The Diamond Concept Enigma: Recent Trends of Its Implementation in Cross-Linked Chitosan-Based Scaffolds For Bone Tissue Engineering

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org Review
The Diamond Concept Enigma: Recent Trends of Its Implementation

in Cross-linked Chitosan-Based Scaffolds for Bone Tissue
Engineering
Celine J. Agnes, Antoine Karoichan, and Maryam Tabrizian*
Cite This: ACS Appl. Bio Mater. 2023, 6, 2515−2545 Read Online
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ABSTRACT: An increasing number of publications over the past ten years have focused on
the development of chitosan-based cross-linked scaffolds to regenerate bone tissue. The design
of biomaterials for bone tissue engineering applications relies heavily on the ideals set forth by
a polytherapy approach called the “Diamond Concept”. This methodology takes into
consideration the mechanical environment, scaffold properties, osteogenic and angiogenic
potential of cells, and benefits of osteoinductive mediator encapsulation. The following review
presents a comprehensive summarization of recent trends in chitosan-based cross-linked
scaffold development within the scope of the Diamond Concept, particularly for nonload-
bearing bone repair. A standardized methodology for material characterization, along with
assessment of in vitro and in vivo potential for bone regeneration, is presented based on
approaches in the literature, and future directions of the field are discussed.
KEYWORDS: Chitosan-Based Scaffolds, Bone Tissue Engineering, Diamond Concept, Cross-linking, Osteogenic Cells, Growth Factors
1. INTRODUCTION In terms of bone regeneration, the scaffold needs to have a

Advancement of the bone tissue engineering field in recent sufficiently high mechanical strength,22−24 in addition to a
years has opened new potentials for the design of alternative biodegradation rate similar to that of new bone tissue
treatments of various orthopedic conditions. Naturally, bone formation.12,25−30 The architecture of the scaffold should be
tissue relies on its self-healing capabilities through direct or porous to allow for cellular migration and vascular network
indirect healing, but this can be hindered following severe infiltration.17,31
Incorporation of cells is also necessary since cells are
traumas or other underlying conditions.1−3 Current conven-
responsible for the secretion of the extracellular matrix along
tional bone graft treatments, while advantageous in some
with different growth factors and cytokines involved in tissue
respects, present many limitations including invasiveness,
healing. 32,33 For bone applications, this aspect often
donor site morbidity, limited graft volume, and long hospital-
encompasses osteoblast lineage cells such as preosteoblasts
ization times.4−10
since they are already lineage committed and require less effort
Interest has since shifted to the design and implementation
in differentiation processes.33,34 More recent work has also
of tissue engineered substitutes. These designs focus on
focused on the usage of mesenchymal stem cells with
targeting the regeneration of native tissue both at the structural
subsequent differentiation once encapsulated in the scaf-
and functional level using a complex polytherapy approach
fold.33,35 Finally, osteoinductive mediators such as bone
called the “Diamond Concept”.9,11−13 This design framework
morphogenetic proteins 2, 4, and 7 (BMP2, 4, 7) are a crucial
established by Giannoudis et al. provides a guideline for the
consideration as their incorporation aids in developing the
minimal requirements needed for efficient bone healing:
scaffold into an optimal environment for cell growth,
osteoconductive scaffolds, osteogenic and angiogenic cells,
differentiation, and new tissue formation.20,36−42 While the
osteoinductive mediators, and a suitable mechanical environ-
ment9−12,14−16 (Figure 1A).
Each component within this approach plays a critical role in Received: February 8, 2023
the overall ability of the biological substitute for bone healing Accepted: April 30, 2023
and regeneration.12,15,17−21 The biomaterial scaffold acts as a Published: June 13, 2023
3D microenvironment for cells by providing structural support
thereby necessitating similarities in mechanical properties
between a chosen biomaterial and native tissue environment.
© 2023 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acsabm.3c00108
2515 ACS Appl. Bio Mater. 2023, 6, 2515−2545
ACS Applied Bio Materials www.acsabm.org Review
Figure 1. Comprehensive overview of the literature review presented in this manuscript. (A) “Diamond Concept” for Bone Regeneration, which
encompasses an osteoconductive scaffold, osteogenic cells, osteoinductive mediators, and vascularization in a suitable mechanical environment.
This builds the foundation for the organization of the review. (B) Increasing number of publications within the chitosan-based cross-linked
scaffold/hydrogel domain for bone tissue engineering applications in the last 20 years. (C) The review’s novelty within the combined domain of
chitosan based cross-linked hydrogel/scaffolds for bone tissue engineering applications implementing the Diamond Concept.
Figure 2. Results of literature search conducted for this manuscript. The search was performed using the keywords: Chitosan, Crosslinked (or
Cross-linked), Bone, and Scaffold or Hydrogels. These results were also limited to publications from the last ten years and written in the English
language. From this search, 188 articles were found to be suitable and screened further to examine the specific target of the study.
three previously discussed components make up the tissue ture.44,48−51 The implementation of cross-linking for chitosan-
engineering triad for the regeneration of many tissues, based scaffolds in bone applications has continued to become
vascularization and mechanical environment are specific more popular in literature studies, thus requiring a more
considerations for bone applications.43 These are added due thorough investigation of its demonstrated benefits (Figure
to the complex nature of bone tissue thereby necessitating a 1B).
more complex design approach. In the scope of this review, we aim to provide in-depth
Polymers represent the largest class of biomaterials comparisons of material and osteogenic properties for
investigated for various biomedical and tissue engineering chitosan-based cross-linked scaffolds within the ideals of the
purposes. These chains of repeating monomers can be sourced
Diamond Concept for bone tissue engineering of nonload
naturally such as chitosan, alginate, and collagen or fabricated
bearing tissues. Each subcategory of the Diamond Concept will
synthetically like poly(glycolic acid) (PGA), poly(lactic-co-
glycolic acid) (PLGA), and poly(methyl methacrylate) be discussed according to the most recent advances in that
(PMMA), among many others.44−47 As evidenced throughout component. This literature review is to our knowledge the first
the literature, chitosan is among the most widely investigated of its kind to review the design of chitosan based cross-linked
polymers for the design of ideal scaffolds for bone tissue scaffolds for bone in this manner (Figure 1C). To accomplish
engineering.44−46,48 Its usage is often compounded with a this, a literature search was conducted using the SCOPUS
physical or chemical cross-linker, which serves to improve database with keywords: chitosan, cross-linked, and bone, and
material properties such as compressive strength, elastic resulting articles were further screened for availability and
modulus, degradation rate, structural stability, and architec- intended application (Figure 2).
2516 https://doi.org/10.1021/acsabm.3c00108
ACS Appl. Bio Mater. 2023, 6, 2515−2545
Table 1. Common Cross-linkers for Chitosan-Based Scaffolds in the Literature
Method of
Cross-link-
Scaffolds ing Cross-linkers Used Influence of Cross-linking on Mechanical/Rheological Properties
EDC/NHS64,65 Increased Young’s modulus for chitosan scaffolds with vanillin (and bioglass) compared to chitosan scaffold.80 Increased resistance to compression
Genipin66−79 for glutaraldehyde cross-linked scaffolds compared to uncross-linked scaffolds, concentration dependent increase in strength for the cross-
Chemical linker.82 No significant difference in Young’s modulus with different genipin concentrations (0.25 and 0.5 M final concentration) under
cross-link Vanillin72,80 compression.72 Increased stiffness for cross-linked scaffold with genipin compared to chitosan scaffold74
− Cova- Glutaraldehyde81−84
lent Schiff base reaction85
bonding
Hexamethylene-1,6-diaminocarboxysulfonate (HDACS)75
ACS Applied Bio Materials
Chitosan Citric acid86
Purines − Guanosine Diphosphate4,51,87−90 Increased compressive modulus with increasing concentrations of TPP in scaffolds.94 Increased compressive strength with TPP cross-linking
Physical β-Glycerophosphate85,91−93 compared to uncross-linked scaffolds.95 Increased stiffness for cross-linked scaffold with pectin compared to chitosan scaffold74
cross-link
Tripoly phosphate (TPP)94−99
− Other
bonding Copper100
Pectin74
EDC/NHS101−103 Increased elastic and loss Modulus with increasing DF-P1000 cross-linker concentration (Schiff Base and EDC dual cross-linking).101 Increased
Schiff base reaction101,104−106 modulus and compressive strength with cross-linking using glutaraldehyde (increasing concentrations and soaking time).109 Increase in
compressive strength for cross-linked scaffolds with glutaraldehyde alone or in combination with calcium cations. 111 Increased compression
Glyoxal102,107,108 modulus with genipin cross-linking (also with graphene oxide addition).121 Increase in compressive strength with glyoxal cross-linking compared
Chemical
cross-link Glutaraldehyde109−117 to uncross-linked scaffolds.107 Increased compressive strength with increasing concentration of inorganic phase (GPTMS)132
− Cova- Genipin113,118−130
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lent N′-Methylene bis(acrylamide)131
bonding
3-Glycidoxypropyl trimethoxysilane (GPTMS)132
2-Hydroxyethyl methacrylate (HEMA)112
Chitosan-
Hexamethylene diisocyanate (HDI)133
composite
Tripoly phosphate (TPP)102,134−136 Decreasing compressive strength with lemon grass oil addition due to decreased H-bonding potential of HPMC.137 Increase in compressive
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Hydroxypropyl methyl cellulose (HPMC)137,138 strength with addition of calcium cations as cross-linker compared to uncross-linked scaffolds.111 Increased elastic modulus (G′) in scaffold with
Mg2+ ions and BMP due to increased cross-linking density.131 Increased compressive strength for DHT treated scaffolds compared to IR treated,
Physical Calcium cations111,114,139 glutaraldehyde, and HEMA cross-linked112
cross-link
Mg2+ ions131
− Other
bonding Dehydrothermal (DHT) Treatment112
Irradiation treatment112
Cu2+ ions140
Review

https://doi.org/10.1021/acsabm.3c00108
Table 2. Common Methodologies for Material Property Testing in Chitosan Cross-linked Scaffolds for Bone (N = 100)
Incorporation in Method Usage in
Material Properties Property Studies (%) Methodology Specific Property (%) Information Obtained
Mechanical 71% Compression 87.3% Compressive strength, Young’s modulus
properties testing64−70,73,74,76,77,80,82,92,94−96,98,99,102−104,106−112,115−118,121,130,132−138,141,142,144−161
Dynamic mechanical analyzer84,131,139,162−164 8.4% Creep and recovery properties
Tensile testing131,165−167 5.6% Tensile strength, Tensile strain
Nanoindentation testing65,86,120 4.2% Nanomechanical properties
Atomic force microscopy80 1.4% Young’s modulus of hydrated samples
Hardness testing98 1.4% Hardness of material

Flexural strength testing84 1.4% Flexural strength
Rheological 28% Frequency sweep66,69,87,90,93,101,104,105,113,124,131,135,145,148,150,159,168,169 64.3% Storage modulus (G′) and Loss modulus (G′′)
properties with angular frequency
Strain sweep93,105,106,119,122,124,126,127,129,169 35.7% Storage modulus (G′) and loss modulus (G′′)
with respect to strain
Time sweep93,101,113,131,159,163 21.4% Determination of gelation
Structural 91% Scanning electron 96.7% Morphology and architecture of the scaffold,
architecture microscopy4,64−68,70−74,76,80−82,84,86,87,90−96,99,100,102,103,105−113,115−122,124,125,127,129−142,146−172 Pore size measurements with ImageJ
properties Liquid Displacement69,74,80,84,95,98,103,106,108,110−112,115−117,133,134,137,139,142,149−151,153−155 28.6% Porosity and density measurement
MicroCT imaging68,88,90,96,121,125 7.7% Porosity and interconnectivity
Mercury porosimeter65,70,76,127,130,159 6.6% Porosity measurement
Physisorption analyzer118 1.1% Measurement of specific surface area and pore
structure
Atomic force microscopy65 1.1% Surface structure
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Stereomicroscope155 1.1% Surface structure
Pycnometer94 1.1% Bulk volume of scaffold to find porosity
Biodegradation 54% In solution (PBS, SBF, or water) with additives (lysozymes, muramidase, collagenase I, 46.3% Percentage of degradation, Weight loss over
properties etc.)64,69,70,73,74,76,86,90,106,112,120,122,125−127,130,131,137,139,141,142,150,156,159,160 time
In PBS73,80,95,102,104,105,107,109−111,113,115,116,126,131−133,135,136,138,152,153,156,165,170 46.3% Weight remaining ratio, Weight loss percentage
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In simulated body fluid96,117,134,146 7.4% Degradation weight

