it to exert mechanical stimulation to the cells through
swelling change in different transition temperatures. The objective of the first stage is to
polymerize a thermo-responsive polymers by incorporating adjuvant molecules such as cationic
and hydrophilic molecules into copolymer in order to develop a sandbox of thermo-responsive
platforms that can be adjusted depending on the desired T trans and applications. We hypothesized
controllable thermo-responsive polymers by thermal induced polymerization method in order to
study the correlation between monomers and cross-linker in soluble formulation, which played a
crucial role in the polymer Ttrans adjustment.
The polymers were characterized by NMR, GPC, and DSC. To obtain the desirable T trans,
comonomer-weight-ratio percentages have been optimized for further experimentations. Overall,
the polymer formulations will be a staging ground for further development for insoluble material-
formulations.
3.2. Results and Discussions
In our first stage, with the objective of establishing a series of thermo-responsive polymers
and determine the effect of adjusting monomer ratios that potentially influences T trans and polymer
properties. In this experimental stage, TMPTA or trimethylolpropane triacrylate, was chosen for
its three-armed reactive sites that allow copolymerization with the functional molecules such as
DMAEA, which are cationic monomers and have potential to be a candidate for adjusting thermo-
responsive polymers.
PAGE 54
3.2.1. Fourier-Transform Infrared Spectroscopy (FT-IR)
The thermo-responsive polymers were further analyzed using FT-IR (Figure3-2). NiPAAm
monomer and PNiPAAm, polymer with and without cross-linker were analyzed to compare the
results. We identified unique NiPAAm spectra from N-H stretching at 3287 cm -1 with notable
peaks that refer to C-H asymmetric stretching at 2970 cm-1, C-H symmetric stretching at 2874 cm-
1
, C=O amide group at 1634 cm-1, N-H bending at 1540 cm-1, C-N stretching at 1366 cm-1, – CH2-
and –CH3 bending vibrations at 1460 and 1386 cm -1, respectively.8 The FT-IR spectroscopy
showed successful cross-linking between the cross-linker TMPTA and the monomers NiPAAm
and DMAEA.
Figure 3-2: FT-IR spectra of NiPAAm and P(NiPAAm), with and without the 15% TMPTA with
15% w/w DMAEA.
Figure 3-4 shows the same spectral patterns indicated polymerization of TMPTA cross-
linked (NiPAAm-co-DMAEA) at 15% w/w TMPTA with different compositions from 20% to
0%w/w DMAEA. This spectral results confirmed polymerization of NiPAAm as its unique peaks
at 1540 cm-1 had disappeared. However, we cannot differentiated FT-IR spectra as cross-linker and
monomer spectrograms overlapped. We also seen the same result in figure 3-5 as polymers with
PAGE 55
TMPTA ratios at 0, 5, and 15w/w TMPTA demonstrated visually no difference. The polymers
were further characterized in a following section.
Figure 3-3: FT-IR spectra of 15% TMPTA cross-linked P(NiPAAm-co-DMAEA) at different
percentages of DMAEA (0–20% w/w).
Figure 3-4: FT-IR spectra of cross-linked P(NiPAAm) at different percentages of TMPTA (0, 5,
and 15% w/w) without DMAEA.
PAGE 56
3.2.2. Nuclear Magnetic Resonance Spectroscopy
1
H-NMR was performed after polymerization and purification to determine a successful
polymerization between the cross-linker and monomers. First, we have identified TMPTA trivalent
alcohol unique proton signals at approximately 0.83 ppm (-CH 3) and 1.69 ppm (-CH2-CH3). The
methylene protons close to the trivalent core were found at 4.0 ppm. The methylene protons at the
end of the three-armed macromer (-CH=CH2) appeared at 5.8, 6.1, and 6.4 ppm. The disappearance
of the signals at those positions (5.8, 6.1, and 6.4 ppm) indicated a successful of polymerization.
PAGE 57
Figure 3-1: TMPTA chemical structure and its H1-NMR spectra. Three reactive molecular sites
and distinctive signal are detected at 5.8, 6.1, 6.4, and 0.8 ppm.
PAGE 58
Thermo-responsive polymer were successfully polymerized by thermally induced
polymerization. The figure 3-2 shows the spectrogram of TMPTA cross-linked (NiPAAm-co-
DMAEA) after polymerization. At this initial stage, we tried to confirm and identify chemical
conformation according to our hypothesis. As shown in figure 3-2, both comonomers, DMAEA
and NiPAAm, were incorporated into copolymer chain. The signals at 5.8, 6.1, 6.4 disappeared,
whereas only the signal at 0.8 ppm (i position) remained that indicated the presence of TMPTA.
The successful incorporation of NiPAAm and DMAEA were confirmed by the peak signals at a,
b, and d positions.
Figure 3-2: 1H-NMR graph of TMPTA cross-linked (NiPAAm-co-DMAEA) with its chemical
structure. After the cross-linking process and purification, the signal was found to be broader
around 3.9–4.1 ppm.
PAGE 59
3.2.3. Gel Permeation Chromatography
Another factor that need to be determined after thermally induced polymerization is. The
molecular weights of thermo-responsive polymers were evaluated to obtain the influences from
cross-linker TMPTA and monomer ratios. The molecular weight of the thermo-responsive polymer
0% w/w TMPTA with 0% to 20% w/w DMAEA found to be varied from 2.4 ± 0.2 kDa (20% w/w
DMAEA) to 3.3 ± 0.1 kDa (0% w/w DMAEA). The depletion of DMAEA component from
polymer chain caused molecular weight reduction at least 1.4, 2, 2.3, and 1.5 times in 0%, 5%,
10%, and 15% w/w TMPTA, respectively. We attributed this phenomena as cationic molecules
could disrupt polymerization, thus disabled further propagation of polymer chain. This also
explained by the reduction of polydispersity index (PDI) as increasing DMAEA ratios could
potentially disrupt polymerization, thus prematurely terminate chain propagation, resulting lower
polymer dispersity. However, we observed no significant difference between M w and PDI with
increasing TMPTA ratios.
PAGE 60
6 2.5
Number average molecular weight
5
2.3
Polydipersity index
4 N80-D20
2.1 N85-D15
(kDa)
3 N90-D10
1.9 N95-D5
2
N100-D0
1.7
1
0 1.5
0%TMPTA 5%TMPTA 10%TMPTA 15%TMPTA
Figure 3-5: Average molecular weight and polydispersity index by GPC analysis (n = 3). The data
presented is relative to the polystyrene standard. The polymer compositions were 0-15% w/w
TMPTA, 80-100% w/w NiPAAm and 0-20% w/w DMAEA.
3.2.4. Transition Temperature Analysis (T trans)
One of the main objectives of this stage was to create thermo-responsive polymers that has
the ability to fine-tune transition temperature. To determine this, differential scanning calorimetry
(DSC) is the most suitable method.9 The DSC technique offers more precise data because it
provides information on the energy released from the cleavage of hydrogen bonds from water and
polymer molecules as hydrogen bonds between water and NH- molecules break; the endothermic
peak is often referred to as the transition temperature point. 10,11
In the preliminary analysis of a 2%TMPTA sample with increasing DMAEA contents (5%,
10%, and 15% w/w), Figure 3-6 displays the corresponding DSC thermogram. The transition
temperature (Ttrans) was determined as the first onset temperature of the observed endothermic
phase transition signal. It was observed that the 5% w/w DMAEA exhibited the lowest T trans at 35.5
PAGE 61
℃, followed by 39.3℃ (10% w/w) and 42.1℃ (15% w/w). Notably, in the case of the 10% w/w
DMAEA, there was an additional endothermic fluctuation prior to reaching the designated
transition temperature. This phenomenon could potentially be attributed to sample impurities or
inhomogeneity, where certain portions of the sample may possess distinct thermal properties
compared to the bulk composition.12
Table 3-1 show the data of Ttrans, increasing DMAEA ratios (2-30% w/w) at 2% w/w
TMTPA, caused in increasing of Ttrans values from 35.2℃ ± 0.3 ℃ to 46.5℃ ± 0.9℃. Increasing
TMPTA ratios from 2 % to 10% w/w at the same DMAEA ratio (15 % w/w) T trans dropped
considerably from 41.9℃ ± 0.5℃ to 31.7℃ ± 0.4℃. DMAEA or cationic monomer was utilized
as our hypothesis to increase the transition temperature of the thermo-responsive polymer. The
results suggested Ttrans can be influenced by incorporation of monomer and cross-linker into the
polymeric networks. We founded that DMAEA elevated T trans as the cationic monomer has the
ability to stabilize coil-globule transition of the copolymeric network, while, TMPTA has an
opposite effect on Ttrans. This phenomenon could be explained by the transition of a with increasing
feed ratios of TMPTA polymer network to become denser, thereby reduced the distance between
polymer segment chains directly influenced hydration capacity, and ultimately, decreased the
polymer’s Ttrans.13 As shown in figure 3-7, a thermo-responsive polymer was successfully formed
at above 37℃ and completely reversed at below transition temperature, whereas thermo-responsive
polymer without the cross-linker only became cloudy solution and failed to form at above 37℃.
PAGE 62
Figure 3-6: The transition temperature of a 2% w/w TMPTA N85-D5, N90-D10, and N85-D15
samples can be seen on the DSC thermogram to be around 35, 39, and 42 °C.
Cross-linker (%) NiPAAm (%) DMAEA (%) T trans (°C)
0 100 0 32.4 ± 0.6
0 90 10 38.7 ± 0.6
2 95 5 35.2 ± 0.3
90 10 39.0 ± 0.2
85 15 41.9 ± 0.5
70 30 46.5 ± 0.9
10 85 15 31.7 ± 0.4
Table 3-1: Interpreted Ttrans of NiPAAm-based thermo-responsive polymer with different
composition of TMPTA (0, 2, and 10%w/w) and DMAEA (0 - 15% w/w).
PAGE 63
Figure 3-7: Visualization and phase transition of thermo-responsive hydrogel with and without
cross-linking (0 and 5% w/w TMPTA, N90-D10-A0) at 25°C and 37°C.
The Ttrans of thermo-responsive polymers were quantified by DSC as presented in Figure 3-
7 in various series of DMAEA (0-20 % w/w) and cross-linker ratios (0-15% w/w). In this study, it
was found that the cationic monomer DMAEA and cross-linker TMPTA had opposite effects on
transition temperature as previously described in preliminary analysis and NiPAAm-based
formulations. Transition temperatures ranged from 16.3°C ± 1.2°C (15% w/w DMAEA, 0% w/w
TMPTA) to 40.0°C ± 1.3°C (20% w/w DMAEA, 0% w/w TMPTA).. In figure 3-8, the same
samples with TMPTA content were compared, increasing DMAEA clearly acted as hydrophilic
comonomer that prevented the PNiPAAm polymeric network from collapsing and thus in an
elevated Ttrans, while increasing TMPTA has an opposed influence on the polymers. For example,
looking at TMPTA within 5%DMAEA Ttrans decreased from 36.0℃ ± 1.2℃ (0% w/w TMPTA) to
19.3℃ ± 0.6℃ (15% w/w TMPTA) by increasing the TMPTA content from 1-15%. In the absence
of DMAEA, Ttrans has decreased from 32.1℃ ± 0.3℃ (0% w/w TMPTA) to 16.3℃ ± 0.7℃ (15%
w/w TMPTA). Both comonomer and macromer, DMAEA and TMPTA, affect the T trans of
PAGE 64
PNiPAAm-based polymeric network. However, one might argued that those also change other key
characteristics of the material, i.e. charge density and network structure. In order to change the
Ttrans only, we incorporated hydrophilic but functionally inert monomer. In this experiment, we
identified 4-acryloylmorpholine (AMO) as potential hydrophilic comonomer. The morpholine
molecules gravitate water molecules into the polymeric network and potentially promote an
increase in material swelling.3,7,14-16 As shown in figure 3-8, added AMO in the cross-linked
copolymer increased Ttrans. For example, we found that the thermo-responsive polymers from 5%
w/w TMPTA, 15% w/w DMAEA and 5% w/w AMO had a T trans of 31.5℃ ± 0.5℃ that gradually
increased to 37.5℃ ± 1.0℃ by increasing the AMO content up to 20 w/w (5% w/w TMPTA, 15%
w/w DMAEA, 20 % w/w AMO). Here, we expected the polymers change its phase transition
between 30°C and 36°C to match for cell survival and proliferation in the selected application.
From the data that we obtained, we identified potential formulations with T trans in our targeted
range, namely the formulations of 1-5% w/w TMPTA, 10-15% w/w DMAEA, and 1-15% w/w
AMO.
A 50
40
0%TMPTA
Ttrans (⁰C)
30
5%TMPTA
10%TMPTA
20
15%TMPTA
10
0
0 5 10 15 20
%DMAEA
PAGE 65
B 50
40
Ttrans (⁰C)
30
20
5%TMPTA
10 10%TMPTA
0
5 10 15 20
%4-Acryloylmorpholine
Figure 3-8: A: Transition temperature of thermal induced NiPAAm-based polymerization with
different ratios of TMPTA (0 - 15% w/w) and DMAEA (0-20% w/w). B: T trans of thermo-
responsive polymer with different ratios of TMPTAs (5% and 10% w/w), 15% w/w DMAEA, and
AMOs (5-20% w/w).
In summary, this experimental stage aimed to study the polymer transition temperature in
soluble state, which was the earliest step to ensure further studies in advanced stages that have been
set in higher concentration. The valuable data were obtained by various methods such as NMR,
GPC, DSC, and visually to prove the concept that the thermo-responsive polymer properties can
be adjusted to desired application purposes.
PAGE 66
3.5. Conclusions
Thermo-responsive polymers from three-armed macromers were successfully synthesized
and characterized in different comonomer ratios. The results suggest an adjustable transition
temperature in the copolymer chain. The characterizations of the thermoresponsive polymers
illustrated correlation between monomers (NiPAAm, DMAEA, and AMO) and cross-linker
(TMPTA) and Ttrans in soluble formulation. The relationship between transition temperature and
macromer and monomers was found to be influenced by the presence of DMAEA and AMO, which
increased Ttrans. In contrast, the presence of a cross-linker had the opposite effect.
PAGE 67
REFERENCES
1. Fitzpatrick, S.D., Jafar Mazumder, M.A., Lasowski, F., Fitzpatrick, L.E. & Sheardown,
H. PNIPAAm-grafted-collagen as an injectable, in situ gelling, bioactive cell delivery
scaffold. Biomacromolecules 11, 2261-2267 (2010).
2. Zanchi, N.E. & Lancha, A.H., Jr. Mechanical stimuli of skeletal muscle: implications on
mTOR/p70s6k and protein synthesis. European journal of applied physiology 102, 253-
263 (2008).
3. 杨恺陈君李松. Method for synthesizing acryloyl morpholine based on anhydride. in
https://patents.google.com/patent/CN104610197A/en (China, 2015).
5. Le Hegarat, L., Huet, S., Pasquier, E. & Charles, S. Impact of solvents on the in vitro
genotoxicity of TMPTA in human HepG2 cells. Toxicology in Vitro 69, 105003 (2020).
7. Rivas, B.L., Maureira, A. & Geckeler, K.E. Novel water-soluble acryloylmorpholine
copolymers: Synthesis, characterization, and metal ion binding properties. J Appl Polym
Sci 101, 180-185 (2006).
8. Khan, A. Preparation and characterization of N-isopropylacrylamide/acrylic acid
copolymer core-shell microgel particles. J Colloid Interface Sci 313, 697-704 (2007).
9. Taylor, D.K., Jayes, F.L., House, A.J. & Ochieng, M.A. Temperature-Responsive
Biocompatible Copolymers Incorporating Hyperbranched Polyglycerols for Adjustable
Functionality. Journal of Functional Biomaterials 2, 173-194 (2011).
10. Constantin, M. Lower critical solution temperature versus volume phase transition
temperature in thermoresponsive drug delivery systems. Express Polymer Letters 5, 839-
848 (2011).
11. Boutris, C., Chatzi, E.G. & Kiparissides, C. Characterization of the LCST behaviour of
aqueous poly(N-isopropylacrylamide) solutions by thermal and cloud point techniques.
Polymer 38, 2567-2570 (1997).
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12. Runt, J. & Huang, J. Chapter 8 - Polymer blends and copolymers. in Handbook of
Thermal Analysis and Calorimetry, Vol. 3 (ed. Cheng, S.Z.D.) 273-294 (Elsevier Science
B.V., 2002).
13. Milichovsky, M. Water¡ªA Key Substance to Comprehension of Stimuli-Responsive
Hydrated Reticular Systems. Journal of Biomaterials and Nanobiotechnology
Vol.01No.01, 14 (2010).
14. Deng, S., Wu, J., Dickey, M.D., Zhao, Q. & Xie, T. Rapid Open-Air Digital Light 3D
Printing of Thermoplastic Polymer. Advanced Materials 31, 1903970 (2019).
15. Anseth, K.S., Wang, C.M. & Bowman, C.N. Reaction behaviour and kinetic constants for
photopolymerizations of multi(meth)acrylate monomers. Polymer 35, 3243-3250 (1994).
16. Schiavon, O., Caliceti, P., Ferruti, P. & Veronese, F.M. Therapeutic proteins: a
comparison of chemical and biological properties of uricase conjugated to linear or
branched poly(ethylene glycol) and poly(N-acryloylmorpholine). Farmaco 55, 264-269
(2000).
PAGE 69
CHAPTER 4. THERMO-RESPONSIVE POLYMER FROM PHOTO SYNTHESIS
CHAPTER IV
Photo-Induced Bulk Polymerization
PAGE 70
4.1 Introduction
Photo-induced bulk polymerization is one alternative fabrication method for biomaterial
fabrication. Biocompatible materials play an important role in tissue engineering. The primary
function of a three-dimensional scaffold is to provide a three-dimensional environment that
resembles an in vivo environment for cell proliferation and migration. Photo-polymerization is the
process whereby liquid-polymerizable formulation is transformed into a solid by the action of light.
Advantages of this technique at fast fabrication, high spatial control, and a relatively economical
price. Overall, this process is faster and more efficient than thermally induced polymerization.
Despite the main disadvantages of oxygen inhibition and the price of monomers, this method has
been used in many fields such as protective coating, the printing industry, and dental filling
applications.1
The key photo-induced polymerization is the photoinitiator (PI). For example, Irg819 is a
photoinitiator that works by absorbing light at a specific wavelength, which leads to the formation
of free radicals. These free radicals can then initiate polymerization reactions. . 2 Generally, the
radical molecule adds to the double bond on the monomer molecules, effectively turning it into a
propagating radical. Then, it reacts with other monomers until no monomer is left or it reaches the
termination process. With a high enough concentration of PI and light intensity, the oxygen
inhibition problem could be mitigated. There are a commercialized PI available in the market.
There are two types of PI: type I PIs mainly consist of aryl ketones, which typically contain
compounds containing benzoyl groups. The photoinitiation begins with the initiator absorbing a
photon. Then, homolytic alpha cleavage occurs between the alpha carbon and the carbonyl. On the
other hand, type II PI initiators use UV light to create an excited molecule and then add electrons
PAGE 71
or hydrogen atoms from the donor molecule. This technique can process photocurable resins into
porous cell carriers via different methods such as solvent casting and film polymerization. 3
In this chapter, the relationship between the monomers NiPAAm, DMAEA, AMO, and the
cross-linker TMPTA from the previous chapter was further investigated in photo-induced bulk
polymerization to obtain a higher cross-linking density to achieve insoluble scaffold formulations.
Highly adjustable thermo-responsive discs were created with three-armed macromers (TMPTA)
incorporating a thermo-responsive backbone (NiPAAm) with cationic (DMAEA) and heterocycle
molecules (AMO). The three-armed macromer facilitates the change of material chemical
properties. The positive charge from the cation molecules could potentially disrupt the coil-globule
effect, thus increasing the energy required for the PNiPAAm network to collapse. 4 The AMO
presence could further increase the energy requirement for the PNiPAAm network by an effect
called “steric hindrance”.5 Consequently, it is crucial to optimize and acquire thermo-responsive
biomaterials with transition temperature close to physiological condition (37 °C). The experiment
covered different ratios of cross-linker, additive monomers, and photoinitiator ratios. Thereafter,
the suitable formulations were optimized, and the cells (mouse fibroblast cells, L929) were seeded
on the polymeric film to test the biocompatibility.
PAGE 72
As mentioned in the previous chapter, there is still crucial knowledge to be guided before
the three-dimensional printing technique can be used with photo-polymerization methods. In this
study, we fabricated a thermo-responsive disc to achieve a higher cross-linking ratio than with
thermally induced polymerization. Photopolymerization was used to create insoluble formulations,
where the new thermo-responsive disc can maintain its shape even at temperatures below the
transition temperature. Utilizing photo-induced polymerization, TMPTA and photoinitiator along
with monomers such as DMAEA and AMO can be polymerized within seconds, thus offering a
new method to fabricate thermo-responsive biomaterials with less time and effort. Whereas typical
polymerization such as thermal induced polymerization includes many steps and complex
purification, the photo-polymerized polymers are simple and easy, leaching out unwanted
substances such as unreacted monomers or solvent residue with step diffusion. For a cross-linked
disc to fabricate, the cross-linker (macromer) and photoinitiator (PI) have to be irradiated from a
UV wavelength source such as a UV lamp. The cross-linked scaffold should be stable enough to
hold shape on demand after polymerization. PI and unreacted monomers should be able to diffuse
out to decrease the chance of cell toxicity. After polymerization, the disc should be able to respond
to surrounding environmental cues such as changing temperature.
Irgacure 819 has been chosen investigated initiators for disc formulations. We used Irg2959
as the photoinitiator. Irg2959 is known to be biocompatible and highly efficient for UV
polymerization. The absorbance wavelength of Irg2959 is 365 nm. It also has been extensively
investigated in many publications as a photoinitiator for biocompatible hydrogels. 6-8 Thermo-
responsive discs were successfully fabricated in a cylindrical shape to test the swelling ratios as
PAGE 73
defined in materials & methods chapter for systematically varied composition ratios. The results
showed that increased cross-linker content reduced the swelling ratios. The results also showed
that the thermo-responsive effect depends on the DMAEA composition ratios. However, the major
obstacle to using Irg2959 as a photoinitiator is our three-dimensional printing system emission
light, which is an LED light source with a very specific wavelength (385 nm) , thus Irg2959 does
not suitable for our further development. Therefore, an alternative photoinitiator was chosen for
photo-polymerization in further experiments. Irgacure819 is another photoinitiator for radical
polymerization and has more efficiency at 385 nm. We reported successful disc fabrication with
both Irgacure2989 and Irgacure819 (Table 4-1). The discs were composed of 3% w/w TMPTA and
compositions as listed. Photocurable solutions without cationic monomers failed to form a cross-
linked disc. This implies the importance of cationic monomers in the photo-polymerization process.
PAGE 74
NiPAAm
CODE AMO(%w/w) DMAEA (%w/w) Cross-linked
(%w/w)
N100-D0-A0 100 0 0 -
N90-D0-A20 90 10 0 -
N80-D0-A0 80 20 0 -
N70-D0-A30 70 30 0 -
N60-D0-A40 60 40 0 -
N50-D0-A50 50 50 0 -
N90-D10-A0 90 0 10 +
N80-D20-A0 80 0 20 +
N70-D30-A0 70 0 30 +
N60-D40-A0 60 0 40 +
N50-D50-A0 50 0 50 +
N90-D50-A5 90 5 5 +
N80-D10-A10 80 10 10 +
N70-D15-A15 70 15 15 +
N60-D20-A20 60 20 20 +
N50-D25-A25 50 25 25 +
Table 4-1: Polymerization Results of Thermo-Responsive Discs with Two Minutes of Photo-
Induced Bulk Polymerization and 3% w/w TMPTA
PAGE 75
4.2.1. DSC Analysis
To find suitable resin formulations, different compositions of monomer mixtures were
cross-linked by photo-induced polymerization into nonporous thermo-responsive discs. The discs
were formed in 2 mL polypropylene molds. DSC measurement was performed to determine the
effect of cross-linker and additive monomers on the transition temperature of the thermo-
responsive disc as shown in figure 4-1. We found that the cationic monomer (DMAEA) and
network former TMPTA both significantly impacted on transition temperatures, ranging from
16.4℃ ± 0.9 ℃ (0% w/w DMAEA, 20% w/w TMPTA) to 40.0℃ ± 0.7℃ (20% w/w DMAEA,
1% w/w TMPTA) (Figure 4-2A). At a constant TMPTA content, we found increasing DMAEA
contents to elevate Ttrans. For instance, for 5% w/w TMPTA the transition temperature increased
in response to addition of 20% DMAEA from 25.9℃ ± 0.8℃ (0% w/w DMAEA) to 32.5℃ ±
2.0℃ (20% w/w DMAEA). In order to investigate the effect of TMPTA content only, we
determined Ttrans of NiPAAm in presence of 1% and 20% TMPTA and found T trans to substantially
decrease from 32.7℃ ± 0.9℃ (0% w/w DMAEA, 1% w/w TMPTA) to 16.4℃ ± 0.8℃ (0% w/w
DMAEA, 20% w/w TMPTA). As reported from thermally induced polymer in the last chapter
increased TMPTA molar feeding caused denser cross-linked network, resulting in decreasing
distance between polymer segment chains due to the trivalency of the monomer which directly
decreased material’s transition temperature.9
DMAEA and TMPTA demonstrated an impact on the transition temperature of the
NiPAAm-based networks. However, both of components also changed other key characteristics of
the materials such as density and network structure. By incorporating of a hydrophilic but
functionally inert monomer, we intended to add a degree of freedom to adjust the transition
PAGE 76
behavior of the polymeric network to the targeted temperature (33 - 36℃). The introduction of 4-
acryloylmorpholine (AMO) was investigated as a potential candidate as the comonomer. The
hydrophilic molecular character promotes water molecules into polymer network, therefore, an
increase in swelling ratio of the materials.10-12 Furthermore, AMO was expected to encourage
solubility of the polymer resin in less hydrophobic and increased biocompatibility. The presence
of AMO in the copolymer with high TMPTA increased transition temperature (Figure 4-2B). For
example, copolymerized discs with 5% w/w TMPTA, 15% w/w DMAEA and 5% w/w AMO
showed a Ttrans at 30.7℃ ± 1.7℃ which gradually increased to 37.1℃ ± 0.6℃ via increasing the
content to 20% w/w DMAEA and 20% w/w AMO. The copolymer formulations with a T trans
between 30℃ to 36℃ was considered with reference to cell survival upon provide temperature
shift.13,14 We considered resin composition with 15% w/w DMAEA as suitable choice to support
cell adhesion, as these formulations gave the best compromise between high amine content and
cell survival in an initial biocompatibility test. AMO ratios were adjusted and transition
temperature was measured. Further investigation of formulations with transition temperatures of
interest focused on those containing 1-5% w/w TMPTA, 10-15% w/w DMAEA, and 1-15% w/w
AMO, as indicated in Figure 4-1.
PAGE 77
50 50
A B
40 40
Ttrans (⁰C)
Ttrans (⁰C)
30 30
20 1%TMPTA 20
5%TMPTA 5%TMPTA
10 10%TMPTA 10
10%TMPTA
0 20%TMPTA
0
0 10 20 30 0 5 10 15 20 25
DMAEA (%w/w) AMO (%w/w)
50
C C
45
Ttrans (⁰C)
40
35
N70-D15-A15
30 N60-D20-A20
N50-D25-A25
25
0 3 6 9 12
TMPTA (%w/w)
Figure 4-1: DSC analysis of Ttrans of thermo-responsive discs with 1-20% w/w TMPTA. 0-20%
w/w DMAEA and 0% w/w AMO (A). The effect of AMO on disc’s transition temperature (5-10%
w/w TMPTA, 15% w/w DMAEA) (B). The effect of TMPTA (1–10% w/w) on transition
temperature in different formulations (N70-D-15-A15, N60-D20-A20, and N50-D25-A25) (C).
PAGE 78
Figure 4-2: DSC thermogram analysis of 3% TMPTA N80-D15-A5 shows the transition
temperature at around 34℃.
4.2.2. Effect of Comonomer on Swelling and Analysis
In order to evaluate of the discs in response to the change of temperature we determined
the swelling ratios of different resin formulation was determined at 25℃ and 37℃ as shown in
Figure 4-3. We began to lay our focus on the impact of DMAEA and AMO in T trans the reference
from 15% to 25% w/w with cross-linker content of 2.5%, 5%, and 7.5% w/w. Discs with 25% w/w
AMO and 25% w/w DMAEA had obviously higher transition temperature than 20% w/w and 15%
w/w DMAEA and AMO formulations for all cross-linker additions. The disc with 2.5% w/w cross-
PAGE 79
linker (N70-D15-A15) had the lowest Ttrans at 34.8℃ ± 0.6℃, which can be increased to 44.2 ℃
± 0.1 ℃ by increasing the DMAEA and AMO contents to 25% w/w.
To more accurately investigate the swelling ratios, we examined discs with varying
amounts of cross-linker at both 37 °C and 25 °C. The discs with 2.5%, 5%, and 7.5% w/w TMPTA
(15% w/w DMAEA and AMO) successfully shrank by (-0.2 ± 0.1) g/g, (-0.8 ± 0.1) g/g, and (-0.8
± 0.1) g/g. Lower TMPTA contents resulted in higher water uptake by the discs as a result of
decreasing cross-linking density. As the swelling changed in response to a decrease in temperature
from 37 °C to 25 °C, we defined the parameter ΔSR(37/25) as the difference in swelling ratios.
The values of ΔSR(37/25) between 15% w/w DMAEA and AMO and 25% w/w DMAEA and
AMO were (11.5 ± 0.8), (1.4 ± 0.8), and (1.0 ± 0.7) at 2.5%, 5%, and 7.5% w/w TMPTA,
respectively. These values represent the difference in ΔSR between the two formulations, not the
ΔSR(37/25) of each individual formulation. The ΔSR(37/25) helps to indicate the changes in
material swelling of the differently composed discs in the relevant temperature range and to identify
the effects of the comonomers.
PAGE 80
Figure 4-3: Swelling ratios (A–C) of thermo-responsive discs from different cross-linker content
(2.5, 5, and 7.5% w/w TMPTA) and monomer compositions (N70-D15-A15, N60-D20-A20, and
N50-D25-A25) between 25℃ and 37℃. The ΔSR(37/25) and the scaffold T trans of N70-D15-A15,
N60-D20-A20, and N50-D25-A25 are shown in (D).
After reviewing the previous results, we focused on optimizing the formulaic scaffold of
thermo-responsive formulations that respond within a physiological range. Specifically, we
compared the transition temperatures of discs with different concentrations of AMO (5% and 10%
w/w) and low concentrations of cross-linker network (1, 2, and 3% w/w). We found that the discs
with the 10% w/w AMO and 15% w/w DMAEA (N75-D15-A10) formulation had significantly
higher transition temperatures than the discs with the 5% w/w AMO (N80-D15-A5) formulation
PAGE 81
for all TMPTA concentrations. The discs with 1% w/w TMPTA and 15% w/w DMAEA and 5%
w/w AMO had the lowest transition temperature at 32.1°C ± 0.3°C, while increasing the AMO
content to 10% w/w resulted in a higher transition temperature of 34.6°C ± 0.9°C.
Swelling ratios between 37℃ and 25℃ were clearly seen affected by the cross-linker
content: Discs with 3% TMPTA and 10% w/w (N75-D15-A10) or 5% w/w (N80-D15-A5) AMO
successfully shrank by -1.2 ± 0.4 g/g and -1.00 ± 0.1 g/g. The other TMPTA formulations (1% and
2% w/w TMPTA) failed to response. This indicates the lower TMPTA ratio increases the
polymeric network density, thus allow more water molecules into scaffold matrix to the point that
the cross-link network unable expulse water molecules. The comparison of absolute relative change
of ΔSR(37/25) between 5% w/w AMO and 10% w/w AMO were (0.6 ± 0.5), (1.1 ± 0.6), and (0.8
± 0.2) times at 1, 2, and 3% w/w TMPTA. AMO has found to be affected on the thermo-responsive
discs through it hydrophilic and non-ionic molecular interaction, resulting facilitation of water
molecules into the polymeric network and increased swelling ratio. 15 The ΔSR(37/25) results
indicated the thermo-responsiveness of disc depends to the compositions such as TMPTA,
DMAEA and AMO.
PAGE 82
Figure 4-4: Swelling ratio at different temperatures (37 °C and 25 °C) and T trans of 1% TMPTA
(A), 2% TMPTA (B), 3% TMPTA cross-linker(C), and SR(37/25) of 1%, 2%, and 3% TMPTA
cross-linker (D). Negative value indicates shrinkage of sample discs upon heating.
PAGE 83
4.2.3. Polymeric Film Cytocompatibility
With the promising results on swelling difference in response to temperature, we chose a
scaffold formulation with 3% w/w TMPTA, 4% w/w PI, which reportedly has T trans of 36.5℃ ±
0.4℃, ΔSR(37/25) of -0.8 ± 0.2 g/g for preliminary biocompatibility determination. The materials
are expected to support the cells with cationic-molecular affiliation. First, the polymeric resin-
mixture were fabricated into a thin film, on the silanized glass slides and seeded with L929 mouse
fibroblast cells and observed for 24 hours. DAPI and Alexa staining were used to test
biocompatibility with the polymer. The result showed that the cells could survive and attach to the
film in 24 hours. The polymeric film showed biocompatibility with no significant difference
observed on the cell number density.
Figure 4-5: Fluorescence image of L929 cells on 3% w/w TMPTA (4% w/w PI) thermo-responsive
polymeric films after 24 hours at constant temperature (37°C). Cells were stained with DAPI,
Alexa-Fluor 488® staining, and white light.
PAGE 84
10
Number of cells (103)/cm²
7.5
2.5
0
N80-D10-A10 N70-D15-A15 N60-D20-D20 N50-D25-A25
Figure 4-5: The average L929 cell numbers per cm 2 on polymeric films containing 3% w/w
TMPTA and 4% w/w PI were investigated under different compositions after 24 hours. The
initial cell density was set at 3 × 103 cells per cm2
4.3. Conclusions
The cross-linked thermoresponsive discs were successfully formulated and determined for
thermo-responsiveness. The photocurable thermo-responsive discs (3% w/w TMPTA, N80-D15-
A5, 4% w/w PI) were identified as most potential to be a candidate for advancement of three-
dimensional printing. Initial biocompatibility results showed promising cell attachment to the
polymer film. The results suggest the thermo-responsive photocurable solution can be tested
toward more complicated scaffold structure such as three-dimensional scaffold for further
investigation toward Digital Light Processing 3D printing and mechanical stress delivery to
adherent cells for tissue engineering applications.
PAGE 85
REFERENCES
1. Peiffer, R.W. Applications of Photopolymer Technology. in Photopolymerization, Vol.

