Thesis Report Hua My Van Bebeiu17021

Vietnam National University – HCMC
International University
School of Biomedical Engineering
FABRICATION OF IN SITU CROSSLINKING HYDROGELS

BASED ON OXIDIZED ALGINATE/N,O-CARBOXYMETHYL
CHITOSAN/β-TRICALCIUM PHOSPHATE FOR BONE
REGENERATION
By
Hua My Van
BEBEIU17021
A thesis submitted to the School of Biomedical Engineering in partial fulfillment of the

requirements for the degree of Engineer
Ho Chi Minh city, Vietnam

Feb – 2022
FABRICATION OF IN SITU CROSSLINKING HYDROGELS BASED ON

OXIDIZED ALGINATE/N,O-CARBOXYMETHYL CHITOSAN/β-TRICALCIUM
PHOSPHATE FOR BONE REGENERATION
APPROVED BY:
_______________________________ , ________________________________,
Nguyen Thi Hiep, Assoc. Prof, Advisor Nguyen Thi Hiep, Assoc. Prof, Chair
______________________________
Huynh Chan Khon, PhD, Reviewer
______________________________
*(Typed Committee name here)., Member
______________________________
*(Typed Committee name here)., Member
______________________________
*(Typed Committee name here)., Secretary
THESIS COMMITTEE
ACKNOWLEDGEMENT
The thesis report on this topic is the result of my continuous efforts and the support
and encouragement of teachers, family, friends, and relatives. Therefore, I would like to
express sincere gratefulness to those who have always been by my side and supported me
during my study and scientific research.
Foremost, I would like to express my deep respect and gratitude to my advisor,
Assoc. Prof. Nguyen Thi Hiep, for her patience and dedication through following and
giving feedback during my work. Moreover, I would like to give special thanks to MS. Vu
Thanh Binh for directly guiding providing the necessary scientific information for this
thesis. Without his willingness and enthusiasm, I probably wouldn’t have gotten to this
point. Besides, I felt fortunate when I received tremendous support from MD. Tang Tuan
Ngan, BS. Tran Minh Chien and Mr. Luong Dai Tin at laboratory A1. 406. Also, I am very
grateful to Ms. Nguyen Duong Tu Quynh, Mr. Hoang Anh Duc, and all my friends who
have always accompanied and encouraged me on this journey. In addition, thanks to the
School of Biomedical Engineering, I have enough favorable conditions to complete my
scientific research.
Last but not least, I would like to give my most profound appreciation to my family
for always being by my side, giving me strength and confidence to overcome difficulties
in studying and thesis working.
TABLE OF CONTENT
LIST OF FIGURES ............................................................................................................ 1
LIST OF TABLES .............................................................................................................. 3
LIST OF ABBREVIATIONS ............................................................................................. 4
ABSTRACT........................................................................................................................ 5
CHAPTER 1: INTRODUCTION ....................................................................................... 6
1.1. Overview of conventional bone defect therapies and the importance of bone tissue
engineering ...................................................................................................................... 6
1.2. Literature review on recent studies in fabricating hydrogels based on N,O-
carboxymethyl chitosan, oxidized alginate, and β-tricalcium phosphate........................ 9
1.2.1. N,O-carboxymethyl chitosan (NOCC) .............................................................. 9
1.2.2. Oxidized alginate (OA) ................................................................................... 11
1.2.3. β-tricalcium phosphate (β-TCP) ...................................................................... 13
1.2.4. Literature review on recent studies in fabricating hydrogels based on NOCC,
OA, and β-TCP .......................................................................................................... 13
1.3. Proposed solution ................................................................................................... 15
1.4. Project goals ........................................................................................................... 16
1.5. Research framework ............................................................................................... 18
CHAPTER 2: METHODOLOGY .................................................................................... 19
2.1. Decision matrix ...................................................................................................... 19
2.2. Materials ................................................................................................................. 21
2.3. Methods .................................................................................................................. 21
2.3.1. Fabrication of OA/NOCC/β-TCP hydrogels ................................................... 21
2.3.1.1. Synthesis of OA ........................................................................................ 21
2.3.1.2. Synthesis of NOCC ................................................................................... 22
2.3.1.3. Fabrication of OA/NOCC/β-TCP hydrogels ............................................ 24
2.3.2. Characterizations of OA/NOCC/β-TCP hydrogels ......................................... 25
2.3.2.1. Gelation time ............................................................................................. 25
2.3.2.2. Cross-sectional surface morphology analysis ........................................... 25
2.3.2.3. Porosity ..................................................................................................... 25
2.3.2.4. Fourier transform infrared spectroscopy (FT-IR) analysis ....................... 26

2.3.2.5. Energy-dispersive X-ray spectroscopy (EDS) analysis ............................ 26
2.3.2.6. Swelling degree measurement .................................................................. 26
2.3.2.7. In vitro degradation assessment ................................................................ 27
2.3.2.8. Compression testing .................................................................................. 27
2.3.3. In vitro testing .................................................................................................. 27
2.3.3.1. Cell culture ................................................................................................ 27
2.3.3.2. In vitro live/dead assay ............................................................................. 28
2.3.3.3. In vitro cytotoxicity................................................................................... 29
2.2.4. Statistical analysis............................................................................................ 30
CHAPTER 3: RESULTS .................................................................................................. 31
3.1. Gelation time .......................................................................................................... 31
3.2. Cross-sectional surface morphology analysis ........................................................ 32
3.3. Porosity................................................................................................................... 35
3.4. Confirmation of hydrogel formation ...................................................................... 36
3.5. Confirmation of preresiquite elements ................................................................... 38
3.6. Swelling degree measurement ................................................................................ 40
3.7. In vitro degradation assessment ............................................................................. 41
3.8. Compression testing ............................................................................................... 42
3.9. In vitro cytocompatibility of OA/NOCC/β-TCP hydrogels ................................... 44
3.9.1. In vitro live/dead assay .................................................................................... 44
3.9.2. In vitro cytotoxicity ......................................................................................... 45
CHAPTER 4: DISCUSSION ............................................................................................ 45
CHAPTER 5: CONCLUSION ......................................................................................... 45
REFERENCES ................................................................................................................. 54
APPENDIX 1: Initial formulations................................................................................... 60
LIST OF FIGURES
Figure 1. A description of BTE utilizing natural polymeric scaffolds. On the scaffolds, stem
cells are seeded, proliferated, and differentiated into osteo-like cells. The scaffold is
then inserted into the bone defect site to repair and restore the tissue's function ............... 9
Figure 2. Chemical structure of chitosan .......................................................................... 10
Figure 3. (A) Structure of alginate monomers; (B) Structural representation of different
monomer blocks in a single alginate polypeptide; (C) Ca2+ crosslink with alginate to form
an “egg-box” structure ...................................................................................................... 12
Figure 4. Chemical structures of (A) Oxidized alginate (OA); (B) N,O-carboxymethyl
chitosan (NOCC); and (C) Schiff’s base crosslinking between OA and NOCC .............. 14
Figure 5. Illustration of OA/NOCC/β-TCP hydrogel and its components ....................... 16
Figure 6. The research framework employed in this study ............................................... 18
Figure 7. Illustration of the synthesis of OA..................................................................... 22
Figure 8. Illustration of the synthesis of NOCC ............................................................... 23
Figure 9. Schematic of OA/NOCC/β-TCP hydrogel fabrication procedure ..................... 24
Figure 10. The procedure of in vitro live/dead assay........................................................ 28
Figure 11. The procedure of in vitro cytotoxicity ............................................................. 29
Figure 12. Gelation time of OA 6%/NOCC/β-TCP hydrogels and OA 3%/NOCC/β-TCP
hydrogels ........................................................................................................................... 31
Figure 13. Images of hydrogels formed with different ratios of OA:NOCC of (A) OA
6%/NOCC/β-TCP and (B) OA 3%/NOCC/β-TCP hydrogel systems .............................. 32
Figure 14. SEM images of (A) OA 6%/NOCC/β-TCP and (B) OA 3%/NOCC/β-TCP
hydrogel samples at magnifications of 100X, 200X, and 10000X. .................................. 33
Figure 15. The pore size of OA 3%/NOCC/β-TCP hydrogel samples ............................. 34
Figure 16. Pore size distribution of OA 3%/NOCC/β-TCP hydrogel samples................. 35
Figure 17. The porosity of OA 3%/NOCC/β-TCP hydrogel samples .............................. 36
Figure 18. FT-IR spectra of OA, NOCC, β-TCP, and OA 3%/NOCC/β-TCP hydrogels at
four different OA:NOCC ratios ........................................................................................ 37
Figure 19. EDS mapping images of OA 3%/NOCC/β-TCP hydrogel samples ................ 39
1
Figure 20. Mass and atom percentage of C, Ca, and P elements in OA 3%/NOCC/β-TCP
hydrogel samples .............................................................................................................. 40
Figure 21. Equilibrium swelling degree of OA 3%/NOCC/β-TCP hydrogel samples ..... 40
Figure 22. Degradation images of OA 3%/NOCC/β-TCP hydrogels within 14 days ...... 41
Figure 23. In vitro degradation assessment of OA 3%/NOCC/β-TCP hydrogel samples 42
Figure 24. Images illustrating the compression testing of OA-1/NO-3 hydrogel sample 43
Figure 25. Stress-strain curve of OA 3%/NOCC/β-TCP hydrogels and OA 3%/NOCC
hydrogels (without β-TCP) at four ratios of OA:NOCC at strain from 0% to 50% ......... 43
Figure 26. FDA/PI staining of L-929 cells after 1-day culturing in the control and OA
3%/NOCC/β-TCP hydrogel samples ................................................................................ 44
Figure 27. Cytotoxicity evaluation of the control and OA 3%/NOCC/β-TCP hydrogel
samples using Resazurin assay ......................................................................................... 45
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LIST OF TABLES
Table 1. Decision matrix of typical crosslinking methods for synthesizing hydrogels .... 19
Table 2. FT-IR band assignment for OA, NOCC, β-TCP, and OA 3%/NOCC/β-TCP
hydrogels spectra .............................................................................................................. 37
Table 3. Mass and atom percentage of C, Ca, and P elements in OA 3%/NOCC/β-TCP
hydrogel samples .............................................................................................................. 39
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LIST OF ABBREVIATIONS
HAp Hydroxyapatite
ECM Extracellular Matrix
BTE Bone Tissue Engineering
3D Three-dimensional
GAG Glycosaminoglycans
SA Sodium Alginate
OA Oxidized Alginate
NOCC N,O-Carboxymethyl Chitosan
β-TCP β-Tricalcium Phosphate
PBS Phosphate Buffer Saline
DW Distilled Water
MWCO Molecular Weight Cut-Off
FT-IR Fourier Transform Infrared Spectroscopy
EDS Energy-dispersive X-ray Spectroscopy
SEM Scanning Electron Microscopy
SD Standard Deviation
CMCS Carboxymethyl Chitosan
BMSCs Bone Marrow Stromal Cells
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ABSTRACT
Recently, bone tissue engineering has been considered as a promising therapeutic
option for bone defects by providing functionally biological substitutes that are analogous
to bone features. Natural polymers and bioceramics are the commonly used biomaterials
for fabricating hydrogel scaffolds in bone regeneration applications. In this study,
hydrogels based on in situ crosslinking between oxidized alginate (OA) and N,O-
carboxymethyl chitosan (NOCC) in combination with β-tricalcium phosphate (β-TCP)
were performed, using different OA concentrations and OA:NOCC ratios. Various
hydrogel characterizations were examined and revealed the strong effect of OA
concentrations and NOCC contents in the ability of hydrogel formation, swelling degree,
degradability, and compression strength. Besides, the Schiff’s base crosslinking between
aldehyde groups of OA and amino groups of NOCC indicating hydrogel formation was
confirmed via Fourier transform infrared spectroscopy (FT-IR) spectra. The presence of β-
TCP in hydrogels was also confirmed by FT-IR, scanning electron microscopy (SEM), and
energy-dispersive X-ray spectroscopy (EDS) images. Furthermore, most hydrogels were
non-cytotoxic and expressed the capability for cells to penetrate, migrate, and exchange
nutrients and oxygens. Generally, the results suggested that hydrogels based on OA,
NOCC, and β-TCP have the potential for bone regeneration. Besides, further experiments
are recommended to analyze their in vivo biocompatibility.
Keywords: β-tricalcium phosphate, bone regeneration, in situ crosslinking

