Bioprinting.

From technical set-

up to biological
NanoInnovation applications
2020
MICHELE CONTI - UNIPV
Rome
 Additive manufacturing?
3D printing?
Bioprinting?
Different words for the same technology?
 Additive manufacturing process

Virtual Model Slicing Printed Model

3
 ISO/ASTM 52900:2015 (ASTM F2792)
FFF: Fused Filament Fabrication Additive manufacturing -- General principles --
SLA: Stereolithography
DLP: Direct Light Processing Terminology
SLS: Selective Laser Sintering
DMLS: Direct Metal Laser Sintering AM/3D printing https://www.iso.org/standard/69669.html
SLM: Selective Laser Melting
technologies
Vat
Material Power bed Material Binder
Polymerizat
Extrusion fusion Jetting Jetting
ion

FFF (or Material

SLA DLP Polymers Metals DOD
FDM) Jetting

SLS DMLS SLM EBM

EBM: Electron Beam Melting

DOD drop on demand
 3DP technologies

Fused Deposition A thermo-plastic filament, pushed

Modeling (FDM) through a heating chamber and
extruded through a small nozzle

Stereolithography (SLA)
3DP technologies

Material Jetting

Binder Jetting

• Material: thermoplastic filaments (PLA,

Selective Laser Melting ABS, HIPS,TPU, TPE, PETG, Nylon,
(SLA) reinforced/charged materials)
• Curing: temperature gradient
• Inexpensive process
Electron Beam Melting
(EBM) • Accuracy and speed are low when
compared to other process
 3DP technologies

Fused Deposition Modeling

(FDM) Uses a container of liquid
photopolymer resin, from which the
model is obtained using an UV light
Stereolithography (SLA) employed to harden the resin
3DP technologies

Material Jetting

Binder Jetting

Selective Laser Melting • Material: photo-polymeric resins

(SLA) hardened by UV light
• Curing: UV laser
Electron Beam Melting • Expensive process, high accuracy
(EBM)
 3DP technologies

Fused Deposition Modeling

(FDM) Printing material is dropped through
small diameter nozzles (similar to
inkjet paper print)
Stereolithography (SLA)
3DP technologies

Material Jetting

Binder Jetting

Selective Laser Melting • Material: photo-polymeric resins

(SLA) hardened by UV light
• Curing: UV light
Electron Beam Melting • Multiple material, parts & colors and
(EBM) high accuracy
 3DP technologies

Fused Deposition Modeling Powder is spread in equal layers &

(FDM)
binder applied through jet nozzles that
“glue" the powder particles
Stereolithography (SLA)
3DP technologies

Material Jetting

Binder Jetting

Selective Laser Melting • Material: binder powder

(SLA) • Curing: binder (glue)
• Parts can be made with a wide range
Electron Beam Melting of different colours
(EBM)
 Additive manufacturing? We have to deal with a class of
3D printing? technologies with their peculiar
Bioprinting? features, aims, and issues
Different words for the same technology? With a common key idea …
 2. STL creation
1. Producing a 4. Removal of 5. Post
and file 3. Printing
3D file prints processing
manipulation

Workflow

10
Michele Conti | CompMech – University of Pavia – michele.conti@unipv.it
 3NTR A4v3
SLA
FORM 2
Desktop SLA
SLA
Renishaw
AM 400 LeapFrog
Creatr HS

Material jet
ObJet 260
Connex 3
FDM

Bioprinting 3NTR A4v2

3D@UniPV

Cellink
Inkredible+

Binder jetting
3DSystems LeapFrog Creatr
ProJet 460 Plus Dual Extruder
http://www-4.unipv.it/3d/
3D4MED - https://www.3d4med.eu

UniPV and IRCCS San Matteo

Stefania Marconi

stefania.marconi@unipv.it
From 3D printing to 3D BIOprinting

Functionalize 3D
printed objects

BIOprinting
Tissue engineering
An interdisciplinary field that applies the principles of
engineering and life sciences towards the development of
biological substitutes that restore, maintain, or improve
biological tissue function or a whole organ.
Langer R, Vacanti JP. Tissue engineering. Science 1993,
260(5110), 920–926

Regenerative medicine
Application of tissue science, tissue engineering, and
related biological and engineering principles that restore
the structure and function of damaged tissues and organs
U.S. department of health and human services, 2006:
A New Vision - A Future for Regenerative Medicine,
 Classic paradigm in TE

• Scaffolds
• Cells
• Growth Factors
 Conventional fabrication technique

Scaffold • Solvent casting and

particulate leaching
• Gas foaming
• Phase separation
• Freeze drying
• …..