In culture media94,151 3.7% Degradation rate
In water158 1.8% Degradation percentage
Review

Figure 3. Material properties to consider following cross-linking of chitosan scaffolds. Each property is presented with common methodology for
testing found in the literature.
Although the need for assessment of scaffold material soluble under dilute acidic conditions, which yields the
properties and bone regenerative capabilities is evident in advantage of fabricating minimally invasive, injectable
many studies found in the literature, no clear set standardized chitosan-based scaffolds. Use of chitosan with a higher DD
methodology has been presented in the literature for bone often correlates with higher solubility and offers more
applications. Thus, our additional goal of this review is to protonated sites that can be exploited to modify the chitosan
highlight the most popular methods of experimental testing to chains or complex them with other biomaterials, lipids,
drive the construction of a more standardized methodology in proteins, and genetic material for a wide range of therapeutic
the development of biomaterial substitutes for bone. applications.57 These chemical modifications of chitosan
solutions such as quaternized, carboxyalkyl, and thiolated
2. CHITOSAN AS A BIOMATERIAL chitosan have more recently emerged as additional options for
base materials in designed bone regenerative scaffolds since
Among the various polymers available for scaffold fabrication, they aid in attaining more optimal material properties.54
chitosan remains one of the most attractive biomaterials in the
field due to various desirable inherent properties such as
excellent biocompatibility and biodegradability, antibacterial 3. CROSS-LINKING FOR CHITOSAN SCAFFOLDS
properties, as well as its mucoadhesive nature and versatility for Although chitosan possesses many beneficial properties for
further chemical and physical modification.46,52,53 Chitosan is consideration as a biomaterial substitute, previous literature
derived from chitin through the deacetylation of N- has shown insufficiencies in certain parameters of the material
acetylglucosamine residues into N-glucosamines following needed to achieve optimal bone healing.58−60 For instance,
treatment with an alkaline compound. Similarities in structure chitosan lacks the necessary mechanical strength for bone
between the attained chitosan and glycosaminoglycans found related applications and exhibits issues with stability and
in the extracellular matrix provide chitosan biomaterials with control of biodegradation rates.44,48−50,61,62 To overcome these
the ability to interact well with cells.54 Numerous studies have limitations, the addition of small molecule cross-linkers is
demonstrated the material’s potential for bone applications explored to improve chitosan’s capability for healing.58
since it can support the proliferation and adhesion of Particularly, in recent years, cross-linking of chitosan has
osteoblastic lineage cells as well as mesenchymal stem become a popular trend allowing for not only the development
cells.22,54 Further to this benefit, the wide abundance of chitin of chitosan scaffolds with optimal properties but also the
in nature designates chitosan as an accessible and cost-effective development of composite scaffolds through cross-linking with
biomaterial for use in many different applications including other materials (Table 1).
both tissue engineering and pharmaceuticals.53 Various studies in the literature have examined the use of
The physicochemical and biological properties of chitosan cross-linkers including genipin, glutaraldehyde, and sodium
are largely influenced by its degree of deacetylation, which is tripolyphosphate as means to ensure stability within the
defined by the percentage of N-glucosamine residues polymer network.48−51,63 The incorporation of these cross-
formed.55,56 Degree of deacetylation (DD) of commercially linkers relies on physical or chemical means through bonding
available chitosan can vary from low (<50%) to full to the polymer’s functional groups either by covalent bonding
deacetylation (100%), but many studies have shown that a or supramolecular interactions.58,60 With respect to chitosan,
higher DD is favorable in bone tissue regeneration.57 These this bonding often implements its amine groups, which can
newly formed amine groups allow for chitosan to become form a bond to hydroxyl groups.46
As discussed previously, this cross-linking can be further time sweeps are performed 35.7% and 21.4% of the time,
improved in chitosan through modifications of the polymer respectively.
backbone by methacrylate or substitution of carboxymethyl With respect to assessment of scaffold morphology and
derivatives.131,141,142 While physical methods of cross-linking architecture changes, scanning electron microscopy (SEM) is
including UV or gamma irradiation treatments tend to allow considered as the most popular method with a percentage of
for a reversible process in certain pH and temperature usage around 96.4% in recent publications of the field. This
environments, the use of chemical treatments for cross-linking technique is so popular as it allows researchers to determine
is more commonly found in regenerative medicine applica- the pore size, interconnectivity, and distribution as well as the
tions.59,62,63,143 Treatment with chemical reagents has been distribution of any other added materials within the scaffold.
shown to result in a higher degree of cross-linking and Furthermore, researchers can also use this technique to
formation of a permanent network rather than a reversible observe and image the surface morphology of the designed
one.60,62 material. Other techniques including liquid displacement
In the selection of a cross-linker for chitosan, many factors (28.6%), Micro CT (7.7%), or porosimeter usage (6.6%)
need to be considered including the intended use of the have also been explored in the literature to a lesser extent,
scaffold and the properties of the cross-linking agent itself. For either alone or in conjunction with SEM, to determine the
tissue engineering applications, the cross-linking agent must percentage of porosity and density. Assessment of biode-
not induce an immunogenic effect and should not affect cell gradation rate in the literature generally encompasses weight
and material interactions.58−60 Within the chemical domain, measurements at multiple time points after incubation of
glutaraldehyde and genipin are the most used cross-linkers for scaffolds in either PBS alone, with enzyme solutions, or in
chitosan-based scaffold applications since their beneficial media. In the scope of chitosan-based scaffolds, the effect of
abilities have been well-documented in the literature.63 cross-linker incorporation is beneficial to optimize and increase
However, the toxicity of the functional aldehyde group of native material properties for bone related application.
glutaraldehyde has resulted in its limited usage in commercial 3.2. Influence of Chitosan Cross-linking on Mechan-
products.58,60 Conversely, genipin has been shown to be less ical Properties. The protectoral function of bones in the
cytotoxic, as it stems from natural roots.58,60,63 body necessitates the consideration of mechanical properties of
3.1. Methodology for Material Property Studies of the material in the design of biological substitutes, that will
Chitosan-Based Scaffolds. Addition of a cross-linking agent provide sufficient mechanical support and integrity throughout
to scaffolds is expected to have significant effects with regards the entire repair process.22,23 This explains why the mechanical
to material properties including degradation rate, structural environment is considered as one of the essential branches of
architecture, and mechanical properties. This notion thus the diamond concept on its own. Insufficiencies in mechanical
requires further testing of non-cross-linked and cross-linked strength have been demonstrated to be detrimental to the
materials for comparison to attain a more thorough under- functionality of encapsulated cells rendering healing unsuc-
standing of its effects (Table 2, Figure 3). Within recent cessful.22,174 For instance, variations in stiffness result in the
literature, 91% of material property studies are shown to differentiation of cells into other lineages, since cells can sense
include some form of evaluation of structural architecture, 71% mechanical changes to their microenvironment through
show an examination of mechanical properties, and 54% look mechanosensation and respond accordingly.22,174 Increased
at biodegradation mechanisms. mechanical properties and stiffness of chitosan-based materials
With respect to mechanical assessment, the most reported with cross-linking can favor the differentiation of mesenchymal
technique (87.3%) in the literature is found to be compression stem cells into the osteogenic lineage compared to others since
testing due to its ability to closely mimic the experience of osteogenic cells favor stiffer substrates.24
bone in vivo. This is expected since it is thought to more Design criteria often suggest that a material’s mechanical
accurately represent the material’s mechanical behavior when properties should be similar to those of the native bone tissue,
placed in the body.22 However, in addition to compressive which tend to vary based on the type of bone.22,23 The
modulus and strength, other parameters such as Young’s compressive strength and Young’s modulus of healthy human
modulus, toughness, fatigue strength, and shear modulus of cortical bone has been illustrated in the literature to be
materials have also been reported in the literature but to a between 130 to 200 MPa and 7 to 30 GPa, respectively,
lesser extent.9,12,30,173 For derivation of these parameters, other whereas trabecular bone values are reported to be between 0.1
methodologies are utilized including a mechanical tester for to 16 MPa and 0.05 to 0.5 GPa.175 Mechanical properties of a
tensile properties, atomic force microscopy or nanoindentation material tend to vary in response to a variety of factors.
for nanoindentation and stiffness, and a dynamic mechanical Changes in structural items such as the incorporation of other
analyzer to measure the response of force versus displacement. polymeric components, 6 4 , 7 0 , 1 0 6 , 1 1 8 , 1 5 5 , 1 5 6 cross-link-
Further mechanical scaffold characterization has focused on ers, 69,76,82,94,95,98,102,107,111,112,118,132,136,153 and addi-
examining the material’s viscoelastic properties under tives64,66,69,82,95,104,106,107,116,121,122,132−134,137,139,144,149,152 can
deformation using rheology. For chitosan-based scaffolds, have dramatic effects on the mechanical properties of the
literature studies often include either one or a combination chitosan-based biomaterial substitute.176 However, comparison
of frequency,87,104,124,135,148,150,168 strain,99,101,113,131,152 or between studies is often difficult since chitosan can be used
time sweep99,105,106,122,127,129,169 tests, which all provide with different modifications, molecular weights, and degree of
complementary information about the material’s mechanical deacetylations.22
properties. However, recent literature has shown a popularity The incorporation of a cross-linker as previously mentioned
in using frequency sweeps especially since they can provide is used with the intention of improving the mechanical
information such as the elastic and loss modulus with respect properties of chitosan scaffolds. Improved compressive
to angular frequency. These particular sweeps are performed strengths and young’s modulus values compared to uncross
64.3% of the time in recent studies, whereas strain sweeps and linked scaffolds have regularly been demonstrated in the
Figure 4. Influence of scaffold design changes on the mechanical properties. (A) Compressive strength measurements of chitosan and collagen
composite scaffolds at different ratios (75:25, 50:50, 25:75) prior to cross-linking. (B) Measurements of the same composite scaffolds after cross-
linking with glyoxal. Both A and B were reproduced with permission from ref 107. Copyright 2021 Royal Society of Chemistry and the Centre
National de la Recherche Scientifique. (C) Elastic modulus and compressive strength of gelatin chitosan composite scaffolds with increasing
concentrations of bioglass (0 to 30%). C was reproduced and adapted under open access conditions (Creative Commons Attribution License) from
ref 116. Copyright 2016 Kanchan Maji et al. and Hindawi.
literature.95,98 A study by Pinto et al. showed that the addition chitosan and collagen scaffolds at varying ratios (75/25, 50/50,
of glutaraldehyde in increasing concentrations to a chitosan and 25/75) before and after cross-linking with glyoxal (Figure
scaffold, produced increasing Young’s modulus values from 4A,B). Results demonstrated that as the concentration of