hydrogel, N,O-carboxymethyl chitosan, oxidized alginate
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CHAPTER 1: INTRODUCTION
1.1. Overview of conventional bone defect therapies and the importance of bone tissue
engineering
Human bones are a vital component of the human skeletal system
that protects organs and allow the body to move freely. Bone is divided into two types:
spongy cancellous part and stiffer cortical part (Kanwar & Vijayavenkataraman, 2021);
(Prasad, 2021). They are made up of both organic constituents, such as protein collagen, in
which type I collagen represents 85-90% (wt%) of extracellular matrix (ECM) protein, and
inorganic mineral phase included mainly by hydroxyapatite (HAp), which support strength
to the whole bone structure (Guo et al., 2021). Other bone ECM components include non-
collagenous proteins (osteonectin, osteocalcin, osteopontin, fibronectin, and sialoprotein)
and polysaccharides (Guo et al., 2021).
Bone has a remarkable capacity to regenerate, but significant bone loss or the
formation of an unfavorable microenvironment, such as in cases of severe trauma, non-
union fractures, developmental malformations, revision operations, intervertebral disk
injuries, or tumor excision, might limit this potential (Neves & Ph, 2011). Besides, bone
characteristics are greatly influenced by their age and location inside the body (Tran et al.,
2020). Injured bone tissue can cause substantial alterations in patients’ life quality, such as
restricting essential duties like walking, leading to social and psychological issues. Current
therapeutically accessible remedies for bone defects rely on bone graft transplants
(autologous, allogeneic, and xenogeneic), bone transport procedures (Ilizarov technique),
and implants made from various materials. Every year, roughly 2.2 million bone graft
surgeries (autologous and banked bone of human cadavers) are performed globally to
repair or regenerate the bone (Neves & Ph, 2011). Autografts are widely regarded as the
gold standard in bone restoration. However, immune rejection by the host tissue or
complications like blood loss may develop, necessitating blood transfusions. Furthermore,
the treatment is not only expensive but also limited in terms of tissue supply, and it causes
high donor-site morbidity (Kalsi et al., 2021). Allografts are generally nonvital (dead) bone
obtained from deceased donors and treated using a freeze-drying technology that removes
all the water content by vacuum drying. These grafts eliminate morbidity at the donor site,
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but they can pose a danger of disease transmission and serious immunological responses
in the recipient. Also, donor shortage is always a challenging problem. Similarly,
xenogeneic bone is nonvital bone generated from other animals, primarily bovine.
Compared with human cadaver bones, xenograft bones pose a greater risk of
immunological rejection and contamination by viral proteins, so they are often treated at
extremely high temperatures. The Ilizarov procedure begins with an osteotomy and
then bone distraction using extensible fixation devices. Although this procedure eliminates
issues associated with bone graft osseointegration, it takes longer treatment times (12–18
months) and can cause lingering pain to the patient (Gonzalez-Fernandez et al., 2021);
(Neves & Ph, 2011).
In these circumstances, bone tissue engineering (BTE) offers a great promise of
potential treatment. By providing functionally engineered biological substitutes mimicking
the bone properties, BTE can become a breakthrough technology needed to address the
bone deficiency in various destructive and malformed clinical conditions (Neves & Ph,
2011). One of the most common approaches in BTE is implanting porous scaffolds in the
defect zone (Sarker et al., 2015). The scaffolds serve as an ECM with analogous structure
and function of original bone and allow cells to adhere, proliferate, and differentiate in a
three-dimensional (3D) environment to ultimately regenerate bone (Kanwar &
Vijayavenkataraman, 2021); (Krishnakumar et al., 2019).
Polymeric hydrogels are vital among scaffolding biomaterials for BTE because of
their inherent structural and compositional similarities to the ECM (Sarker et al., 2015).
Hydrogels are hydrophilic and jelly-like materials that hold large amounts of water and
biological fluids (Reakasame & Boccaccini, 2018). Thus, all of which are essential for cell
growth, including nutrients, signaling molecules, and oxygen, can permeate into the
hydrogel (Zhang et al., 2021). In addition, hydrogels have good biocompatibility,
biodegradability, highly porous network, controllable physical characteristics, and flexible
fabrication methods, making them an ideal biomaterial to provide proper support and
interaction with the natural bone ECM (Tran et al., 2020). Also, owing to the network of
3D cross-linked polymer inside the fluid, hydrogels have no flow in the steady-state, giving
them unique features comparable to human tissues (Axpe & Oyen, 2016). Therefore,
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hydrogels have attracted significant attention for tissue engineering, particularly for BTE
applications.
Typically, hydrogels are classified into two main types: naturally- and
synthetically- derived hydrogels. Synthetic polymers have been regarded as a promising
biomaterial for bone regeneration due to their high mechanical properties, consistent
characteristics, and long lifetime, but the lack of critical elements for cell adhesion and
migration, low biocompatibility, and their acidic degraded residues prevents their
widespread use (Zhang et al., 2021); (Tran et al., 2020). On the other hand, natural
hydrogels are preferred over synthetical ones due to their wide availability, better
biofunction, biocompatibility, and biodegradability (Reakasame & Boccaccini, 2018)
(Tabriz, 2017). An overview of bone regeneration by employing a natural polymeric
scaffold can be seen in Figure 1. Commonly used natural polymers for fabricating scaffolds
in BTE include collagen type I, chitosan, hyaluronic acid, alginate, agarose, gelatin, and
silk fibroin (Guo et al., 2021). Among them, chitosan and alginate are two frequently used
biomaterials that possess the potential for fabricating hydrogels in BTE applications.
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Figure 1. A description of BTE utilizing natural polymeric scaffolds. On the scaffolds,

stem cells are seeded, proliferated, and differentiated into osteo-like cells. The scaffold is
then inserted into the bone defect site to repair and restore the tissue's function (Guo et
al., 2021)
Besides hydrogels, ceramics play an essential role in bone substitute biomaterials
that have been utilized in medical applications to repair and restore injured skeletal and
body parts for many years (Kim et al., 2019). Ceramics are generally bioactive and release
calcium ions, resulting in increased bioactivity and stimulation of bone formation (Kanwar
& Vijayavenkataraman, 2021). One of the most widely used ceramics bone substitutes is
calcium phosphates (CaP) due to its similar composition to bone minerals, superior
biocompatibility (Bose & Tarafder, 2012), and capacity to stimulate osteoblastic
differentiation in progenitor cells (Samavedi et al., 2013). CaPs have been found to enhance
bone growth in vivo and attract bone marrow stromal cells (BMSCs) to ectopic regions for
bone formation (Samavedi et al., 2013). CaP can appear in various phases depending on
temperature, contaminants, and water content. CaP forms have different biological
activities and degradation rates depending on the Ca/P ratio, crystallinity, and phase purity.
Due to their osteogenic features and capacity to build strong links with host bone tissues,
β-tricalcium phosphate (β-TCP) is one of the most often employed phases among different
CaP (Bose & Tarafder, 2012).
1.2. Literature review on recent studies in fabricating hydrogels based on N,O-
carboxymethyl chitosan, oxidized alginate, and β-tricalcium phosphate:
1.2.1. N,O-carboxymethyl chitosan (NOCC)
Chitosan is a linear polysaccharide synthesized by deacetylation of chitin, the
second most abundant polysaccharide in the world, following cellulose (Kim et al., 2019).
Chitosan is a cationic polymer comprising of D-glucosamine (deacetylated unit) and N-
acetyl glucosamine (acetylated unit) (Figure 2) that is generally found in the exoskeleton
of crustaceans such as shrimp or crab, the cell wall of fungi, and coral (Tran et al., 2020).
Based on its origin and processing techniques, the molecular weight of chitosan can vary
from 300 to more than 1000 kDa (Neves & Ph, 2011). Enzymes like lysozymes perform
chitosan degradation, and the rate of degradation is determined by the number of acetylated
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units (Guo et al., 2021); (Kalsi et al., 2021). Chitosan is structurally homologous to
glycosaminoglycans (GAG), one of the ECM components that interact with collagen fibers
and plays a crucial contribution in cell-cell adhesion (Logithkumar et al., 2016). Chitosan
has a notable osteoconductivity but a low level of osteoinductivity. It promotes osteoblast
and mesenchymal cell proliferation as well as in vivo neovascularization (Logithkumar et
al., 2016). With unique biological features such as non-toxicity, biodegradability,
biocompatibility, bioadhesive property, and immuno-enhancing (Tran et al., 2020); (Neves
& Ph, 2011), chitosan has received a lot of interest in the biomedical area, particularly in
bone repair and implant.
Figure 2. Chemical structure of chitosan