673 1-14 (American Chemical Society, 1997).
2. Meereis, C.T., et al. BAPO as an alternative photoinitiator for the radical polymerization
of dental resins. Dent Mater 30, 945-953 (2014).
3. Rueggeberg, F.A., Ergle, J.W. & Lockwood, P.E. Effect of photoinitiator level on
properties of a light-cured and post-cure heated model resin system. Dent Mater 13, 360-
364 (1997).
15096 (2019).
5. Voronenkov, V.V. & Osokin, Y.G. Effects of Steric Hindrance in Molecules on the
Properties and Synthesis of Hydrocarbons. Russian Chemical Reviews 41, 616-629
(1972).
6. Coimbra, P., Fernandes, D., Ferreira, P., Gil, M.H. & de Sousa, H.C. Solubility of
Irgacure (R) 2959 photoinitiator in supercritical carbon dioxide: Experimental
determination and correlation. J Supercrit Fluid 45, 272-281 (2008).
7. Xu, H., Casillas, J., Krishnamoorthy, S. & Xu, C. Effect of Irgacure 2959 and lithium
phenyl-2,4,6-trimethylbenzoylphosphinate on cell viability, physical properties, and
microstructure in 3D bioprinting of vascular-like constructs. Biomed Mater (2020).
8. Liu, M., Li, M.D., Xue, J. & Phillips, D.L. Time-resolved spectroscopic and density
functional theory study of the photochemistry of Irgacure-2959 in an aqueous solution. J
Phys Chem A 118, 8701-8707 (2014).
Vol.01No.01, 14 (2010).
Sci 101, 180-185 (2006).
PAGE 86
13. Yoon, S.K., Kim, S.H. & Lee, G.M. Effect of low culture temperature on specific
productivity and transcription level of anti-4-1BB antibody in recombinant Chinese
hamster ovary cells. Biotechnol Prog 19, 1383-1386 (2003).
14. Weidemann, R., Ludwig, A. & Kretzmer, G. Low temperature cultivation--a step towards
process optimisation. Cytotechnology 15, 111-116 (1994).
15. Hülya Efe, M.B., Memet Vezir Kahraman and Nilhan Kayaman-Apohan. Synthesis of 4-
acryloylmorpholine-based hydrogels and investigation of their drug release behaviors.
Journal of the Brazilian Chemical Society 24, 814-820 (2013).
PAGE 87
CHAPTER 5. Fabrication of Thermo-Responsive Scaffolds from DLP Printing
CHAPTER V
Fabrication of Three-dimensional Thermo-Responsive
Scaffolds from continuous Digital Light Processing Printing
PAGE 88
5.1. Introductions
One of the major challenges in medical treatment is the repair or replacement of damaged
tissues or organs due to the limited availability of such tissues and the immune response they may
trigger.1 Despite the promising results of tissue engineering research, there are still many obstacles
to overcome due to the complex nature of tissues. In vitro studies, which are conducted in a
laboratory setting, have provided valuable insights, but there is a need to translate these findings to
in vivo settings, where tissues function in a living organism. Three-dimensional scaffolds offer the
potential to mimic in vivo conditions through the use of 3D cell culture. However, these scaffolds
still lack many of the key factors that are present in vivo, such as mechanical stimulation. The
incorporation of active stimuli-responsive materials, or smart materials, that respond to
environmental cues such as pH and temperature, could provide a way to mimic the mechanical
stimulation that tissues experience in vivo. Such materials have the potential to be used in various
applications in bioscience and regenerative medicine, including scaffolds, to provide the necessary
stimulation for tissue growth and repair.2-7
Thermo-responsive monomers such as NiPAAm are used in polymeric network chains to
create thermo-sensing scaffolds via various techniques as described in a number of publications. 8-