0.27 MPa with 0% to 1.7 MPa in 10% resulting in a more rigid chitosan decreased, the compressive strength of the material
and stable structure.82 Since many different cross-linking also decreased (with cross-linking: CH−CO 75/25:19.3 kPa to
methods exist, some authors have chosen to compare the CH−CO 25/75:9.3 kPa).107 This is promising as it suggests
effects of multiple cross-linkers on the mechanical properties to the possible benefit of chitosan for mechanical properties
determine which methodology is most efficient. Karakecili et al. compared to collagen. Another study was also able to support
evaluated different cross-linkers including EDAC/NHS, this possible benefit, but this time examining changes in
tripolyphosphate, and glyoxal in chitosan and collagen type I concentration of N,O-carboxymethyl chitosan with aldehyde
composite scaffolds, with results indicating a significant effect containing hyaluronate acid in a scaffold cross-linked through
of cross-linking method on mechanical strength.102 The glyoxal Schiff base reactions.106 Increasing the concentration of N,O-
cross-linking showed the highest percentage of increase for the carboxymethyl chitosan in the scaffold resulted in a significant
compression modulus, whereas EDAC/NHS was found to increase in compressive modulus from 4.35 kPa with 4% to
have the lowest values of the three cross-linkers. Interestingly, 20.99 kPa in 8%.
significant differences between cross-linkers are not always As the addition of compounds and mediators have been
observed within the literature. A study comparing tripoly- widely studied to improve the material and osteogenic
phosphate and genipin cross-linkers in a chitosan and nano properties of scaffolds, the influence of these additions on
beta-tricalcium phosphate scaffold suggested a bimodal trend mechanical properties must also be considered. Some additives
in concentrations with no significant differences between the are included for the purpose of improving mechanical
cross-linkers.76 Demonstration of both these cross-linker properties while others are not expected to have a significant
modality effects indicates the necessity of significant testing effect. One of the main methods for improving the mechanical
for optimal composition when designing scaffolds prior to strength is the incorporation of hydroxyapatite or the
exploration of in vitro and in vivo osteogenic potential of induction of scaffold mineralization . A study by Lu et al.
materials. examined compressive stress values of mineralized and
In addition to the influence of cross-linking, some studies of nonmineralized N,O-carboxymethyl chitosan scaffolds with
composite chitosan cross-linked scaffolds have also examined cross-linking through amidation reactions using EDC/NHS.142
the effect of different polymeric ratios within the scaffold. Nair The mineralization of the scaffold presented a marked increase
et al. tested for compressive strength differences between in ultimate compressive stress from 19.5 to 91.6 kPa. This
Figure 5. Rheological property evaluation. (A) Effect of increasing chitosan-cysteine (CH−CY) solutions (1.5 to 2.5%) and difunctionalized PEG
cross-linkers (0.3 to 0.6 w/v %) on G′ and G′′ values. Reproduced and adapted under open access conditions (Creative Commons Attribution
License) from ref 101. Copyright 2022 Qing Min et al. and MDPI. (B) Percentages of published articles for inclusion of mechanical properties,
rheological properties, both, and none.
effect was expected, as n-HA is known for its reinforcing examined through comparison to a scaffold without beta
ability. Fucoidan was also examined in this study as a potential tricalcium phosphate (β-TCP). While the loss tangent values
drug delivery vehicle, but authors suggest that its addition were below one for this study, confirming the materials
could improve the ability of the scaffold to mineralize as well. viscoelasticity, the elastic modulus values (at 1 Hz: G′ of 55 Pa
Fucoidan incorporated scaffolds showed increased nonminer- for β-TCP 1% and 75 Pa for β-TCP 1.5%) seem relatively low
alized and mineralized values of compressive stress compared compared to the use of other cross-linkers and additives
to pure N,O-carboxymethyl chitosan scaffolds alone. Another reported in the literature. Another study examining scaffolds of
study demonstrated that through the addition of increasing chitosan-cysteine conjugates with difunctionalized PEG cross-
concentrations of bioactive glass (0, 10, 20, 30 wt %) in linkers demonstrated a range of elastic modulus values between
chitosan and gelatin scaffolds, the compressive strength and 962.5 to 1561.8 Pa with increasing weight to volume
elastic modulus were able to increase (Figure 4C).116 percentages of chitosan-cysteine solutions from 1.5 to 2.5%
However, not all additives have been shown to have a (Figure 5A).101 Results presented in this study suggest a
beneficial effect. An example of this is the addition of beneficial effect of increasing concentrations of chitosan-
lemongrass oil, which was found to produce a decrease in cysteine solutions as well as an increased concentration of
compressive strength with increasing concentrations.137 This difunctionalized PEG cross-linkers (0.3 to 0.6 w/v %). The
undesirable effect was explained to be due to a disruption in large difference (one to 2 orders of magnitude) between G′
cross-linking of hydroxypropyl methyl cellulose and chitosan and G′′ was observed as well, indicating again the stability of
with this addition. the scaffold. The null effect of pyrophosphatase addition to a
These results therefore suggest extensive testing for additive chitosan-based guanosine diphosphate cross-linked scaffold on
effects in scaffold design studies. It is important to note that an its rheological properties has recently been demonstrated using
intimate relationship exists between mechanical properties and this methodology.87
multiple other material parameters, including the structural Time sweeps can also be beneficial in providing knowledge
architecture. Incorporation of higher porosity and larger pores, about the elastic modulus as a function of time. This particular
which are often necessary for cellular functionality and vascular technique has been well implemented in the literature as
network infiltration, often are a result of decreased cross- means to determine gelation time measurements. For example,
linking and thus the materials present decreased mechanical An et al. were able to tune the gelation time of a methyl
strength.174 Therefore, a balance must be struck between the acrylate chitosan and aldehyde containing oxidized hyaluronate
architectural design and the corresponding scaffold mechanical acid using this technique.105 The gelation time was determined
properties. as the time point where G′ and G′′ crossed over, as an
3.3. Influence of Chitosan Cross-linking on Rheo- indication of the transition from a liquid to a solid (more
logical Properties. Strain sweep tests are often conducted as elastic) scaffold. Other authors have used this method to
a preliminary step to frequency sweeps to determine in what establish the elastic modulus changes throughout different time
amplitude range the material exhibits viscoelastic properties by periods after cross-linking.129 From reviewing the literature for
applying a strain signal with increasing amplitudes. The chitosan-based cross-linked scaffolds in the last ten years, it is
frequency sweep test is often performed over a range of evident that rheological properties of materials have been less
frequencies (0.01 to 100 Hz) with a constant strain to attain explored compared to mechanical and compressive studies
important parameter values including the shear elastic modulus (Figure 5B). The viscoelasticity of a material is however an
(G′), loss/viscous modulus (G′′) and loss tangent (tan(δ)) important consideration as it also affects the osteogenic
values. Together, these values give further insight to the differentiation of cells and their spreading behavior.24 Rapid
stability of cross-linked scaffolds. A study by Dessi et al. used relaxation of the scaffold following an applied stress encourages
frequency sweep testing to demonstrate the deformation differentiation of the mesenchymal stem cells.24 Therefore,
behavior of Beta tricalcium phosphate (1 and 1.5%) and although it is more common to find studies that examine both
chitosan cross-linked composite scaffolds.99 Since the G′ elastic recently, this combination of studies should be a set standard
modulus was higher than the loss modulus G′′ throughout the in implementation of Diamond Concept in bone repair, as it
frequency range, authors were able to conclude that the allows for a more concrete understanding of the material and
behavior was primarily elastic indicating a stable structure. possible interactions with body environment and cells.
Interestingly, no significant difference was observed due to 3.4. Influence of Chitosan Cross-linking on Structural
concentration changes, but this conclusion should be further Architecture. The microarchitecture of scaffolds plays a vital
Figure 6. Characterization of structural architecture for various modifications of chitosan scaffold. (A) SEM images of chitosan scaffolds with
increasing concentrations of genipin (left −3.75%, right −7.5%). Reproduced and adapted under open access conditions (Creative Commons
Attribution License) from ref 73. Copyright 2017 Simona Dimida et al. and Hindawi. (B) Total porosity measurements (T.Po), structure thickness
(St.Th), and specific surface (Sp.S) for chitosan scaffolds with increasing graphene oxide (GO) percentages (0, 0.5, 3%). Reproduced and adapted
under open access conditions (Creative Commons Attribution License) from ref 178. Copyright 2019 Sorina Dinescu et al. and MDPI. (C)
Percentage of porosity for sodium alginate and chitosan composite scaffolds with and without the addition of collagen and graphene oxide using
liquid displacement method. Reproduced and adapted with permission from ref 139. Copyright 2018 American Chemical Society.
role in supporting various functions. Careful consideration is Hydroxyapatite (HA) is one such frequent additive to
needed when developing scaffolds to achieve the optimal chitosan-based cross-linked scaffolds.71,89,96,167 As a major
structural architecture, depending on the intended therapeutic constituent of natural bone, HA not only increases the
application. Bone is a highly porous structure; as such, porosity mechanical strength of scaffolds but also their biocompatibility
is an important parameter of a scaffold to better mimic the and bioactivity.102
native tissue.177 An interconnected porous microstructure This, however, can often be accompanied by changes to the
would favor higher cell infiltration and activity, vascularization, overall porosity or pore size.65,153 Compared to the control
proper exchange of nutrients, and diffusion of therapeutic scaffold with only tripolyphosphate (TPP) cross-linking,
agents.17,31 Shemshad et al. observed a decrease in pore size following
In general, chitosan-based scaffolds demonstrate excellent incorporation of nanohydroxyapatite along with bioactive
porosity, with a commonly reported range of 80−90%.69,111 silicate diopside. Nonetheless, the size remained within the
Moreover, pore diameter sizes tend to often be >100 μm, range of 40 to 250 μm often recommended for bone tissue
which is reported to be optimal for efficacious tissue infiltration engineering applications. The incorporation of these factors
and ingrowth.117 However, cross-linking in efforts to enhance did also yield a rougher pore morphology, which can be
the mechanical properties might be detrimental to the desired advantageous for better cell and tissue adhesion.95 β-TCP is
scaffold architecture, and thus, it is important to determine a another ceramic that can be added to confer to scaffolds similar
right balance between polymer and cross-linker concentrations. properties as HA does. However, Jahan et al. observed that β-
Genipin is among the most commonly used cross-linkers TCP had a larger particle size compared to HA, resulting in
when fabricating chitosan-based scaffolds. While increasing the more occluded scaffold pores, thereby decreasing their size.51
concentration of this cross-linker can slow down the To enhance the overall morphology of chitosan-based
degradation rate of the scaffold as a consequence of higher scaffolds, the addition of graphene oxide (GO) as an additive
cross-linking, Dimida et al. observed no significant change was attempted. This carbon-based material can aid in cross-
neither in the structural architecture of the formed scaffolds linking different polymer chains together or polymers and
nor in their mechanical properties (Figure 6A).73 Similarly, other biomolecules, as well as increasing the mechanical
increasing concentrations of glutaraldehyde, another popular properties of a scaffold and its biocompatibility, osteogenic