ADDITIVE MANUFACTURING

• 3D and porous
• to mimic host tissue with its mechanical • 3D printing
• properties • Light based techniques
• to promote cell activities • Extrusion based technique
• to give cells suitable and ECM-like cues • Electrospinning
• … • ….
BIOFABRICATION

Generation of biologically functional

products with structural organization
from living cells, micro-tissues or
hybrid tissue constructs, bioactive
molecules or biomaterials either
through top-down (Bioprinting) or
bottom-up (Bioassembly) strategies
and subsequent tissue maturation
processes

Groll J., et al. Biofabrication, 2016

Moroni L et al., Biofabrication: A Guide to Technology and Terminology. Trends
Biotechnol. 2017 Nov 11. pii: S0167-7799(17)30279-2
 BioP: key elements

Bioprinting

Bio-ink Bio-plotter
 Bioink vs Biomaterial ink

Groll, J., et al. "A definition of bioinks and their distinction from biomaterial inks." Biofabrication 11.1
(2018): 013001.
 BioP: bio-ink

Biological
components
Bio-ink

Hydrogel
 BioP: bio-ink BioP: bio-ink

Growth Factors
Cells (ESCs MSCs
iPSCs)
Biological
Chemicals
components
Additives
Bio-ink

Microcarriers

Hydrogel Drugs
 BioP: bio-ink

Biological
components
Natural
Bio-ink

Hydrogel

Synthetic
 BioP: bio-ink

Alginate

Agarose

Collagen
Biological
components Natural Hyaluronic acid
Bio-ink

Fibrin

Cellulose
Hydrogel
Extracellular matrix

Pluronic

Crosslinking Synthetic Poly(ethyleneglycol)

Polycaprolactone
 Hydrogel crosslinking

Polymer Hydrogel

+ Crosslinker

Ionic or covalent crosslinking

Photo crosslinking
Thermic
BioP: bio-ink
 Bio-ink features for 3D BioP

Printability

Degradation
kinetics and Biocompatibility
byproducts

Permeability of
Need
oxigen anda multidisciplinary
Hydrogel approach
Crosslinking
nutriens

Structure Viscoelastic
(pores) properties

Mechanical
properties
 BioP: bio-plotter

Bio-plotter

Ink-jet Extrusion Laser Assisted Stereolithography

 BioP: our bio-plotter

Bio-plotter

Ink-jet Extrusion Laser Assisted Stereolithography

Cellink INKREDIBLE+ (Cellink AB)

Our pneumatic extrusion-based 3D bio-plotter
BioP process
Hydrogel

1 Bio-ink
Biological
components, Drugs
2 Bio-plotter

3 Bioprinting

4 Crosslinking

5 Cell Colture at 37 °C

Transplantation
Bioprinted Drug Discovery
6 construct In-vitro biological study
 Bioprinting: one word for a complex process

Printability

Growth and
differentiatio Crosslinking
n

Nutrient Mechanical/
transport, structural
proliferation properties

Cell adhesion and

migration Material/
•cell migration structural
•cell scaffold degradation
interaction
 Bioprinting: one word for a complex process

• Printing resolution
Printability
• nozzle diameter
• speed head
• Z-distance Growth and
differentiatio Crosslinking
• pressure n
• Viscosity (shear-
thinning)

Nutrient
* Mechanical/
transport, structural
proliferation properties

Cell adhesion and

migration Material/
•cell migration structural
•cell scaffold degradation
interaction

*Paxton, Naomi, et al. "Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability." Biofabrication 9.4 (2017): 044107.
 Polymer
Bioprinting: one word for a complex process

Printability

Growth and
differentiatio
n
Crosslinking
+ Crosslinker
• Crosslinker • Ionic crosslinking
concentration • Photocrosslinking
• Model size • Thermic
• Exposure time
• Primary and Nutrient
* Mechanical/
secondary transport, structural
proliferation properties