11
Three-dimensional printing technologies have gained significant attention in biomaterial
research for their ability to create complex hierarchical structures. The potential for these
technologies to meet clinical standards presents promising opportunities for organ transplantation
and tissue engineering, and has attracted interest from medical professionals and researchers
worldwide as a way to improve patient quality of life.9,11,12 Furthermore, the three-dimensional
printing market is expected to double in size from 16 to 40.8 billion dollars by 2024. 13
PAGE 89
In order to achieve this, continuous Digital Light Processing (cDLP) is one of the three-
dimensional lithography printing techniques that enables photocurable solutions to fabricate
complex structural scaffolds with UV light irradiation layer by layer. This technique allows for a
fabrication speed faster than nozzle or laser techniques and considerably precise as much as 20 µm.
Three-dimensional lithography printers are widely available in the market and can work with
various photocurable materials, including poly(caprolactone) (PCL) 14-16, poly(lactic acid)17, and
NiPAAm.18 However, numbers of publication pointed out that biocompatible materials for photo-
polymerization are limited.8,19,20
The study presented a biocompatible alternative material for photocurable solvent-based
drug delivery systems. The material has been demonstrated to have the potential for improved
scaffold resolution and biocompatibility in both human and animal subjects. 21-23 In this study, a
thermoresponsive scaffold material was developed using a combination of a biocompatible cross-
linker and monomers through a process known as continuous digital light processing (cDLP). The
resulting photocurable resin had the ability to create a three-dimensional platform that was
adjustable in its swelling properties. The addition of a positive charge monomer to the platform
allowed for improved cell adhesion through the use of electrostatic interactions between the
positively charged surface of the scaffold and negatively charged cells.24 These results can be
further explored through biocompatibility tests with different cell types from animals and humans.
All photoresist formulations were prepared using NiPAAm as back-bone monomer,
trimethylolpropane triacrylate (TMPTA) as cross-linker, dimethylaminoethyl acrylate and AMO
as active molecules, and Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide as photoinitiator. All
PAGE 90
of the substances described above were dissolved in absolute ethanol or glycofurol. For the scaffold
to be fabricated, the photocurable solution was transferred into the building tray of the printer. The
PNiPAAm-based scaffolds were printed as mentioned by the continuous digital light processing
method (or cDLP). The configurations feature a lattice-shaped scaffold (height × width × height =
7.6 × 7.8 × 2.5 mm) and raft-shaped structure comprising 50 and 30 layers, respectively. The layer
thickness of printing was 50 µm per layer. Each layer was used at various curing times as shown
in the figures.
Initially, ethanol was used as to prepare a photocurable solution. While this allowed for the
creation and testing of complex structural scaffolds, we faced difficulties during the printing
process, such as premature detachment from the printing head and imperfections in the scaffold
structure.
In order to improve the printing quality of the scaffolds, we decided to switch to a new
solvent medium, glycofurol (GF), which has been widely used in the pharmaceutical industry and
has shown good biocompatibility with animal and human tissues and cells. GF has been
demonstrated to be biocompatible in vivo and is also used in drug delivery systems, making it a
suitable choice for printing scaffolds with improved biocompatibility. 21-23,25 As it has been
demonstrated GF in 50:50 mixture with PBS was injected into rat brain tissue and was well
tolerated and showed minimal inflammatory.23 Furthermore, GF has higher viscosity than ethanol.
Increasing viscosity of GF could improve scaffold resolution while printing due to damping
effect.21,26 Although there may be some residual GF released from the scaffolds, it is expected to
be present at concentrations lower than those tested in previous studies and is not expected to have
a negative impact on cellular response. However, future research should include a chromatographic
evaluation of the residual GF to confirm this.Scaffolds fabricated with glycofurol as the solvent
PAGE 91
showed a more uniform shape and no premature detachment of scaffolds was observed. In addition,
it was possible to reduce the printing time per layer to as low as 10 seconds per layer using
glycofurol.
5.2.1. Thermo-responsiveness of three-dimensional Scaffolds
The swelling ratio of the scaffolds was determined using the method described in a previous
chapter. This involved washing the scaffolds with water to remove unreacted monomers and
solvent, and then measuring the scaffold weights under different temperature conditions at 37°C
and 25°C. The results showed that the scaffolds with a higher percentage of Irg819 had a lower
swelling ratio due to the stronger cross-linking of the network. This finding is consistent with the
longer printing time, which causes stronger cross-linking network and lower swelling ratio. 27
Figure 5-1: The 3D raft and lattice scaffold visualization of the 3% w/w TMPTA (N80-D15-A5)
in hydrated and lyophilized structures (scale bar: 1 cm) compared to the three-dimensional design.
The voxel size are: 7.6 × 7.7 × 1.8 mm and 7.6 × 7.7 × 2 mm. The raft structure is created using
side-by-side fibers to create a visible surface for cell adhesion, preventing cells from penetrating
PAGE 92
deeper layers. The 3D lattice scaffold offers an open, porous structure for cells to grow and attach
in three dimensions.
Based on the previous results, we selected a promising formulation (3% w/w TMPTA) for
the processing with cDLP. In these first experiments, we investigate the effect of exposure time
per layer, photo-initiator ratio (PI), and the solvent on the scaffolds properties. The raft
configuration was fabricated for the purpose of evaluating the biocompatibility of the material. The
planar structure of the scaffold facilitates the monitoring of cellular proliferation. Conversely, the
lattice configuration possesses a macroporous architecture, comprising 50 layers, each layer
possessing a thickness of 50 µm, with an overall dimension of 7.6 x 7.8 x 2.5 mm (L x W x H).
The macroporous lattice design allowing good cell adhesion and efficient nutrient supply via
interconnected channels.28,29 The model and picture of the scaffold are demonstrated in figure 5-
1. The resin formulations were processed with various exposure time per layer (10, 20, and 30
seconds).
The crucial property for the thermo-responsive scaffold is to respond to changing
temperature. In order to assess these property we measured the scaffold swelling ratios in response
to variable factor such as concentration of PI and exposure time, that are known to affect the
network modulus of the scaffold. Higher PI concentration and longer exposure time per layer where
hypothesized to swelling ratio of the materials (figure 5-2) as higher exposure time and PI
concentration causes increased polymeric network density that reduces interactions between
polymer chains and water molecules when the temperature reaches T trans. We observed strongest
thermo-responsiveness in swelling with 4% w/w PI at exposure time of 10 seconds, which have
1.3 ± 0.03 times stronger ΔSR(37/25) than 2% PI, 10 seconds. The thermo-responsiveness of the
PAGE 93
scaffolds between 25°C and 37°C increased with decreased exposure time and increase PI
concentration. We found no significant difference of T trans between PI content and exposure time.
Figure 5-2: The swelling ratio of a 3% TMPTA N80-D15-A5 scaffold is shown for two
formulations with 2% (A) and 4% (B) w/w photo-initiator (Irg819), respectively. The swelling ratio
is expressed in terms of the weight of the scaffold before and after swelling (g/g). The T trans values
for the scaffold, as determined by differential scanning calorimetry (DSC), are also shown on the
graphs. (C) The ΔSR (37/25) values for these formulations are also included.
5.2.2. Three-dimensional Scaffold Fabrication by Glycofurol (GF)
In previous experiments, using ethanol as a solvent or diluent hindered the printability of
scaffolds made from photocurable resins. This was due to issues such as premature detachment of
the scaffold from the printing head and low structural resolution due to low resin viscosity. The
introduction of GF, an amphiphilic biocompatible solvent, can minimize or even overcome these
PAGE 94
challenges.30 GF has been widely used as a biocompatible solvent in biomedical applications and
also has the potential to be used as a 3D printing medium. 23,26,30-32
Using GF-containing resins allowed us to maintain the thermo-responsive properties of the
scaffold and significantly reduce the exposure time per layer from 20 to 10 seconds. Additionally,
the problem of scaffold detachment from the printing head during the printing process was
resolved. Increasing concentrations of the cross-linker have improved the strength of the polymeric
network. The difference in TMPTA concentrations in the scaffolds are clearly shown in Figure 5-
3, with lower TMPTA scaffolds appearing more transparent than those with 10% and 20% TMPTA
above the transition temperature.
Figure 5-3: GF-based raft scaffold with 5, 10, and 20% w/w TMPTA in cell culture medium prior
to cell seeding at 37℃ (N80-D15-A5, 10 sec/layer, 4% Irg819). At the same temperature, the
scaffolds with higher TMPTA ratio showed smaller and more compact configuration compared to
5% TMPTA. The differences in scaffold size and structure are highlighted by the gray dash pattern.
PAGE 95
GF-diluted resins compared to ethanol-printed resin allowed for the halving of exposure
time per layer from 20 seconds to 10 seconds. Additionally, the use of these resins prevented
premature detachment of the printed construct from the printing head during the printing process.
The swelling ratios of GF-based scaffolds with various ratios of DMAEA and AMO were
determined at two temperatures (25°C and 37°C). Lattice scaffolds fabricated from resins without
DMAEA and AMO (3% TMPTA N100-D0-A0) displayed the highest change in swelling due to a
lowest transition temperature of 28.0°C, which is consistent with the expected results for a pure
cross-linked NiPAAm matrix. As anticipated, the addition of AMO and DMAEA increased the
transition temperature as already described for the ethanol-based resins. The AMO content
effectively increased Ttrans from 33.9°C ± 0.3°C (N85-D15-A0) to 38.6°C ± 1.2°C (N65-D15-A20).
The transition temperature of the scaffolds with different ratios of DMAEA and an AMO content
of 15% was elevated from 30.9°C ± 0.5°C (N85-D0-A15) to 39.5°C ± 0.7°C (N65-D20-A15). The
corresponding values of ΔSR (37/25) decreased from -4.5 ± 0.1 g/g (N85-D0-A15) to -0.8 ± 0.2
g/g in (N65-D20-A15). These results demonstrate the effect of DMAEA that increased T trans and
stabilized NiPAAm-based networks, thereby reducing ΔSR (37/25) and improving the material's
suitability for cell adhesion. It is desirable for ΔSR (37/25) values to be negative but in a moderate
range (-3 to -2 g/g) to maintain cell adhesion to the biomaterial surface.
PAGE 96
Figure 5-4: The swelling behavior of 3D scaffolds printed from GF-based resins (3% w/w TMPTA,
4% w/w PI, printing time 10 s/layer) was studied at different temperatures (25°C and 37°C) and
with different ratios of (A) 0-20% w/w AMO (15% w/w DMAEA) and (C) 0-20% w/w DMAEA
(15% w/w AMO). The transition temperature of the scaffolds was also evaluated. The resulting
ΔSR (37/25) is shown in charts B and D.
We selected the optimized formulation of DMAEAs and evaluated the impact of AMO on
scaffold properties. We varied the percentage of AMO within a fixed range of DMAEA and
constant ratio percentage, and thoroughly analyzed the effect of AMO on the scaffolds (Figure 5-
5). The scaffolds that did not contain AMO exhibited a decline in T trans from 34.9 °C to 33.5 °C,
when compared to the (N80-D15-A5) and (N85-D15-A0) formulations that had 5% AMO. These
results were consistent with previous experiments that showed that AMO can increase the transition
temperature in TMPTA scaffolds due to a steric hindrance effect. While we did not observe a
significant difference in the swelling ratio between those in 20% w/w DMAEA group, the influence
of AMO on the transition temperature was still detectable.
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Figure 5-5: The swelling behavior of 3D scaffold from GF-based resins (3% w/w TMPTA, 0-5%
w/w AMO, 15% w/w DMAEA, 4% w/w PI, printing time 10 sec/layer) at different temperatures
(25℃ and 37℃) with the transition temperature. The resulting ΔSR (37/25) is shown in the table.
5.2.3. Rheological Properties of Scaffolds
Altering the cultivation temperature is expected to alter the swelling of the scaffold and
apply mechanical stimulation, both of which are important factors that can affect cell fate. The
scaffold's rheological properties were characterized using oscillation rheology, which showed that
the scaffold's moduli were within the range of the cellular stiffness reported in the literature
(myoblasts: 2 kPa, fibroblasts: 1-10 kPa).33,34 As shown in Figure 5-6, the storage moduli of the
printed scaffolds increased with the exposure time per layer (5-6A) and the concentration of the
photo-initiator (5-6b). For example, at Ttrans, the storage moduli of the samples fabricated with 4%
photo-initiator and 10 seconds of exposure per layer were 1.2 and 1.5 times higher than the samples
with 3% and 2% photo-initiator, respectively. Increasing the exposure time per layer from 10
seconds to 20 and 30 seconds doubled the moduli. The transition temperature remained unchanged
PAGE 98
as the altered parameters (concentration of photo-initiator and exposure time per layer) did not
affect the chemical composition of the network, but did affect the network density as observed by
the changes in swelling. The addition of DMAEA significantly decreased the scaffold moduli
(Figure 5-7). At 20% w/w DMAEA, the hydrophilic contribution of the cationic monomer was
strong enough to completely suppress the coil-globule transition of the copolymeric network and
no further changes in storage modulus were observed within the investigated range, which
correlates with the observed increase in transition temperature. With the exception of N93-D2-A5,
the investigated samples (Figure 5-7) showed very little temperature-induced change in the bulk
storage moduli. However, it is important to consider that the volume change of the construct and
the corresponding displacement of the focal adhesion points with the cells are the primary causes
of mechanical stress on the adhered cells. Therefore, the relationship between the volume changes
that maintain cell adherence and cell survival and the change in storage modulus of the bulk
hydrogel is not clear and requires further investigation.
PAGE 99
PAGE 100
Figure 5-6: Rheological temperature sweep measurements from 20°C to 50°C (3 K min-1) were
conducted to investigate the influence of (A) different printing times per layer (10, 20, and 30
sec/layer) and (B) photo-initiator ratios (2, 3, and 4% w/w) on the storage modulus of printed
lattice-type scaffolds (N80-D15-A5, 3% TMPTA, diluent: glycofurol). The transition temperatures
of the scaffolds, as determined by differential scanning calorimetry (DSC), are summarized in the
bottom table. The moduli increased with the exposure time per layer and the photo-cross-linker
content. During the temperature sweeps, the moduli increased when the transition temperature was
passed due to increased polymer-polymer interactions and decreased hydration.
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Figure 5-7: The oscillatory rheological storage modulus of 3% TMPTA scaffolds printed with
different ratios of DMAEA (5% AMO, diluent: glycofurol, 4% Irg819, printing time 10 sec/layer)
was measured using a temperature sweep at 1Hz and an amplitude of 1% from 20°C to 50°C and
back down to 20°C with a ramp of 3 or -3 K min-1. The Ttrans of the scaffolds, as measured by
differential scanning calorimetry (DSC), is shown in the table below and indicated in red arrow.
As the content of DMAEA increased, the hydration of the scaffold increased, the transition
temperature increased, and the modulus decreased. As a result, the phase transition-induced
increase in storage modulus during the temperature sweep was less pronounced in materials with
higher DMAEA content.
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The results of this study show that the macromer, photoinitiator, monomer compositions,
and UV exposure time per layer can all affect the transition temperature and mechanical strength
of thermo-responsive scaffolds during photo-polymerization. To more fully understand the factors
that impact scaffold properties and identify technical challenges, it is recommend to conduct
comparative studies using different printing techniques and optimized formulations for different
photocurable resin. Additionally, advances in three-dimensional fabrication strategies, such as
biofabrication and bioprinting, may be useful for optimizing the biocompatibility of scaffolds. 9,12
5.3. Conclusions
Three-dimensional thermo-sensitive scaffolds composed of P(NiPAAm-co-DMAEA-co-
AMO) cross-linked with TMPTA were successfully fabricated through the use of a three-
dimensional printing technique. The inclusion of glycofurol as a photo-polymerization solvent
enabled the production of scaffolds that were capable of undergoing mechanical stimulation via
water uptake and changes in morphology. The optimal composition of glycofurol for three-
dimensional photo-polymerization was determined to be 5% w/w AMO, 15% w/w DMAEA, and
3% w/w TMPTA, with a transition temperature of 36.3°C ± 0.9°C. Additionally, the effects of
various parameters, including curing time and monomer composition, on scaffold properties were
investigated. These findings provide valuable insights towards the development of mechanical
stimulating platforms and their potential applications.
PAGE 103
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14. Yeong, W.Y., et al. Porous polycaprolactone scaffold for cardiac tissue engineering
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20. Zhang, J. & Xiao, P. 3D printing of photopolymers. Polymer Chemistry 9, 1530-1540
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PAGE 106
CHAPTER 6. THREE-DIMENSIONAL SCAFFOLD BIOCOMPATIBILITY
CHAPTER VI
Three-dimensional Thermo-responsive Scaffold
Biocompatibility
PAGE 107
6.1 Introduction
Stimuli-responsive biomaterials, which can respond to changing external stimuli, offer a
way to alter the surrounding cell microenvironment on demand. The first report of such a
responsive material was in 1945 for barium titanate, an inorganic compound that exhibits the
piezoelectric effect.1 The class of stimuli-responsive materials is broadly diverse and based on the
category of stimuli. The responsive materials can be categorized as thermo-2, photo-3, pH-4,
mechanical-5, potential-6, magneto-7, and biochemical-responsive materials.8 The ability of
responsive materials to be programmed on demand can provide crucial biodynamic and
biochemical effects for cells and surrounding tissues. One example is the thermo-responsive
poly(N-isopropylacrylamide), or P(NiPAAm) hydrogel. This material has been used to generate
compressive or expansive tension for cells based on the hydrogel swelling or shrinking in response
to different temperature conditions. P(NiPAAm) is also known to be easily chemically
manipulated.9
Cell adhesion on the scaffolds is a critical property to consider when evaluating 3D
thermo-responsive materials.10 Such material was conservatively designed for the cells to detach
from material’s surfaces at below material’s transition temperature.11 In this material, however, we
propose an alternative strategy for mechanical stimulation on adherent cells via a stimulus-
responsive scaffold. A platform using N-isopropylacrylamide (NiPAAm)-based thermo-responsive
material is thoroughly investigated.12,13 As it has been shown that mammalian cells can be cultured
between 30 to 37 °C without any negative effect on cell development. 14-16
The surface architecture and morphology of biomaterials are known to have important
functions in biological systems, as they influence the behavior of cells and tissues. 10,17,18 There are
PAGE 108
many factors that must be considered in order to improve the compatibility of biomaterials with
physiological tissue. For example, the surface topology or roughness of the material can be divided
into three categories: nano-roughness (<100 nm), micro-roughness (100 nm–100 μm), and macro-
roughness (from 100 μm to several millimeters).19 Cellular responses to material surfaces can vary
and depend on the cell type and roughness scale. Therefore, roughness can regulate and influence
the biological response of cells and tissues in contact with the material. There are reports that cells
can proliferate and spread better on micro-rough surfaces than on smooth ones, and that cells can
align themselves in the direction of grooves and grow faster on these surfaces. 20 In addition to the
morphology of biomaterials, porosity is also an important requirement for scaffolds in tissue
engineering applications. The tailored, open-pore and interconnected structure allows cells to
attach and proliferate, improving cell ingrowth on the scaffold, which is essential for new tissue
formation and overall improved wound healing.18,21 Another strategy to improve cell attachment
to scaffolds is to modify the surface properties, such as hydrophobicity. The hydrophobicity of a
biomaterial surface can affect the ability of cells to attach to it and can regulate the organization of
the cell cytoskeleton and cell morphology as well as their function. In general, cells prefer
hydrophilic surfaces over hydrophobic ones. However, more hydrophobic surfaces tend to have
more protein adsorption due to the interaction between the hydrophobic molecules on the material
surface and the hydrophobic groups of proteins, which their contact and cell into action. 22
In chapter fifth, the three-dimensional thermo-responsive scaffolds and their transition
temperatures were characterized. A specific focus was laid on the GF-printed thermo-responsive
material (3% w/w/ TMPAT, 15% w/w DMAEA, 5% w/w AMO) with exposure time 10 seconds
per layer that has moderate swelling ratio and transition temperature within the range of
physiological temperature. To examine cell adhesion on the scaffolds, various cell types (L929s,
PAGE 109
C2C12s, hMSCs, hASCs) were seeded directly on the thermo-responsive scaffold surfaces and
cultivated in static and periodic temperature. The ability to control scaffold morphology and
porosity through three-dimensional printing was also utilized to manipulate the cells. In this way,
the thermo-responsive polymers were used to deliver mechanical force to the cells in periodic
temperature cultivation.
In a first step we choose a well-known mouse fibroblast cell line (L929) to use as a model
at the static cultivation temperature of 37 °C. The cells were tested in the raft and lattice
configurations over a seven-day period. We hypothesized that the positive charge from the
DMAEA molecules in the scaffolds would provide an anchor for the cells to attach to that could
withstand the mechanical stress from changing temperature conditions. Various parameters that
could affect biocompatibility were also evaluated, including exposure time per layer, post-
treatment, photo-initiator concentrations, and monomer concentrations. The next experiment
included the scaffolds with seeded cells cultivated in periodic temperature conditions to observe
the mechanical stimulation between scaffolds and cells. Finally, the scaffolds were modified with
poly(L-lysine) to further improve their biocompatibility with human-derived cells.
In the first step, we have chosen the most promising resin formulation (3% w/w TMPTA
N80-D15-A5. The formulation modulus is well aligned within the range of 1-10 kPa that is
described as being physiological for the L929s.23 The formulation is hypothesized to support cell
proliferation and viability through shifting cultivation temperature and thereby modulus. First, the
effect of exposure time per layer (10 to 30 sec/layer) and PI concentration (1 - 4% w/w) was tested
with L929 cells (105 cells) at a constant temperature of 37°C (Figure 6-1). All cell were stained
PAGE 110
with calcein-AM and ethidium homodimer to differentiate live (green) and dead (red) cells. At the
end of the experiment (7 days), all formulations showed cell coverage with live cells and high cell
density, indicating cell viability and proliferation. No noticeable differences between the
formulations were observed only few dead cells were detected (Figure 6-1). Fibroblasts in the
scaffolds displayed a well-spread morphology. The diffuse character of the fluorescence signal and
some degree of autofluorescence from the material were also noted. These results indicate that
scaffold biocompatibility was not influenced by photoinitiator concentrations and curing time in
the printing process.
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Figure 6-1: Fluorescence microscopy images of L929 fibroblasts on 3% TMPTA, N80-D15-A5
scaffolds at 37℃ are shown after 7 days of culture. The images show the effects of different
photoinitiator ratios (horizontal) and exposure times (vertical) on the cells after live/dead staining.
Small images in each group show the green fluorescence channel for calcein (green dash box) and
the fluorescence wavelength for ethidium homodimer in red color (blue dash box). The large
images in each group show an overlay of both channels (red dash box). Scale bars represent 500
µm
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6.2.1. With/Without After Treatment
In the process of lithography, the scaffolds are typically treated with UV-light after photo-
induced polymerization to strengthen the bond between molecules and polymerize any unreacted