chitosan cross-linker, do not show a great effect on the general properties, and in some instances, the total porosity of chitosan
microarchitecture but did aid in enhancing the mechanical scaffolds.139 Dinescu et al. reported enhanced porosity with the
behavior of the scaffold. On the other hand, the compressive addition of 3% wt. GO in a freeze-dried chitosan scaffold. They
modulus of sodium tripolyphosphate cross-linked chitosan also observed that the addition of GO favored the formation of
scaffold showed significant increase with higher cross-linking larger pores (Figure 6B).178 In other instances where chemical
concentrations, but the porosity decreased to as low as cross-linking was completed, the addition of GO slightly
17.2%.94 Such results might suggest glutaraldehyde as an decreased the pore size of an alginate−chitosan−collagen
optimal cross-linker to combine improved mechanical composite scaffold chemically cross-linked with Ca2+ (Figure
strengths with maintained porous microstructure. However, 6C).139 Nonetheless, addition of GO commonly yields a more
increasing the concentrations of glutaraldehyde can risk an homogeneous architecture with more defined pores, thick pore
increase in cytotoxic effects. Therefore, more focus should be walls, and rougher morphology all which beneficially influence
placed on finding a suitable cross-linker that supports all cell attachment and osteogenic differentiation and improve the
necessary properties relevant to the Diamond Concept. overall efficacy of the scaffold for bone tissue engineer-
To complement cross-linking with the enhancement of ing.81,121,179
mechanical and osteogenic properties of a scaffold, the 3.5. Influence of Chitosan Cross-linking on Degrada-
addition of apatites and other ceramics is commonly employed. tion Rate. Since one of the expectations of bone healing in
Figure 7. Degradation profiles of composite chitosan scaffolds in lysozyme. (A) Chitosan (CS) and polycaprolactone (PCL) scaffolds with different
concentrations of polymers (100:0, 20:80, 40:60, 0:100) cross-linked with genipin. Reproduced and adapted with permission from ref 120.
Copyright 2022 Elsevier. (B) Chitosan/hydroxyapatite/collagen composite (Ha-Col1-CS) scaffolds before and after cross-linking with different
methodologies: (1) DHT − dehydro-thermal treatment, (2) IR − irradiation, (3) GTA − glutaraldehyde, and (4) HEMA − 2-hydroxyethyl
methacrylate. Reproduced and adapted under open access conditions (Creative Commons Attribution License) from ref 112. Copyright 2019 Md.
Shaifur Rahman et al. and Springer.
defects is the regeneration of bone tissue over the course of HEMA composites. Results indicated that the glutaraldehyde
time, the biodegradability of the material is an important cross-linked scaffold exhibited a 16% degradation on day 21,
consideration of scaffold design. When the scaffold is placed in whereas the scaffold without the cross-linker was 55%
vivo, it is expected that its degradation rate will match that of degraded at this same time point (Figure 7B). Interestingly,
the native bone tissue ingrowth thus allowing the gradual this improvement in degradation rate using cross-linkers was
transfer of mechanical load.12,25−30 The scaffold’s role is to act not shown when adding a fibrin coating to a chitosan
as a temporary bridge between both ends of the defect helping scaffold.70
cells, as well as acting as a platform to create an optimal Since composite scaffolds are often formed with chitosan
microenvironment.25 Degradation rates that are too fast or too and other polymers such as collagen, the biodegradation of
slow result in inadequate healing and lead to insufficiencies in scaffolds in collagenase solution is also sometimes assessed in
mechanical support throughout the healing process.27 the literature.108,122,126,127 Grabska-Zielinska et al. examined
Due to the necessity for control, assessment of this property the degradation profile of a chitosan and fish gelatin scaffold
has become a standard practice in material design and with genipin cross-linking and the benefits of graphene oxide
characterization, including the design of chitosan-based incorporation. Interestingly, when cultured in collagenase 2
scaffolds.28 Within the body, degradation of polymers typically solution, the scaffold without graphene oxide mostly degraded
occurs through a chain scission process of the polymer induced (72%) within the first 24 h. However, the addition of graphene
by enzymes such as lysozymes, which results in the hydrolysis oxide in doses above 1 wt % was able to withstand some of this
of chitosan and the cleavage of its β-(1−4)-glycosidic degradation showing percentage values around 44% to 52%
bonds.26,51 A study by Kim et al. demonstrated this concept depending on concentration.108 Similarly, the addition of
through the incorporation of varying concentrations of amino group functionalized silica particles with alendronate
lysozymes in a methacrylated glycol chitosan hydrogel.141 served to slow down the degradation process of chitosan
Increases in lysozyme concentration from 0.1 to 10 mg/mL scaffolds with collagen and hyaluronic acid.122 However, the
resulted in accelerated degradation rates of the hydrogel with a beneficial effects of biodegradation mediators should be further
final degradation of around 30% left after 14 days when a explored for better understanding of mediator-mediated
lysozyme concentration of 10 mg/mL was used. biodegradation.
Work within the literature on chitosan cross-linked scaffolds In addition to the importance of the biodegradation profile
has often embraced the lysozyme technique to study in vitro itself, the byproducts of this degradation must also be
degradation behavior.69,70,73,74,130,139,141 Formation of a considered in aspects of design. These should be nontoxic
and nonteratogenic and do not elicit any undesirable
composite chitosan and polycaprolactone (PCL) scaffold
immunogenic response when degradation oc-
with genipin cross-linking showed decreased degradation
curs.12,20,25,27,29,180 Often, byproducts can be beneficial in the
rates compared to chitosan alone cross-linked control groups
development of an adequate microenvironment for healing and
(Figure 7A).120 The dependence of the chitosan amount on
can improve bioactivity within the defect site.25,51
the degradation rate was not surprising since the lysozyme is
expected to target the chitosan specifically. Another study by
Rahman et al. further demonstrated chemical cross-linking’s 4. CELL ENCAPSULATION IN CHITOSAN
influence on biodegradation rates and stability using a CROSS-LINKED SCAFFOLDS
chitosan/hydroxyapatite/collagen composite scaffold.112 This In addition to modifications of the scaffold material itself, the
scaffold was cross-linked with either glutaraldehyde (GTA), Diamond Concept also stresses the necessity of an
dehydrothermal treatment (DTH), irradiation (IR) or 2- encapsulated viable cell population. Incorporation of cells is
hydroxyethyl methacrylate (HEMA), resulting in Ha·Col1·Cs- important in successful bone regenerative therapies since bone
GTA, Ha·Col1·Cs-IR, Ha·Col1·Cs-DTH, and Ha·Col1·Cs- cells such as osteoblasts, osteoclasts, and osteocytes are
Figure 8. Common methodologies for in vitro studies using cell cultures in the literature. These steps include testing for biocompatibility,
osteogenic differentiation, and biomineralization.
responsible for the production, remodeling, and maintenance thought to be further divided into three subgroups, namely
of bone.181 Previous studies with empty scaffolds have shown biocompatibility, osteogenic differentiation, and mineralization
almost no cellular ingrowth from the native tissue, which (Figure 8).
negatively affects the regenerative ability.182 With relevant cell The initial aspect of this often encompasses a quantification
encapsulation, both osteogenic differentiation of the cells and of cellular proliferation and metabolic activity using commer-
angiogenesis are encouraged when present at the target site, cial assays such as MTT/XTT/MTS (41.9%), alamarBlue
thereby resulting in the secretion of varying growth factors, (21.5%), or Cell Counting Kits (18.3%). While these listed
matrix proteins and cytokines such as vascular endothelial tend to be the most popular in terms of quantitative assays,
growth factor and interleukin 6.32,33,180,183 Culmination of many other techniques encompassing a visual assessment
these secretion activities with successful cellular differentiation component have also been demonstrated in the literature. For
can improve the healing outcomes. example, around 33.3% of recent articles have examined
4.1. Methodology for Cellular In Vitro Studies in cellular distribution and morphology through staining using
Chitosan Scaffolds. The first step in designing an antibodies for phalloidin and nucleic acid. Another common
encapsulated cell-based substitute is to examine the material’s staining found in the literature is Live/Dead or calcein based
ability to support cellular activity. While cross-linking of assays, which stain live and dead cells different colors making it
chitosan-based scaffolds can result in improved mechanical easy to quantify cytotoxicity and overall percentage of viability.
properties which is beneficial in supporting loading, it can be Furthermore, in many studies SEM is used to visually
accompanied by a decrease in porosity and pore size. Since the determine cell adhesion properties on the surface of the
functionality of cells once encapsulated is known to be heavily scaffold. All these methods for biocompatibility provide
dependent on their access to nutrients and oxygen, such researchers with a more thorough understanding of whether
significant changes to the internal structure with cross-linking their material can support generic cellular activities without
can lead to drastic changes in cellular viability and activity. significant adverse effects. However, not all these methods will
Another important consideration is the effect of the method provide the same information and therefore it is crucial to
of cross-linking itself on cellular viability and functionality. understand the differences between testing methods. Table 3 is
Some cross-linking techniques, like physical cross-linking, can presented to help differentiate these differences and serve as a
have low cytotoxicity to cells however, they might require resource for experiment planning in future studies.
harsh conditions such as changes in temperature or pH and do In addition to simply testing biocompatibility, the ability of a
not always provide the optimal material properties necessary material to support osteogenic differentiation is another
for bone applications.58,184 On the other hand, chemical cross- important consideration to address since the target application
linking might be more beneficial with respect to material in many of these studies is bone regeneration. Around 63.8% of
properties due to stronger bonding but can have higher costs relevant literature have included this aspect into their In vitro
and higher risk of cytotoxicity, especially with cross-linkers studies, with the most common technique for studying this
such as glutaraldehyde.58 Genipin cross-linking, which is being the detection of an early osteogenic marker, alkaline
commonly used as a cross-linker for chitosan-based materials, phosphatase (84.7%). Bone ALP is known to regulate the
is known to be biocompatible and less cytotoxic than other mineralization process since it provides a phosphate reservoir
chemical cross-linkers.58 to the matrix through hydrolysis of the mineralization
With all the considerations of cross-linker effects on cellular inhibitor, pyrophosphate. Thus, it is arguably a necessary
activity, this addition necessitates a thorough evaluation of methodology with regards to claiming successful differentiation
cellular functionality in chitosan-based scaffolds. For bone potential. This assay is sometimes complemented with other
applications, the in vitro assessment of cellular activity is techniques such as qPCR, which serves to assess gene
Table 3. Common Methodologies for In Vitro Testing in Chitosan Cross-linked Scaffolds for Bone (N = 94)
Usage in
In Vitro Incorporation in In Specific In Vitro
Experiments Vitro Studies (%) Information Obtained Methodology (%)
Cellular 98.9% Cellular Metabolic Activity, MTT/XTT/MTS Assay69,70,73,76,80,91,94−96,99,106,107,109,111,116,117,124,125,129−133,136,138,139,142,149−151,153−156,158−160,171,172 41.9%
Biocompatibility Growth and Proliferation IF Staining − DAPI with or without Phalloidin65,66,70,75−78,80,82,86−90,99,105,107,113,120,124,130,131,138,140,145,152,153,162,167,169,170 33.3%
Alamar Blue Assay73,77,82,87,88,90,93,108,119,122,126−128,135,137,141,144,145,153,165,171 21.5%
Cellular Counting Kits64,65,71,75,101,104,105,115,118,120,134,140,148,152,164,167,170 18.3%
PrestoBlue Kit102,110,113 3.2%
EdU and BrdU Staining162,185 2.2%
Resazurin Assay68,103 2.2%