Cell adhesion and

migration Material/
•cell migration structural
•cell scaffold degradation
interaction
Hydrogel network
*Paxton, Naomi, et al. "Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability." Biofabrication 9.4 (2017): 044107.
 Bioprinting: one word for a complex process

Day 2-21 ‘ho

wm
Printability an
ym
illio
ns
of
cel
Growth and l ss
differentiatio Crosslinking ho
ul d
n Iu
se?
’

Day 1
Nutrient
* Mechanical/
transport, structural
proliferation properties

Cell adhesion and

migration Material/
• Bioink composition
•cell migration structural • Bioink concentration
•cell scaffold degradation
interaction • Holding temperature
• Holding time
Zhao, Yu, et al. "The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology." Biofabrication 7.4 (2015): 045002.
 Bioprinting: one word for a complex process

Matrix stiffness is Modulation of

a key determinant [alginate] bioink
Printability
[of mechanical
mesenchymal properties
stem cell (MSC) ] Growth and
differentiation differentiatio Crosslinking
n

*
Nutrient Mechanical/
transport, structural
proliferation properties

Cell adhesion and Spatial regulation of

migration Material/
[MSC] fate within
•cell migration structural
•cell scaffold degradation bioprinted
interaction
tissues

Freeman, Fiona E., and Daniel J. Kelly. "Tuning alginate bioink stiffness and composition for controlled growth factor delivery and to spatially direct MSC fate within bioprinted tissues." Scientific reports 7.1 (2017): 17042.
 Bioprinting: one word for a complex process

Printability

Growth and
differentiatio Crosslinking
n

How crosslink is
related to the
stiffness of the
Nutrient printed Mechanical/
transport,
proliferation construct? structural
properties

Cell adhesion and

migration Material/
•cell migration structural
•cell scaffold degradation
interaction
 Overall Goal
Fantini, V., Bordoni, M.,
Scocozza, F., Conti, M.,
3D printing of NeuroMuscular Junction (NMJ) Scarian, E., Carelli, S., ...
& Cereda, C. (2019).
Bioink composition and
printing parameters for 3D
modeling neural
tissue. Cells, 8(8), 830.

To create a model for the analysis of

neurodegenerative disease

3D printing of muscle cells 3D printing of neurons

for tissue engineering Collaboration with: C. Cereda,
applications M. Bordoni, V. Fantini,
Collaboration with: G. Cusella, Laboratorio di Neurobiologia
G. Ceccarelli, F. Ronzoni, M. Sperimentale, Inst. C.
Sampaolesi - Human Anatomy Mondino, PV
Unit, Dept. of PHEMF UniPV
 BioP process

Large number
Cells of cell

Proliferation

Bio-ink

Cell colture

Crosslinking Bio-plotter

• Viability
• Proliferation
• Differentiation
 BioP process

Large number
Cells of cell

Proliferation

Bio-ink

Cell colture

Crosslinking Bio-plotter

• Viability
• Proliferation
• Differentiation
 3DP of muscle cells for tissue engineering applications

Commitment
Microfibril
Study goal
Fibril
3D bioprinting mouse
Activation/ myoblast cell line (C2C12)
Fascicle Proliferation to study cells
differentiation into bio-ink
and 3D modelling muscle
fiber physiology
Tendon
Differentiation

Bone
 3DP of muscle cells for tissue engineering applications

Biological
Material Crosslinking 3D BioP # of Cell
tests

Sodium
10x106
1 Alginate and CaCl2 Cube
cells/ml
Gelatin

Proliferation
FIBRIN Bio-ink Thrombin/ Differentiation
2 Line
(Cellink AB) CaCl2 Morphological
analysis
Gene
GelMa Bio-ink 25x106
3 UV (406 nm) Line expression
(Cellink AB) cells/ml
analysis

GelXA CaCl2+ UV
4 Line
(Cellink AB) (406 nm)

C2C12 cell line (immortal line of mouse skeletal myoblasts)

 Proliferation test results

LIVE/DEAD
24 Hours Day 4 Day 7 Day 14

4x 4x 4x 4x

10x 10x 10x 10x

Ø Good cell distribution and viability at 24 Hours

Ø At day 4 increases cell death, keeping round shape – not differentiated as in 2D
Ø At day 7 and day 14 cell differentiation is limited
 3DP of muscle cells for tissue engineering applications