Besides these outstanding advantages, chitosan also has disadvantages, notably its
insolubility in a neutral aqueous environment or many organic solvents that make it
challenging to handle and process (Narayanan et al., 2014). One of the strategies for
improving chitosan’s solubility is modification its chemical structure by adding small
chemical groups like alkyl or carboxymethyl units. This method can dramatically boost the
solubility of chitosan at various pH values without influencing its properties (Fonseca-
Santos & Chorilli, 2017). Recently, carboxymethyl chitosan (CMCS) has attracted superior
attention due to its increased water solubility, antibacterial properties, biocompatibility,
human safety, and very low cytotoxicity (Upadhyaya et al., 2013). There are several
derivatives of CMCS; one of them is N,O-carboxymethyl chitosan (NOCC), in which the
carboxymethyl substituents are introduced into N- and O- positions in amino and primary
hydroxyl groups of chitosan (Upadhyaya et al., 2013) (Figure 4B). The carboxymethylation
can be prepared by using sodium hydroxide, chloroacetic acid, and isopropyl alcohol (Chen
et al., 2004). The most appealing physico-chemical features of NOCC are distinct gel-
10
forming potential, strong water-retentive ability, and high biocompatibility, making this
hydrophilic and amphoteric polyelectrolyte with a predominant cationic profile appropriate
for biomedical applications, including BTE (Logithkumar et al., 2016).
1.2.2. Oxidized alginate (OA)
Alginate, also known as algin or alginic acid, is a low-cost biopolymer commonly
acquired from calcium, barium, magnesium, and sodium alginate salts extracted from
various brown marine algae cell walls and intracellularly regions (M. Xu et al., 2021).
Alginate is a linear polysaccharide consisting of (1–4)-β-D-mannuronic acids (M) and
(1,4)-α-L-guluronic acids (G) (the monomers in Figure 3A) that can be arranged in
homopolymeric (-MMM- or -GGG-) or heteropolymeric (-MGM- or -GMG-)
configurations (Figure 3B) to form a long anionic polymer chain (M. Xu et al., 2021). To
fasten the gelation process, long alginate chains are ionic crosslinked (ionotropic gelation)
with each other by divalent cations (Uǧuz et al., 2020). Calcium ion (Ca2+) is the most
widely used for this strategy regarding its non-toxicity, while others cations ( Pb2+, Cu2+,
Sr2+, and Ba2+) have restrictions due to their slight toxicity (Aguero et al., 2021). When
combined with divalent cations, alginate solutions provide quick gelling by forming ionic
interchain bridges between adjacent polymer chains (Reakasame & Boccaccini, 2018). The
crosslinking process happens via the interaction of Ca2+ with sodium ions on carboxylate
groups in G block monomers and the electronegative oxygen atoms of the hydroxyl groups
to create an “egg-box” model (M. Xu et al., 2021) (Figure 3C). Alginate materials have
many outstanding features, including the ability to perform in situ gelations, water-
solubility, cytocompatibility, mucoadhesion, degradability, adjustable viscosity with
concentration, release prolongation of active substances, promote cell growth, and protect
the release system of cells and particles (Hernández-González et al., 2020); (Uǧuz et al.,
2020). Also, alginate is abundant, highly biocompatible, tunable, non-toxic, and non-
immunogenic (Reakasame & Boccaccini, 2018). These primary benefits have led to the
extensive use of alginate in many bio-related applications, particularly in bone
regeneration.
However, one of the main drawbacks of alginate hydrogels is their relatively low
in vivo degradability (Reakasame & Boccaccini, 2018). High molecular weight alginate
11
may be hard to remove from the body since humans lack alginate breakdown enzymes.
Furthermore, due to its lack of specialized molecular interaction capabilities with
mammalian cells, alginate has weak cell attachment and penetration (Reakasame &
Boccaccini, 2018). Once again, the chemical modification by oxidizing alginate can
overcome these problems. Sodium periodate is a common oxidizing agent to functionalize
polysaccharides like alginate (Emami et al., 2018). The alginate oxidation with sodium
periodate occurs on hydroxyl groups at C-2 and C-3 positions of the uronic, forming the
aldehyde groups on the alginate backbone (Figure 4A) (Sarker et al., 2015). Hence,
oxidized alginate (OA) has more reactive groups and faster biodegradability than alginate,
thanks to its lower molecular weight (Kong et al., 2021).
Figure 3. (A) Structure of alginate monomers; (B) Structural representation of different

monomer blocks in a single alginate polypeptide; (C) Ca2+ crosslink with alginate to
form an “egg-box” structure
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1.2.3. β-tricalcium phosphate (β-TCP)

β-TCP (Ca3(PO4)2) (Ca/P ratio is 1.5) has become one of the most popular
biomaterials for bone graft substitutes since it has both osteoconductivity and
osteoinductivity and shows superior biocompatibility, bioactivity, and thermodynamically
stability. β-TCP is less stable and has lower mechanical strength than HAp, another
bioceramic closest to bone mineral composition (Campana et al., 2014), making it more
soluble in the aqueous environment (Samavedi et al., 2013). Also, β-TCP is a bioresorbable
ceramic that can be resorbed by osteoclasts and easily regenerated by new bone (Bose &
Tarafder, 2012). However, the brittleness and poor strength of β-TCP materials can hinder
their use in hard tissue regeneration. Therefore, β-TCP in combination with organic
polymers such as polysaccharides have been investigated to strengthen their mechanical
properties and expand their applications in BTE. The high density of hydroxyl groups in
the backbone of the polysaccharides chain can enhance its hydrogen bonding with
bioceramic nanoparticles (Salama, 2021).
1.2.4. Literature review on recent studies in fabricating hydrogels based on NOCC,
OA, and β-TCP:
Because bone ECM comprises various compositions, bone scaffolds with a single
component such as alginate, chitosan, or β-TCP alone might not provide essential signals
for cellular proliferation. Nonetheless, the combination of two or more components for
scaffold fabrication may have a synergistic effect that improves the scaffold’s mechanical
strength while also facilitating cell adhesion, proliferation, and differentiation (Sharma et
al., 2016).
Aldehyde groups in OA and the free amino groups in NOCC can perform in situ
crosslinking through Schiff’s based reaction (Figure 4C) (Zhao et al., 2016), obtaining the
gelation. Moreover, Schiff’s base reaction is moderate and does not require any additional
hazardous crosslinkers or external stimulations, making it ideal for various biological
applications, and it has been widely applied in BTE (Ma et al., 2020). In addition, divalent
cation Ca2+ in β-TCP can crosslink with aldehyde groups in OA to achieve physical
hydrogel (Emami et al., 2018). Therefore, many researchers have been working on multi-
component hydrogel scaffolds by combining biomaterials like OA, NOCC, and β-TCP
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bioceramics to get over the weak mechanical characteristics of these biopolymers and the
brittleness of bioceramics and create promising hydrogels for applications in bone
substitutes.
Figure 4. Chemical structures of (A) Oxidized alginate (OA); (B) N,O-carboxymethyl

chitosan (NOCC); and (C) Schiff’s base crosslinking between OA and NOCC
Ma et al. fabricated injectable hydrogels based on OA hybrid HAp nanoparticles
and CMCS injectable hydrogels by Schiff’s base reaction (Ma et al., 2020). Rheological
testing confirmed the hydrogel formation based on dynamic imine linkages. The gelation
time revealed a negative correlation to OHAH content and oxidation time but a positive
correlation to the storage moduli. The porous structures of the lyophilized hydrogels were
visible, with many HA nanoparticles spread over the pore wall surface. The self-healing
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ability and cytocompatibility of these hydrogels were also demonstrated. The obtained
results showed that these hydrogels are potential and promising in bone repair applications.
In the research of Li’s team, a series of NOCC/OA was successfully fabricated (Li
et al., 2012). After 24 h of culturing with NIH-3T3 cells, the in vitro cytotoxicity testing
reported that the hydrogels were non-toxic. Furthermore, bovine serum albumin was first
released from the hydrogels by diffusion, then through a degradation-dependent process at
later phases. In general, the formed hydrogel might be promising for drug delivery and
tissue engineering applications.
In another research, Cai’s group performed novel hydrogel composites based on
OA, gelatin, and β-TCP (Cai et al., 2007). These hydrogels were shown to have
interconnecting pores with an average pore size surrounding 205 ± 58 µm allowing for cell
encapsulation. Moreover, in vitro research using osteoblast encapsulation reported that
they were non-toxic and biocompatible.
In a study of Zhou and his coworkers, gelatin/CMCS/β-TCP composite scaffolds
for BTE by radiation-induced crosslinking at room temperature were developed (Zhou et
al., 2012). The fabricated scaffolds showed crosslinked network with uniform distribution
of β-TCP, highly interconnected porous structure, swelling ability, and proper mechanical
strength. The β-TCP amount significantly affected the crosslinking and swelling behavior
of different scaffolds. The scaffolds also expressed excellent biocompatibility and bone
regeneration boosting ability with the effect of β-TCP through in vivo implantation in the
mandible of the beagle dog. The results demonstrated these composite scaffolds as a
potential biomaterial for BTE.
1.3. Proposed solution
The abovementioned studies illustrate that many hydrogels based on the
combination of OA and NOCC, OA and CMCS, or β-TCP with OA or CMCS have been
developed and analyzed. Most of them showed possible results for these hydrogel systems
to be ideal candidates for bone substitute biomaterials. Meanwhile, the research on
hydrogels consisting of all the above polymer components with β-TCP is still relatively
few, especially so far there have been no studies on hydrogels composed of OA, NOCC,
and β-TCP in BTE. The ability of in situ crosslinking by Schiff’s based reaction between
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aldehyde group on OA and amino group on NOCC without additional crosslinkers was
demonstrated (Li et al., 2012). β-TCP with good biodegradability properties and can
crosslink with the aldehyde group of OA is expected to give well dispersion in OA/NOCC
hydrogel and help develop a novel scaffold for bone substitutes. Therefore, in this project,
a novel in situ crosslinking hydrogel based on OA, NOCC, and β-TCP, forming
OA/NOCC/β-TCP hydrogels were fabricated and analyzed to drive the potential
formulation for bone regeneration applications. The illustration of the hydrogel formation
can be shown in Figure 5.
Figure 5. Illustration of OA/NOCC/β-TCP hydrogel and its components