monomers.24 However, with our scaffolds formulations that already contained molecules that
susceptible to high energy excitation such as the cationic DMAEA, which may have been severely
degraded under UV radiation, reducing the scaffold's ability to support cells. 25 We found that the
scaffolds with UV treatment had lower cell density compared to the scaffolds without UV treatment.
At the start of the experiment, the cells on the treated scaffolds were found to attach less to the
scaffolds. This difference became more noticeable at the end of the experiment after one week,
when there was a higher live-cell signal on the scaffolds without UV treatment than on the scaffolds
with UV treatment.
PAGE 113
Figure 6-2: Fluorescence images of L929s on 3% TMPTA, N80-D15-A5 scaffold with raft
architecture (37 ℃) with and without UV treatment between day 1 and 7 are shown. The images
show an overlay of the fluorescence channel for calcein (green) and the fluorescence wavelength
for ethidium homodimer (red). Scale bar = 500 µm.
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Figure 6-3: Fluorescence images of L929s on 3% TMPTA, N80-D15-A5 scaffold (37 ℃) with and
without UV treatment between day 1, 3, 5, and 7 are shown. The images show an overlay of the
fluorescence channel for calcein (green) and the fluorescence wavelength for ethidium homodimer
(red). Scale bar = 500 µm.
6.2.2. The Scaffold Fiber Sizes and Photoinitiator Concentrations
There are several reports that suggest that the fiber in the scaffold structure can have an
impact on the scaffold's compatibility.26-28 This may be due to the mechanical properties of the
scaffold, such as its stiffness or strength, which can influence the behavior of the cells or tissues.
Additionally, the size of the beam-like structures or fibers may also affect the scaffold's ability to
transport nutrients and oxygen to the cells, as well as its ability to remove waste products. 29
Therefore, it is important to carefully consider the size of these structures when designing scaffolds
for specific applications.30 In our experiment, we observed that the fiber size in the scaffold
significantly impacted the growth of fibroblasts after a 7-day cultivation period (Figures 6-3 and
6-4). Cell density on scaffolds with thinner fibers (240 µm) was highercompared to those with
thicker fibers (470 µm). It is possible that the increased surface area of the scaffolds with thinner
fibers contributed to their greater cell proliferation.30 It is also worth noting that there were
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differences between the treated and untreated scaffolds, with the treated scaffolds showing less cell
proliferation. These results support the findings from the previous paragraph, which showed that
the post-treatment of the scaffolds with photo-induced polymerization had a negative impact on
their cytocompatibility.
Figure 6-4: Fluorescence microscopy images of L929 fibroblasts on 3% TMPTA, N80-D15-A5,
and 10 s/layer scaffolds with a raft architecture (37°C) at day 7. The horizontal axis shows the
scaffolds with thin (upper) and thick fibers (lower). The vertical axis shows scaffolds with (left)
and without UV (right) treatment. The images show an overlay of the fluorescence channel for
calcein (green) and the fluorescence wavelength for ethidium homodimer (red). Scale bar: 500 µm
(4x) and 50 µm (10x). Size: 7.6 × 7.6 × 1.35 mm.
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6.2.3. GF as Solvent for Three-Dimensional Printing
With ethanol as a solvent in printing process, we observed non-uniform configuration of
the scaffolds because ethanol is water soluble and has a very low viscosity equal to water. Little
movement or vibration from the printing procedure may cause the printed layer to dislocate, thus
affecting scaffold integrity or causing premature scaffold detachment altogether. Glycofurol
(tetrahydrofurfuryl alcohol polyethylene glycol ether, described herein as GF) is an alternative
water-soluble solvent and is one of the biocompatible solvents utilized in particular pharmaceutical
formulations.31-36 To the best of our knowledge, such use of GF has never been described anywhere
for cDLP photosensitive printing. Hence, glycofurol has been introduced as a solvent in this
experiment for its biocompatibility and viscosity that is suitable for three-dimensional printing. As
we have shown in the previous chapter, the glycofurol-printed scaffolds have similar properties
compared to the ethanol-printed scaffolds.
The C2C12 mouse myoblasts have been widely recognized as a widely accepted model
for the investigation of mechanical stimulation.37,38 In line with this, we deemed it appropriate to
investigate the fate of C2C12 cells on the printed formulations within our study. In the experiment,
we observed the cellularity of L929 fibroblasts and C2C12s on GF-printed raft architecture
scaffolds (N80-D15-A5, 4% PI, and 3% TMPTA) at a constant temperature of 37°C. The results
are shown in Figures 6-5 and 6-6. We found that the scaffolds allowed L929s to enter and
proliferate within them. On the other hand, C2C12s had a tendency to aggregate together, forming
clumps that showed minimal signs of proliferation at the end of the experiment.
PAGE 117
A B
C D
Figure 6-5: The cellularity (number of cells) of L9292 fibroblasts on GF-based scaffolds with raft
architecture (made with N80-D15-A5, 4% PI, and 3% TMPTA) was examined at different time
points (1-A, 3-B, 5-C, and 7-D days) following the scaffold's creation with exposure time per
layer (10 s). Scale bars equal 500 µm (low magnification (4x), left images) or 200 µm (higher
magnification (10x), right images). Size of the scaffolds: 7.6 × 7.6 × 1.35 mm.
PAGE 118
A B
C D
Figure 6-6: The cellularity (number of cells) of C2C12 myoblasts on GF-based scaffolds with raft
architecture (made with N80-D15-A5, 4% PI, and 3% TMPTA) was examined at different time
points (1-A, 3-B, 5-C, and 7-D days) following the scaffold's creation with exposure time per
layer (10 s). Scale bars equal 500 µm (low magnification (4x), left images) or 200 µm (higher
magnification (10x), right images). Size of the scaffolds: 7.6 × 7.6 × 1.35 mm.
The raft architecture scaffolds, provide a flat surface for cells to adhere and facilitate
microscopic evaluation of biocompatibility. To test the viability of L929 fibroblasts, we
microscopically evaluated cells on scaffolds fabricated from formulation N80-D15-A5 with 3%
w/w TMPTA and 4% w/w PI, with either 10 or 20 s of exposure time per layer to ensure cell
proliferate on GF-printed scaffolds. Fluorescence microscopic images of the cells on the scaffolds
after cultivation for 1, 3, 5, and 7 days at a constant temperature (37 °C) are shown in Figure 6-7.
We found that cell proliferation was visible to a similar degree on all the samples. No significant
difference in compatibility was observed between the scaffolds fabricated with 10 s and 20 s
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exposure times per layer (Figure 6-7A). Based on these positive results, we printed the raft
architecture scaffolds with DMAEA content of 15% and 10% w/w using an exposure time of 10
s/layer and evaluated them further. The normalized intensities of the calcein fluorescence signals
(NFI) were plotted, and we found that NFI was 1.5-fold (p = 0.07) higher for N80-D15-A5
compared to N85-D10-A5 at day 7 (Figure 6-7B). This suggests that a DMAEA content of 15%
may be optimal for promoting cell proliferation and survival, which has been further investigated
in the next section.
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Figure 6-7: (A) The viability of L929 fibroblasts on scaffolds fabricated from formulation N80-
D15-A5 with 3% w/w TMPTA and 4% w/w PI and with exposure times of 10 or 20 s per layer
on 1, 3, 5, and 7 days at a constant temperature of 37 °C. (B) The normalized intensities of the
calcein fluorescence signals (NFI) were plotted for N85-D10-A5 compared to N80-D15-A5 day
1, 3, and 7. Scale bars equal 500 µm (low magnification (4×), left images) or 200 µm (higher
magnification (10×), right images). p-value is denoted as * p.
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6.2.4. The Biocompatibility in Periodic Temperature Cultivate Conditions
The cells were seeded on scaffolds of the raft architecture fabricated from resins with
different ratios of DMAEA and were subjected to periodic changes in cultivation temperature in
order to demonstrate the scaffold’s ability to sustain cell adhesion above and below transition
temperature and to exert mechanical stimulation to the adherent cells. The ability of thermo-
responsive scaffolds to deliver mechanical stress to targeted cells was demonstrated under periodic
temperature conditions. The GF-printed scaffolds were seeded with cells and cultured at a
temperature of 30°C for one hour per day after 48 hours of cultivation. The cells were seeded on
scaffolds with raft architecture, which were printed from resins with different DMAEA
formulations. The cell seeded scaffolds with periodic cultivation were investigated in order to
demonstrate scaffold’s ability to maintain cell adhesion at above and below transition temperature
and to apply mechanical stimulation to the cells. In figure 6-8 and 6-9, shown cellularity on
scaffolds seeded for 1, 3, and 7 days with periodic cultivation temperature 30°C for one hour. The
L929s-seeded scaffolds maintained cell adherence to the scaffolds with a DMAEA content of at
least 10% w/w, while the scaffolds with less than 10% DMAEA showed almost no cell attachment.
The L929-seeded scaffolds with higher DMAEA demonstrated cell proliferation to the end of the
experiment. The cells were found to stick to the grooves of the scaffold and spread to cover the
surface after initial seeding and growth. At 10% or lower levels of DMAEA, the cells detached
during temperature changes, indicating the positive effect of DMAEA's charged moieties on cell
adhesion. The detachment may also have been caused by changes in the swelling ratio of the
material with a lower transition temperature. Interestingly, the C2C12-seeded scaffolds
that were exposed to periodic temperature conditions. We demonstrated that the C2C12 cultivated
on the raft scaffolds at constant temperature (37 °C for up to 7 days) showed almost no cell
PAGE 122
adherence (Figure 6-6). The scaffolds fabricated from resins N85-D10-A5, N80-D15-A5, and N75-
D20-A5, however, demonstrated sign of cell proliferation under periodic temperature. In
conclusion, we observed significant result between the isothermal and periodically changed
cultivation conditions, which shows a successful mechanical stimulation of the adherent cells by
the thermo-responsive materials. As it has been stated the mechanical stress, influenced and up-
regulate molecules such as calmodulin, nNOS, MMP-2, HGF, c-Met, and mitogen-activated
protein kinase.37,39 This possibility warrants further investigation.
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Figure 6-8: Cellularity of L929s on printed scaffolds (3% w/w TMPTA, 5% w/w AMO, 4% w/w
PI, exposure time 10 s/layer) in raft architecture cultivated under periodically changed
temperature (cycle: 23 h at 37 °C and 1 h at 30 °C). The fluorescent micrographs showed overlaid
live/dead staining and were recorded at day 1, 3, and 7. Scale bars represent 500 µm (low
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magnification (4×), left images) or 200 µm (higher magnification (10×), right images).
Figure 6-9: Cellularity of C2C12s on printed scaffolds (3% w/w TMPTA, 5% w/w AMO, 4%
w/w PI, exposure time 10 s/layer) in raft architecture cultivated under periodically changed
live/dead staining and were recorded at day 1, 3, and 7. Scale bars represent 500 µm (low
PAGE 125
Qualitative fluorescent analysis was used to further characterize the effect of different
cationic monomer contents. The scaffolds were tested in periodic temperature conditions and
stained with calcein-AM at the end of each time point (one, three, and seven days). The graph
(Figure 6-10) shows a significant increase in NFI signal from scaffolds with 10% and 15%
DMAEA with C2C12s. On day 1, we observed no significant fluorescence differences between
these groups. However, the difference becomes clearer at days 3 and 7, where scaffolds with less
than 5% w/w DMAEA show almost no NFI signal, indicating no cell growth on the scaffolds. At
day 3, NFIs of L929 cells on scaffolds with 10%, 15%, and 20% w/w DMAEA increased 9.2, 10.2,
and 19.8 folds, respectively, compared to cells on scaffolds without cationic presence. The
scaffolds with 15% and 20% w/w DMAEA showed a steadily upward trend in intensity over the
investigated time period. At day 8, the difference in NFI became clearer, with the scaffolds with
20% w/w DMAEA demonstrating the highest intensities (11.9 ± 1.0). The experiment with C2C12
myoblasts revealed similar results in periodic cultivated temperature. The scaffolds with less than
5% w/w DMAEA showed no evidence of cell proliferation support. Interestingly, the scaffolds
with 10% w/w DMAEA had an NFI that increased between day 1 and 3 and then decreased between
day 3 and 7. We attribute this result to the lack of sufficient cationic adhesion moieties to maintain
cell adhesion over numerous swelling/deswelling cycles. In comparison to L929 fibroblasts,
C2C12 cells have been described as more sensitive to material changes. 40 In comparison, at a
constant temperature condition, the growth of C2C12 cells on 15% w/w DMAEA was low and the
cell morphology was rounded, with no significant cell-cell contacts (Figure 6-6). C2C12 on
scaffolds (15% and 20% w/w DMAEA) cultivated with periodic temperature conditions showed
signs of proliferation, as the intensities increased 8 fold (15% DMAEA) and 15 fold (20%
DMAEA) from day 1 to 7.
PAGE 126
Figure 6-10: The normalized fluorescence intensity (NFI) of calcein-stained L929 cells (top) and
C2C12 cells (bottom) on raft scaffolds (3% w/w TMPTA, 4% w/w PI, printing time 10 s/layer) of
different DMAEA compositions (0–20% w/w) was measured at days 1, 3, and 7 during cultivation
with periodic changes in temperature. Statistically significant differences are indicated by *.
PAGE 127
In this chapter, the investigation mainly focuses on demonstrating cell lines interaction with
thermo-responsive scaffolds that show a moderate change in swelling while incorporating
functional groups. This property is not found in conventional thermally responsive materials. The
effect of periodic cell cultivation on the thermo-responsive scaffolds and biocompatibility are
demonstrated. The materials have shown promising results in this study, which subsequent work
will have to carefully examine the amount and frequency of mechanical stimulation needed and
their cellular effects in detail.
6.2.5. Toward Human Cell Augmentation
The biocompatibility of thermo-responsive scaffolds was evaluated using primary human
cells to assess their potential for use in practical applications. Human mesenchymal cells (hMSC)
were seeded on the scaffolds and cultured under both constant and periodic temperature conditions.
However, as illustrated in Figures 6-11, the hMSC cells failed to proliferate on the scaffolds and
fewer live cells with circular morphology were observed in all formulations. On day 1, the scaffolds
in different formulations showed minimal observable living cells, which may be due to the cells'
inability to settle onto the scaffold surface and subsequent removal by the culture medium. In
contrast, a different cell type, hASC (Figure 6-12), had better results in terms of detection on the
scaffold surfaces, but still demonstrated no signs of proliferation and a decrease in cell number at
the end of the experiment. These findings suggest that certain cell types may have preferred
conditions for growth on thermo-responsive scaffolds and highlight the challenges in developing
this type of platform.41 This demonstrates the challenges in developing a thermo-responsive cell
carrier platform and the need for further research.
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Figure 6-11: : Cellularity of hMSCs on printed scaffolds(3% w/w TMPTA, 5% w/w AMO, 4%
live/dead staining and were recorded at day 1, 3, 5 and 7. Scale bars represent 500 µm (low
PAGE 129
Figure 6-12: Cellularity of hASCs on printed scaffolds(3% w/w TMPTA, 5% w/w AMO, 4%
live/dead staining and were recorded at day 1, 3, 5 and 7. Scale bars represent 500 µm (low
6.2.6. Poly-L-lysine Impregnation
The challenged results from previous experiments with human-derived cells prompted the
search for a solution to the unsatisfactory outcomes. The scaffolds were further enhanced with
poly-L-lysine, a substance commonly used to coat cultureware and improve cell adhesion through
nonspecific electrostatic interactions with the cell membrane. 42-44 At day 1, a high number of cells
were observed on 20% w/w DMAEA scaffolds in the resin with poly-L-lysine, while cellularity
was decreased on 15% w/w DMAEA scaffolds, indicating the further benefit of the presence of
PAGE 130
cationic moieties from poly-L-lysine and DMAEA. At day 3, the cells were laterally spread on
both 15% and 20% w/w DMAEA with poly-L-lysine scaffolds. The normalized fluorescence
intensity (NFI) at day 4 and 7 increased, indicating cell growth within the scaffolds. The NFI of
poly-L-lysine coated groups revealed that N75-D20-A5 had 41% more intensity than the N80-D15-
A5 scaffolds. This difference became clearer at the end of the experiment, as shown in Figure 6-
14, where the NFI from the N75-D20-A5 scaffolds was more than 2.2 times the intensity of the
N80-D15-A5 scaffolds. As expected, hASC-seeded scaffolds without poly-L-lysine coating
showed no cell proliferation or maturation. This again confirms the challenges in developing
biocompatible scaffolds.
PAGE 131
Figure 6-13: Biocompatibility of hASCs on printed scaffolds (3% w/w TMPTA, 5% w/w AMO,
4% w/w PI, exposure time 10 s/layer) in raft architecture with and without poly-L-lysine
impregnation cultivated under periodically changed temperature (cycle: 23 h at 37 °C and 1 h at 30
°C). The fluorescent micrographs showed overlaid live/dead staining and were recorded at day 1,
3, 5 and 7. Scale bars represent 500 µm (low magnification (4×), left images) or 200 µm (higher
magnification (10×), right images).
PAGE 132
Figure 6-14: NFI from calcein staining of GF-printed poly-L-lysine impregnation raft scaffolds
(3% TMPTA, 5% AMO, 4% Irg819, and printing time 10 sec/layer) in periodic temperatures with
15% and 20% DMAEA at day 1, 3, and 7. The qualitative evaluation of poly-L-lysine impregnation
is clearly observed. The poly-L-lysine-treated scaffold with DMAEA significantly improved cell-
scaffold adhesion for both scaffold formulations (N80-D15-A5 and N75-D20-A5).
PAGE 133
6.3. Conclusion
Biocompatibility is a major factor in determining the functionality of scaffolds in tissue
engineering applications. In this study, we successfully characterized Poly(NiPAAm-co-DMAEA-
co-AMO) cross-linked TMPTA (N80-D15-A5, 3% TMPTA, 10 seconds/layer) scaffolds with
different configurations (raft and lattice) in a direct contact experiment using GF as a biocompatible
printing solvent. The scaffolds demonstrated the ability to support cell attachment in periodic
temperatures and promote proliferation. This strategy provides an on-demand thermosensitive
platform for cell mechanostimulation and presents a promising alternative for 4D printing scaffold
technology.
PAGE 134
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CHAPTER 7. DISCUSSION AND CONCLUSIONS
CHAPTER VII
Discussion & Conclusions
PAGE 139
7.1 Discussion
Mechanical stimulation during cell cultivation on scaffolds has been shown to improve
cell and tissue development. This is because mechanical force is considered essential for tissue
formation and development.1 There are various methods reported such as compressive loading2,
longitudinal stretching3, substrate bending4,5, bi-axial6,7, and shear stress systems.8,9 However,
these approaches require the use of a bioreactor and complex setup. 10 Additionally, it is crucial to
maintain the supply of oxygen and nutrients from the microenvironment to cells within the complex
structure of tissue engineering, which remains a significant challenge. 11,12 To address these
challenges, we propose an alternative strategy for cell stimulation that utilizes a thermo-responsive
material. This material has the ability to apply mechanical force to cells and can be fabricated into
three-dimensional scaffolds, which can efficiently provide the nutrients into the cells. The main
objectives of this thesis aim to create a cell carrier platform of thermo-responsive polymers that
respond to the environmental stimulus and are compatible with cell cultivation and maintain cell
adherence in both swollen and de-swollen state.
In order to achieve our goal of thermo-responsive materials, PolyNiPAAm was considered
to be a building block of the polymer. NiPAAm polymers have been reported in various
applications such as smart surfaces and cell stimulation platforms. 13-15 Utilizing prior experience,
a thermoresponsive material based on N-isopropylacrylamide (NiPAAm) is proposed in this
study.16,17 Various studies have shown that mammalian cells can be grown without any difficulty
at a temperature range of 30 to 37 °C.18-20 Our target transition temperature range was 30-36°C. To
achieve this, the lower critical solution temperature (LCST) of NiPAAm-based material was
adjusted through copolymerization. The addition of hydrophilic comonomers raises the transition
PAGE 140
temperature, while hydrophobic comonomers have the opposite effect. 21-23 Next, the TMPTA or
cross-linker has been chosen as it has been used also in biomedical applications and shown
biocompatibility with tissues.24-26
Traditional thermo-responsive materials, such as PNiPAAm, have been found to exhibit
unique surface characteristics that can affect cell adhesion. These materials are insoluble in water
above their lower critical solution temperature (LCST) of around 32°C, but can be solubilized
reversibly below the LCST. Previous studies have shown that below the LCST, the wettability of
PNiPAAm changes, which can affect cell adhesion and lead to cell detachment from the material. 27
To improve cell adhesion ability below the LCST, our study explores the use of DMAEA as a
potential molecule. DMAEA possesses a cationic charge that allows it to bind to the negatively
charged surface of cells, thereby creating a more favorable environment for cell attachment and
growth during episodes below the LCST.28,29
Our study aimed to investigate the potential of a new cell carrier platform made from
thermo-responsive polymers, including TMPTA, NiPAAm, and a cationic monomer, to stimulate
cells through cyclic changes in temperature around the LCST, resulting in increased or decreased
swelling of the network. We achieved adjustable thermo-responsive polymer properties, such as
transition temperature and hydrophilicity, through additive monomer compositions. Our
copolymerized thermo-responsive network contained a cationic comonomer. The material's
development was divided into three stages, including the creation of a three-dimensional
biomaterial structure with controllable porosity, which is an important factor in biomaterials. With
three-dimensional printing technology, we could program the porosity of the structure. We
conducted biocompatibility testing using the three-dimensional continuous digital light processing
PAGE 141
(cDLP) method, staining with live/dead dyes to evaluate the compatibility of the scaffolds with
both animal and human cells in constant and periodic temperature conditions. We describe these
stages and tests in detail in the chapters.
7.1.1 Synthesis and Characterization of Thermally Induced Solution Polymerization of
Monomer Mixtures
At the first stage, the aim was to explore the relationship between the transition
temperature and compositions. Solution polymerization was carried out using TMPTA and
monomers by thermal-induced solution polymerization and characterized to assess the success of
cross-polymerization. This method was considered to be suitable approach for thermo-responsive
platform as it has been widely used in numerous polymerizations including thermo-responsive
materials.30 To modulate the transition temperature range of 30-36 °C, as per the reports stating the
feasibility of mammalian cell development between 30 °C and physiological temperature, without
any hindrance, adjustments are possible.18-20 AMO was introduced in a later stage of the
experiment. It was possible to modify transition temperature of the materials with DMAEA and
AMO as it was found that the increasing content of DMAEA and AMO increased the transition
temperature, which was attributed to charge-ionic stabilization31 and steric hindrance effect.32
In details, charge-ionic stabilization in polymer science is the binding affinity exhibited
by cationic ions towards water molecules in a polymer matrix. The phenomenon can have a
significant impact on the behavior of the polymeric network. 33,34 Steric hindrance effect, on the
other hand, refers to the hindrance caused by bulky or large substituents towards the approach of
other molecules or moieties. The steric hindrance effect can affect the orientation of polymer
PAGE 142
segments and the conformation of polymer chains, leading to changes in their physical properties
and behavior.35 The increase in TMPTA feed ratio led to denser polymer networks, resulting in a
decrease in distance between the polymer chains. This was caused by the trivalent chemistry of the
monomer, which in turn reduced the material's hydration capacity and transition temperature. 36
The overall effect correlated with cross-linker and monomers have been summarized in
Table 7-1. The finding also collaborated with other publications as well as it also reported similar
effects were found in these materials, for example, Heydarifard S. et al. reported cationic monomers
significantly influence hydrogel properties such as water uptake and swelling. 31 E. Molly Frazar
and colleagues reported PNIPAAm copolymerized with cationic comonomers to target and attract
negatively charged pollutants.37 Karadağ and colleagues cross-linked TMPTA at 1% concentration