Biological
Material Crosslinking 3D BioP # of Cell
tests

Sodium
10x106
Alginate and CaCl2 Cube
cells/ml
Gelatin

Proliferation
FIBRIN Bio-ink Thrombin/ Differentiation
2 Line
(Cellink AB) CaCl2 Morphological
analysis
Gene
GelMa Bio-ink 25x106
3 UV (406 nm) Line expression
(Cellink AB) cells/ml
analysis

GelXA CaCl2+ UV
4 Line
(Cellink AB) (406 nm)

C2C12 cell line (immortal line of mouse skeletal myoblasts)

(https://cellink.com/)

CELLINK FIBRIN Proliferation test results

(https://cellink.com/wp-content/uploads/2019/03/cellink_!brin.png)
Based on CELLINK® Bioink, CELLINK FIBRIN contains !brinogen to ensure consistent
printability during the fabrication process. CELLINK FIBRIN includes an enhanced
crosslinking solution that uses thrombin and an ionic binding agent to develop a
compound network with unparalleled stability. After crosslinking, CELLINK FIBRIN

LIVE/DEAD - 24 Hours
 Differentiation test results

LIVE/DEAD
Day 14 Day 28

Ø Uniform cell distribution,

Ø Optimal printability and
good cell viability, even in
50 µm 100 µm
long term
Ø Better cell differentiation
on crosslinked edges
Ø Live/Dead Assay shows
myotubes formation

50 µm 100 µm
 Gene expression
7 day
3 day
3
3 MyOD
MyOD
MCK
MCK

fold increase
2
fold increase
2

*
1 1

0 0
2D 3D Prol 3D Diff 2D Diff 3D Prol 3D Diff

15 day 21 day
3 3
MyOD MyOD
MCK M-Cadherin
fold increase

fold increase
2 2

** **
1 1

0 0
2D Prol 3D Prol 2D Diff 3D Diff 2D Prol 3D Prol 2D Diff 3D Diff

Ø Expression of two important master genes related to muscle differentiation

(MyOD – late diff. and MCK – early diff.) highlight a positive effect of the 3D
construct on the differentiation process
 Immunofluorescence results

Phalloidin/MHC – Day 28 differentiation condition

3D BioP
50 µm 100 µm
2D control

50 µm 100 µm

Ø Analysis revealed myotubes laden into CELLINK FIBRIN bioink

https://www.cellink.com/technology/collaborator-
* Phalloidin: actin filament. MHC: myosin filaments
data/university-of-pavia-collaboration/
 3DP of muscle cells for tissue engineering applications

Biological
Material Crosslinking 3D BioP # of Cell
tests

Sodium
10x106
Alginate and CaCl2 Cube
cells/ml
Gelatin

Proliferation
FIBRIN Bio-ink Thrombin/ Differentiation
Line
(Cellink AB) CaCl2 Morphological
analysis
Gene
GelMa Bio-ink 25x106
3 UV (406 nm) Line expression
(Cellink AB) cells/ml
analysis

GelXA CaCl2+ UV
4 Line
(Cellink AB) (406 nm)

Goal. Use UV to get an uniform crosslinking in the core in order to

achieve also a differentiation in this region
 3DP of muscle cells for tissue engineering applications

Biological
Material Crosslinking 3D BioP # of Cell
tests

Sodium
10x106
Alginate and CaCl2 Cube
cells/ml
Gelatin

Proliferation
FIBRIN Bio-ink Thrombin/ Differentiation
Line
(Cellink AB) CaCl2 Morphological
analysis
Gene
GelMa Bio-ink 25x106
UV (406 nm) Line expression
(Cellink AB) cells/ml
analysis

GelXA CaCl2+ UV
4 Line
(Cellink AB) (406 nm)

3D printing process extremely cumbersome – limited repeatability

 3DP of muscle cells for tissue engineering applications

Biological
Material Crosslinking 3D BioP # of Cell
tests

Sodium
10x106
Alginate and CaCl2 Cube
cells/ml
Gelatin

Proliferation
FIBRIN Bio-ink Thrombin/ Differentiation
Line
(Cellink AB) CaCl2 Morphological
analysis
Gene
GelMa Bio-ink 25x106
UV (406 nm) Line expression
(Cellink AB) cells/ml
analysis