1.4. Project goals
With the increasing demand for bone substitutes in BTE, continuous research is
required to find potential biomaterials to satisfy the requirements for bone scaffolds.
16
Herein, the goal of this study is the fabrication of a novel OA/NOCC/β-TCP hydrogel at
different concentrations of OA and volume ratios between OA and NOCC to analyze their
influence on hydrogels’ structure. The developed hydrogels then are characterized through
several factors, including gelation time, surface morphology, porosity, confirmations of
hydrogel formation and precursor elements, swelling and degradation degree, and
compression. Finally, OA/NOCC/β-TCP scaffolds undergo in vitro cytotoxicity and
live/dead assays to identify their cytocompatibility and cell viability. All the results are
taken into consideration to eventually evaluate and find the hydrogel with the most feasible
formulations for bone regeneration applications.
In addition, the utilization of naturally occurring polymers such as alginate and
chitosan in the hydrogel fabrication also brings many benefits in the environmental,
economic, social, and global contexts. Thanks to the developed fisheries and the long
coastline bordering the East Sea, Vietnam has the advantage of abundant aquatic and
seafood resources. Among them, crustaceans such as shrimp and crabs, coral, and seaweeds
account for a vast amount. They are harvested and applied widely in many fields, including
food, health, cosmetology, etc. However, they also negatively impact the environment due
to considerable waste after processing and use. Therefore, the extraction of biological
compounds such as chitin from coral or crustacean shells or alginate from marine algae
effectively reduces the waste of valuable biomaterials and offers potential in areas of
research and application. Moreover, biodegradable polymers like OA and NOCC are
gaining much interest due to their friendly nature. Besides, these inexpensive materials also
have the advantage of a straightforward extraction and synthesis process, allowing a
potentially global biomaterial market. This approach also helps solve the job demand for
coastal people and researchers when the need for aquaculture as well as the extraction and
fabrication of biomaterials keeps increasing. In general, this research was developed not
only to create a potential hydrogel system in BTE application but also to contribute to
solving environmental, economic, social, and global problems to provide long-term
sustainable values.
17
1.5. Research framework

The research framework of this project is divided into three main stages (Figure 6).
The first stage is the fabrication of hydrogels. To do this, OA and NOCC need to be
prepared in advance by the oxidation of sodium alginate with sodium periodate and the
carboxymethylation of chitosan with sodium hydroxide, chloroacetic acid, and isopropyl
alcohol, respectively. After that, β-TCP is dispersed into an OA solution, and the mixture
is then mixed with NOCC to form hydrogels. Hydrogel characterization is the second
stage. In this stage, the fabricated hydrogel with different ratios and contents will
experience several tests to characterize their physical and chemical properties. These tests
include gelation time, cross-sectional surface morphology analysis, porosity, Fourier
transform infrared spectroscopy (FT-IR) analysis, energy-dispersive X-ray spectroscopy
(EDS), swelling degree measurement, in vitro degradation assessment, and compression
test. In the third stage, in vitro testing composed of live/dead assay and cytotoxicity is
performed on L929 fibroblast to evaluate the cell viability and cytocompatibility of
OA/NOCC/β-TCP hydrogels.
Figure 6. The research framework employed in this study
18
CHAPTER 2: METHODOLOGY
2.1. Decision matrix
Table 1. Decision matrix of typical crosslinking methods for synthesizing hydrogels
Photo- Schiff’s base

Criteria Crosslinkers
polymerization reaction
- Low molecular - Crosslinker is the - Reaction between
weight monomers or chemical employed to electrophilic carbon
oligomers are generate a cross- atoms of aldehydes or
exposed to radiations linked fluid system by ketones and
Mechanism (like ultra-violet (UV) connecting separate nucleophilic amines
or visible light polymers to create imine
radiations) to form a linkages under
crosslinked polymeric physiological
network conditions
- Fast hydrogel - Can link multiple - Simple method
formation at room molecules together - Construct injectable
temperature under and enhance viscosity and in situ forming
mild condition - Increase elastic hydrogel
- Controllable modulus - Hydrogel can have
mechanical properties self-healing
Positive
capability
outcomes
- No need for catalyst
or crosslinkers
- High crosslinking
degree results in
strong mechanical
properties of hydrogel
- Require photo- - Potential
Limitations
initiators and solvents cytotoxicity
19
that might be - Increase friction

cytotoxic pressure
- Only useful when
employing thin/
transparent scaffolds
due to their
ineffectiveness in
penetrating through
solid 3D scaffolds
- Long term exposure
to UV can induce
undesired
crosslinking, produce
cytotoxic free
radicals, affect cell
functionality,
stimulate local
inflammation
- Non-specific
reaction sites
- Photo-initiators can - Some crosslinkers - Environmental-
Environmental induce environmental can cause friendly
issues impacts environmental
impacts
Economics - High synthesis cost - Expensive - Low cost
(Hu et al., 2019); (Belyadi et al., 2019) (Hu et al., 2019);
(Ramiah et al., 2020); (Tran et al., 2020)
References (Krishnakumar et al.,
2019); (Choi & Cha,
2019)
20
Among various crosslinking strategies for synthesizing hydrogels,

photopolymerization, crosslinker, and Schiff’s based reaction are the typical choices.
According to the decision matrix displayed in Table 1, Schiff’s based crosslinking shows
outstanding advantages of a simple, easy to implement, low cost, and environmental-
friendly procedure compared to the remaining methods, allowing for its wide application
in hydrogel fabrication.
2.2. Materials
Chitosan (degree of deacetylation ≥ 80 %) was purchased from Vietnam Food Joint
Stock Company. Sodium alginate was provided by Nhatrang Institute of Technology
Research and Application, Vietnam Academy of Science and Technology. β-TCP granules
were provided by the Laboratory of Tissue Engineering and Regenerative Medicine,
International University, Vietnam National University, HCMC. Chloroacetic acid was
purchased from HiMedia Laboratories Pvt. Ltd. (India). Sodium metaperiodate (NaIO4) (≥
99%) was purchased from Thermo Fisher Scientific Inc. (UK). Isopropyl alcohol was
purchased from Guangdong Goanghua Sci-Tech Co., Ltd. (China). Sodium hydroxide
(NaOH), hydrochloric acid (HCl), and ethylene glycol were purchased from Xilong
Chemical Ltd (China). Dialysis tubing cellulose membrane (D9652, MWCO of 14 kDa)
and phosphate-buffered saline (PBS) tablets were purchased from Sigma–Aldrich Co.,
LLC (USA).
Trypsin/Ethylenediaminetetraacetic Acid (EDTA), Dulbecco's Modified Eagle
Medium (DMEM), Fetal Bovine Serum (FBS), antibiotics (penicillin and streptomycin)
(100 mL, 100X) were purchased from Thermo Fisher Scientific Inc. (USA).
2.3. Methods
2.3.1. Fabrication of OA/NOCC/β-TCP hydrogels
2.3.1.1. Synthesis of OA
OA was synthesized by following previous research with minor modifications (Ma
et al., 2020). The illustration of the procedure is shown in Figure 7. Firstly, 1 g of sodium
alginate (SA) was added to 100 mL distilled water and stirred until it dissolved completely,
forming a 1% (w/v) solution. Next, 0.432 g of NaIO4, corresponding to 40% molar
equivalent of alginate monomers, was dissolved in 5 mL distilled water and then added
21
dropwise to the SA solution. This step was conducted in the dark. The mixture was kept
stirring at room temperature for 2 h in a wholly covered container to avoid light exposure.
After that, 0.2 mL of ethylene glycol was added to the mixture to stop the reaction by
quenching any remaining NaIO4. The mixture was stirred for another hour under a similar
condition. The achieved solution then was dialyzed for 3 days using a dialysis bag (MWCO
of 14 kDa) against distilled water, with the water being replaced three times per day.
Subsequently, the solution was neutralized by 1.5 M NaOH solution, then frozen overnight
and lyophilized by Freezone 6 L Benchtop Freeze Dry System (Labconco, USA) to obtain
the final product. OA was eventually stored at 2-4 oC for further use.
Figure 7. Illustration of the synthesis of OA

2.3.1.2. Synthesis of NOCC
NOCC was synthesized from chitosan as described in previous studies (Nguyen et
al., 2019); (Nguyen-My Le et al., 2020) with few modifications. Briefly, 2 g of chitosan
22
was immersed in 10 mL isopropyl alcohol at room temperature. After that, 6.25 g of NaOH
was dissolved in 10 mL distilled water, and then the NaOH solution was added to the
chitosan suspension. The mixture was put at rest for 1 hour with manual stirring every 15
minutes. Next, 6.25 g of chloroacetic acid was dissolved in 5 mL isopropyl alcohol, and
the combination was supplemented to the above mixture in 5 equal portions at 5-min
intervals. The mixture was continually applied with manual stirring every 15 min for the
next 3 h at 60 °C to promote the carboxymethylation. Subsequently, the reaction mixture
was filtered to collect the solid residue product. Since the solid sample had high pH, it was
dissolved entirely in distilled water (66.67 mL) and neutralized by 2.5 M HCl solution. The
neutral solution then was dialyzed for 3 days using a dialysis bag (MWCO of 14 kDa)
against distilled water, with the water being replaced three times a day. After that, the
solution was frozen overnight and then lyophilized by Freezone 6 L Benchtop Freeze Dry
System (Labconco, USA) to obtain the final product. Lastly, NOCC was stored at 2-4 oC
for further use. Figure 8 illustrates the synthesis of NOCC as described above.
Figure 8. Illustration of the synthesis of NOCC
23
2.3.1.3. Fabrication of OA/NOCC/β-TCP hydrogels

The achieved product of OA after freeze-drying was dissolved in distilled water at
3% and 6% (w/v) concentrations to form OA solutions. Similarly, NOCC solution was
prepared by dissolving freeze-dried NOCC product in distilled water at 3% (w/v)
concentration. Since OA and NOCC crosslink with each other via Schiff’s base reaction
and β-TCP can exhibit ionic crosslinking between Ca2+ with the carboxylate group of OA,
β-TCP was first uniformly dispersed in OA solutions, and the mixture was then mixed with
NOCC to form hydrogels. Hence, β-TCP was fixed at 20% (w/w) of the total hydrogel
weight in both OA 3%/NOCC and OA 6%/NOCC combinations, and it was mixed well
with OA solutions. After that, NOCC 3% was added to the mixture of OA 3% and OA 6%
with β-TCP to form OA 3%/NOCC/β-TCP and OA 6%/NOCC/β-TCP hydrogels. The
NOCC content in each hydrogel system was increased, corresponding to the OA:NOCC
volume ratios are 1:1, 1:2, 1:3, and 1:4, namely OA-1/NO-1, OA-1/NO-2, OA-1/NO-3,
and OA-1/NO-4, respectively. Hence, 8 hydrogel samples were formed in total. The
hydrogel fabrication procedure and the sample code can be shown in Figure 9.
Figure 9. Schematic of OA/NOCC/β-TCP hydrogel fabrication procedure
24
2.3.2. Characterizations of OA/NOCC/β-TCP hydrogels