into hydrogels composed of acrylamide/crotonic acid (AAm/CA), and observed that the resulting
hydrogels retained up to 96.7% of water at room temperature. 38
Based on the transition temperature profiles obtained at this stage, it was concluded that
formulations with suitable transition temperatures were obtained by incorporating 1-5% w/w
TMPTA, 10-15% w/w DMAEA, and 1-15% w/w AMO. However, it was observed that the cross-
linking density of the polymer was lower than the desired level. To attain a higher cross-linking
density, the formulations underwent bulk photo-induced polymerization in the second stage instead
of thermal-induced polymerization. The use of photo-induced polymerization facilitated the
conversion of a highly viscous liquid to a bulk solid polymer in mere seconds. 39
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Impact on Transition
temperature
TMPTA ↓
DMAEA ↑
MMA ↓
AMO ↑*
Table7-1: Overview of influence of macromer and monomers on T trans on thermo-responsive
polymer. *Increase of AMO ratio correlates with TMPTA presence as steric hindrance disrupts the
coil-globule transition from the cross-linked PNiPAAm network. 35 All the parameters mentioned
above were optimized to obtain the polymeric transition temperature at the physiological level. Up
(↑) and down (↓) arrows indicate an increase or decrease in the impact on the transition temperature.
7.1.2. Photo-Induced Bulk Polymerization of Resins
After obtaining the data from the first stage, our focus was on bulk polymerization to
enhance the cross-linking density of the thermo-responsive polymers. We specifically explored UV
light-induced resin copolymerization and produced non-porous structures through photo-induced
bulk polymerization, which has been widely reported in biomedical material fabrication 40 such as
hydrogels41 and 3D scaffolds.42-45 Photocurable polymers possess several advantageous attributes,
such as ease of production, the possibility of photo-polymerization in vivo or ex vivo, and the ability
to incorporate a variety of substances and cells into their formulations and applications. 40,46,47 For
example, Hydrogel films made from the NiPAAm monomer were photopolymerized using both
hydrophilic and hydrophobic photo-initiators in either water or a water/ethanol mixture. Hydrogels
prepared at approximately 7°C using the hydrophobic photo-initiator and a water/ethanol (50:50)
PAGE 144
solvent displayed significantly higher degrees of swelling at all levels of crosslink density when
compared to hydrogels prepared at the same temperature using a hydrophilic photo-initiator and
water as the solvent.48. However, not many biocompatible photocurable resin were reported. 44,49,50
In this stage, higher cross-linking density at was successfully achieved through bulk
photo-induced polymerization. After the transition temperature of the polymer formulations was
confirmed, the focus shifted to investigate the swelling ratio of non-porous cylindrical scaffolds.
To identify if a phase transition occurred between these two temperatures, we introduced a new
parameter, ΔSR (37/25), as defined in the methods section. This parameter is a useful tool for
observing differences in material swelling behavior and characteristics between temperatures of
25°C and 37°C. A negative value of ΔSR (37/25) indicates that the polymeric network expels
water, and vice versa. The thermo-responsive discs with 3% TMPTA and either 10% (N75-D15-
A10) or 5% (N80-D15-A5) AMO were found to shrink, resulting in a negative ΔSR (37/25) value.
Our findings showed that increasing the content of DMAEA and AMO resulted in reduced
swelling and disc change between 25°C and 37°C. This can be attributed to the attraction of water
molecules into the polymeric discs by the cationic charge from DMAEA and morpholine residue,
leading to an increase in the material's swelling.35,51,52 To reduce potential cytotoxicity from the
cross-linker, the TMPTA content was kept at a low value of 3% w/w. Based on the report by Lai
J-Y. et al., it has been found that increasing the concentration of the crosslinker, 1-ethyl-3-(3-
dimethyl aminopropyl) carbodiimide hydrochloride (EDC), beyond 50 mM can have negative
effects on animal cornea cells, leading to severe ocular inflammatory responses. 53 From the data
obtained in this stage, promising formulations with 3% TMPTA, 10-15% DMAEA, and 5% AMO
PAGE 145
that met the target phase transition temperature were found. This formulation was identified for
further experiments.
7.1.3 Continuous Digital Light Processing
7.1.3.1 Three-Dimensional Scaffold Fabrication In the third stage, we
successfully fabricated three-dimensional scaffolds utilizing the cDLP technique, which has also
been extensively employed in the fabrication of biomedical scaffolds. 54-58 The impact of
formulation parameters related to 3D printing, such as exposure time per layer, photo-initiator
content, and solvent, on print quality and the mechanical properties of the scaffold were also
investigated, as these parameters have been mentioned to have a key role in scaffold mechanical
properties.59,60 The selected monomer formulations (3% w/w TMPTA and 15% w/w DMAEA)
were dissolved with ethanol to yield a concentration of 10% (w/v). Investigated formulations were
printed into two different configurations: a raft and a three-dimensional lattice. Raft-configuration
scaffolds were selected to evaluate the biocompatibility of the material because their geometry
enables observation of cells on the impermeable scaffold surface. Cell proliferation can be easily
monitored using microscopy. The second design is a macroporous lattice of 7.6 × 7.8 × 2.5 mm
(length × width × height). The macroporous scaffold design was chosen to allow the cells for
adhesion and proliferation, while facilitate nutrient via interconnecting channels.61,62
The findings of this study are consistent with our previous research on bulk photo-
polymerization of TMPTA, DMAEA, and AMO. Specifically in this stage, we observed that lower
photoinitiator (PI) contents and shorter exposure times resulted in materials with reduced cross-
linking, which in turn led to a smaller response in swelling change between 25°C and 37°C, as
PAGE 146
indicated by a low value of ΔSR (37/25).The results of ΔSR(37/25) showed the influence of
DMAEA in elevating the transition temperature and stabilizing the thermo-responsive polymer
network within the investigated temperature range, leading to a decrease in ΔSR(37/25). Scaffolds
with lower photoinitiator ratios and shorter curing times per layer showed an increase in the
swelling ratio, with no significant observable change in transition temperature between those
groups. These findings are consistent with the report by Yang Y. et al., found that longer exposure
times can cause overgrowth of the construct due to light scattering and increasing printing ratio
(the printed/designed height ratio). In contrast, shorter exposure times were found to improve the
printability of vertical channels, with an increased layer height.63,64
7.1.3.2 Three-Dimensional Scaffold Fabrication with Glycofurol as Solvent
The low viscosity of ethanol as a solvent for the monomer resin mix used for scaffold
fabrication by cDLP resulted in limitations to the printability of the resin, including premature
detachment of the sample and low structure resolution. In this set of experiments, we aim to address
these challenges. Glycofurol (GF) with higher viscosity than ethanol was utilized. Through the
“damping effect”65-67 scaffold resolution was improved. The damping effect is a phenomenon in
which energy, such as vibration, is dissipated in a viscous fluid.68 GF has been described as
biocompatible for many application in pharmaceutical industry. GF in a 50:50 mixed with PBS
was directly injected into rat’s brain and was well tolerated with minor inflammatory response. 65
Therefore, we expected the residue GF in the scaffolds have no effect on cellular response but
chromatographic evaluation of the residual GF is recommended in further studies.
PAGE 147
We developed a method to dilute GF resins while preserving their thermo-responsive properties.
This resulted in a 50% reduction in exposure time per printed layer from 20 to 10 seconds, and
prevented premature detachment of the printing from the printing head during the printing process.
We were able to fabricate GF-based scaffolds in both raft and lattice geometries by varying the
ratios of DMAEA and AMO at different temperatures (25 °C and 37 °C). The AMO and DMAEA
contents could be increased to a similar effect as the ethanol-printed constructs. We suggest that
the ΔSR (37/25) values should be within a moderate range of -3 to -2 g/g and negative to facilitate
cell adhesion to the biomaterial surface. In addition, the moduli of GF-printed lattice scaffold
have been investigated. It is a crucial factor as the scaffolds are expected to switch the swelling and
apply mechanical stimulation to the cells.69,70 Rheological characterization of the scaffold moduli
by oscillation rheology show the fabricated scaffold moduli are within the range of the cellular
stiffness reports (myoblasts: 2 kPa, fibroblasts: 1–10 kPa).71,72 The scaffolds with a higher content
of PI showed higher storage moduli than those with a lower content of PI. Increasing the exposure
time per layer from 10 s/layer to 20 s/layer and 30 s/layer also increased the moduli. We found that
these parameters did not affect the transition temperature, as the chemical composition remained
unchanged. However, the density of the polymeric network was clearly impacted by these
parameters, as a change in network swelling was observed. A higher content of DMAEA was also
found to decrease the scaffold moduli, as the cationic charge in the polymer chain was strong
enough to suppress the coil-globule transition, which corresponded to an increase in the transition
temperature.37,73,74 It is important to note that the change in the volume of the construct and the
resulting movement of the cells' focal adhesion points cause mechanical stress on the cells. Thus,
the relationship between the volume changes that support cell adherence and survival and the
change in storage modulus in the hydrogel is not clear and requires further investigation.
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7.1.4. Cytocompatibility Evaluation of the Scaffolds
The scaffold formulation with promising results, which has a transition temperature of
36.3 °C ± 0.9 °C and the ΔSR (37/25) of (−1.3 ± 0.2) g/g with a change in material modulus from
2.8 kPa (30 °C) to 7.1 kPa (37 °C), both of which fall within the physiological range of 1-10
kPa.71,72 The biocompatibility of the 3D scaffolds was first evaluated using murine cell lines (L929)
cultivated under isothermal temperature conditions at 37 °C. Cell adherence was assessed using
calcein-AM and ethidium homodimer staining to differentiate between live and dead cells. 75 The
results showed that parameters such as increasing fiber thickness and UV after-treatment negatively
affected cell biocompatibility through changes in surface morphology76 and damage to cationic
molecules, respectively.77 The effect of exposure time and PI concentration were tested. There was
no significant difference between tested formulations. On cellular level, the cells were well-spread
and a low number of dead cells were found. The fluorescence signal's diffuse nature suggests a
degree of autofluorescence from the materials. Despite this, the low number of visibly stained dead
cells indicates that the material's toxicity is insignificant, and any observed changes in cell numbers
are likely due to normal cell turnover. The raft architecture scaffolds were evaluated for cell
viability and one plane for better microscopic evaluation with L929 fibroblasts. The results
revealed comparable cell proliferation with 10 s or 20 s of exposure time per layer. The scaffolds
with 15% DMAEA showed a 1.5-fold higher normalized fluorescence intensity of the calcein
signal, indicating improved cell proliferation and survival. These results suggest that 15% DMAEA
content is beneficial for optimizing cell growth on the scaffolds.
In this study, murine myoblast cells (C2C12s) were also seeded in raft-architecture
scaffolds to evaluate the fate of cells on the printed formulation. It is well established that myoblasts
PAGE 149
can benefit from mechanical stimulation and have been previously used as a model for such
stimulation.78,79 However, we observed almost no cell proliferation in C2C12 cells cultivated on
our raft scaffolds at a constant temperature. We attribute this finding to the increased sensitivity of
C2C12 cells to the changes in the material.80
7.1.5. Cellular Response to Scaffolds under Periodic Changes in Cultivation
Temperature
In order to demonstrate the scaffold’s ability to sustain cell adhesion above and below
transition temperature and to exert mechanical stimulation. Raft scaffolds in different ratios of
DMAEA were seeded with L929s and C2C12s and cultivated in periodic changes in cultivation
temperature. The L929s were found to maintain adherence to the scaffolds with at least 10%
DMAEA. Cells on higher DMAEA scaffolds also proliferated over experiment course. At lower
DMAEA content than 10% w/w, the cells detached from the scaffold surfaces upon periodical
shifts in cultivation temperatures. The results of this study indicate the significant impact of
DMAEA on cell adhesion and align with previous reports on the positive influence of cationic
groups on cell attachment. For instance, Courtenay J. C. et al. have demonstrated that chemical
modification of cellulose with positively charged trimethylammonium groups increases cell
attachment by more than 90%.81 Sallouh M. reported on the synergistic effect of cationic moieties
(amino ethylmethacrylate) and specific cell adhesion moieties (GRGDSF-peptides) on embryonic
neural stem cells when cultured on a hydrogel.28 Furthermore, an extensive change in swelling ratio
with lower transition temperature possibly contributed to the cell detachment episode in periodic
cultivation.
PAGE 150
The study of the scaffolds cultivated with C2C12 murine myoblast cells revealed that
those containing 10%, 15%, and 20% w/w DMAEA showed cell proliferation when cultivated
under periodic temperature conditions. This is in contrast to the C2C12 scaffolds cultivated in
isothermal conditions at 37°C. This difference suggests that mechanical stimulation was achieved,
as it has been previously reported that mechanical forces generated by material swelling can up-
regulate molecules such as calmodulin, nNOS, MMP-2, HGF, c-Met, and mitogen-activated
protein kinase.70,79
Moreover, we conducted a quantified Net Fluorescence Intensity (NFI) analysis to
investigate the impact of cationic monomer ratios on cultivated cells. The scaffolds containing 10%
DMAEA showed promising results. Results showed that the scaffolds with 0-5% DMAEA did not
demonstrate an increase in average NFI. The same findings were also observed in C2C12
myoblasts where 0-5% DMAEA failed to support cell adherence. Interestingly, at 10% DMAEA,
the NFI increased from day 1, but declined between day 3 and 7. The primary reasons behind this
decline seem to be the cells' sensitivity to changes in the mechanical properties of the material and
a deficiency of cationic molecules that can sustain cell viability during the cycles of swelling and
deswelling.80 Furthermore, an assessment was conducted to evaluate the biocompatibility of the
scaffold within a three-dimensional lattice structure, which offers a more physiologically relevant
template for cell-cell interactions,82,83 with results indicating that cells covered the scaffold at the
end of the experiment. The results indicated that the three-dimensional scaffolds promoted cell
attachment and proliferation with lower DMAEA concentration, potentially due to a favorable
microenvironment that facilitated efficient cell seeding, nutrient distribution, and enhanced cell
survival during cultivation.76,82-86
PAGE 151
The examination of primary human cells (hMSCs and hASCs) on thermo-responsive
scaffolds was conducted, but faced challenges as the cells struggled to adhere and proliferate on
the scaffold surfaces, leading to cell loss through washing with the medium. These results contrast
with the observations made using animal cell lines, suggesting that primary human cells might
require additional electrostatic interaction with a cationic molecular charge for successful
attachment to the scaffold. This demonstrates the importance of extracellular matrices in
facilitating cell attachment to the scaffold, as well as the challenges posed by cellular complexity
in the development of thermo-responsive scaffolds.15,87,88 To address these challenges, poly-L-
lysine was coated onto the scaffold surface. This resulted in excellent biocompatibility with
primary cells, compared to uncoated scaffolds.
7.2. Conclusion
This study reports on the synthesis of thermo-sensitive scaffolds using TMPTA-cross-
linked poly(NiPAAm-co-DMAEA-co-AMO) networks. The scaffolds were created through
thermally induced polymerization, photo-induced polymerization, and three-dimensional cDLP
printing. The resulting discs/scaffolds were thermo-responsive, with the ability to control swelling
ratio and transition temperature. Glycofurol, a biocompatible solvent, was also integrated to
enhance printing quality. After testing multiple formulations, the most promising combination for
cell cultivation with periodic temperature changes was found to be 3% w/w TMPTA, N80-D15-
A5, and 4% w/w PI, with an exposure time of 10 seconds per layer at 36.3 ± 0.9 °C. This novel
approach provides a customizable platform for cell mechano-stimulation that offers a viable
alternative to traditional three-dimensional thermo-sensitive discs/scaffolds. The findings suggest
PAGE 152
that this material platform holds significant potential for controlled and cell type-specific
mechanical stimulation and could be used to engineer a mechanical stimulation device for various
biomedical applications.
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CHAPTER 8. SUMMARY
CHAPTER VIII
Summary
PAGE 161
CHAPTER 8. SUMMARY
Mechanical stimulation plays a crucial role in promoting cell differentiation. However,
applying physical force directly to cells requires complex equipment and a sterile environment,
posing challenges. To overcome this, stimuli-responsive biomaterials or 4D scaffolds can serve as
an alternative platform for mechanical stimulation. These scaffolds, fabricated using advanced 3D
printing techniques, can apply the necessary force to cells. To optimize their functionality,
bioactive molecules or extracellular matrices can be incorporated or decorated on their surfaces.
This thesis proposal focuses on developing a versatile material platform that allows customization
through systematic composition adjustment and on-demand printing, while also offering surface
modification capabilities. The primary objective is to create a novel cell carrier platform using
thermo-responsive polymers. By manipulating the additive monomer compositions, we can finely
adjust properties such as the transition temperature of the polymers, tailoring them to specific
requirements. Furthermore, this platform will enable the fabrication of complex three-dimensional
biomaterial structures with controllable porosity, a critical aspect of biomaterial design. Leveraging
the capabilities of three-dimensional printing technology, we can program and achieve desired
porosity levels in the printed structures, providing enhanced flexibility for biomaterial design.
The development of thermo-responsive scaffolds involved three distinct stages aimed at
designing an optimized platform that effectively operates within the physiological range while
ensuring cell viability. One of the key challenges was to achieve a balance between
thermoresponsive behavior and biocompatibility. In the initial stage, we investigated the interplay
between a crosslinkable three-armed macromer (trimethylolpropane triacrylate-TMPTA) and
various monomers (N-isopropylacrylamide-NiPAAm, methyl methacrylate-MMA,
dimethylaminoethyl acrylate-DMAEA, 4-acryloylmorpholine-AMO) using thermally induced
solution polymerization. NiPAAm, known for its thermoresponsive properties, was selected
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CHAPTER 8. SUMMARY
despite its limited biocompatibility. DMAEA was chosen to adjust the polymer network transition
temperature by introducing cationic charge, which disrupts the coil-globule effect of PNiPAAm
and provides cell adhesiveness of the composition. Additionally, the hydrophilic monomer AMO
was incorporated to further fine-tune the polymeric network. We examined the behavior of these
components within the physiological range and their integration into the PNiPAAm network,
establishing significant correlations between the transition temperature of the polymer and the
crosslinker and monomers in their soluble condition.
In the second stage of our research, we introduced photo-induced polymerization to
enhance the crosslinking ratio. By utilizing this method, we successfully fabricated photo-
polymerized mixtures (photoresists) into thermo-responsive discs, enabling us to study their
swelling behavior between 37℃ and 25℃. Our findings revealed that the swelling behavior could
be adjusted by varying the ratios of the crosslinker and monomers in the experimental groups.
Through careful experimentation, we identified a suitable composition (3% w/w TMPTA, 80%
w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator(PI)) that required
minimal crosslinking incorporation while still retaining thermo-responsiveness. Furthermore, we
conducted a preliminary biocompatibility study by fabricating the mixture into thin-films and
cultivating them with L929 fibroblast cells.
In the third and final stage, we utilized the optimized formulations from the previous stage
to build thermo-responsive 3D scaffolds using continuous Digital Light Processing (cDLP) printing.
We investigated the effects of various parameters, such as curing time and monomer composition,
on the swelling property of the scaffolds. Additionally, we introduced glycofurol (GF) as a photo-
polymerization solvent, which allowed us to produce scaffolds with improved resolution and
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CHAPTER 8. SUMMARY
reduced printing time. The resulting optimized scaffolds, with a composition of 3% w/w TMPTA,
80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, 4% w/w PI, and 10 seconds per layer,
exhibited the desired thermo-responsiveness. To further understand the mechanical properties and
thermal dependencies of these scaffolds, we conducted rheological analysis. This analysis helped
establish a relationship between the mechanical properties of the scaffolds and their response to
temperature changes.
To investigate the potential of cell stimulation through periodic changes, we conducted an
experiment involving the seeding of L929 fibroblasts and C2C12 myoblasts on thermo-responsive
3D scaffolds. Our objective was to assess the ability of cells to proliferate on scaffolds with
different compositions. Specifically, we examined two types of scaffolds: lattice scaffolds,
characterized by a porous structure with a periodic network that enables cells to inhabit a 3D
environment, and raft scaffolds, which feature a dense 3D structure designed for cells to reside on
the surface for observation and evaluation. The lattice scaffolds were composed of ≥2% w/w
DMAEA, while the raft scaffolds consisted of ≥5% w/w DMAEA. To evaluate cell proliferation,
we conducted direct contact experiments and employed live/dead assays, subjecting the scaffolds
to temperature switching conditions at 31℃ and 37℃. These experimental setups aimed to provide
insights into the response and behavior of cells in the presence of thermo-responsive scaffolds with
varying compositions. The results revealed favorable adhesion and spreading of the cells on the
scaffolds. Interestingly, in our dynamic temperature experiment, we observed that myoblasts
seeded on the scaffolds exhibited both proliferation and spreading, whereas myoblasts subjected to
constant-temperature conditions did not show the same behavior. This suggests that the expansion
and contraction of the scaffold, observed in previous experiments, may impact cell viability.
Further investigation is needed to better understand this phenomenon. Additionally, we enhanced
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CHAPTER 8. SUMMARY
cell adhesiveness of the scaffolds by impregnating the scaffolds with poly-L-lysine and tested them
with hASCs (human adipose-derived stem cells). Significant differences were observed between
scaffolds with and without poly-L-lysine, highlighting the effectiveness of this approach.
In conclusion, we have successfully developed a thermo-responsive 3D scaffold that
exhibits a transition temperature within the physiological range, ensuring cell survival, and
provides mechanical stimulation to the cells through the coil-globule effect without causing cell
detachment. Among the formulations tested, the GF-printed formulation (3% w/w TMPTA, 80%
w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator) with an exposure
time of 10 seconds per layer showed the most promising results for cell cultivation under periodic
changes in temperature, with a transition temperature of 36.3 °C ± 0.9 °C. Furthermore, we
conducted direct cell contact experiments and confirmed the biocompatibility of the thermo-
responsive macromer-based scaffolds. These findings demonstrate that this material platform
offers a versatile and responsive material for mechanical stimulation of cells on three-dimensional
scaffolds. These promising results suggest that this approach holds significant potential for tissue
engineering applications and can be utilized to develop mechanical stimulation devices for various
biomedical applications.
PAGE 165
APPENDIX
Bibliography
List of Publication
Curriculum Vitae
Declaration of Authorship
Acknowledgements
Related publication
APPENDIX. CURRICULUM VITAE
Bibliography
Name: Ketpat Vejjasilpa
Date of birth: May 5, 1989
Place of birth: Phetchaburi, Thailand
Nationality: Thai
Marital status: Single
Education:
Present PhD research student in Institution of Pharmacy, Pharmaceutical Technology, medical