IF Staining − Ki6765,120 2.2%
Cellular Cytotoxicity and Live Dead Assay/Calcein AM Staining64−66,76,88,90,96,101,105,106,118,124,125,138,139,141,148,151,160,161,164,168−170 25.8%
Counting of Viable Cell Lactic Dehydrogenase Assay125,157,170 3.2%
Numbers
Trypan Blue Staining94,166 2.2%
FDA Staining71,94 2.2%
Acridine orange/Ethidium bromide staining152 1.1%
Flow Cytometry − Annexin V,64 Propidium Iodide64 1.1%
DNA Content PicoGreen total DNA Assay4,70,74,76,102,124,163,171 8.6%
Cellular Biocompatibility, Scanning Electron Microscopy64−66,70,74−76,78,82,86,88,89,95,99,102,104,109,116,118,122,126−128,132,133,138,140,151,153,158,167,170,171 35.5%
Morphology and Adhesion Light Microscopy91,158,166,172 5.4%
HE Staining74 1.1%
Osteogenic 63.8% Osteoblast Differentiation Alkaline Phosphatase 84.7%
2526
Differentiation Specific Protein Secretion Testing4,64−66,69−71,74,76−78,80,82,86,87,89,90,94,95,99,101,102,109,110,115,119,120,122,124,126−128,131,132,134,136,138,140−142,145,152,156−158,160,162,164,170,171
ELISA - Osteocalcin,71,86,164 Type 1 Collagen,101 Osteopontin164 5.1%
Sirius Red Dye Assay113/SirCol Collagen Assay74 3.4%
Osteoblast Differentiation Related IHC, ICC, IF Staining for different markers − RUNX2,64,116,121 OPN,121 BSP,64 OCN,87,116,185 OSX89 11.9%
Activity Stainings
Gene Expression of Osteoblast qPCR − ALP,64,65,70,75,82,90,113,130,131,145,148,167,170 BSP,64,65,70,80,130 COL1A1,64,70,82,102,120,124,128,131,140,141,145,152,158,170 44.1%
Related Genes OCN,64,65,78,80,82,102,109,120,124,128,131,140,141,145,158 RUNX2,64,78,80,82,90,102,109,113,121,128,140,141,145,148,152,158,163,170
www.acsabm.org
OPN,65,78,82,102,120,121,124,130,131 BMPs,82,152 ON,70 OSX65