GelXA CaCl2+ UV
Line
(Cellink AB) (406 nm)

There is still not uniform differentiation inside the strand core

3DP of muscle cells for tissue engineering applications

NOT differentiated

differentiated
 Bioink. Crosslinking, stiffness, and biology

crosslinker Du

Crosslinked
(with a Stiffer
gradient)

Not
Softer
Crosslinked

NOT differentiated
Study hypothesis: increasing GelMa concentration (and photocrosslinker
differentiated concentration) increases 3D construct stiffness
 Modeling bioprinting process

Study goal
Computational modeling of crosslinked sodium alginate and gelatin
hydrogel used for bioprinting applications

Crosslinked
(with a
gradient)

Barrier for
crosslinker
perfusion

Not
Crosslinked
a) SA-Gel b) SA-Gel + c) After 24h -
CaCl2 CaCl2 removal

Measure of CaCl2 absorbed by SA-Gel was performed at 2, 5, 10, 15 , 20 minutes, 2, 24

and 48 hours. Test was repeated for 3 times.
* SA: Sodium Alginate; Gel: Gelatin; CaCl2: Calcium Chloride
* SA: Sodium Alginate; Gel: Gelatin; CaCl2: Calcium Chloride; CL: Crosslinked
Modeling bioprinting process

Modeling reactive-diffusive processes in

sodium alginate hydrogels stabilized by CaCl2
and accounting for the effects of cross-linking
on the diffusion of gelation agents

Solved in a 3D domain
by Finite element
scheme
(1D problem)
 Modeling bioprinting process

Inclusion of a diffusion Crosslinker distribution is not

coefficient as function of uniform in space and does not
gelation allows to get diffuse linearly in time
experimental data
 Modeling bioprinting process

Fully crosslinked
region
SEM of crosslinked hydrogel

Counts of Ca atoms Boundary Partially crosslinked

layer region

In collaboration with Dr. Michele Marino (UniTov) and Ilenia Tredici (UniPV)
 Measuring percentage of hydrogel crosslinking -
Experimental set-up
Materials

Hydrogel: 8% Sodium alginate (SA) and 4% Gelatin (Gel)

Crosslinking: 2% and 5% Calcium Chloride (CaCl2)

Methods

• We 3D printed a cylinder (d: 8 mm, h: 2.1 mm) using Cellink INKREDIBLE+

• Crosslinking

o We added 0,5 ml of CaCl2 for crosslinking 3D printed construct edges

(construct top face was not immersed)

o We crosslinked construct for 2 and 5 minutes

• For each case we printed 5 samples (n_tot=20)

• Samples were dried for 24 h before microanalysis

 Measuring percentage of hydrogel crosslinking -
Experimental set-up
• First, we analyzed constructs with stereomicroscope and then we choose the
best 2 samples for SEM and microanalysis (n_tot=8)

2% CaCl2 5% CaCl2
5 min A1, A2 C1,C2
10 min B1, B2 D1,D2

• For each sample, we run microanalysis along two diameters (n_tot=16) of the
same sample to extract chemical elements that make up the constructs (e.g.
Ca, Cl, S, O, C, etc.)
SEM Microanalysis
Diameter-1

Diameter-2

• Calcium (Ca) were normalized with Sulfur (S) for avoiding shadow areas
Measuring percentage of hydrogel crosslinking -
Experimental set-up
Fully crosslinked
region
SEM of crosslinked hydrogel

Counts of Ca atoms Boundary Partially crosslinked

layer region

2% CaCl2 – 5 min 2% CaCl2 – 10 min 5% CaCl2 – 5 min 5% CaCl2 – 10 min

 Take home messages

3D printing is NOT (yet) a There is NOT ONE a 3D 3DP is an ENABLING

ONE-CLICK technology printing technology FOR technology
ALL the applications
But there are a number of CHANGE YOUR WAY OF
technologies and DESIGNING
materials that can give
you a solution PERSONALIZATION

X
 www.unipv.it/compmech/bioprinting_home.html

michele.conti@unipv.it
 n
it o
n
tt e
r a
ou
y Bioprinting.
r From technical set-
s fo
k up to biological
an applications
h
NanoInnovation
2020
Rome
T MICHELE CONTI - UNIPV
michele.conti@unipv.it