faculty, Leipzig university, Germany
- Adjustable Thermo-Responsive cell carrier and implants from biodegradable macromers

- Advisor: Jun.-Prof. Dr. Michael C. Hacker
- Received Royal Thai Government (RTG) scholarship
2011-2015 M.Eng. in Biomedical engineering, Mahidol University, Bangkok, Thailand
- Intratumoral distribution of SN-38 from polymeric depots in glioblastoma tumor model

- Advisor: Asst. Prof. Norased Nasongkla, Ph.D.
- Received PERCH-CIC and BMES scholarship
2014-2015 Student exchange program (JASSO scholarship) at Sophia University, Tokyo, Japan
- Research topic: β-Tri calcium phosphate for drug delivery and bone cement
- Advisor: Kiyoshi Itatani
2007- 2011 B.Eng. in Biomedical engineering, Mahidol University, Bangkok, Thailand
- Drug Delivery system for minimal invasive brain cancer Chemotherapy

Advisor: Asst. Prof. Norased Nasongkla, Ph.D.
2003-2007 Triamudomsuksa Pattanakarn high school, Patumwan, Bangkok, Thailand
PAGE 167
APPENDIX. LIST OF PUBLICATIONS
LIST OF PUBLICATIONS
Publications List
- Vejjasilpa, K.; Maqsood, I.; Schulz-Siegmund, M.; Hacker, M.C. Adjustable
Thermo-Responsive, Cell-Adhesive Tissue Engineering Scaffolds for Cell
Stimulation through Periodic Changes in Culture Temperature. Int. J. Mol. Sci. 2023,
24, 572. https://doi.org/10.3390/ijms24010572
- A. Koenig, J. Schmidtke, L. Schmohl, S. Schneider-Feyrer, M. Rosentritt, H. Hoelzig,
G. Kloess, K.Vejjasilpa, M. Schulz-Siegmund, F. Fuchs, S. Hahnel, Characterisation
of the filler fraction in CAD/CAM resin-based composites, Advanced Composites,
2021
- Manaspon C., Nasongkla N., Chaimongkolnukul K., Nittayacharn P., Vejjasilpa K.,
Kengkoom K., Boongird A., Hongeng S., Injectable SN-38-loaded Polymeric Depots
for Cancer Chemotherapy of Glioblastoma Multiforme. Pharm Res, 2016. 33(12): p.
2891-2903.
- Vejjasilpa K., Manaspon C., Boongird A., Larbchareonsub N.,Hongeng S., Israsena
N. and Nasongkla N. Intratumoral SN-38 distribution profiles from biocompatibility
polymeric depots against glioblastoma, Exp Biol Med (Maywood) December 2015
vol. 240 no. 12 1640-1647
- Manaspon, C., Nittayacharn, P., Vejjasilpa, K.,Fongsuk, C., Nasongkla, N. SN-38:β-
cyclodextrin inclusion complex for in situ solidifying injectable polymer implants,
Proceedings of the Annual International Conference of the IEEE Engineering in
Medicine and Biology Society, 2011, IEEE EMBS, art. no. 6090881, pp. 3241-3244.
PAGE 168
APPENDIX. LIST OF PUBLICATIONS
Conference List
- K. Vejjasilpa, Maqsood I., M. Schulz-Siegmund, M. C. Hacker “Adjustable thermo-
responsive adhesive cell carriers for cell stimulation through periodic changes of
culture temperature”, German Pharmaceutical Society (DPhG) Annual meeting,
Leipzig, 2021
- Vejjasilpa K., I. Maqsood, M. Schulz-Siegmund, M. C. Hacker, Adjustable Thermo-
Responsive Materials from Three-armed Macromers for Cell Carrier and Implant
Design, 2020, 16th Research Festival of Life Sciences, Leipzig, Germany, 2020
- Vejjasilpa K.,I. Maqsood, M. Gronbach, M. Schulz-Siegmund, M. C. Hacker,
Adjustable Thermo-Responsive Materials from Three-armed Macromers for Cell
Carrier and Implant Design,30th Annual Conference of the European Society for
Biomaterials, Dresden, Germany, 2019
N. and Nasongkla N. Study of high blood perfusion organs after injection of SN-38-
loaded polymeric depots, International Congress on Chemical, Biological and
Environmental Sciences (ICCBES), Kyoto, Japan, 2015
- Ketpat Vejjasilpa, et al. Biocompatibility Study of Injectable PolymericGels in Rat
Brains, 4th East Asian Pacific Student Workshop on Nano-Biomedical Engineering at
National University of Singapore (NUS), 2010
- Theerasilp M, Torapimai N, , Swatdipakidi D, Vejjasilpa K, Pungkom H, Siraugson
S, Pobunsong P, Akarajirathun P and Nasongkla N, SN-38 containing polymeric rods
as implantable drug delivery systems for brain cancer therapy, ISBME, Bangkok,
2009.
PAGE 169
Ketpat Vejjasilpa
Date of Birth: 5th May 1989
187 Pttanakarn 65 Rd. Prawat Bangkok Thailand 10250
Tel: +66-85-138-4408
E-mail: ketpat.vejjasilpa@medizin.uni-leipzig.de, ketpat1@hotmail.com
Education
Present PhD research student in Institution of Pharmacy, Pharmaceutical Technology,
medical Faculty, Leipzig University, Germany (expected to graduate at the end of
Oct 2021)
- Adjustable thermo-responsive adhesive cell carriers for cell stimulation through

periodic changes of culture temperature
- Advisor: Jun.-Prof. Dr. Michael C. Hacker
- Received Royal Thai Government (RTG) scholarship
2011-2015 M.Eng. (Biomedical engineering), Mahidol University, Bangkok, Thailand
- Intratumoral distribution of SN-38 from polymeric depots in glioblastoma tumor

model
- Advisor: Asst. Prof. Norased Nasongkla, Ph.D.
- Received PERCH-CIC and BMES scholarship
2014-2015 Student exchange program (JASSO scholarship) at Sophia University, Tokyo, Japan
- Research topic: β-Tri calcium phosphate for drug delivery and bone cement
- Advisor: Kiyoshi Itatani
2007- 2011 B.Eng. in Biomedical engineering, Mahidol University, Bangkok, Thailand
- Drug Delivery system for minimal invasive brain cancer Chemotherapy

Advisor: Asst. Prof. Norased Nasongkla, Ph.D.
2003-2007 Triamudomsuksa Pattanakarn high school, Bangkok, Thailand
RESEARCH INTEREST
 4D bioprinting  Polymer synthesis  Stereolithography

 Biomaterials  Tissue engineering  In vivo imaging
 Biosensor  Theranostics research
 Drug delivery  Smart material
PAGE 170
Publications List
- Vejjasilpa, K.; Maqsood, I.; Schulz-Siegmund, M.; Hacker, M.C. Adjustable
Thermo-Responsive, Cell-Adhesive Tissue Engineering Scaffolds for Cell
Stimulation through Periodic Changes in Culture Temperature. Int. J. Mol. Sci.
2023, 24, 572. https://doi.org/10.3390/ijms24010572
- A. Koenig, J. Schmidtke, L. Schmohl, S. Schneider-Feyrer, M. Rosentritt, H.
Hoelzig, G. Kloess, K.Vejjasilpa, M. Schulz-Siegmund, F. Fuchs, S. Hahnel,
Characterisation of the filler fraction in CAD/CAM resin-based composites,
Advanced Composites, 2021
- Manaspon C., Nasongkla N., Chaimongkolnukul K., Nittayacharn P., Vejjasilpa
K., Kengkoom K., Boongird A., Hongeng S., Injectable SN-38-loaded Polymeric
Depots for Cancer Chemotherapy of Glioblastoma Multiforme. Pharm Res, 2016.
33(12): p. 2891-2903.
- Vejjasilpa K., Manaspon C., Boongird A., Larbchareonsub N.,Hongeng S.,
Israsena N. and Nasongkla N. Intratumoral SN-38 distribution profiles from
biocompatibility polymeric depots against glioblastoma, Exp Biol Med (Maywood)
December 2015 vol. 240 no. 12 1640-1647
- Manaspon, C., Nittayacharn, P., Vejjasilpa, K.,Fongsuk, C., Nasongkla, N. SN-
38:β-cyclodextrin inclusion complex for in situ solidifying injectable polymer
implants, Proceedings of the Annual International Conference of the IEEE
Engineering in Medicine and Biology Society, 2011, IEEE EMBS, art. no. 6090881,
pp. 3241-3244.
PAGE 171
Conference List
- K. Vejjasilpa, Maqsood I., M. Schulz-Siegmund, M. C. Hacker “Adjustable
thermo-responsive adhesive cell carriers for cell stimulation through periodic
changes of culture temperature”, German Pharmaceutical Society (DPhG) Annual
meeting, Leipzig, 2021
- Vejjasilpa K., I. Maqsood, M. Schulz-Siegmund, M. C. Hacker, Adjustable Thermo-
Responsive Materials from Three-armed Macromers for Cell Carrier and Implant
Design, 2020, 16th Research Festival of Life Sciences, Leipzig, Germany, 2020
- Vejjasilpa K.,I. Maqsood, M. Gronbach, M. Schulz-Siegmund, M. C. Hacker,
Adjustable Thermo-Responsive Materials from Three-armed Macromers for Cell
Carrier and Implant Design,30th Annual Conference of the European Society for
Biomaterials, Dresden, Germany, 2019
N. and Nasongkla N. Study of high blood perfusion organs after injection of SN-38-
loaded polymeric depots, International Congress on Chemical, Biological and
Environmental Sciences (ICCBES), Kyoto, Japan, 2015
- Ketpat Vejjasilpa, et al. Biocompatibility Study of Injectable PolymericGels in Rat
Brains, 4th East Asian Pacific Student Workshop on Nano-Biomedical Engineering at
National University of Singapore (NUS), 2010
- Theerasilp M, Torapimai N, , Swatdipakidi D, Vejjasilpa K, Pungkom H, Siraugson
S, Pobunsong P, Akarajirathun P and Nasongkla N, SN-38 containing polymeric rods
as implantable drug delivery systems for brain cancer therapy, ISBME, Bangkok,
2009.
PAGE 172
APPENDIX. DECLARATION OF AUTHORSHIP
DECLARATION OF AUTHORSHIP
Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig und ohne unzulässige Hilfe oder
Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere, dass Dritte von mir
weder unmittelbar noch mittelbar eine Vergütung oder geldwerte Leistungen für Arbeiten erhalten haben,
die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen, und dass die vorgelegte Arbeit
weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde zum
Zweck einer Promotion oder eines anderen Prüfungsverfahrens vorgelegt wurde. Alles aus anderen Quellen
und von anderen Personen übernommene Material, das in der Arbeit verwendet wurde oder auf das direkt
Bezug genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden alle Personen genannt,
die direkt an der Entstehung der vorliegenden Arbeit beteiligt waren. Die aktuellen gesetzlichen Vorgaben
in Bezug auf die Zulassung der klinischen Studien, die Bestimmungen des Tierschutzgesetzes, die
Bestimmungen des Gentechnikgesetzes und die allgemeinen Datenschutzbestimmungen wurden
eingehalten. Ich versichere, dass ich die Regelungen der Satzung der Universität Leipzig zur Sicherung
guter wissenschaftlicher Praxis kenne und eingehalten habe.
15.06.2023 ....................................
(KETPAT VEJJASILPA)
Datum Unterschrift
PAGE 173
APPENDIX. ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
This dissertation and research achievements would not have been possible without the
support of my family and friends, who have taken good care of me and taught me many lessons.
I am truly grateful to Prof. Dr. Michaela Schulz-Siegmund for providing me with the opportunity
to join her research team and for her ongoing support throughout my educational journey. Her
guidance and mentorship have been invaluable in helping me to grow as a researcher and achieve
my academic goals. I cannot express enough how much I appreciate her belief in my potential and
her dedication to helping me succeed. Thank you, Prof. Schulz-Siegmund, for your unwavering
support and encouragement.
I am sincerely grateful to Jprof. Dr. rer. nat. habil. Michael C. Hacker for his guidance and
invaluable insights into biomaterials that have shaped my work on this project. I also thank him for
his support in the writing of this dissertation and the publication of my research.
I am grateful to the Deutsche Forschungsgemeinschaft (DFG, project number: 59307082 and
TRR3-A-P01) and the Royal Thai Government (RTG) for providing financial support and
scholarships that have enabled me to complete this work.
I would also like to express my special thanks to Prof. Dr. Tilo Pompe (Institute of Biochemistry,
University of Leipzig) for generously providing me with hvFbs for the scaffold experiments, which
have expanded my knowledge of scaffold-cell interactions.
I am grateful to Ms. Lisa Franz at the Faculty of Medicine, Leipzig University, for providing access
to and guidance with the FTIR device. I would also like to thank Mr. Dipl.-phys. Jörg Lenzner
(Faculty of Physics and Geosciences, Institute for Physics II) for his help in accessing the SEM
equipment and pictures.
I would like to express my personal gratitude to Dr. Iram Modsooq for her help with L929s
cultivation on polymeric film biocompatibility experiments.
PAGE 174
APPENDIX. ACKNOWLEDGEMENTS
I would also like to give special thanks to Annett Starke (Institute for Pharmacy, Pharmaceutical
Technology, Leipzig University) for her work in maintaining the lab and its equipment in good
working order.
I am deeply grateful to all of my colleagues for the enjoyable time we shared at the institute, the
pleasant working atmosphere, and the constructive discussions. I will always cherish the good
times we had together during our research endeavors in Leipzig, Germany. Thank you all for your
invaluable contributions and support. I hope to see you all again sometime soon.
KETPAT VEJJASILPA
PAGE 175
6.208
Article
Adjustable Thermo-Responsive,
Cell-Adhesive Tissue Engineering
Scaffolds for Cell Stimulation
through Periodic Changes in
Culture Temperature
Ketpat Vejjasilpa , Iram Maqsood, Michaela Schulz-Siegmund and Michael C. Hacker
Special Issue
Smart Gels and Their Applications
Edited by
Prof. Dr. Vijay Kumar Thakur
https://doi.org/10.3390/ijms24010572
International Journal of
Molecular Sciences
Article
Adjustable Thermo-Responsive, Cell-Adhesive Tissue
Engineering Scaffolds for Cell Stimulation through Periodic
Changes in Culture Temperature
Ketpat Vejjasilpa 1 , Iram Maqsood 1,2,3 , Michaela Schulz-Siegmund 1 and Michael C. Hacker 1,4, *
1 Pharmaceutical Technology, Institute of Pharmacy, Medical Faculty, Leipzig University, Eilenburger Str. 15A,
04317 Leipzig, Germany
2 Riphah Institute of Pharmaceutical Sciences (RIPS), Riphah International University (RIU),
Lahore 54000, Pakistan
3 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street,
Baltimore, MD 21201, USA
4 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, Universitaetsstrasse 1,
40225 Duesseldorf, Germany
* Correspondence: michael.hacker@hhu.de or mch@mchlab.de
Abstract: A three-dimensional (3D) scaffold ideally provides hierarchical complexity and imitates the
chemistry and mechanical properties of the natural cell environment. Here, we report on a stimuli-
responsive photo-cross-linkable resin formulation for the fabrication of scaffolds by continuous
digital light processing (cDLP), which allows for the mechano-stimulation of adherent cells. The resin
comprises a network-forming trifunctional acrylate ester monomer (trimethylolpropane triacrylate,
or TMPTA), N-isopropyl acrylamide (NiPAAm), cationic dimethylaminoethyl acrylate (DMAEA)
for enhanced cell interaction, and 4-acryloyl morpholine (AMO) to adjust the phase transition
temperature (Ttrans ) of the equilibrium swollen cross-polymerized scaffold. With glycofurol as a
biocompatible solvent, controlled three-dimensional structures were fabricated and the transition
temperatures were adjusted by resin composition. The effects of the thermally induced mechano-
Citation: Vejjasilpa, K.; Maqsood, I.;
stimulation were investigated with mouse fibroblasts (L929) and myoblasts (C2C12) on printed
Schulz-Siegmund, M.; Hacker, M.C.
constructs. Periodic changes in the culture temperature stimulated the myoblast proliferation.
Adjustable Thermo-Responsive,
Cell-Adhesive Tissue Engineering
Keywords: thermally responsive biomaterial; stimulus-responsive biomaterial; mechanical
Scaffolds for Cell Stimulation
stimulation; smart hydrogel; dynamic light processing; photo-polymerization
through Periodic Changes in Culture
Temperature. Int. J. Mol. Sci. 2023, 24,
572. https://doi.org/10.3390/
ijms24010572
1. Introduction
Academic Editor: Alexandre
Mironov
Mechanical stimulation during the cultivation of tissue-engineered constructs can pro-
mote tissue development and quality [1]. Different techniques have been applied to exert
Received: 11 October 2022 external stimulation, such as compressive loading systems [2], longitudinal stretch systems
Revised: 21 December 2022 that provide uniaxial tension, [3] systems utilizing substrate bending, [4,5] bi-axial traction
Accepted: 26 December 2022 systems, [6,7] and shear stress input systems [8,9]. However, those systems require complex
Published: 29 December 2022
bioreactor setups to create a suitable microenvironment. [10] We propose an alternative
strategy for exerting mechanical stimulation on adherent cells via a stimulus-responsive
scaffold. Such a strategy requires a smart biomaterial that changes its swelling degree
Copyright: © 2022 by the authors.
upon an environmental stimulus that is compatible with the cell culture and that maintains
Licensee MDPI, Basel, Switzerland. interaction with adherent cells in both the swollen and the de-swollen state. Different
This article is an open access article stimuli to initiate such a phase transition have been investigated, and the temperature re-
distributed under the terms and mains the most thoroughly investigated trigger that has been used for the design of various
conditions of the Creative Commons multifunctional materials [11–18]. Based on previous expertise, an N-isopropylacrylamide
Attribution (CC BY) license (https:// (NiPAAm)-based thermo-responsive material is envisioned here [19,20]. As it has been
creativecommons.org/licenses/by/ reported that mammalian cells can be cultured between 30 ◦ C and a physiological tempera-
4.0/). ture without impeding development [21–23], a target range for the transition temperature
Int. J. Mol. Sci. 2023, 24, 572. https://doi.org/10.3390/ijms24010572 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 572 2 of 17
of 30–36 ◦ C was set. The lower critical solution temperature (LCST) of NiPAAm-based
materials can be adjusted through copolymerization, whereby hydrophobic comonomers
decrease the transition temperature and hydrophilic comonomers have the opposite ef-
fect [24–26]. PolyNiPAAm has been used in various applications, such as smart surfaces,
from which a cultivated tissue layer can be harvested without trypsinization [27]. When
the temperature is decreased below a lower critical phase transition temperature (LCST),
the pNiPAAm surface layers swell and the cultured cell layer, together with the deposited
extracellular matrix, detaches. In order to utilize a thermo-responsive material for cell
stimulation, it is necessary that the cells remain adhered to the material body both above
and below the transition temperature. We hypothesize that cell adhesion can be sufficiently
mediated by introducing positive charges to the material network [28,29]. Hence, we inves-
tigate whether this strategy can also be utilized for cell mechanical stimulation through the
increased/decreased swelling of the network upon cyclic temperature changes around the
LCST with a copolymerized thermo-responsive network comprising a cationic comonomer.
To this end, a resin formulation for the three-dimensional printing of the tissue en-
gineering scaffolds that exhibits thermo-responsive behavior in the targeted temperature
range and maintains interaction with adhered cells in the swollen and de-swollen state is
warranted. Trimethylolpropane triacrylate (TMPTA) is known as a biocompatible trifunc-
tional macromer and is used as a cross-linker for the formation of an insoluble network
of copolymerized NiPAAm [30]. The cationic comonomer dimethylaminoethyl acrylate
(DMAEA) is introduced as the functional component to mediate cell adhesion independent
of the swelling degree of the material. 4-acryloylmorpholine (AMO) is selected to adjust
the transition temperature (Ttrans ) of the copolymer network [14].
This work focused on UV light-induced resin copolymerization, first in bulk and
thereafter by cDLP printing, and the characterization of the resulting networks with regard
to transition temperature and material swelling. Continuous DLP is one of many three-
dimensional printing techniques that have been used for medical scaffold fabrication
with high resolution [31,32]; it uses a photo-curable resin that is polymerized in layers
by projected light. However, not many biocompatible resins for photo-polymerization
are available [33–35]. In this work, we additionally introduce tetrahydrofurfuryl alcohol
polyethylene glycol ether (glycofurol, GF) as an alternative solvent for resin formulation.
GF has already been utilized as a biocompatible solvent or diluent for drug delivery to both
human and animal tissue. Cytocompatibility of the resin-derived networks is evaluated
with L929 and C2C12 cells. Promising formulations are cultivated and the response of the
adherent cells to the periodic changes in cultivation temperature are compared.
2. Results and Discussion