Protein Expression of Osteoblast Western Blot − Col1A1,64,120,131 RUNX2,64,131 ALP,131 OPN,64,65,120,131 BSP,64,65 OSX,64 OCN,64,65,70,120 ECM Proteins,120 B- 6.8%
Related Genes Catenin,131 Smad1,131 p-smad1/5/8,131 cell growth factors120
Mineralization 36.2% Quantification of Mineral and Alizarin Red Staining4,64−66,69,70,87,99,102,105,107,112,115,120,121,124,131,139,141,142,144,148,152,156,157,160,162 45.8%
Calcium Deposition Von Kossa Staining74,89,107,145,185 8.5%
Total Calcium Kit70,76,80,157 6.8%
O-Cresol phthalein complexone method74,110 3.4%
Review

expression of other early and late-stage markers in a Table 4. Common Cell Lineages and Lines Presented in the
comprehensive manner. Literature for Chitosan-Based Scaffolds
With respect to mineralization of the material, this is often
Category Cells in Literature Scaffold
the least considered and evaluated aspect of in vitro studies, 4,80,88,89,138
present in only around 36.2% of publications. The most Lineage MC3T3 preosteoblast cells Chitosan
committed (murine) Chitosan/
common techniques for this involve mineral staining using
gelatin102,110,113,121,125
either Alizarin Red or Von Kossa but a few studies have also Chitosan/collagen136
shown promising results using quantitative calcium assays. Chitosan/collagen/
4.2. Cell Sources for Bone Tissue Engineering in alginate139
Chitosan Scaffolds. A variety of factors need to be Chitosan-cysteine
considered in the decision of cell source including aspects conjugate101
such as the ease of availability for harvesting, low morbidity of Methacrylic anhydride
modified chitosan170
donor site, high efficiency of isolation, adequate cell
Methacrylic anhydride
proliferative ability, and sufficient osteogenic differentiation modified chitosan/
potential. While many different options for cell sources are poly(acrylamide)131
currently being explored in chitosan-based cross-linked Carboxymethyl chitosan/
scaffolds for bone tissue engineering, the focus remains on alginate140
finding a reliable cell source that will meet many of the criteria Chitosan/hyaluronic acid130
listed above. Currently, these consist of either osteogenic cells 7F2 osteoblast cells (murine) Chitosan74
that are already lineage committed or stem cells, which allow Carboxymethyl chitosan/
fucoidan142
for control of differentiation (Table 4). MG-63 osteoblast like cells Chitosan82,94,95,99
4.2.1. Osteogenic Lineage Committed Cells in Chitosan from osteosarcoma (murine) Chitosan/collagen/
Scaffolds. The first category often discussed when addressing hyaluronic
this challenge is osteogenic cells such as osteo-progenitor cells, acid119,122,126,127
osteoblasts, and osteocytes.30,33 Although these cells are still Chitosan/gelatin144
derived from mesenchymal cells, their previous commitment to Chitosan/polyvinyl
alcohol132
the osteogenic lineage allows them to begin secreting bone
Chitosan/
matrix components almost immediately upon implanta- polycaprolactone171
tion.33,34 Thus, osteogenic markers such as type 1 collagen Stem cells Mesenchymal stem cells Chitosan66,76−78,86,87,157
and alkaline phosphatase are expected to be present sooner (MSCs) − attained from Chitosan/gelatin116,134,148
into the regenerative process, therefore improving the rate of multiple sources
Chitosan/agarose/gelatin115
bone healing.33 In recent years, the MC3T3 preosteoblastic Chitosan/collagen112,128
cell line has become a standard model regarding lineage Thiolated chitosan/
committed cells for applications in bone tissue engineer- gelatin152
ing.186,187 In many studies with chitosan-based cross-linked Carboxymethyl
scaffolds, these cells have thus been included for evaluation of chitosan64,65,71
osteogenic differentiation and mineralization potential in the Chitosan/gelatin109
materials. For example, in a genipin cross-linked chitosan- Methylacrylylated chitosan/
hyaluronic acid105
gelatin scaffold with GO incorporated, Selaru et al. were able to Chitosan/
use MC3T3 cells to demonstrate enhanced osteogenic polycaprolactone120
differentiation and mineral deposition compared to scaffolds Fatty acid modified
without GO.121 Similarly, Karakeçili et al. observed enhanced chitosan/decellularized
bone ECM124
MC3T3 mineralization aided by the addition of n-HA.102
Methacrylated glycol
While these committed lineage cells provide many benefits in chitosan141
the healing cascade, they are often time-consuming with regard Chitosan/fibrin70
to the culturing process and their potential for expansion is Others C2C12 myoblasts (murine) Chitosan/alginate162
limited in vitro.30 Furthermore, collection and harvesting Chitosan/lactide156
processes for primary cells are invasive by nature, therefore
encouraging scientists to commonly look at alternatives for cell
types such as the incorporation of stem cells.33 MG-63 cells, whereas Pati et al. demonstrated the benefits of
Another common category of cells for various in vitro cross-linking with an increase in mineralization (Figure
osteogenic models includes osteosarcomas such as MG-63 and 9).99,171 However, while osteosarcomas can be useful in
Saos-2. These cell lines are derived from bone tumors and can providing valuable information regarding biocompatibility and
display similar properties as osteogenic cells like alkaline osteogenic potential in vitro, their clinical translatability is
phosphatase (ALP) production and mineral deposition.188 hindered by their tumoral nature. Therefore, it can be argued
Osteosarcomas have been widely used to investigate the that these cells are not ideal within the scope of the Diamond
osteogenic suitability of a various range of chitosan-based Concept and an alternative source should be considered or
cross-linked scaffolds.93,96,129,133,153,155,165 In the study by tested simultaneously with these cells.
Shemshad et al., osteogenic differentiation of MG-63 cells 4.2.2. Stem Cells in Chitosan-Based Scaffolds. Stem cells
seeded on a TPP cross-linked chitosan scaffold with addition present many advantages for use, since they have self-renewal
of hydroxyapatite and bioactive silicate diopside is reported.95 capabilities and can easily be induced to differentiate into
By adding the osteogenic factor strontium to their scaffold, various lineages.180,189,190 Although this broad categorization
́
Rodriguez-Mé ndez et al. observed increased ALP activity of encompasses several different types, adult mesenchymal stem
Figure 9. Assessment of (A) osteoblastic differentiation (ALP activity) and (B) mineralization potential (Alizarin Red Staining) from MG-63 cells
over 21 days for chitosan scaffolds with and without tripolyphosphate cross-linker. Reproduced and adapted with permission from ref 99. Copyright
2012 Wiley Periodicals, Inc.
Figure 10. Common osteoinductive mediators in the literature for chitosan-based cross-linked scaffolds.
cells (MSCs) are most extensively used in the literature for be retrieved in high abundance from harvesting, and their
bone regeneration application as they are not associated with differentiation from fibroblast-like morphology makes them
ethical or safety constraints.33,35 Adult MSCs can be isolated very interesting for rapid formation of connective tis-
from tissues such as bone marrow and adipose, which are sue.42,182,183,189,192,193 However, their osteogenic ability has
vascularized.30,180,183 They possess beneficial properties been questioned since some studies have shown lower
including multilineage potential for differentiation (adipogenic, capability for differentiation compared to bone marrow derived
chondrogenic, and osteogenic), high proliferative abilities, and stem cells.33,189 Others have demonstrated that their differ-
immunomodulatory capabilities.180,181,183 Bone marrow de- entiation potential is adequate and can be enhanced in this cell
rived stem cells are the frequent cell choice for bone tissue type through culture with osteogenic medium.182
engineering applications and treatment of bone disease since
they have demonstrated the highest potential for osteogenic 5. OSTEOINDUCTIVE MEDIATORS’ ENCAPSULATION
differentiation and bone repair.41,182,183 These cells are IN CROSS-LINKED CHITOSAN SCAFFOLDS
harvested during bone marrow biopsies, often from the iliac The third major aspect in the design of biological substitutes
crest, sternum, or proximal tibia, which all are rich in marrow for bone tissue regeneration involves the incorporation of
and are induced for differentiation through exposure of cells to bioactive molecules, drugs, and growth factors with osteoin-
a high phosphate environment.182,183,191 ductive properties. These components are responsible for
Although bone marrow derived stem cells are commonly influencing cellular functionality including the attraction of
employed and have been extensively characterized, they only cells to the injured site, and stimulation of cells to undergo
compose around 0.001 to 0.1% of cells present in the marrow, osteogenic differentiation in turn resulting in bone regener-
which make their isolation in large quantities very difficult.182 ation.194−197 While in cell cultures growth factors can be added
The cell number for isolation is dependent on various to media, it is often more effective to encapsulate them in the
parameters including the donor’s age and associated donor scaffold or conjugate them to the polymer backbone through
site morbidity and their limited quantity is a significant chemical modification.195,198 Within the scaffold, factors can be
disadvantage to their use.189 locally delivered to the injury site in a controlled release
An alternative cell source for osteogenic applications manner, allowing for the creation of a similar microenviron-
includes adipose derived stem cells (ADSCs). These cells are ment to the natural healing cascade.194,199,200
attained from adipose tissue through minimally invasive Many studies have been conducted within the literature
surgical procedures such as liposuction.183,189,191 They can examining the application of different osteoinductive mediators
with respect to cross-linked chitosan scaffolds including growth even higher ALP activity. The influence of HA incorporation
factors, mediators, enzymes, drugs, compounds, and nano- either alone or in combination with fucoidan also showed
particles (Figure 10). increased calcium deposition in alizarin red staining, signifying
5.1. Encapsulation of Growth Factors in Cross-linked its benefits in mineralization induction. Combination of HA
Chitosan Scaffolds. Traditional growth factors, such as and β-TCP in varying ratios has also been tested in chitosan-
BMPs, have great potential in bone regeneration applications based scaffolds68 ,82 ,96 ,134 since this biphasic calcium
since they are known to have a physiological role in the phosphate composite is expected to have increased osteocon-
fracture healing cascade.200 BMPs residing within the trans- ductivity to HA alone.
forming growth factor-beta (TGF-β) family are one of the More recently, studies have focused on this criterial element
most popular osteogenic growth factors currently. These by incorporating enzymes, natural compounds, and drugs in
factors encourage bone formation through downstream events their chitosan-based scaffolds. A few studies used pyrophos-
that occur due to receptor binding.194−196,200 BMPs 2 and 7 phatase in combination with the purine cross-linker guanosine
specifically are known to be responsible for bone regeneration diphosphate to promote mineralization.4,87,89 Nayef et al. have
and have been demonstrated in the literature for controlled shown that due to the enzymatic cleavage of the purine during
delivery both alone and in conjunction with other mediators the degradation process, a large quantity of pyrophosphate is
from chitosan-based scaffolds.64,78,115,130,131,162,170,194−196 A found in the culture media.4 Incorporation of pyrophosphatase
study by Nath et al. demonstrated the osteogenic effects of served to cleave observed pyrophosphates into phosphate ions
BMP2 incorporation into a chitosan and hyaluronic acid allowing for an increased ratio of phosphate to pyrophosphate,
scaffold cross-linked with 3 mg of genipin. Results showed that thus producing mineralization at levels similar to those treated
MC3T3 preosteoblast cells in the scaffolds coencapsulated with direct BMP7 injection in MC3T3 cell cultures.4
with BMP2 exhibited higher expression levels of early Additional studies have expanded the pyrophosphatase
differentiation markers ALP and osteopontin (OPN) at day encapsulation in conjunction with adipose derived stem
7 compared to control groups, as well as significantly higher cells87 in the chitosan scaffold and reported on its beneficial
late-stage expression of OPN at day 28. While this study effect in vivo using mouse tibial defect models.89
showed the benefits of coencapsulation, the osteogenic effect The loading of bisphosphonate drugs such as Alendronate
of sustained release of BMP2 was not considered for cells have also been explored for promoting osteogenic activity in
outside the scaffold.130 This missing aspect was however chitosan cross-linked scaffolds.120,122 Alendronate has been
explored in the scope of a chitosan-agarose-gelatin scaffold shown to increase BMP2 expression, thereby stimulating
with chitosan oligosaccharide/heparin nanoparticles. Through osteogenic differentiation and mineralization. A recent study
indirect culture of MSCs, Wang et al. demonstrated that the by Shi et al. examined these possible beneficial effects in a
loading of BMP2 within the nanoparticles resulted in a chitosan and polycaprolactone scaffold cross-linked with
sustained release over time leading to increased ALP activity genipin.120 Through seeding of ectomesenchyme cells, they
and osteogenic induction throughout 21 days. These BMP2 demonstrated increased osteogenic induction (ALP activity)
loaded nanoparticles in the chitosan-agarose-gelatin scaffold and calcium deposition with Alendronate compared to the
showed significantly higher ALP levels compared to the control scaffolds. Additionally, results showed an increased
nanoparticles or scaffold alone and demonstrated similar levels level of BMP2 proteins within this test group, which could
to the group with BMP2 alone at early time points. At later suggest Alendronate as a possible lower-cost alternative to
stages, this BMP2 loaded test group also exhibited more BMP2 growth factor. Since bisphosphonate drugs are often
positive alizarin red calcium staining compared to the BMP2 more commonly associated with their roles in osteoclast
alone control group.115 Interestingly, while BMPs are present activity inhibition, their usage proves beneficial in diseases such
in a variety of studies in the literature for chitosan-based as osteoporosis or Paget’s disease where additional bone
scaffolds due to their demonstrated beneficial effects, other turnover is observed.
alternatives are considered due to their high cost and possible Natural medicinal compounds derived from herbs and
side effects. minerals have also been considered as an alternate option for
5.2. Encapsulation of Bioactive Compounds in Cross- promotion of osteogenic differentiation to mitigate high costs
linked Chitosan Scaffolds. In addition to traditional growth and long-lasting side effects.84,101,149 Two flavonoid com-
factors, studies within the bone tissue engineering domain pounds, Quercetin and Icariin, have been explored recently
heavily rely on the incorporation of osteoinductive mediators with encapsulation into chitosan-based cross-linked scaffolds as
such as bioceramics, namely possible agents for promotion of bone regeneration and
HA51,65,69,71,77,82,84,89,94,95,102,104,112,137,142,146,147,153,165−167 formation. Both compounds exhibit similar effects in bone
and β-TCP.70,76,93,146 Both ceramics contain calcium and metabolism to estrogen, thus inhibiting osteoclast differ-
phosphate, which make up the main components of entiation and promoting osteoblast formation. A study by
mineralized bone.9,201−204 Many studies have included HA Min et al. showed a synergistic osteogenic effect of amino-
within their designed chitosan-based scaffolds due to its functionalized mesoporous bioglass nanoparticles loaded with
biocompatibility with cells and its known bioactivity both in Quercetin in a chitosan cysteine conjugate composite (CH−
vitro and in vivo.205 Recent work has explored the combination CY) with difunctionalized PEG cross-linkers.101 Hydrogels
of HA with other compounds for added promotion of with loaded nanoparticles exhibited increased ALP activity and
osteogenic activities. Lu et al. incorporated n-HA with fucoidan Collagen I content at 14 days compared to CH−CY gels alone.
into a hydroxypropyl chitosan scaffold cross-linked with The authors suggested that the synergistic effect was a result of
genipin.69 While the incorporation of n-HA alone in these both Si and Ca ions released due to nanoparticle degradation
scaffolds resulted in a marked concentration-dependent as well as quercetin release. While these encouraging results of
increase in ALP activity for 7F2 osteoblasts compared to natural compounds are outlined clearly in the literature, their
controls, the combination of n-HA with fucoidan produced an tested incorporation in the scaffolds serves to design a drug
Figure 11. General mechanisms that mediate the release of factors from scaffolds and common methodologies used to assess this release.
Table 5. Common Methodologies for Encapsulation Efficiency and Release Kinetic Studies in Chitosan Cross-linked Scaffolds