2.3.2.1. Gelation time
In brief, 300 µL of each OA/NOCC/β-TCP hydrogel sample was fabricated
following the abovementioned procedure. After adding NOCC solution to each sample and
starting to mix the hydrogel, the time was immediately recorded until there was no fluid
flow and the samples completely formed hydrogel at a steady state. The experiment was
conducted for 5 minutes in quadruple. The results were expressed as mean ± standard
deviation (SD).
2.3.2.2. Cross-sectional surface morphology analysis
Scanning electron microscopy (SEM) was used to observe the cross-sectional
surface morphology of the OA/NOCC/β-TCP hydrogels. Briefly, 8 hydrogel samples were
prepared, frozen overnight at -80 oC, and freeze-dried for at least 6 hours to yield the
scaffolds. Each sample was then sliced to show the interior cross-sectional surface, which
was next coated with a thin gold layer by Smart Coater (JEOL, Japan) and observed with
a JSMIT100 microscope (JEOL, Japan) at 15 kV. The hydrogels’ pore size at 30 and 100
different points in each sample was analyzed by ImageJ.
2.3.2.3. Porosity
The solvent replacement approach was employed to determine the porosity of
OA/NOCC/β-TCP scaffolds. At first, the volume of freeze-dried scaffolds’ geometry (𝑉𝑠 )
was calculated from their height and diameter. Next, each scaffold was initially weighted
(𝑊𝑜 ) and then immersed in absolute ethanol overnight at room temperature. After that, the
samples were taken out of ethanol, blotted to remove ethanol on the surface, and weighed
immediately (𝑊𝑒 ). Volumes of the pores (𝑉𝑝 ) are calculated as:
𝑊𝑒 − 𝑊𝑜
𝑉𝑝 = × 𝜌𝑒
𝑊𝑜
where 𝜌𝑒 is ethanol density at room temperature (𝜌𝑒 = 789 mg/mL). Finally, the porosity
of each scaffold sample was calculated as:
𝑉𝑝
Porosity (%) = × 100%
𝑉𝑠
The experiment was conducted in triplicate. The results were shown as mean ± SD.
25
2.3.2.4. Fourier transform infrared spectroscopy (FT-IR) analysis

The FT-IR technique was employed to identify the presence of the phosphate
groups in β-TCP, the aldehyde groups in OA, the added carboxymethyl groups in NOCC,
as well as the Schiff’s base, crosslinking between OA and NOCC. The results were
obtained by the Vertex 70v FT-IR spectrometer (Bruker, USA) at the Center for Innovative
Materials & Architectures (INOMAR, Vietnam). The measurement was conducted on
freeze-dried samples in the wavenumber range of 4000-400 cm-1.
2.3.2.5. Energy-dispersive X-ray spectroscopy (EDS) analysis
The EDS technology is used to detect and quantify the elemental compositions in
samples. In a correctly equipped SEM, the electron beam excites the atoms on the sample’s
surface, resulting in the emission of specific X-ray wavelengths that are characteristic of
the element’s atomic structure. An energy-dispersive detector, which distinguishes various
X-ray energies, is used to analyze the X-ray emissions and thus assign the appropriate
elements that make up the atom compositions on the sample surface. Hence, the EDS
analysis was used in this project to detect and quantify carbon (C), nitrogen (N), calcium
(Ca), and phosphor (P) elements that existed in the hydrogel samples. The procedure was
the same as described in the cross-sectional surface morphology testing. Briefly, the
lyophilized hydrogels were sliced and coated with a thin gold layer by Smart Coater (JEOL,
Japan) and observed with a JSMIT100 microscope (JEOL, Japan) at 15 kV. The presence
of the above elements in each sample is recorded by mapping images. The amount of each
element at four random points was detected, measured, and calculated as mean ± SD.
2.3.2.6. Swelling degree measurement
The swelling degree of hydrogel samples in the hydrated condition was obtained
by gravimetric measurement. In brief, 1.2 mL of each hydrogel sample was prepared,
measured the initial weight, and then placed in a self-made supporting holder with a mesh
bottom (the holes have a side length of 1 mm). Utilizing supporting holders can help
minimize possible errors induced by human factors when handling, blotting, and weighing
hydrogels. Each supporting holder was placed in one well of a 6-well plate and filled with
10 mL of PBS solution (1X, pH = 7.4) to immerse the hydrogels completely. All the
26
samples were incubated at 37 oC. Each sample was carefully taken from PBS and weighed
at particular periods. The swelling degree was calculated using the below equation:
𝑊𝑡 − 𝑊𝑜
Swelling degree (%) = × 100%
𝑊𝑜
where 𝑊𝑜 and 𝑊𝑡 is the initial weight and the weight at time t of the hydrogel samples,
respectively. The experiment was performed in triplicate. The results were presented as
mean ± SD.
2.3.2.7. In vitro degradation assessment
Similar to the swelling test, the degradation of OA/NOCC/β-TCP hydrogel samples
were assessed gravimetrically under imitated physiological conditions of PBS (1X, pH =
7.4) solution and 37 oC of incubation. The initial weight of each sample was recorded as
𝑊𝑜 and at specific day-interval, the samples were weighted and denoted as 𝑊𝑡 . The
experiment was carried out in triplicate and lasted for 14 days. The remaining amount of
each hydrogel sample 𝑊𝑑 (%) at each time point was calculated by the following equation:
𝑊𝑡
𝑊𝑑 (%) = × 100%
𝑊𝑜
The results were presented as mean ± SD.
2.3.2.8. Compression testing
The compression testing was conducted to evaluate the compressive strength of
OA/NOCC/β-TCP hydrogels. In brief, cylindrical hydrogel samples with a diameter of 6
mm and a height of 5 mm were prepared and let gelling for 1 hour at room temperature.
After that, the experiment was performed by using the Texture Analyser (TA.Xtplus, Stable
Micro Systems, USA) in unconfined compression up to 50% strain at room temperature.
The experiment was performed in quadruple. The results were calculated as mean ± SD.
2.3.3. In vitro testing
2.3.3.1. Cell culture
Cryotubes containing L929 mouse fibroblasts were thawed. Then cells were
cultured in T-75 flasks using Dulbecco's Modified Eagle Medium (DMEM) supplemented
with 10% FBS and 1X antibiotics (penicillin and streptomycin) until they reached a
confluency of 70%. After that, cells were harvested by Trypsin/EDTA 0.25%.
27
Hemocytometer was used to count cell numbers, and then cells were seeded in a 96-well
culture plate at a density of 104 cells per well.
2.3.3.2. In vitro live/dead assay
The in vitro biocompatibility of OA/NOCC/β-TCP hydrogels was investigated by
using L929 mouse fibroblasts. The procedure illustration is shown in Figure 10. Firstly, the
freeze-dried scaffolds of each hydrogel sample were prepared and sterilized. Meantime,
L929 fibroblast cells were seeded in a 96-well culture plate at 104 cell densities in 100 µL
of culture medium per well and then incubated for 24 hours at 37°C. Next, the sterilized
scaffolds were placed in the above cell-containing wells and incubated for the next 24
hours. After that, the culture media was removed, and the samples were washed with PBS
solution. The working solution was prepared by adding 8 µL of fluorescein diacetate (FDA)
(5 mg/mL) and 50 µL of propidium iodide (PI) (5 mg/mL) into every 5 mL PBS to stain
the viable and dead cells, respectively. 100 µL of the working solution was added to each
well with 15 minutes of incubation and then washed with PBS again. Lastly, using a
fluorescent microscope (Nikon, Japan), live and dead cells on the scaffold samples were
detected. The experiment was performed in quadruple.
Figure 10. The procedure of in vitro live/dead assay

28
2.3.3.3. In vitro cytotoxicity

The hydrogels’ cytotoxicity was examined by Resazurin assay following the ISO
10993-5 Standard Test as displayed in Figure 11. Firstly, 0.1 g/mL extracts of hydrogels
were prepared by incubating lyophilized samples in a cell culture medium at 37°C for 24
hours to achieve extracted solutions. The hydrogel extracts were diluted at different
concentrations, including 100% and 50%. DMEM was employed as a control (0%
extracted solution). Meanwhile, L929 fibroblasts were seeded in a 96-well culture plate at
a density of 104 cells in 100 µL of culture medium for each well and then incubated at 37
°C for 24 hours. After that, the culture media in each well was replaced with 100 µL of
hydrogel extracts and continually incubated at 37 °C for the next 24 hours. Next, replacing
hydrogel extracts in each well with 100 μL Resazurin 1X in culture medium and the plate
was further incubated at 37 °C for 4 hours. Ultimately, the absorbance of fluorescent
signals was measured at 530 nm wavelength by VarioskanTM Multiplate Reader. The
results were expressed as mean ± SD of computed cell viability percentage compared to
untreated control cells. The experiment was conducted in sextuple.
Figure 11. The procedure of in vitro cytotoxicity
29
2.2.4. Statistical analysis

All experiments were performed in at least triplicate. The results were expressed as
mean ± standard deviation (SD). The statistical significance was assessed using one-way
ANOVA by GraphPad Prism 9.0 software at a significance level of p < 0.05.
30
CHAPTER 3: RESULTS
3.1. Gelation time
In most samples at different OA:NOCC ratios of OA 6%/NOCC/β-TCP and OA
3%/NOCC/β-TCP combinations, hydrogels were formed in a relatively short time. As
shown in Figure 12, the gelation time gradually decreased with an increasing amount of
NOCC, which means that the gelation time at OA-1/NO-1 samples was the longest and at
OA-1/NO-4 samples was the fastest in both hydrogel systems. However, there was a
difference in gelation time between the OA 6%/NOCC/β-TCP and OA 3%/NOCC/β-TCP
hydrogels at the respective OA:NOCC ratios. According to the recorded results, at the same
OA:NOCC ratios, the OA 6%/NOCC/β-TCP hydrogels were more difficult to form a gel
and took longer gelation time. In particular, the OA-1/NO-4 samples were able to form a
gel in around 72 s while most trials of OA-1/NO-1 sample did not or hardly form a gel after
5 min of mixing hydrogel components. The fabricated structures of the remaining ratios
were also weak, wet, and unstable. On the other hand, the OA 3%/NOCC/β-TCP hydrogels
showed a much faster gelation time when all the samples could quickly form stable
structures in less than 2 min. Significantly, the fastest gelation time was exhibited in OA-
1/NO-3 and OA-1/NO-4 ratios, around 60 s. Also, the OA 3%/NOCC/β-TCP samples at
each OA:NOCC ratio seem to have a steadier structure than that of OA 6%/NOCC/β-TCP
samples. The images of the fabricated hydrogel samples can be seen in Figure 13.
Figure 12. Gelation time of OA 6%/NOCC/β-TCP hydrogels (left) and OA 3%/NOCC/β-