Discs and a three-dimensional scaffold from thermo-responsive networks composed of
NiPAAm, DMAEA, TMPTA, and AMO were successfully polymerized by photo-induced
bulk polymerization and cDLP, respectively. In order to identify a suitable resin formula-
tion, different series of monomer mixtures were cross-polymerized into nonporous discs by
photo-induced bulk polymerization. The transition temperatures of the water-swollen discs
were quantified by DSC, as presented in Figure 1. All the DSC analyses were performed in
aqueous solutions. Although the transition temperature is generally affected by osmolarity,
pH value, electrolyte type, and the concentration of the solution [36], we did not intend to
simulate the exact cell culture conditions with the medium serum and additives to obtain
a clearer signal only affected by the polymer network composition. It was found that the
cationic monomer DMAEA and the network former TMPTA significantly influenced the
transition temperature, and the NiPAAm-based formulations with transition temperatures
ranging from 16.4 ◦ C ± 0.9 ◦ C (0% w/w DMAEA, 1% w/w TMPTA) to 40.0 ◦ C ± 0.7 ◦ C
(20% w/w DMAEA, 20% w/w TMPTA) were obtained (Figure 1A). When the samples with
the same TMPTA content were compared, the DMAEA acted as hydrophilic comonomer
and an increasing content resulted in an increasing Ttrans . For example, within the 5%
w/w TMPTA discs the transitional temperature increased from 25.9 ◦ C ± 0.8 ◦ C (0% w/w
Int. J. Mol. Sci. 2023, 24, 572 3 of 17
DMAEA) to 32.5 ◦ C ± 2.0 ◦ C (20% w/w DMAEA). Looking at the TMPTA effect in the ab-
sence of DMAEA, the transition temperature considerably decreased from 32.7 ◦ C ± 0.9 ◦ C
(0% w/w DMAEA, 1% w/w TMPTA) to 16.4 ◦ C ± 0.8 ◦ C (0% w/w DMAEA, 20% w/w
TMPTA). An increase in the feed ratio of TMPTA created denser networks, thus decreas-
ing the distance between the polymer segment chains due to the trivalent chemistry of
the monomer which directly affected hydration capacity, subsequently decreasing the
material’s transition temperature [37]. e material’s transition temperature
Figure 1. Transition temperatures of photo-polymerized discs as obtained by DSC. (A) Resins

–
with 1–20% w/w TMPTA, –
0–20% w/w DMAEA, and 0% w/w AMO. (B) Effect of AMO on transition
– –
temperature (5–10% w/w TMPTA, 15% w/w DMAEA). (C) Effect of TMPTA (1–10% w/w) on transition
temperature in different resin formulations (N70-D15-A15, N60-D20-A20, and N50-D25-A25).
Both comonomers, DMAEA and TMPTA, affect the transition temperature of the
NiPAAm-based networks, but both monomers also change other key characteristics of
the material, i.e., the charge and cross-linking density. With the implementation of a
hydrophilic but functionally inert monomer, 4-acryloylmorpholine (AMO), we intended
to further adjust the transition temperature of the cross-polymerized networks to the
desired range of 33 ◦ C to 36 ◦ C without changing the charge and cross-linking. The
molecular character of the morpholine residue attracts water molecules into the polymeric
network and promotes an increase in material swelling [14,38,39]. In addition, AMO
was also expected to promote the solubility of the resin in less hydrophobic and more
biocompatible solvents. As illustrated in Figure 1B, the presence of AMO in the TMPTA-
cross-linked copolymer increased the transitional temperature. For example, the discs
copolymerized from 5% w/w TMPTA, 15% w/w DMAEA, and 5% w/w AMO had a Ttrans
of 30.7 ◦ C ± 1.7 ◦ C. The transition temperatures gradually increased to 37.1 ◦ C ± 0.6 ◦ C
(5% w/w TMPTA, 15% w/w DMAEA, 20% w/w AMO) with the increasing AMO content.
The resin composition was optimized at a DMAEA content of 15% (w/w), which was
the best compromise of a high amine content and cell survival in the initial compatibility
tests. With regard to the desired phase transition temperature, we identified promising
formulations from the data presented in Figure 1 that consist of 1–5% w/w TMPTA, 10–15%
w/w DMAEA, and 1–15% w/w AMO. These formulations were investigated in more detail.
Int. J. Mol. Sci. 2023, 24, 572
– – 4 of 17
–
2.1. Swelling Characteristics of Photo-Polymerized Discs

We postulate that mechanical stress will be exerted by the dimensional changes in
the materials as a result of different material water contents in response to the changes in
the environmental temperature. Consequently, the swelling ratios of the different resin
compositions were evaluated at 25 ◦ C and 37 ◦ C (Figure 2). A more specific focus was
given to the transition temperatures of the formulations with 5% and 10% w/w AMO and
low contents of the network-forming comonomer TMPTA (1%, 2%, and 3% w/w). The
discs with 10% w/w AMO and 15% w/w DMAEA (N75-D15-A10) had significantly higher
transitional temperatures than the formulations with 5% w/w AMO (N80-D15-A5) for
all the TMPTA additions. The discs with 1% w/w TMPTA (15% w/w DMAEA and 5%
w/w AMO, Figure 2A) had the lowest Ttrans at 32.1 ◦ C ± 0.3 ◦ C, which was increased to
34.6 ◦ C ± 0.9 ◦ C by increasing the AMO content to 10% w/w.
Figure 2. Swelling ratio at different temperatures and Ttrans of 1% TMPTA (A), 2% TMPTA (B), 3%
TMPTA cross-linker (C),),and
and ΔSR
∆SR (37/25)
(37/25) of of
1%,1%,
2%,2%,
andand 3% TMPTA
3% TMPTA cross (D). Negative value
cross-linker
indicates shrinkage of sample discs.
We also observed a clear change in the swelling ratios at the investigated temperatures
of 37 ◦ C and 25 ◦ C when different TMPTA concentrations were compared. In order to show
if water is expelled from the formulation by thermally induced gelation, we defined the
parameter ∆SR (37/25) (as defined in the methods section), which becomes negative if the
ΔSR
phase transition occurs between the two temperatures and the cross-linked networks expel
water, while positive values indicate an increase in swelling at 37 ◦ C. The discs with 3%
TMPTA and 10% w/w (N75-D15-A10) or 5% w/w (N80-D15-A5) AMO successfully shrank
by −1.2 ± 0.4 g/g and − −1.0 ± 0.1 g/g.− In contrast, the lower TMPTA contents resulted
in positive values for ∆SR (37/25)
ΔSR a result of a reduced cross-linking density. We also
as
observed the expected effect of AMO on network hydrophilicity [40].
2.2. Three-Dimensional Scaffold Fabrication

From the described resin formulations that were processed into disc-shaped sam-
ples by the bulk photo-polymerization, selected formulations (3% w/w TMPTA) were
transferred to processing by a three-dimensional printer using the concept of dynamic
light processing (DLP). We investigated formulation parameters that are specific to the
applied three-dimensional printing technique, namely exposure time per layer, the photo-
initiator content (PI), and the solvent, as these parameters have been reported before to
influence printing quality and the scaffold’s mechanical properties [41,42]. The selected
Int. J. Mol. Sci. 2023, 24, 572 5 of 17
scaffold’s mechanical properties
monomer formulations (3% w/w TMPTA and 15% w/w DMAEA) with an optimized
Ttrans (0% AMO: 34.6 ◦ C ± 0.9 ◦ C and 5% AMO: 35.6 ◦ C ± 0.3 ◦ C) determined via disc
polymerization were prepared and diluted with ethanol to yield a concentration of 10%
(w/v). Two different scaffold designs, namely a raft and a three-dimensional lattice, were
printed (Figures S1 and S2). The raft-configuration scaffolds were selected to investigate
the biocompatibility of the material as the geometry allows the observation of the cells on
the cell impermeable scaffold surface. Cell proliferation can be conveniently observed by
microscopy. The second design is a macroporous lattice of 7.6 × 7.8 × 2.5 mm (length ×
width × height), with fiber diameters of 240 µm. A fiber arrangement that used thicker
fibers (470 µm) as the scaffold foundation (first layer) was used. In the subsequent nine
fiber layers, the fiber orientation was rotated by 90◦ per layer. The scaffolds (lattice and raft)
were composed of 50 layers with a thickness of 50 µm each. The macroporous structure of
the lattice was selected to enable cell adhesion and proliferation and allow for efficient nu-
trient exchange via interconnecting channels [43,44]. The resins were printed with various
exposure times per layer (10, 20, and 30 s).
The intended property of the materials presented here is to respond to temperature
and to exert a mechanical stimulus on adherent cells by volume change. The type and
concentration of PI as well as the exposure time are known to influence the network
characteristics [45]. Lower contents of PI and shorter exposure times are expected to
yield materials with reduced cross-linking and a smaller response in the swelling change
a lowby
between 25 ◦ C and 37 ◦ C, as expressed value
a lowofvalue
ΔSR of ∆SR (37/25) (Figure 3). Based on a
comparison of the corresponding resin formulations processed into discs (N80-D15-A5), we
observed the strongest change in swelling with 4% PI at an exposure time of 10 s (Figure 3C).
polymerized
As for the photo-polymerized discs,
discs, ∆SRΔSR(37/25) was also compared for different scaffold
formulations. We observed no significant change in transition temperature as a result of PI
content and exposure time.
Figure 3. Swelling ratio (g/g) of 3% TMPTA N80-D15-A5 scaffold with (A) 2% w/w and (B) 4% w/w
photo-initiator (Irg819). Scaffold Ttrans values as determined by DSC added to the graphs. (C)ΔSR
∆SR
(37/25) values of the presented formulations.
2.3. Three-Dimensional Scaffold Fabrication with Glycofurol as Solvent

In the experiments using ethanol as a solvent/diluent for cDLP, certain limitations
concerning the printability of the resins, such as the premature detachment of the sample
and low structural resolution due to low resin viscosity, became apparent. With the
objective being to address these issues, glycofurol (GF), which has a higher viscosity than
ethanol, was utilized [46,47]. The increased viscosity of GF in comparison to ethanol could
potentially improve scaffold resolution during printing due to damping effects [46,48,49].
Int. J. Mol. Sci. 2023, 24, 572 6 of 17
GF has been introduced as a biocompatible solvent for biomedical applications and as a

pharmaceutical excipient. GF in a 50:50-mixture with PBS was administered to rat brain
tissue and was well tolerated and provoked only a minor inflammatory response [46]. The
possible residual amount of GF that might be released from the washed scaffolds over
time is considered to be much less than the concentrations tested in the mentioned study.
Therefore, residual GF is not expected to affect the cellular response but a chromatographic
evaluation of the amount of residual GF will become necessary in future studies.
It was possible to print GF-diluted resins with preserved thermo-responsive properties
and effectively halve the exposure time per printed layer from 20 to 10 s. Furthermore,
premature detachment of the construct from the printing head during printing could also be
avoided. The swelling ratios of the GF-based scaffold for both geometries (raft and lattice)
were determined by varying different DMAEA and AMO ratios at different temperatures
(25 ◦ C and 37 ◦ C) (Figure S3). The lattice scaffolds fabricated from resins without DMAEA
and AMO (3% TMPTA N100-D0-A0) showed the strongest change in swelling due to a
low transition temperature of 28.0 ◦ C ± 0.1 ◦ C, which corresponds to the expectations for
a pure cross-linked NiPAAm matrix (Figure 4). As expected, the addition of AMO and
DMAEA increased the transition temperature that was established for the ethanol-based
resins. The increased AMO content effectively increased the transition temperature of the
cross-linked bulks from 33.9 ◦ C ± 0.3 ◦ C (N85-D15-A0) to 38.6 ◦ C ± 1.2 ◦ C (N65-D15-A20).
The transition temperature of the scaffolds with different ratios of DMAEA and an AMO
content of 15% was elevated from 30.9 ◦ C ± 0.5 ◦ C (N85-D0-A15) to 39.5 ◦ C ± 0.7 ◦ C
(N65-D20-A15). The. The corresponding
corresponding valuesvalues
of ∆SRof (37/25)
ΔSR decreased from (−4.5 ± − 0.1) g/g
− . The results of ΔSR
(N85-D0-A15) to (−0.8 ± 0.2) g/g in (N65-D20-A15). The results of ∆SR (37/25) showed
the effect of DMAEA, which could increase the transition temperature and stabilize the
NiPAAm-based networks from extensive collapse in the investigated temperature range,
thuss decreasing
decreasing∆SR
ΔSR(37/25). From the authors’’ perspective,
perspective,the
the∆SR
ΔSR(37/25) values should
be measurable and negative but in a moderate range (−−3 to −−2 g/g) in order to maintain
cell adhesion to the biomaterial surface.
Figure 4. Swelling behavior of three-dimensional scaffold printed from GF-based resins (3% w/w
TMPTA, 4% w/w PI, printing time 10 s/layer) at different temperatures (25 ◦ C and 37 ◦ C) with the
transition temperature with different ratios of (A) AMO and (B) DMAEA. The corresponding ∆SR ΔSR
(37/25) is shown in charts (C,D).
2.4. Rheological Properties of Scaffolds

Changes in cultivation temperature are expected to switch the scaffold swelling and
exert mechanical stimulation, which is an important factor that influences cell fate [50,51].
Rheological characterization of the scaffold moduli by oscillation rheology revealed that
the moduli are in the range of the literature data on cellular stiffness (myoblasts: 2 kPa,
fibroblasts: 1–10 kPa) [52,53]. As shown in Figure S4, the determined storage moduli of the
Int. J. Mol. Sci. 2023, 24, 572 7 of 17
printed scaffolds increased with the exposure time (S4a) and photo-initiator concentration
(S4b). For example, at the transition temperature, the storage moduli of the samples
fabricated with 4% PI and 10 s of exposure per layer were 1.2 and 1.5 times higher as
compared to the samples with 3% PI and 2% PI, respectively. An increase in exposure
time per layer from 10 s/layer to 20 and 30 s/layer doubled the moduli. The transition
temperature remained almost unchanged as the altered parameters (concentration of PI and
exposure time per layer) do not affect the chemical composition of the network. Network
density, however, is affected by these parameters, as observed by the changes in the network
swelling. The incorporation of DMAEA strongly decreases the scaffold moduli (Figure S5).
At 20% w/w DMAEA, the hydrophilic contribution of the cationic monomer was strong
enough to fully suppress the coil-globule transition of the copolymeric network and no
change in storage modulus could be observed any more in the investigated range, which
correlates with the observed increase in transition temperature. Apart from N93-D2-A5,
the investigated samples (Figure S5) showed very low temperature-induced changes in
the bulk storage moduli. In this context, it should be considered that the construct volume
change and the corresponding displacement of the focal adhesion points with the cells are
the primary causes of the mechanical stress on the adhered cells. Therefore, the correlation
between the volume changes that are compatible with the maintenance of cell adherence
and cell survival and the storage modulus change in the bulk hydrogel is not obvious and
needs further experimental evaluation.
2.5. Biocompatibility Evaluation

We chose a promising, photo-polymerizable thermo-responsive formulation (3% w/w
TMPTA N80-D15-A5, 4% w/w PI, exposure time 10 s per layer), which has a transition
temperature of 36.3 ◦ C ± 0.9 ◦ C and the ∆SR (37/25) of (−1.3 ± 0.2) g/g and shows a
change in material modulus from 2.8 kPa (30 ◦ C) to 7.1 kPa (37 ◦ C). Both moduli are within
the physiological range of 1–10 kPa and are reported as the best suited for fibroblasts [54].
Therefore, the material is expected to support cell viability at both temperatures, and the
periodic change in modulus is expected to stimulate cell proliferation and differentiation.
First, we tested the effect of exposure time and PI concentration on the biocompatibility of
the resin formulation with murine fibroblasts (L929) at the static cultivation temperature
of 37 ◦ C (Figure 5). The cells were stained with calcein-AM and ethidium homodimer on
printed scaffolds. All of the formulations showed coverage with live cells at day seven,
which, in comparison to the cell density after seeding, indicates a considerable proliferation.
There was no discernible difference between the different tested formulations. On a cellular
level, the fibroblasts maintained a well-spread morphology. A low number of cells were
found to be dead, as indicated by the fluorescence signal in the red channel due to the
DNA-bound ethidium homodimer (Figure 5). The diffuse character of the fluorescence
signal also indicates some degree of autofluorescence of the materials. The low number of
distinctly stained dead cells suggests no explicit material toxicity, but rather an expected
turnover of the cells.
The scaffolds of the raft architecture, as illustrated in supplementary Figure S1, provide
cell adherence in one plane for better microscopic evaluation. The L929 fibroblast viabil-
ity was microscopically evaluated on scaffolds fabricated from formulation N80-D15-A5
with 3% w/w TMPTA and 4% w/w PI, with 10 or 20 s of exposure time per layer. The
fluorescence microscopic images of the cells on the scaffolds after cultivation for 1, 3, 5,
and 7 days at a constant temperature (37 ◦ C) are summarized in Figure 6. On all the
samples, cell proliferation was visible to a comparable degree. No significant difference in
compatibility on the scaffolds fabricated with exposure times of 10 and 20 s per layer was
visible (Figure 6A). Based on these positive results, the scaffolds of the raft architecture with
a DMAEA content of 15% and 10% w/w were printed with an exposure time of 10 s/layer
and evaluated. The normalized intensities of the calcein fluorescence signals (NFI) were
plotted and a 1.5-fold (p = 0.07) higher NFI was observed for N80-D15-A5 as compared
Int. J. Mol. Sci. 2023, 24, 572 8 of 17
to N85-D10-A5 at day 7 (Figure 6B). We consider this as an indication of the benefit of a

content of 15% DMAEA to optimize cell proliferation and survival.
of L929 on 3% TMPTA, N80-D15-A5 scaffolds (37 ◦ C) at

Figure 5. Fluorescence images of L929 fibroblasts
different photo-initiator ratios (horizontal) and exposure times (vertical) after live/dead staining
at day 7. In each group, one small picture shows the fluorescence channel for calcein (green) and
another the fluorescence wavelength for ethidium homodimer (red). The large picture in each group
shows an overlay of both channels. Scale bars represent 500 µm.
2.6. Cellular Response to Scaffolds under Periodic Changes in Cultivation Temperature

The cells were seeded on scaffolds of the raft architecture fabricated from resins
with different ratios of DMAEA and were subjected to periodic changes in cultivation
temperature in order to demonstrate the scaffold’s ability to sustain cell adhesion above
and below transition temperature and to exert mechanical stimulation to the adherent
cells. Figure 7 shows cellularity on scaffolds cultivated for 1, 3, and 7 days with a daily
reduction in cultivation temperature to 30 ◦ C for one hour. The L929 cells maintained
adherence to the scaffolds fabricated from the resins with a DMAEA content of at least 10%
w/w. The scaffolds with less than 10% w/w DMAEA showed almost no cell attachment.
The L929-seeded scaffolds with a higher amount of DMAEA showed a proliferation of
adherent cells over the cultivation time of 7 days. Microscopically, the cells tended to
initially adhere to the grooves of the scaffold structure, filling the accessible grooves after
gravitational seeding. Upon proliferation, the cells grew outward and eventually covered
the full surface of the scaffold. Below a threshold value of 10% DMAEA, the cells detached
from the scaffolds during the temperature cycles. This confirms the cell adhesive effect
of the positively charged moieties in DMAEA. In addition to this chemical effect, more
extensive changes in the swelling ratio in the material with a lower transition temperature
possibly contributed to the observed cell detachment.
Another cell type that should benefit from mechanical stimulation is the myoblast
cell. C2C12 mouse myoblasts have previously been utilized as a model for mechanical
stimulation [55,56]. For this reason, we also observed the fate of C2C12 cells on printed
formulations. Here, we demonstrated that the C2C12s cultivated on the raft scaffolds at
constant temperature (37 ◦ C for up to 7 days) showed almost no cell growth (Figure S6).
The scaffolds fabricated from resins N85-D10-A5, N80-D15-A5, and N75-D20-A5, how-
ever, showed cell proliferation when cultured under a periodically changed cultivation
temperature. Overall, the differences observed between the isothermal and periodically
changed cultivation conditions indicate a successful mechanical stimulation of the adherent
cells by the material as the mechanical stress from the material swelling is considered to
influence and up-regulate molecules such as calmodulin, nNOS, MMP-2, HGF, c-Met, and
mitogen-activated protein kinase [51,56].
Int. J. Mol. Sci. 2023, 24, 572 9 of 17
ature in order to demonstrate the scaffold’s ability to sustain cell adhesion above and
Figure 6. (A) Cellularity (L9292 fibroblasts) on GF-based scaffolds with raft architecture (N80-D15-A5,
4% PI, 3% TMPTA) printed with different exposure times per layer (10 and 20 s/layer) at different
time points (1, 3, 5, and 7 days). (B) Normalized intensities of green fluorescence emitted from live
cells on two formulations, N85-D10-A5 and N80-D15-A5 (3% TMPTA, 10 s/layer), at day 1, 3, and 7.
Scale bars equal 500 µm (low magnification (4×), left images) or 200 µm (higher magnification (10×),
right images). p-value is denoted as * p.
ature in order to demonstrate the scaffold’s ability to sustain cell adhesion above and
Figure 7. Cellularity of L929 and C2C12 cells on printed scaffolds (3% w/w TMPTA, 5% w/w
AMO, 4% w/w PI, exposure time 10 s/layer) in raft architecture cultivated under periodically
changed temperature (cycle: 23 h at 37 ◦ C and 1 h at 30 ◦ C). The fluorescent micrographs showed
overlaid live/dead staining and were recorded at day 1, 3, and 7. Scale bars represent 500 µm (low
2.7. Effect of DMAEA Content on Cell Proliferation on the Scaffolds