for Bone (N = 31)
Growth Factor Incorporation in Growth Usage in Specific Property
Properties Factor Studies (%) Information Obtained Methodology Category testing (%)
Encapsulation 45.2% Drug loading rate UV−vis/ spectrophotometer−absorbance 35.7%
efficiency encapsulation efficiency readings67,84,101,104,122
ELISA4,97,115,168 28.6%
HPLC120 7.1%
Weight measurements of drug106 7.1%
Drug specific testing BCA protein assay−used commonly for BSA 14.3%
loading92,130
Toluidine Blue staining (presence of heparin)97,147 14.3%
Release kinetics 96.7% Release rate of drug UV−vis/ spectrophotometer−absorbance 50%
percentage of drug readings65,68,74,84,88,101,104,106,120,122,133,148,154,158,162,170
released ELISA4,64,78,97,115,130,168 26.7%
Inductively coupled plasma mass spectrometry131,140 6.7%
Aluminum trichloride method159 3.3%
HPLC136 3.3%
Drug specific testing BCA protein assay−used commonly for BSA 10%
loading92,156,163
Toluidine Blue Assay (concentration of Heparin)147 3.3%
delivery system whereby drug release can be carefully collagen composite scaffold.128 In groups with silica particles,
controlled. Often, loading of these compounds relies on the the mRNA expression levels of osteogenic related genes
usage of nano or microparticles within the scaffold as can be (RUNX2, COL-1, and osteocalcin) were significantly higher
seen in the previous example. than the control group at day 14. Interestingly, results
5.3. Inorganic Mediators Encapsulation in Micro- and demonstrated a dependency of the mRNA expression on the
Nanoparticles. Nanoparticle encapsulation in chitosan-based size of the particles at this early stage where the smaller
cross-linked scaffolds has incorporated the usage of mostly particles exhibited higher levels. In addition, SEM micrographs
inorganic materials including silica,91,106,119,122,123,126,128 cop- of both silica particle groups showed aggregation on the
per,140 zinc oxide,138,149 strontium,171 and titanium oxide.129 surface of the cell as a sign of mineralization and apatite
Silica is one of the components that make up bioactive glass, formation compared to the smooth surface found in the
thus making its incorporation in studies popular. Unlike the control collagen and chitosan scaffold. EDS analysis for
previous compounds and additives, silica has been shown to calcium and phosphorus on these surfaces illustrated silica’s
promote apatite formation in cultures of simulated body fluid effect on calcium and phosphate production. These two test
as well as osteogenic differentiation. Filipowska et al. groups had Ca/P ratios around 1.65, which is similar to that of
demonstrates both effects using hBMSCs in a chitosan and native crystalline hydroxyapatite. Other inorganic materials
Table 6. Common Osteogenic Mediators Examined in the Literature for Release Kinetics from Chitosan-Based Scaffolds
Mediator in
Literature Scaffold Tested Cross-linking Method Observed Outcomes
BMP-2 Chitosan/alginate EDC/NHS Lower cross-linking levels showed higher initial burst release, as well as absolute amount of BMP-2
released over 1 month162
Chitosan/ Genipin Initial burst release was observed that became more gradual and sustained after 3 days and lasted
hyaluronic acid over 4 weeks130
Chitosan/collagen Sodium Initial burst release followed by a linear stage for 3 days after which the kinetics reached a slow-
tripolyphosphate release stage136
(STPP)
Chitosan Sodium No initial burst release was observed. Release was sustained over 45 days, after which 90% of BMP-2
tripolyphosphate was released97
(STPP)
Chitosan/agarose/ Glutaraldehyde Zero-order-like release sustained over 15 days115
gelatin
Alendronate Chitosan/ Genipin Addition of polycaprolactone to the scaffold demonstrated a more sustained release of alendronate
polycaprolactone compared to the control which had faster release. More than 90% of the drug was released from
either scaffold after 7 days120
Icariin Chitosan Glutaraldehyde 50% of icariin was released after 8 h. By the 24 h mark, 73% of loaded icariin was released, after
which the rate of release became slow and sustained84
Ascorbic acid Chitosan Guanosine A burst release was observed in the first 5 h, in which 50% of ascorbic acid was released. Afterward,
diphosphate the release became more gradual up to 6 days88
Gentamicin Chitosan Β-Glycerophosphate 100% of the drug was released in 4 h154
Levofloxacin Chitosan/biphasic Genipin Initial fast release that begins to plateau after 10 min. This burst release is more pronounced with a
calcium higher Ca/P ratio. More than half the drug is released after 30 min, regardless of Ca/P ratio68
phosphates
Figure 12. Cumulative release profiles of different encapsulants from scaffolds. (A) Release percentage of pentoxifylline at the 6 h time point for
chitosan scaffolds cross-linked with genipin, pectin, or freeze-dried. Reproduced and adapted with permission from ref 74. Copyright 2015 IOP
Publishing. (B) Cumulative release of rhBMP2 from PLGA microspheres, which will be placed in composite chitosan scaffolds with mineralized
collagen I. Reproduced and adapted with permission from ref 136. Copyright 2017 Wiley Periodicals, Inc. (C) Release patterns of rhBMP-2 and
vancomycin (VAN) from methacrylated chitosan photo-cross-linked scaffolds with or without PLGA microspheres. Reproduced and adapted with
permission from ref 170. Copyright 2021 Elsevier. (D) Cumulative release percentage of tetracycline hydrochloride from carboxymethyl chitosan/
oxidized gellan gum gels with hydroxyapatite (HAp/gel) and gels with magnetic hydroxyapatite/gelatin microspheres (MHGMs). Reproduced and
adapted with permission from ref 104. Copyright 2022 Elsevier.
listed above have also been tested within these scaffolds, that the incorporation of copper nanoparticles in a
however to a much lesser extent. Copper is one of these less carboxymethyl chitosan and alginate scaffold resulted in
explored elements but has been shown to promote bone increased ALP activity at day 7, and increased COL-1 and
formation in the literature. A study by Lu et al. demonstrated osteocalcin mRNA expression at day 14.140 Interestingly, the
copper nanoparticles were used in place of Cu2+ ions in this has been demonstrated in other studies in the litera-
study to slow down the cross-linking process of the ture.78,84,88,94,106,115,122,130,156,168 However, it would be inter-
carboxymethyl chitosan and alginate scaffold. It has been esting in this scenario to determine whether the kinetics
shown that the Cu2+ ion alone can serve as a cross-linker, thus becomes more gradual at later points. A study by Ji et al. has
allowing the scaffold to cross-link slowly as ions are released. indicated that while this burst trend is often observed, it is
5.4. Investigation of Release Kinetics of Encapsulated usually accompanied by two additional stages: a line and slow
Compounds. Since one of the main challenges in the design stage. This was evident in the results of their study, where the
of biological substitutes with mediators, growth factors, or release of BMP2 from composite chitosan scaffolds with
drugs is the achievement of a sustained release profile, kinetic mineralized COL-I and poly lactic-co-glycolic acid (plga)
release studies are often conducted in vitro as part of scaffold microspheres was high at day 3 (162.8 pg rhbmp2 per mg) but
characterization.22,206−208 To perform these tests, scaffolds are gradually reached 361.06 pg at the 28th day of culture (Figure
loaded with a model component and its concentration in the 12B).136 This notion of stages for release profiles was also
supernatant is measured over a certain amount of time (Figure found in other studies where an initial burst release occurred.88
11, Table 5). The most common method to assess the release In other instances, authors have focused their drug delivery
kinetics includes UV−vis spectrophotometry and absorbance studies to the encapsulation of antibacterial or anti-
readings, with half of the relevant articles having reported on inflammatory agents rather than those to promote bone
this methodology. Other techniques like enzyme linked healing.65,68,104,170 Incorporation of these drugs, while not
immunosorbent assays, HPLC, or protein assays can also be directly influencing osteogenic differentiation and mineraliza-
used. Ideally, the release of growth factors and mediators for tion, still can have a beneficial effect in these applications since
osteogenic promotion needs to be sustained throughout the inflammation can hinder successful bone regeneration. Since
healing process. Thus, research incorporating compounds such fracture healing starts with a preliminary inflammatory phase, it
as BMPs, bisphosphonates, or inorganic materials in chitosan could potentially be more useful to deliver these drugs faster
scaffolds often tests this kinetics throughout a designated such as in the Pentoxifylline study mentioned above. Song et
period. Some studies have examined a short duration of time al. also showed early burst release patterns of an antibacterial
up to 2 weeks,64,92,136,152,156 while others have examined agent, Vancomycin, with more than 80% released in the first 2
release past 4 or 5 weeks 4,97,131 enabling a more days in a chitosan hydrogel with a coencapsulation of PLGA
comprehensive understanding of the mediator’s possible effect microparticles loaded with rhBMP2 (Figure 12C).170 While
over time. this quick release was expected based on other studies, authors
Osteogenic mediators and factors can be added to chitosan suggested that this rate needs to be further tuned to match that
scaffolds mainly through covalent or noncovalent bind- of the antibacterial stage.
ing.209,210 The latter is more prominent in bone-oriented Loading of drugs within nano or micro spheres or particles
applications and involves the adsorption of the factors onto the prior to encapsulation into a chitosan scaffold could serve to
scaffolds or their entrapment within the scaffold mesh.22,211 delay the release of drugs or compounds and encourage a more
Electrostatic interactions, hydrophobic interactions, hydrogen gradual release. The control of release for antibacterial agents,
bonding, or van der Waals forces play a major role in silver sulfadiazine and tetracycline hydrochloride, has recently
immobilizing factors noncovalently onto the scaffolds, and been explored using magnetic hydroxyapatite and gelatin
disruption of these interactions can influence the release microspheres cross-linked with glutaraldehyde.104 Chen et al.
kinetics.209,210 Generally, different mechanisms mediate the demonstrated that due to the solubility of tetracycline
release of factors from scaffolds including desorption and hydrochloride in water, the release rate tends to be accelerated
diffusion of factors outside of the scaffold, swelling of the by diffusion in the aqueous environment (Figure 12D). While
scaffold, or degradation of the scaffold’s structure, and they release kinetics from the microspheres and carboxymethyl
have been previously reviewed in a publication by Herdiana et chitosan and oxidized gellan gum gel alone resulted in a burst
al.212 In addition to the type of factor added, the release release, the incorporation of the nanoparticles within the gel
mechanism also depends on how the factor interacts with and was shown to provide a more sustained release rate. At the 21-
is bound to the scaffold as well as the properties of the scaffold day time point, cumulative release was 16.1%, which was
itself. Thus, changes in the composition of the scaffold can decreased compared to the gel alone. Similarly, Dorati et al.
allow for the rate of release to be tuned and controlled more observed complete release of gentamicin from a β-glycer-
precisely.211 ophosphate cross-linked chitosan scaffold within only 4 h,
As discussed in the previous sections, cross-linking of which extended to 80 days following the loading of the drugs
chitosan greatly influences the material properties of the within PLGA−PEG microparticles.154
formed scaffold, which in turn can affect the release of factors
depending on the cross-linker used and its concentration 6. VASCULARIZATION IN BONE TISSUE
(Table 6). Therefore, it is necessary to take that into ENGINEERING
consideration when developing cross-linked chitosan scaffolds The fifth element of the diamond concept for proper bone
in order to yield optimal kinetics. As an example, the addition healing is the development of an adequate vascular network
of a model drug, Pentoxifylline, was examined in multiple throughout.42 Osteogenesis and angiogenesis are closely
chitosan scaffold formulations with either genipin (CG) or associated processes, sharing some essential mediators. Skeletal
pectin cross-linkers (CP).74 Results indicated a significant vasculature plays a significant role in the process of bone
difference in release kinetics among all tested groups with the development (endochondral and intramembranous ossifica-
genipin cross-linker showing the most promise with a lower tion), regeneration, and remodeling.213,214 As known, bone is
cumulative release percentage after 6 h (Figure 12A). highly vascular tissue and the presence of a vascular network
However, the high percentage values at 6 h (around 80% in aids in providing oxygen and nutrients to cells, calcium and
CG scaffolds) seems to indicate an initial burst release, which phosphate for mineralization as well as removing waste
Figure 13. Effect of sphingosine 1-phosphate (S1P) loaded silica nanoparticles (MSNs) addition to N,O-carboxymethyl chitosan (NOCC) and
aldehyde hyaluronic acid (aHa) nanocomposite hydrogels on capillary network formation in vitro and in vivo. (A, B) Results of CAM assay for
examination of angiogenesis and quantification of vascular area. (C, D) HE staining images at the 2-week time point after implantation into a
mouse. Arrows present the blood vessels. D presents the counted number of vessels shown. Reproduced and adapted with permission from ref 106.
Copyright 2022 Royal Society of Chemistry.
products from the site.213,215−217 Formation of these vascular acid (aHa) nanocomposite hydrogels resulted in the formation
networks within biomaterial substitutes has been one of the of a dense capillary network compared to control scaffolds
main challenges in the bone tissue engineering domain and is (Figure 13A,B).106 The difference between these groups was
often overlooked in literature studies. Lack of sufficient shown to be significant with respect to the area of vessels
vascularization in tested scaffolds has been shown to result in counted from 30.03% in S1P loaded hydrogels to 16.26% in
cell hypoxia and death since nutrients and oxygen can only control. This significant effect of S1P addition was also
travel around 150 μm from surrounding networks.216,218−220 confirmed in vivo subcutaneously in mice (Figure 13C,D).
Vasculogenesis and angiogenesis are thought to be the main Another chitosan-based composite scaffold was assessed for
mechanisms for vascular network formation in bone. While the vascularization potential in vivo as well, but this time using a
vasculogenesis mechanism relies on progenitor cells forming critical size defect model in the femoral head of a rabbit.134
the vasculature without an existing network, angiogenesis
This study only examined one configuration of their scaffold
builds off pre-existing networks surrounding the defect
(2:1:3 mass ratio of chitosan, gelatin, and biphasic calcium
site.213,221 Assessment of vascular formation within the scaffold
phosphate nanoparticles), but hematoxylin and eosin (HE)
in vitro and in vivo relies on measurement of vascular
markers.222 These can include vascular endothelial growth staining results demonstrated improved vascularity over the
factor, von Willebrand factor, vascular endothelial cadherin, course of time from one to three months. No clear pattern
and the platelet endothelial cell adhesion molecule among emerges from the literature regarding which encapsulants
others.222 significantly improve vascularization of the scaffold, but some
Recent studies of chitosan-based scaffolds have incorporated publications have proposed the incorporation of a coculture
a chicken chorioallantois membrane (CAM) assay to examine system to overcome this aspect.88,216,221,223 This involves the
the induction of angiogenesis. Through this method, Zhang et coencapsulation of an osteoblast precursor in direct or indirect
al. were able to demonstrate that the addition of sphingosine 1- contact with endothelial cells, allowing for the regeneration of
phosphate (S1P) loaded silica nanoparticles (MSNs) to N,O- a tissue more similar to the native environment.224 However,
carboxymethyl chitosan (NOCC) and aldehyde hyaluronic the specifics of a coculture platform for healing, while
Table 7. Cross-linked Chitosan-Based Scaffold Formulations That Demonstrated Beneficial Bone Regeneration In Vivo
Species Defect Model Scaffold Additions Tested
Chitosan81,100 Carbon nanotubes and BMP-2,64, Alendronate,120 Graphene oxide and titanium oxide,81
Carboxymethyl chitosan64,65 Bisphosphonates and BMP-2,131 Magnesium ions and polyhedral oligomeric silsesquiox-
Cranial/mandi- ane (POSS) nanoparticles,152 Graphene oxide and nanohydroxyapatite,65 Copper100
Chitosan/polycaprolactone120
ble defect
model Methacrylic modified chitosan131
Rats Chitosan/collagen73
Thiolated chitosan/gelatin152
Subcutaneous Chitosan97 Silicon-substituted calcium phosphate, rhBMP-2 and heparin97