TCP hydrogels (right)
31
Figure 13. Images of hydrogels formed with different ratios of OA:NOCC of (A) OA
6%/NOCC/β-TCP and (B) OA 3%/NOCC/β-TCP hydrogel systems
3.2. Cross-sectional surface morphology analysis
Cross-sectional surface morphology of lyophilized OA 6%/NOCC/β-TCP and OA
3%/NOCC/β-TCP hydrogel samples was observed by SEM technique and shown in Figure
14. Generally, most OA 6%/NOCC/β-TCP samples formed incomplete pore structures,
except for the OA-1/NO-4 sample. The distinct pores were hardly discernible in the
samples at the remaining ratios. Also, their surface was rough, messy, and had many torn
pieces that seemed to be easily ruptured. On the contrary, in OA 3%/NOCC/β-TCP
samples, hydrogels were formed with a discernable 3D porous structure, allowing for
assessing their pore size distribution as presented in Figure 13. Their surface was smoother,
more continuous and organized, and had fewer fragments, but there were also areas
interrupted by multiple small pores. Among them, the OA-1/NO-3 sample showed the most
apparent porous structure with large and distinct pores distributed more uniformly and
thick pore walls indicating a more robust structure. The above descriptions were more
firmly established at 200X magnification. Additionally, at 10000X magnification, the
presence of crystal β-TCP was detected in both hydrogel systems, demonstrating that β-
TCP was successfully incorporated into the hydrogels.
32
Figure 14. SEM images of (A) OA 6%/NOCC/β-TCP and (B) OA 3%/NOCC/β-TCP

hydrogel samples at magnifications of 100X, 200X (scale bars are 100 µm), and 10000X
(scales bars are 1 µm).
The porous OA 3%/NOCC/β-TCP samples allowed for the assessment of their pore
size distribution as presented in Figure 15 and Figure 16. The pore size distribution was
33
calculated by the average pore diameter at 100 different pores in each sample. The results
showed that when the content of NOCC increased, the pore size grew bigger, ranging from
99.50 µm to 144.73 µm, and the pore walls became more stable with fewer fragile wall
pieces. The OA-1/NO-3 and OA-1/NO-4 samples had the largest pore size and were
approximately the same, which were 144.73 ± 11.00 µm and 141.04 ± 16.32 µm,
respectively. The lower SD in the OA-1/NO-3 sample demonstrated a more homogeneous
pore size than the OA-1/NO-4 sample. In general, the OA-1/NO-3 sample of the OA
3%/NOCC/β-TCP hydrogel system showed a stronger porous structure with the largest
pore size, stable pore wall, and relatively
Pore uniform pore distribution
size of OA/NOCC/β-TCP among all samples.
scaffolds
180
150
120
Pore size (μm)
90
60
30
0
OA-1/NO-1 OA-1/NO-2 OA-1/NO-3 OA-1/NO-4
Figure 15. The pore size of OA 3%/NOCC/β-TCP hydrogel samples (n=30)
34
Figure 16. Pore size distribution of OA 3%/NOCC/β-TCP hydrogel samples (n=100)

Since the OA 3%/NOCC/β-TCP hydrogels exhibited significantly superior gelation
time as well as surface morphology compared to the OA 6%/NOCC/β-TCP hydrogels, they
showed more potential in further experiments. Therefore, the OA 6%/NOCC/β-TCP
hydrogels were dropped, and the work was totally focused on the OA 3%/NOCC/β-TCP
hydrogels from the following experiment to characterize and evaluate four samples of
different OA:NOCC ratios.
3.3. Porosity
As given in Figure 17, the total void space percentage of scaffold samples was
found. The OA-1/NO-3 sample displayed the highest porosity, close to 47.31%.
Meanwhile, the pore volume in other ratios was slightly lower, around 40%. This result is
compatible with the abovementioned SEM observation of the hydrogels, where the OA-
1/NO-3 sample showed the most homogeneous porous structure while the other samples
had a more messy and less uniform pore formation.
35
Figure 17. The porosity of OA 3%/NOCC/β-TCP hydrogel samples

(ns: non-significant, *: p<0.05)
3.4. Confirmation of hydrogel formation
FT-IR spectra of β-TCP and freeze-dried OA, NOCC, and hydrogel samples were
analyzed and shown in Figure 18 and Table 2. Most of the peaks in the hydrogel spectrum
appeared to come from its components. The sharp peaks at 947 cm-1 and 1012 cm-1 in the
945-1087 cm-1 broad band can be assigned to the phosphate group (PO43-) in β-TCP
(Berzina-Cimdina & Borodajenko, 2012). A tiny absorption peak of OA that appeared at
1735 cm−1 could be attributed to the C=O stretching vibration of the aldehyde group (Kong
et al., 2021). However, this peak could barely be observed in the hydrogel samples, which
might be because of the lower amount of OA in the OA/NOCC ratios as well as the weak
absorption band of aldehyde groups in OA (Ma et al., 2020); (Reakasame & Boccaccini,
2018). Besides, an appearing peak at 1411 cm-1 and 1601 cm-1 for associated symmetric
and asymmetric stretching vibrations of COO- indicating that carboxymethyl groups were
presenting on NOCC and in the hydrogel samples (Chen et al., 2004). The absorption peak
at 1156 cm-1 demonstrates the C-O stretching bands (Zhu et al., 2018). The O-H and N-H
stretching bonds are represented by the broad peak centered at roughly 3320 cm-1, whereas
36
the nearby smaller peaks at 2915 cm-1 and 2855 cm-1 belong to C-H and C-2 bond stretching
vibrations, respectively (Nguyen-My Le et al., 2020). The expected C=N linkage peaks
were observable at 1641 cm-1 in all hydrogel samples (Cinarli et al., 2011), but the peak
was small due to the overlap by adjacent stronger and broader -COO- stretching peak of
carboxymethyl groups. Overall, the FT-IR spectra confirmed that all the hydrogel samples
were formed by Schiff’s base crosslinking between aldehyde groups of OA and
carboxymethyl groups of NOCC with the presence of β-TCP.
Figure 18. FT-IR spectra of OA, NOCC, β-TCP, and OA 3%/NOCC/β-TCP hydrogels at
four different OA:NOCC ratios
Table 2. FT-IR band assignment for OA, NOCC, β-TCP, and OA 3%/NOCC/β-TCP
hydrogels spectra
Functional group Wavenumber (cm-1)
3-
PO4 stretching vibration 947; 1012
C=O stretching vibration of aldehyde 1735
COO- symmetric stretching vibration 1411
COO- antisymmetric stretching vibration 1601
37
C-O stretching vibration 1156

O-H and N-H stretching vibration 3320 (broad)
C-H stretching vibration 2915
C-H2 stretching vibration 2855
C=N stretching vibration 1641
3.5. Confirmation of prerequisite elements

The elemental component analysis of OA 3%/NOCC/β-TCP hydrogel samples was
obtained by the EDS method. Figure 19 shows the mapping images of C, N, Ca, and P in
four hydrogel samples. Almost precursor elements that make up the hydrogel components
were detected in all samples, except for the N element in the OA-1/NO-1 and OA-1/NO-2
samples. In addition, the element quantification was also analyzed as mass and atom
percentage, which was presented in Table 3 and Figure 20 displayed the corresponding
charts. The result showed that there was a high percentage of C (37.70-53.23%) and a small
content of Ca (2.20-6.79%) and P (0.79-4.70%) that existed in the samples. However, the
amount of N element could not be detected that might be because N accounted for a small
content in the hydrogel system, and the signal of the other elements was too high while N
had a much weaker signal. In general, EDS analysis demonstrated the presence of
preresiquite elements in the formation of hydrogel composition, including C, N, Ca, and P
especially expressed most clearly at the ratio OA-1/NO-3 and OA-1/NO-4 through
mapping images.
38
Figure 19. EDS mapping images of OA 3%/NOCC/β-TCP hydrogel samples (1000X

magnification, all scale bars are 30 µm)
Table 3. Mass and atom percentage of C, Ca, and P elements in OA 3%/NOCC/β-TCP
hydrogel samples
OA-1/NO-1 OA-1/NO-2 OA-1/NO-3 OA-1/NO-4

Element
Mass Atom Mass Atom Mass Atom Mass Atom

(%) (%) (%) (%) (%) (%) (%) (%)
C 44.71 ± 1.5 53.23 ± 2.63 26.96 ± 2.48 37.70 ± 2.70 43.11 ± 0.82 52.11 ± 0.64 44.45 ± 1.27 47.03 ± 1.07
Ca 12.34 ± 2.18 5.00 ± 0.98 15.34 ± 1.98 6.79 ± 0.74 6.05 ± 1.13 2.20 ± 0.43 12.36 ± 1.24 4.74 ± 0.62
39
P 2.27 ± 1.10 1.94 ± 0.60 8.62 ± 1.26 4.70 ± 0.76 2.78 ± 0.40 2.30 ± 1.38 1.62 ± 0.42 0.79 ± 0.21
Figure 20. Mass and atom percentage of C, Ca, and P elements in OA 3%/NOCC/β-TCP
hydrogel samples
3.6. Swelling degree measurement
Figure 21. Equilibrium swelling degree of OA 3%/NOCC/β-TCP hydrogel samples

(*: p<0.05, ***: p<0.001)
The equilibrium swelling behavior of different OA 3%/NOCC/β-TCP hydrogel
samples was investigated and calculated as shown in Figure 21. The samples were checked
and weighed after 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours of being immersed in PBS solution
at 37 oC. All the samples started to swell after 1 hour of incubation. The results revealed
that the degree of swelling was higher in the samples with higher NOCC content at almost
40
all time points. Furthermore, most ratios showed the highest swelling degrees at 12 h, in
which the OA-1/NO-4 sample gave the highest value, which was close to 72.51%, and
gradually decreased to around 56.71%, 52.90%, and 42.69% at the OA-1/NO-3, OA-1/NO-
2, and OA-1/NO-1 sample, respectively.
3.7. In vitro degradation assessment
In a simulated physiological condition at 37 °C for 14 days, degradation kinetics
were recorded for all hydrogel samples as displayed in Figure 22, and the calculated weight
remaining percentage is presented in Figure 23. From the beginning to the 6th day, the OA-
1/NO-4 sample remained the highest weight. However, from the 6th to 8th day, it degraded
with the fastest rate and appeared to degrade almost completely from 109.60% to 1.71%.
Meanwhile, the remaining ratios still partially retained their structures at this point, with
the weight remaining in samples OA-1/NO-1 to OA-1/NO-3 was 38.17%, 69.95%, and
26.60%, respectively. They showed a longer degradation time than the OA-1/NO-4 sample,
and the OA-1/NO-1 and OA-1/NO-2 samples exhibited a slower and more stable
degradation degree. However, after 14 days of incubation, all hydrogel samples degraded
entirely.
Figure 22. Degradation images of OA 3%/NOCC/β-TCP hydrogels within 14 days
41
Figure 23. In vitro degradation assessment of OA 3%/NOCC/β-TCP hydrogel samples