Fluorescence intensity was quantified to further characterize the effect of different
cationic monomer feeds on the investigated cells (Figure 8). As expected, the materials
with a low DMAEA content showed low fluorescence signal intensities. The scaffold with
Int. J. Mol. Sci. 2023, 24, 572 10 of 17
DMAEA feeds of more than 10% in the printing resins yielded promising results. The NFI
at day 1 on different formulations showed no statistical difference between the different
materials for both cell lines. However, we observed an increase in the average NFI with the
increasing DMAEA content. At day 3, the scaffolds with 0–5% DMAEA showed no increase
in the intensities, whereas the cells on scaffolds with 10% and more DMAEA showed a
–
significant increase in NFI. The signal intensities of the L929 cells on 10%, 15%, and 20%
DMAEA on the raft scaffold increased 9.2-, 10.2-, and 19.8-fold as compared to the cells
on scaffolds from resins without DMAEA on the same day. On scaffolds with 15% and
20% DMAEA, the NFI increased steadily over the investigated time points. At day 8, the
differences in NFI became more obvious, and the L929 cells on the scaffolds with 20%
DMAEA displayed the highest calcein stain (11.9 ± 1.0).
Figure 8. Normalized fluorescence intensity (NFI) at day 1, 3, and 7 of calcein-stained L929 (top) and
C2C12 cells (bottom) on raft
– scaffolds (3% w/w TMPTA, 4% w/w PI, printing time 10 s/layer) of
different DMAEA compositions (0–20% w/w) upon cultivation with periodic changes in cultivation
temperature. Statistically significant differences are denoted by *.
The equivalent experiment with C2C12 myoblasts revealed similar results in response
to periodic changes in the cultivation temperature. On scaffolds with less than 5% w/w
DMAEA, cell proliferation was not supported. An interesting behavior of the C2C12 cells
was seen on 10% DMAEA scaffolds. The NFI increased from day 1 to 3 but then declined
between day 3 and 7. We attribute this to an insufficient number of cationic adhesion
moieties to maintain cell adhesion over a larger number of swelling/deswelling cycles. In
– the C2C12 have been described as more sensitive to
comparison to the L929 fibroblasts,
material changes [57]. At a constant cultivation temperature, the cellularity of the C2C12
cells on the 15% w/w DMAEA scaffolds was low and the cells appeared to be predominantly
rounded without significant cell–cell contacts (Figure S6). The C2C12 on scaffolds (15% and
20% w/w DMAEA) cultivated with periodic temperature changes– showed proliferation,
–
Int. J. Mol. Sci. 2023, 24, 572 11 of 17
as indicated by a fold increase in fluorescence intensity of 8.8 (15% DMAEA) and 15 (20%
DMAEA) from day 1 to 7.
The promising results of the raft-type scaffolds motivated an evaluation of the L929
cells on a lattice-type scaffold that provides a more physiological template for the cell–
cell interactions (Figure 9) [58,59]. The resins were processed into lattice-type scaffolds
that were designed to allow for effective cell seeding and nutrient distribution through
the porous architecture [60–62]. At day 1, a low number of cells was observed on the
scaffolds with low DMAEA contents in the resins, while the cellularity was increased on
the 15% w/w DMAEA scaffolds, indicating the benefit of cationic moieties for interactions
with the polyanionic cell membranes. At day 3, the cells began to spread on the scaffold
with at least 10% DMAEA. The NFIs at day 5 and 7 were increased, which suggests cell
proliferation within the scaffolds. A reconstruction of the confocal fluorescence microscope
slices recorded from the calcein-AM stained L929 cells on a N80-D15-A5 scaffold at day 7
demonstrated high cellularity on the material strands (Figure 9).
Figure 9. Fluorescence micrographs of three-dimensional lattice-type scaffolds seeded with L929

fibroblasts (2, 10, and 15% w/w DMAEA, 5% w/w AMO, 3% TMPTA, 10 s/layer, 4% w/w PI) and
cultivated with periodic temperature changes at different time points (1, 3, 5, and 7 days). Scale bars
equal 500 µm (low magnification (4×), left images) or 200 µm (higher magnification (10×), right
images). Three-dimensional reconstruction of fluorescent images of N80-D15-A5 at 7 days.
Even the scaffolds with low DMAEA (2% w/w) showed cell proliferation and good cell
viability at day 7, but the DMAEA contents of 10% and 15% again proved more effective. A
possible explanation for the increased cell survival on the three-dimensional porous scaffold
could be the more physiological microenvironment generated during three-dimensional
cultivation [58,59,63,64].
In this study, the specific focus was to identify a material that would a show phase
change shortly below physiological temperature and exhibit a moderate change in swelling
while incorporating functional groups that mediated cell-adhesion above and below the
phase transition temperature. This property is not inherent to conventional thermally
responsive materials but key to our intended use as tissue engineering scaffold material
that could exert a mechanical stimulation on cells cultivated on the material under periodic
changes in cultivation temperature encompassing the transition temperature of the material.
Material formulations are presented that realize these properties. The effects of the swelling
changes on the proliferation and survival of the fibroblasts and myoblasts are shown. With
the material development successfully demonstrated in this study, subsequent work will
Int. J. Mol. Sci. 2023, 24, 572 12 of 17
have to elucidate the extent and frequency of the mechanical stimulation and the cellular
effects in comprehensive detail.
3. Materials and Methods

3.1. Materials
N-Isopropylacrylamide (NiPAAm) (Tokyo Chemical Industry [TCI], Tokyo, Japan),
hexane (Sigma-Aldrich), trimethylolpropane triacrylate (TMPTA) (Sigma-Aldrich), dimethy-
laminoethyl acrylate (DMAEA) (Sigma-Aldrich, Taufkirchen, Germany), 4-acryloylmorpholine
(AMO) (Sigma-Aldrich), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO or Ir-
gacure819) (Sigma-Aldrich), absolute ethanol (EtOH) (Sigma-Aldrich), tetrahydrofurfuryl
alcohol polyethylene glycol ether (glycofurol or GF) (Sigma-Aldrich), Dulbecco’s phosphate
buffered saline (PBS) (Sigma-Aldrich), calcein-AM (Thermo Fisher Scientific, Waltham,
MA, USA), ethidium homodimer (EthD-1) (Thermo Fisher Scientific), low-glucose Dul-
becco’s modified Eagle’s medium (DMEM) (Merk), penicillin streptomycin (pen/strep)
(Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and fetal bovine serum
(FBS) (Sigma-Aldrich) were purchased and used as received. L929 fibroblasts and C2C12
myoblasts were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).
The NiPAAm was purified before use. In brief, the NiPAAm was dissolved in dried
warm hexane under constant magnetic stirring. The solution was placed at −20 ◦ C to
encourage recrystallization of the NiPAAm. The recrystallized NiPAAm was then separated
with a cylindrical funnel and washed again with cold hexane and dried under reduced
atmospheric pressure for 72 h.
3.2. Methodology
Nomenclature. The resin formulations are represented as x% NiPAAm, y% DMAEA,
and z% AMO (all w/w) and labeled by the code Nx-Dy-Az. The intended amount of
TMTPA was added to the monomer solution as exemplarily described here: the resin 3%
TMPTA 80N-15D-5A was prepared by mixing 80% w/w NiPAAm, 15% w/w DMAEA, and
5% w/w AMO. To this mix, 3% w/w TMPTA was finally added with respect to the mass of
the monomer solution. In a typical batch, such a resin mix would finally be composed of
2.0 g NiPAAm, 0.39 g DMAEA, 0.11 g AMO, and 0.075 g TMPTA. All the formulations are
summarized in Tables S1 and S2 in the Supplementary Materials.
Synthesis of hydrogel discs by photo-induced polymerization. In order to test the photo-
induced copolymerization of the monomer mixtures and investigate the effects of the
comonomer ratios (Table S1) on the material properties before three-dimensional fabrica-
tion, the resins were prepared by vigorously mixing the corresponding ratios of monomers
in ethanol to obtain a concentration of 10% (w/v) with 4% (w/w) photo-initiator (PI, Ir-
gacure819) relative to the mass of the monomer mix. A commercial UV chamber (Melody
Susie Violet II, Model DR-301C, 36W) was used for photo-cross-linked disc fabrication.
Briefly, 200 µL of the monomer-PI-solution was pipetted into caps of polypropylene micro-
centrifuge tubes (Eppendorf, Germany) and exposed to UV light (380 nm) for two minutes.
The cross-linked discs were removed from the caps and washed with diluted ethanol (70%)
and distilled water for 24 h each (Figure 10).
Three-dimensional printing of tissue engineering scaffold. A commercial cDLP three-dimensional
printer (PICO2 HD27 UV, Asiga, Erfurt, Germany) was used for three-dimensional printing.
The monomer mix (Table S2) was prepared as described above and subsequently dissolved
with either ethanol or glycofurol at a ratio of 2:1 and finally transferred into a building tray,
of which the volume was reduced by a customized silicon and Teflon insert. The scaffolds
were printed at room temperature with a light intensity of 36.0 mW·cm−2 with a layer
thickness of 50 µm. The scaffold structures (raft and lattice) (Figure S1) were constructed
by the Composer program (Version 1.2.5, Asiga). The exposure time per layer (10, 20, and
30 s per layer) was varied along with the different ratios of photo-initiators. The freshly
printed scaffolds were immersed in ethanol (70%) for 48 h to extract solvents and unreacted
monomers and subsequently in distilled water or culture medium for further use.
Int. J. Mol. Sci. 2023, 24, 572 𝐼𝑎 − 𝐼𝑏 13 of 17
NFI =
𝐼𝑏
Figure 10. Stimuli-responsive fabrication of temperature-sensitive biomaterials by photo-induced

bulk polymerization and cDLP printing. A monomer mixture with PI is irradiated with UV light to
cross-link the resin in disc shape or as scaffold produced by cDLP. The cross-linked matrices were
washed with aqueous ethanol (70% (V/V)) and water.
Swelling ratio analysis. For evaluation of the water uptake by the materials, the thermo-
responsive discs and three-dimensional scaffolds were analyzed for mass change after
incubation at 25 ◦ C and 37 ◦ C (n = 4) in water. Each incubation interval was 24 h. At both
times, the wet weights of the samples were recorded after excess water was absorbed by
placing the samples on cellulose filter paper. Thereafter, the materials were freeze dried for
48 h before recording the dry weight. The swelling was calculated as follows:
Weight of swollen construct(g)

Swelling ratio (g/g) =
Dry weight of construct (g)
Delta swelling ratio: In order to elucidate the changes in the swelling ratio at tempera-
tures surrounding the temperature range of interest, the difference in the swelling ratios
(g/g) determined at 37 ◦ C and 25 ◦ C was determined by subtracting the swelling ratio
determined at 25 ◦ C from the value determined after equilibration at 37 ◦ C and reported as
∆SR (37/25).
∆SR (37/25) = swelling ratio at 37 ◦ C − swelling ratio at 25 ◦ C
When thermo-gelation occurred in the covered temperature range, a negative value

was expected for this parameter as water is expelled from the material when the temper-
ature exceeds the phase transition temperature. A positive value indicated that swelling
Int. J. Mol. Sci. 2023, 24, 572 14 of 17
increased with incubation temperature and no phase transition affecting the swelling ration
occurred between 25 ◦ C and 37 ◦ C.
Rheological properties of printed constructs. An oscillatory rheometer (MCR301, Anton
Paar, Graz, Austria) was used to determine the responses of the selected three-dimensional
scaffolds to changes in environmental temperature using the following analytical protocol:
the equilibrium swollen samples were kept under constant normal force (0.2 N) with an
8 mm parallel plate geometry and analyzed in a temperature sweep experiment with
an oscillation frequency of 1 Hz and an amplitude of 1% strain. After an equilibration
time of 10 min, the temperature was ramped from 20 ◦ C to 50 ◦ C and vice versa with a
heating/cooling rate of 3 K/min. Storage and loss moduli were recorded by the RHEOPLUS
software (Anton Paar, Version 3.4).
Differential scanning calorimetry (DSC). The transition temperatures of the discs and
scaffolds were determined by DSC (DSC1, Mettler Toledo, Greifensee, Switzerland). The
samples were equilibrated in distilled water for 48 h and cut into small pieces, from which a
sample was precisely weighted (10–15 mg) into a 40 µL aluminum crucible. A crucible filled
with water (15 mg) was used as a reference. The heat flux DSC was performed between
10 and 60 ◦ C with a heating rate of 3 ◦ C/min. The first onset temperature of the recorded
endothermic phase transition signal was designated as the transition temperature (Ttrans ).
Cell Culture Studies. L929 fibroblasts and C2C12 myoblasts cells were cultured on
printed scaffolds. Selected scaffolds were seeded with 2 × 105 cells per construct in
standard 12-well plates (Corning Inc., Corning, NY, USA). The plates were placed into an
incubator at 37 ◦ C for the cells to settle for two minutes before the wells were carefully
filled with cell culture medium to completely cover the seeded constructs (2 mL). The cell
culture medium (37 ◦ C) was refreshed every 48 h after the temperature shift period was
completed. A mechanical stimulation of the cells on the materials was induced through
periodic temperature changes. In brief, after 24 h of cell seeding, the plates were taken out
of the incubator (37 ◦ C) and placed in the laminar air flow bench for 10 min to decrease the
medium temperature. The plates were then placed in an incubator thermostat at 30 ◦ C (5%
CO2 ) for one hour before the plates were returned to the incubator at 37 ◦ C. The periodic
cycle was repeated every 24 h until the designated time point.
Live/Dead Assay. The viability of the cells on the material surfaces was qualitatively
assessed by staining the cell-laden constructs with calcein-acetoxymethylester (calcein-AM)
(λex = 488 nm, λem = 520 nm) and ethidium homodimer-1 (λex = 528 nm, λem = 617 nm)
(both from Molecular Probes™, Invitrogen, Waltham, MA, USA) under light exclusion
for 45 min. The live/dead staining solution was prepared by mixing 1 µL of calcein-AM
and 2 µL of ethidium homodimer-1 with 2 mL of PBS. The samples were washed with
PBS to remove excess dye and analyzed by fluorescence microscopy at λex = 488 nm and
λem = 520 nm for calcein in the live cells and at λex = 528 nm and λem = 617 nm for ethidium
homodimer-1 in the dead cells (Nikon DS-Ri2, Japan). Cell viability was documented at
days 1, 3, and 7.
Live cell staining was quantified by analyzing the micrographs (TIFF format, recorded
with an exposure time of 600 ms) (n = 3). The images underwent a registration process and
three-dimensional reconstruction by Fiji software (ImageJ, NIH, Bethesda, MD, USA) [65].
The normalized fluorescence intensity (NFI) of each micrograph was calculated by aver-
aging the fluorescence intensity (Ia ) and subtracting the average background intensity (Ib )
from a scaffold without a cell presence and dividing again by the average background
intensity. The equation is shown below:
Ia − Ib
NFI =
Ib
4. Conclusions
This work presents thermo-sensitive scaffolds composed of TMPTA-cross-linked
poly(NiPAAm-co-DMAEA-co-AMO) networks fabricated via photo-induced polymeriza-
tion and three-dimensional cDLP printing methods from monomer resins. The resulting
Int. J. Mol. Sci. 2023, 24, 572 15 of 17
thermo-responsive disc/scaffold had a controllable swelling ratio and transition temper-

ature. Simultaneously, as a printing solvent, we introduced glycofurol, which has good
biocompatibility and improved the overall product printing quality. The formulation of
3% w/w TMPTA, N80-D15-A5, and 4% w/w PI, with an exposure time of 10 s per layer,
was identified as the most promising for cell cultivation under periodic changes in the
cultivation temperature at 36.3 ◦ C ± 0.9 ◦ C. This strategy provides an on-demand thermo-
sensitive platform for cell mechano-stimulation and thus presents a promising alternative
for the three-dimensional thermo-sensitive discs/scaffolds. These results suggest that
the material platform should be further elucidated for controlled and cell type-adjusted
mechanical stimulation so that it could finally be used for the engineering of a mechanical
stimulation device for biomedical applications.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms24010572/s1.
Author Contributions: Conceptualization and planning, M.C.H. and K.V.; investigation, K.V. and
I.M.; methodology, M.C.H. and K.V.; supervision, M.C.H. and M.S.-S.; writing—original draft, K.V.;
writing—review and editing, M.C.H. and M.S.-S. All authors have read and agreed to the published
version of the manuscript.
Funding: This research is partially funded by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation), project 59307082, TRR67 A1. The authors gratefully acknowledge the financial
support of Ketpat Vejjasilpa by the Royal Thai Government.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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Universität Leipzig

Adjustable Thermo-Responsive Cell Carrier and Implants from

an der Medizinischen Fakultät

Beschluss über die Verleihung des Doktorgrades vom:

Material and Methods

Thermo-Responsive Polymer from Thermal Synthesis Studies

An Adjustable Thermo-Responsive Polymer from Photo Synthesis

Fabrication of Thermo-Responsive Scaffolds from DLP Printing

3D Scaffold Biocompatibility Studies

Bibliography, List of Publications, CV, Declaration of Authorship, Acknowledgements,

BAPO…………………….. UV initiator Irgacure 819

C2C12 cells………………..myoblast cell line

Col-HA…………………….collagen hydroxyapatite composite

CIJ………………………… continuous Inkjet printing

DAPI………………………cell nucleic staining solution (4',6-diamidino-2-phenylindole)

DDS………………………. drug delivery systems

DFG……………………….German Research Institute (Deutsche Forschungsgemeinschaft)

DLP………………………..Digital Light Processing (3D printing technique)

DMEM………………….... Dulbecco’s Modified Eagle Medium

DSC……………………….differential scanning calorimetry

ECM (EZM)…… …………extracellular matrix

EDTA…………………….. ethylenediaminetetraacetic acid

et al. ………………………et alii = and others

FBS………………………. fetal bovine serum

FDA……………………….Food and Drug administration

FTIR………………………fourier-transform infrared spectroscopy

GF…………………………glycofurol, tetraglycol, BioXtra

GPC……………………….gel permeation chromatography

hASCs………………….....human adipose tissue derived stem cells

hvFb……………,,,……… human vascular fibroblast

HPLC……………………..high-performance liquid chromatography

Irgacure2959……………...photo initiator, α-Hydroxy-4-(2-hydroxyethoxy)-α-methylpropiophenone

L929 cells…………………fibroblast cell line

LA………………………...lactic acid = D,L-lactide acid

LCST…………………….. lower critical solution temperature

LIFT………………………laser-induced forward transfer

MNP……………,,……… magnetic nanoparticles

Mn…………………...........number average molecular weight

MSCs……………………..mesenchymal stem cells

NFI…………………..…....net fluorescence intensity

NiPAAm…………….……. N-isopropyl acrylamide

PBS…………..…………….phosphate buffer saline

pKa………….….…………..acid dissociation constant

PGA…………..………...…. polyglycolic acid

PLGA……………….…….. poly(lactic acid-co-glycolic acid)

PMMA…………….………. poly(methyl methacrylate)

SEM……………….……….scanning electron microscope

TCP……………………...…thin Conducting Polymer

TPO…………..…………..... diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

an establishment of stimulus-responsive materials could provide the essential missing piece

such as mechanical stimulation during cultivation to establish new tissue. 3

1.1 Mechanical Cell Stimulation

mechano-stimulus experiment was reported in 1939 by Gluckmann, stated the impact of

experiments investigating the influence of mechanical stimulation on tissue formation were

endothelial cell functionality and endothelial cell markers.

1.1.1 Stimuli-Responsive Materials

up, which may increas the possibility of contamination.10

elements without in vivo experimentation.

functions for tissue regeneration in a valuable way.13