3.8. Compression testing
Compression testing was used to analyze the mechanical properties of hydrogel
samples. Figure 25 exhibited the typical stress-strain curve of OA 3%/NOCC/β-TCP
hydrogel samples with the applied strain up to 50%. At the strain of 50% (i.e., compression
to 50% of the hydrogel’s height), the OA-1/NO-3 showed the highest compressive strength
(the process in Figure 24), which was 2.72 ± 0.22 kPa, followed by the OA-1/NO-2 and
OA-1/NO-4 samples with 2.61 ± 0.04 kPa and 2.38 ± 0.11 kPa, respectively. The
compressive strength was lowest in the OA-1/NO-1 sample with 2.24 ± 0.08 kPa.
Compression strength was also compared between the hydrogel samples with and without
β-TCP at the same ratios of OA:NOCC (Figure 25). The results showed that without β-
TCP supplementation, the OA-1/NO-1 hydrogel only gave a value of around 1.03 kPa, and
the other ratios showed their compressive strength at surrounding 1.65-1.75 kPa. On the
contrary, the hydrogels with the participation of β-TCP displayed much higher in
compression as mentioned above. This result indicated that the presence of β-TCP in the
hydrogels containing OA and NOCC helps enhance the mechanical strength of the formed
hydrogels. Although the samples did not provide high mechanical compression, they
remained their structures without cracking or fracture after finishing the test.
42
Figure 24. Images illustrating the compression testing of OA-1/NO-3 hydrogel sample
Figure 25. Stress-strain curve of OA 3%/NOCC/β-TCP hydrogels and OA 3%/NOCC

hydrogels (without β-TCP) at four ratios of OA:NOCC at strain from 0% to 50%
43
3.9. In vitro cytocompatibility of OA/NOCC/β-TCP hydrogels

3.9.1. In vitro live/dead assay
Figure 26. FDA/PI staining of L929 cells after 1-day culturing in the control and OA
3%/NOCC/β-TCP hydrogel samples (All scale bars are 100 µm)
The live/dead assay was conducted to examine the cell adhesion on the surface of
hydrogels. The control sample had cells seeded on a tissue culture plate, and the tested
hydrogel samples had cells cultured on their surface. Live and dead cells were indicated by
FDA-labeled green fluorescence and PI-labeled red fluorescence, respectively. After 1 day
44
of culturing cells, the results were observed by a fluorescent microscope and expressed in
Figure 26. In all hydrogel samples, the number of alive cells presented by green dots is
much superior. In contrast, the appearance of very few red dots demonstrated very few
dead cells, which is competitive with the control. Thus, these results revealed that all
hydrogel samples possess the potential ability for cell adhesion and proliferation.
3.9.2. In vitro cytotoxicity
Figure 27. Cytotoxicity evaluation of the control and OA 3%/NOCC/β-TCP hydrogel

samples using Resazurin assay
Resazurin assay method was used to evaluate the cytotoxicity of OA 3%/NOCC/β-
TCP hydrogel samples on L929 mouse fibroblasts compared to the control. The control
sample had the cell-seeded on a tissue culture plate, while the cells were cultured on the
hydrogel surface in the experimental samples. The cell viability percentage of the control
and four hydrogel samples is shown in Figure 27. According to the ISO 10993-5 Standard,
a cytotoxic impact is characterized by a reduction in cell viability of greater than 30%. All
samples met the standard with over 70% in cell viability for both hydrogel extract
concentrations and were thus considered non-cytotoxic and biocompatible. Notably, at the
highest extract concentration of 100%, the viability of L929 cells in four samples increased
45
when NOCC content increased, ranging from 83.44% to 99.78%. By reducing the extract
concentration by half to reach 50%, almost hydrogels showed their cell viability close to
100%, which is competitive with the control.
46
CHAPTER 4: DISCUSSION
The initial step in this project was to prepare the hydrogel components, which
included OA and NOCC. According to the modification in the NOCC preparation method
of a previous study to gain better solubility and biocompatibility, the high basicity of
NOCC was minimized by neutralizing the excess NaOH content in the reaction solution
with HCl (Nguyen et al., 2019). This approach was also applied in this study and yielded
a similar result that NOCC was totally soluble in distilled water at 3% (w/v) concentration,
demonstrating that neutralization had no notable effect on the solubility of NOCC while
reducing its basicity.
OA is another critical component in fabricated hydrogels. The oxidation degree of
alginate was calculated based on the molar equivalence between oxidizing reagent, which
is sodium periodate NaIO4, and the alginate monomer repeated units (Zhao et al., 2016);
(Kong et al., 2021). Alginate’s oxidation degree considerably impacts crosslinking ability,
mechanical characteristics, degradation behavior, and swelling capacity of the covalently
bonded material network. The oxidation degree rises resulted in an increase in crosslinking
ability and network density (Kong et al., 2021) but simultaneously causes alginate
breakdown, leading to a reduced molecular weight product (Reakasame & Boccaccini,
2018). In this project, a moderate oxidation degree at 40% was chosen to synthesize
oxidized alginate in the hope that it would have adequate reactive groups without degrading
too quickly due to the decreased molecular weight. Furthermore, much OA-based research
also conducted an intermediate oxidation degree of roughly 40% and showed a potential
of oxidized alginate in designing hydrogel formulations (Emami et al., 2018); (Li et al.,
2012). The OA solutions were prepared at two different concentrations and the first being
3% (w/v), which was the same as NOCC concentration. Besides, since OA showed a lower
viscosity and was easier to dissolve quickly in distilled water, a double concentration of
OA at 6% (w/v) was performed to investigate its gelling ability with NOCC. This
concentration was also applied in a study (Zhao et al., 2016).
The third component of the hydrogel systems in this study is β-TCP. β-TCP has
been extensively explored as a bone repair material due to its outstanding biocompatibility
and bioactivity, as well as high osteoconductivity, especially in combination with polymers
47
to improve its mechanical strength. Therefore, a fixed amount of β-TCP at 20% (w/w) was
chosen based on a previous study (Ho et al., 2020).
Subsequently, two hydrogel systems containing three components, namely OA
3%/NOCC/β-TCP and OA 6%/NOCC/β-TCP at different ratios of OA:NOCC, were
fabricated. At first, various OA:NOCC ratios were conducted at 1:1, 1:2, 1:4, 2:1, and 4:1
[Appendix 1]. After 5 minutes of mixing the components, the formed constructions in both
systems showed that the samples with a higher amount of OA (i.e., 2:1 and 4:1 ratios) have
very weak structures that collapsed easily when removed from the container or could barely
form hydrogels, especially in OA 6%/NOCC/β-TCP system. Meanwhile, hydrogels were
formed in most of the remaining ratios revealing that they tend to form hydrogels when
increasing the amount of NOCC. This might be because when raising the OA content, the
aldehyde groups in OA seem to appear much more than the presence of amine groups in
NOCC, causing insufficient Schiff's base crosslinking for hydrogel formation. Therefore,
the samples with a higher amount of OA were eliminated, and two hydrogel systems were
fabricated and characterized by increasing NOCC in four OA:NOCC ratios from 1:1 to 1:4
to find the most appropriate formulation for hydrogel formation.
Most of the samples were able to form a hydrogel, however, the gelation time and
cross-sectional surface morphology showed remarkable differences between the two
hydrogel systems. At the same ratios of OA:NOCC, samples with higher OA concentration
were more difficult to mix the components, took longer gelation time, and produced
relatively weak and wet structures not until OA-1/NO-4 was made. In contrast, samples
with lower OA concentration expressed easier and faster gelation with a seemingly stiffer
and more stable structure, especially with increasing amounts of NOCC. This observation
complied with SEM images of the hydrogels. The results revealed that almost samples with
an OA concentration of 6% (w/v) could not show discrete pores with turbulent distribution
and displayed rough surface and thin pore walls with many torn pieces. These properties,
however, were improved when the OA:NOCC ratio reached 1:4. Conversely, the
interconnected porous structures with noticeable pores arranged in a more homogeneous
and continuity organization and thicker pore walls were easily observed in the samples
with an OA concentration of 3% (w/v). This trend was also better in higher NOCC-content
48
samples and was most dominant in OA-1/NO-3. Therefore, a lower concentration of OA

and higher volume of NOCC would fasten gelation time and generate a more porous and
stable hydrogel structure due to enhanced crosslinking activity (Li et al., 2012). The lack
of hydrogel-forming potential made the OA 6%/NOCC/β-TCP samples omitted from
further experiments. The OA 3%/NOCC/β-TCP hydrogels at four ratios of OA:NOCC
would be the main subjects of investigation. In the pore size measurement, almost OA
3%/NOCC/β-TCP hydrogels yielded an average pore size of greater than 100 µm that could
be advantageous for cell ingrowth in applications for bone regeneration (Tran et al., 2020).
The highest pore diameters were recorded in OA-1/NO-3 and OA-1/NO-4 ratios with
around 140-145 µm, which is favorable for mineralized bone formation (Kanwar &
Vijayavenkataraman, 2021). Overall, it is inferred that the interconnected pores in OA-
1/NO-3 and OA-1/NO-4 ratios might aid cell migration into the hydrogel, permeation of
nutrients, waste disposal, and blood vessel and nerve infiltration, and large pore sizes might
promote nutrition transport throughout the hydrogel network, supporting tissue
regeneration and blood vessel ingrowth (Tran et al., 2020). In addition, the porosity was
highest in the OA 3%/NOCC/β-TCP hydrogel at the OA-1/NO-3 ratio with around 47.31%.
This result is consistent with SEM images as well, in which the OA-1/NO-3 sample showed
the clearest and most distinct and uniform pores with the most enormous pore size,
allowing for its most remarkable void space to support cell penetration and migration
among the samples. On the other hand, the remaining ratios exhibited a relatively lower
porosity at about 40% due to their poorer pore distribution and smaller porous structures.
The high porosity of 30-97% is preferred for BTE applications (Tran et al., 2020). Hence,
all the OA 3%/NOCC/β-TCP hydrogels can be favorable for applying in this field,
particularly at the OA-1/NO-3 ratio.
The hydrogels were crosslinked by Schiff’s base reaction between the aldehyde
groups of OA and the amino groups of NOCC, and the hydrogel formation was confirmed
through FT-IR analysis of the C=N peak. The FT-IR spectra of typical absorption of PO43-
and EDS mapping images of Ca and P elements also indicated the presence of β-TCP in
four samples. Furthermore, the SEM images at 10000X magnification showed the crystal
β-TCP nanoparticles distributed on the pore wall surface of hydrogels. Altogether, the
49
results demonstrated that β-TCP had been successfully incorporated into the hydrogel
system.
The swelling behavior and degradability of hydrogels were also determined. The
liquid absorption ability of hydrogels, which is considered by the swelling ratio, is required
in tissue engineering to enrich nutrients that stimulate cell proliferation and create a moist
environment for tissue regeneration (Ma et al., 2020). In this experiment, the swelling
degree raised when increasing NOCC volume and reached the maximum after 12 h of
incubation for almost all samples. The reason might be because of the highly swollen
NOCC due to the electrostatic repulsion between the negatively charged carboxymethyl
groups substituted on NOCC as investigated in a study (Chen et al., 2004). Besides, it
seems that due to more uniform and tight pore distribution, less interruption by small pore
clusters, and larger pore sizes of the surface morphology discussed above, the sample with
higher NOCC content might have a greater water uptake capacity, resulting in higher
swelling degree, and vice versa with lower NOCC-volume samples. On the other hand, the
biodegradability of polymeric hydrogels is an essential property in BTE since they should
be eliminated from the site after completing their function (Tran et al., 2020); (Kanwar &
Vijayavenkataraman, 2021). The degradation rate should be managed to make room for
newly produced ECM while maintaining mechanical support (Tran et al., 2020). Thus, the
in vitro degradation assessment was conducted. The OA-1/NO-4 sample showed the fastest
degradation rate and almost degraded entirely after 8 days of incubation under the
simulated physiological condition at 37 °C, while the remaining ratios could maintain their
structure and totally degraded after 14 days. It is thought that the denser crosslinking
network would help control the degradability of hydrogel in a longer time, but the presence
of many amino groups in NOCC of OA-1/NO-4 sample might influence this characteristic.
Another critical feature of polymeric hydrogels for bone regeneration applications
is their compressive strength. Ideally, hydrogels should be engineered to have sufficient
mechanical properties to withstand loads while also allowing bone tissue formation. The
compressive strength of hydrogels should be 100-200 MPa for cortical bone and 2-20 MPa
for cancellous bone (Campana et al., 2014). Nevertheless, the compression strength of four
OA 3%/NOCC/β-TCP hydrogel samples performed in this test showed relatively low
50
values, which was surrounding 2.24-2.72 kPa, in which the highest result was expressed in
the OA-1/NO-3 sample. The rather low OA molecular weight and the raising chain
flexibility once the bonds between C-2 and C-3 were broken might contribute to the low
mechanical strength of OA (Reakasame & Boccaccini, 2018), leading to the influence on
compression strength of the hydrogels. Besides, this test was also carried out on the original
hydrogel samples without β-TCP supplementation. The results reported a significant
improvement in compressibility when β-TCP was added and mixed with the polymers,
demonstrating its presence was effective in enhancing the mechanical strength of the
formed hydrogels. However, β-TCP content in hydrogel might be responsible for the low
compression results as mentioned above. According to an experiment fabricating
gelatin/CMCS/β-TCP scaffold for BTE with different fractions of β-TCP (0%, 5%, 10%,
20%, 30%, and 40%), the mechanical characteristics deteriorated when β-TCP fraction was
greater than 10% resulting from more extensive agglomeration and stress concentrations
(Lafuente-Merchan et al., 2021); (Zhou et al., 2012). Hence, the 20% (w/w) of β-TCP
chosen in this study might not be appropriate for producing hydrogels with desirable
compression strength. Additionally, in the above research or some studies fabricated
hydrogels based on OA and biphasic calcium phosphate (Paul et al., 2015); (Sarker et al.,
2015), the compression test was conducted for both wet and dry conditions of hydrogels
and gave significant difference values. Wet hydrogels yielded the results in kPa, while
freeze-dried hydrogels displayed values up to MPa. Therefore, hydrogels in dry conditions
are supposed to provide significantly higher compression strength. From these aspects,
further experiments such as investigating different β-TCP fractions, especially in OA-
1/NO-3 samples and performing compression tests on the lyophilized hydrogels should be
conducted to analyze, evaluate, and find the most optimal ratio of β-TCP for better
compressive strength.
The biocompatibility of hydrogels was assessed by the in vitro testing, including
live/dead staining and Resazurin assay. The detection by FDA/PI staining exhibited a
competitive result between the hydrogel samples and the control when the number of viable
cells was observed to be significantly superior compared to dead cells in all four samples.
This result indicated that these hydrogels have the potential for cell attachment. Moreover,
51
in the Resazurin assay, all samples at both extract concentrations were recorded to be non-
cytotoxic according to the ISO 10993-5 Standard since they provided viability of L929
fibroblasts greater than 70%. This result has complied with the live/dead assay. At 100%
extract concentration, the more NOCC content, the more cell viability in four samples. This
can be explained by the absence of the aldehyde group in OA, affecting its biocompatibility
(Reakasame & Boccaccini, 2018); (Jin et al., 2021). At the same volume of the samples,
the increasing amount of NOCC means a decreasing amount of OA, i.e., OA-1/NO-1 and
OA-1/NO-4 had the highest and lowest OA content, leading to their highest and lowest
aldehyde groups, respectively. This might explain the rising of cell viability from OA-
1/NO-1 to OA-1/NO-4 hydrogels. When reducing the extract concentration to 50%, the
viability values in all hydrogels were enhanced to reach nearly 100%, which is competitive
with the control. In general, the in vitro testing revealed that all the OA 3%/NOCC/β-TCP
hydrogels were non-cytotoxic and biocompatible.
52
CHAPTER 5: CONCLUSION
Overall, in this work, the in situ cross-linking hydrogels based on OA, NOCC, and
β-TCP were conducted with two hydrogel systems of OA 6%/NOCC/β-TCP and OA
3%/NOCC/β-TCP at four ratios of OA:NOCC in each system, ranging from 1:1 to 1:4. The
hydrogels based on OA 3% (w/v), NOCC 3% (w/v), and β-TCP 20% (w/w) with different
OA:NOCC ratios were successfully fabricated. Concentrations of OA and different
OA:NOCC ratios have shown their effects on hydrogel formation, characterization, and in
vitro cytocompatibility. The OA 3%/NOCC/β-TCP hydrogels exhibited faster gelation
time and better surface morphology at almost ratios, leading to their further implementation
in the rest tests. Since then, the formation of Schiff’s base linkages was confirmed by FT-
IR analysis, indicating that all hydrogels were successfully formed, and the integrating of
β-TCP into hydrogel was also proven. The procedure and results demonstrate the quick
and easy gelation of in situ crosslinking in most ratios, indicating its benefits among
crosslinking strategies. Generally, most hydrogels were biodegradable, biocompatible,
non-cytotoxic, and showed porous structures. In almost results of the tested samples, the
OA 3%/NOCC/β-TCP hydrogel at OA-1/NO-3 ratio yielded the most superior outcomes,
including quick gelation time, interconnected porous structure with the highest porosity,
acceptable swelling capacity and degradability, high compressive strength, and high in
vitro cytocompatibility, demonstrating its potential in BTE applications. Hence,
contributing to the waste reduction to the environment, creating more job opportunities for
people, or developing science and the national economy are promising potentials if this
research is further developed.
However, since this project was carried out during the latter half of this semester
due to the hinder of the COVID-19 pandemic, there are some incomplete results, such as
the X-Ray Diffraction (XRD) for analyzing the crystal structure of β-TCP. XRD of
hydrogel samples has been measured, but results have not been obtained due to time
constraints. Moreover, the in vivo biocompatibility examination should be performed to
investigate the ability of these hydrogels in bone regeneration, especially with the OA
3%/NOCC/β-TCP hydrogel at OA-1/NO-3 ratio. Also, different concentrations of β-TCP
should be conducted to examine their influences on the hydrogel properties.
53
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APPENDIX 1: Initial formulations
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Thesis Report Hua My Van Bebeiu17021

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Thesis Report Hua My Van Bebeiu17021

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Vietnam National University – HCMC

FABRICATION OF IN SITU CROSSLINKING HYDROGELS

A thesis submitted to the School of Biomedical Engineering in partial fulfillment of the

Ho Chi Minh city, Vietnam

FABRICATION OF IN SITU CROSSLINKING HYDROGELS BASED ON

2.3.2.4. Fourier transform infrared spectroscopy (FT-IR) analysis ....................... 26

Keywords: β-tricalcium phosphate, bone regeneration, in situ crosslinking

Figure 1. A description of BTE utilizing natural polymeric scaffolds. On the scaffolds,

Figure 2. Chemical structure of chitosan

Figure 3. (A) Structure of alginate monomers; (B) Structural representation of different

1.2.3. β-tricalcium phosphate (β-TCP)

Figure 4. Chemical structures of (A) Oxidized alginate (OA); (B) N,O-carboxymethyl

Figure 5. Illustration of OA/NOCC/β-TCP hydrogel and its components

1.5. Research framework

Figure 6. The research framework employed in this study

Photo- Schiff’s base

that might be - Increase friction

Among various crosslinking strategies for synthesizing hydrogels,

Figure 7. Illustration of the synthesis of OA

Figure 8. Illustration of the synthesis of NOCC

2.3.1.3. Fabrication of OA/NOCC/β-TCP hydrogels

Figure 9. Schematic of OA/NOCC/β-TCP hydrogel fabrication procedure

2.3.2. Characterizations of OA/NOCC/β-TCP hydrogels

2.3.2.4. Fourier transform infrared spectroscopy (FT-IR) analysis

Figure 10. The procedure of in vitro live/dead assay

2.3.3.3. In vitro cytotoxicity

Figure 11. The procedure of in vitro cytotoxicity

2.2.4. Statistical analysis

Figure 12. Gelation time of OA 6%/NOCC/β-TCP hydrogels (left) and OA 3%/NOCC/β-

Figure 14. SEM images of (A) OA 6%/NOCC/β-TCP and (B) OA 3%/NOCC/β-TCP

Figure 15. The pore size of OA 3%/NOCC/β-TCP hydrogel samples (n=30)

Figure 16. Pore size distribution of OA 3%/NOCC/β-TCP hydrogel samples (n=100)

Figure 17. The porosity of OA 3%/NOCC/β-TCP hydrogel samples

C-O stretching vibration 1156

3.5. Confirmation of prerequisite elements

Figure 19. EDS mapping images of OA 3%/NOCC/β-TCP hydrogel samples (1000X

OA-1/NO-1 OA-1/NO-2 OA-1/NO-3 OA-1/NO-4

Mass Atom Mass Atom Mass Atom Mass Atom

Figure 21. Equilibrium swelling degree of OA 3%/NOCC/β-TCP hydrogel samples

Figure 22. Degradation images of OA 3%/NOCC/β-TCP hydrogels within 14 days

Figure 23. In vitro degradation assessment of OA 3%/NOCC/β-TCP hydrogel samples

Figure 25. Stress-strain curve of OA 3%/NOCC/β-TCP hydrogels and OA 3%/NOCC

3.9. In vitro cytocompatibility of OA/NOCC/β-TCP hydrogels

Figure 27. Cytotoxicity evaluation of the control and OA 3%/NOCC/β-TCP hydrogel

samples and was most dominant in OA-1/NO-3. Therefore, a lower concentration of OA

Research, 23(1), 1–7. https://doi.org/10.1186/s40824-019-0168-8

hydrogels based on oxidized alginate-hybrid-hydroxyapatite nanoparticles and

APPENDIX 1: Initial formulations

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