insertion
Cranial/mandi- Chitosan77 Lysozyme,141 Hydroxyapatite77

ble defect Methacrylated glycol chitosan141
model Phenol conjugated glycol168
Tibial model Chitosan89 Hydroxyapatite and pyrophosphatase89

Mice
Chitosan80,121 Graphene oxide,121,125 Bioglass nanoparticles and BMP-2,80 Hydroxyapatite -decorated silica
Chitosan/collagen/hyaluronic acid122 beads with alendronate,122 BMP-2162
Subcutaneous
insertion Chitosan/gelatin125
Chitosan/alginate162
Cranial/mandi- Chitosan/collagen112 Hydroxyapatite112

ble defect
model
Rabbits Femoral model Chitosan/gelatin134 Biphasic calcium phosphate nanoparticles134
Chitosan86 Citric acid86
Tibial model
Oleyol chitosan/decellularized bone matrix124
important, are considered to be outside of the scope of this provided a thorough understanding of cross-linking’s role in
review. bone formation. By comparing two types of chitosan-based
fibers treated with either a citric acid bath for cross-linking or a
7. PRE-CLINICAL IMPLEMENTATION OF DIAMOND dual cross-linking using a citric acid bath and NHS/EDC, the
CONCEPT USING CHITOSAN-BASED authors were able to demonstrate that the dual cross-linked
CROSS-LINKED SCAFFOLDS chitosan fibers showed more collagen and mineral deposi-
While in vitro experiments are a necessary step in the tion.86 This finding indicated better osteogenic activity and
assessment of osteogenic differentiation and regeneration bone regeneration with higher amounts of cross-linking. In
potential for designed substitutes, the clinical translation of addition to tibial defects, other studies in rabbits with chitosan
in vitro findings require preclinical in vivo validations. Often, scaffolds have used mandible defects112 and femoral head134
the in vitro models are not convoluted enough to accurately defects models, but these models tend to be less common in
represent the complexity of the in vivo healing environment.225 both large and small animals.
As such, incorporating animal studies is an essential part of Use of small animal models such as rat and mouse, is
biological substitute development and evaluation in the significantly more common than large animal studies since
framework of the Diamond Concept.226 Within the literature, these animals are low cost and can be kept in large quantities,
this has included studies of fracture healing models on many allowing for more groups to be tested and more statistically
mammals, both large and small, with implantation or injection relevant information to be obtained on differences.226,227 In
of chitosan-based cross-linked scaffolds (Table 7).226,227 addition, many of the commercially available monoclonal
As large animal models, rabbits are used in multiple studies antibodies used to conduct a more comprehensive evaluation
for chitosan-based cross-linked scaffolds’ evaluation in of healing are targeted for rat and mouse.227 Within these rat
vivo.86,112,124,134 These studies have included examinations of and mouse models, fracture healing is completed within 4 to 6
both the cross-linking mechanisms and the necessity for cell weeks, which is faster than the timeline for healing in large
encapsulation for in vivo studies. Datta et al. examined the animal models.226,227 Assessment of healing in these models is
influence of human amnion derived MSC encapsulation using conducted at an early phase (1−2 weeks) and a late phase (3−
a combinatorial genipin cross-linked fatty acid modified 5 weeks in mice, 4−6 weeks in rats).227
chitosan and decellularized bone ECM hydrogel in a rabbit Among these murine models, researchers tend to examine
tibial defect model.124 Results demonstrated the positive effect their material’s effect on osteogenic differentiation and bone
of cellular addition, as cell encapsulated scaffolds produced healing through usage of cranial de-
highly mineralized tissue densities compared to cell free fects64,77,81,83,100,120,131,136,141,152,168 or subcutaneous incorpo-
groups. In addition, staining showed that this incorporation of ration.65,80,97,121,122,140,162 However, a few studies have instead
cells resulted in similar morphological features to that of the chosen to use tibial89 or femoral defect113 models to better
native bone tissue. This reinforces the notion of osteogenic understand the fate of their scaffold for a load bearing bone
cells as a crucial subcategory of the Diamond Concept. application. Often, incorporation of chitosan-based scaffolds in
Another study using a similar rabbit tibial defect model these cases is done in combination with a fixator or mechanical
Figure 14. In vivo studies using (A−C) MicroCT and (D) HE staining. (A) Images of mouse tibial fractures treated with nothing (SHAM) or
chitosan scaffolds with hydroxyapatite (HA) and pyrophosphatase (P). Reproduced and adapted under open access conditions (Creative
Commons Attribution License) from ref 89. Copyright 2020 Kaushar Jahan et al. and Springer Nature. (B, C) Imaging and quantitative analysis of a
rat cranial defect model at 12 weeks postsurgery treated with control, chitosan and PCL composite scaffolds, and composite scaffolds with
Alendronate. (C) Quantification of the mature bone area, bone volume/tissue volume, and bone mineral density for these samples. Both B and C
were reproduced and adapted with permission from ref 120. Copyright 2022 Elsevier. (D) Staining for new bone formation in defects treated with
nothing (top), chitosan scaffold (middle), or a chitosan scaffold loaded with copper (bottom). Within this figure, the new bone (NB) and old bone
(OB) are distinguished. Reproduced and adapted with permission from ref 100. Copyright 2014 Wiley Periodicals, Inc.
stabilizer to bear the load while the bone is healing. Jahan et al. available imaging techniques since it allows for the
demonstrated the potential of a chitosan-based GDP cross- quantification different parameter values including bone
linked scaffold with hydroxyapatite and pyrophosphatase volume to tissue volume or trabecular spacing, among others.
incorporation in vivo using a rod fixated tibia fracture model These scans are typically performed ex vivo to encourage the
of mice (Figure 14A).89 However, although tibial fracture examination of newly formed tissue in a higher resolution but
models are well-established in murine models, often, femur the technique could also be adapted for monitoring in vivo
fracture models are more beneficial since they provide more throughout the healing process.226,228 Other scanning
consistent results with respect to biomechanical evaluation.226 techniques have also been found in recent literature including
7.1. Methodology of In Vivo Bone Healing Assess- X-ray imaging (10.5%) or SEM (5.3%); however, these are
ment in Chitosan-Based Scaffolds. Assessment of the significantly less common compared to MicroCT imaging
scaffold’s capability for healing in vivo commonly involves three (78.9%).
different components: bone imaging, histological staining and Histomorphometry of samples by different staining methods
quantitative assays226,227 (Figure 15). Table 8 serves to is another technique that can be applied to acquire specific
demonstrate the available techniques for each of these desired information on the role of the scaffold on the bone
components, as well as the percentage of use within recent healing process.226 For example, Georgopoulou et al. used HE
work on chitosan-based scaffolds for bone. and Masson Trichrome stainings to assess the ability of a
Scanning techniques are implemented around 57.6% of the glutaraldehyde cross-linked chitosan and gelatin scaffold to
time in the literature and are known to aid researchers in fully support bone regeneration and formation of the extracellular
assessing newly formed bone tissue prior to sectioning. Micro- matrix.229 HE staining is one of the primary methods for
CT imaging has been shown to be the most popular of the assessing bone healing in vivo, with a reported usage of 86.2%
Figure 15. Common methodologies for in vivo studies using animal models in the literature. These are divided into 3 components: imaging studies,
histological staining, and quantitative assays.
Table 8. Common Methodologies for In Vivo Testing in Chitosan Cross-linked Scaffolds for Bone (N = 33)
Incorporation in In Vivo Usage in Specific In Vivo
In Vivo Experiments Studies (%) Methodology Testing (%)
Imaging techniques 57.6% MicroCT and CT imaging64,65,77,80,89,100,120,124,131,136,140,141,162,164,185 78.9%
X-ray imaging112,168 10.5%
Radiography83,136 10.5%
Scanning electron microscopy81 5.3%
Stereoscopic imaging81 5.3%
Staining techniques 87.9% HE 86.2%
staining77,80,81,83,86,97,100,106,112,113,120−122,124,131,134,136,140,141,152,159,164,169,171,185
Masson’s trichrome staining77,81,86,113,120,122,124,131,134,140,141,152,164 44.8%
IHC/IF osteocalcin (OCN)120,131,152,164 13.8%
Alizarin Red staining64,97,121,122 13.8%
Von Kossa staining65,86,89 10.4%
CD31 Staining106,124,152 10.4%
Toluidine Blue/Alcian Blue staining89,124 10.4%
Gomori’s Trichrome staining121,125 6.9%
IHC/IF RUNX2121,152 6.9%
IHC/IF Collagen 1152,164 6.9%
IHC/IF osteopontin (OPN)65,121 6.9%
ALP staining164,168 6.9%
Giemsa staining140 3.4%
Von Willebrand factor staining106 3.4%
Alpha smooth muscle actin staining152 3.4%
Calcein staining64 3.4%
TRAP staining168 3.4%
Tetracycline staining64 3.4%
IHC/IF bone sialo protein (BSP)65 3.4%
Assays and quantitative 9.1% Quantification of inflammatory cytokines122,169 66.7%
assessments ALP quantification in serum121 33.3%
qPCR for gene expression121 33.3%
in publications with some sort of staining technique. While literature recently since they allow for a more in-depth
used for histology of many different tissues, the bone structure observation of the callus tissue composition with only one
in HE-stained images tends to have a dark pink color, whereas
required staining.230,231 Other less common staining options
connective tissue will be a noticeably lighter shade of pink. Use
of other stains such as Masson Trichrome and Movat include Von Kossa/Van Gieson which aims to look at
Pentachrome have become a more set standard in the mineralization and Alcian Blue to examine cartilage.
Quantification of different parameters using assays does not the material with the cells.22,54 Both chitosan parameters have
appear to be an increasingly common trend with respect to in shown an inverse correlation with degradation thus necessitat-
vivo studies as it is with in vitro studies (9.1%). A few studies ing precise control of these to tune the rate of scaffold
have used ELISA or kits to measure the production of degradation as new bone formation occurs.22,232 With respect
inflammatory cytokines, which could in future be an important to the use of chemically modified chitosan derivatives for bone
test to further understanding the full biocompatibility of the specific applications, no clear trend has emerged thus far and
designed material. further analysis on this should be conducted.
7.2. Influence of Chitosan Cross-linking on In Vivo Studies have adequately demonstrated the importance of
Studies. The combination of these techniques provides a cross-linking, with many publications using genipin or
more accurate representation of the full healing process. glutaraldehyde, yet no clear cross-linking agent has emerged
Within the literature in the past ten years, very few studies have as optimal with respect to material properties. Future studies
examined the effect of scaffold formulation, such as polymer should therefore focus on eliciting a clear trend between
addition or cross-linker concentration geared toward improv- different cross-linkers. Concerns within the literature have
ing the material properties, in the in vivo osteogenic potential been raised regarding the usage of synthetic chemical cross-
experiments. Often, the number of tested groups are linkers such as glutaraldehyde with respect to cytotoxicity.
minimized based on prior investigation of material properties Thus, a balance should be struck between benefits to material
and in vitro cell experiments to reduce the cost for animal properties and effects on cellular functionality. Cross-linking
studies. This means that only the optimal groups, per se, are alone however has not been shown to be sufficient for bone
considered for in vivo experiments, to determine what healing and the encapsulation of bioceramics such as
encapsulants, or factors play a promotional osteogenic role. hydroxyapatite can be argued as an integral component to
Incorporation of the bisphosphonate, Alendronate, into a meet necessary mechanical and osteogenic properties for bone
Chitosan/polylactic acid scaffold was demonstrated to support engineering applications.
bony bridging within the cranial defect model compared to the There has been no set standardized methodology regarding
scaffold alone in micro-CT images (Figure 15B,C).120 This the assessment of material properties for chitosan-based cross-
beneficial trend in growth factor addition was also evident with linked materials, with many studies using varying techniques
the incorporation of BMP2 in carboxymethyl chitosan scaffolds that may not necessarily yield the same information. This, in
with carbon nanotubes. While the initial intention of this in turn, makes comparisons between studies significantly more
vivo study was to assess specifically at the carbon nanotube challenging. Future work in this domain should investigate the
effect, the addition of BMP2 resulted in a nearly fully covered material properties of the designed scaffold in the following
defect at week 8, whereas the carbon nanotubes alone in the presented manner. First, mechanical properties should be
scaffold had significantly reduced bone tissue production.64 assessed using compression testing since it resembles the
The influence of inorganic materials as mediators was also experience of the scaffold in the native tissue environment.22
established in a rat critical size defect model using a chitosan This method should be supplemented with rheological testing
scaffold with copper cross-linking.100 Results showed improved as viscoelasticity plays an important role in promoting
mineralized tissue formation in groups with copper compared differentiation of encapsulated cells.24 Second, the structural
to controls and chitosan alone (Figure 15D). This supportive architecture examination should be completed scanning
effect was also confirmed using HE staining. Generally, results electron microscopy for surface morphology and comple-
presented here illustrate the necessity of osteogenic com- mented with micro-CT for internal structure parameters.
pounds in the designed scaffold and encourage the develop- While liquid displacement is more popular for porosity
ment of additional and alternative growth factors for these assessment, it can be argued that, if not inhibited by cost
applications. and availability, micro-CT provides a more comprehensive
evaluation of this internal architecture. Finally, biodegradation
8. FUTURE PERSPECTIVES AND CONCLUSION rates of the chitosan-based scaffolds should be assessed
This review serves to provide a thorough comparison of through incubations in solutions of PBS or water with
modifications made to cross-linked chitosan-based scaffolds enzymes. This method more accurately serves to simulate
with respect to the Diamond Concept including changes of the natural body conditions experienced by implanted
cross-linkers, cell types and growth factors. Based on the scaffolds. Although these degradation patterns have been
research results in the development of these scaffolds within extensively characterized within the literature for various
the last ten years, it is evident that the synergistic combination scaffold fabrications in different buffers such as PBS, water,
of all aspects of the Diamond Concept are necessary to simulated body fluid,76,96,117,134,146 or culture media71,151 no
achieving successful bone healing. However, consideration for clear standard has been established with regard to the
all these aspects in literature studies of cross-linked chitosan appropriate degradation timeline needed for striking a balance
materials remains lacking hence why no clinically relevant with the rate of new bone tissue formation. A more precise
chitosan scaffold-based solution has been achieved. The base timeline for material degradation should be explored further in
polymer chitosan, often found in many different variations future work to allow for the design of optimal chitosan-based
commercially, should be the preliminary consideration in the biomaterials.
design process with emphasis placed on chitosan solutions with With respect to cellular encapsulation, research should focus
high DD and molecular weight (MW). Chitosan has been on using more clinically relevant cell lines such as stem cells.
demonstrated in the literature to have sufficient biocompati- The last 10 years of chitosan-based scaffold studies
bility with cells and presents many beneficial properties with demonstrate good advancement in this regard. Assignment of
regards to osteogenic activities.54 The large DD allows for the a standardized methodology for cellular biocompatibility
prominence of positive charges in the amine groups assessment is challenging since many techniques have been
throughout the structure thus improving the interaction of proven effective for attaining similar information regarding
metabolic activity, cytotoxicity, and adhesion. However, the Authors

addition of a cross-linker to scaffolds makes the necessity for Celine J. Agnes − Department of Biomedical Engineering,
this assessment evident in all future work. Techniques for McGill University, Montreal, Quebec H3A 2B4, Canada;
studying the potential for cellular differentiation and Shriner’s Hospital for Children, Montreal, Quebec H4A 0A9,
mineralization should include an evaluation of both early and Canada; orcid.org/0000-0002-2689-0991
late stage markers and would benefit greatly from gene Antoine Karoichan − Shriner’s Hospital for Children,
expression studies on top of secretion and staining techniques. Montreal, Quebec H4A 0A9, Canada; Faculty of Dental
Few studies of cellular activity in vitro have sought to Medicine and Oral Health Sciences, McGill University,
regenerate the complex aspects of the bone structure such as Montreal, Quebec H3A 1G1, Canada; orcid.org/0000-
vascularization. This aspect is known to be important for 0002-0657-7746
successful bone tissue engineering since it serves to provide Complete contact information is available at:
oxygen and nutrient to cells within the injured sites, but its https://pubs.acs.org/10.1021/acsabm.3c00108
success in vitro and in vivo remains a challenge in terms of
achievement.218,233 The coculture of osteoblastic lineage cells Notes
with endothelial cells is a seemingly promising method for this The authors declare no competing financial interest.
endeavor. Future studies on chitosan-based bone tissue
engineering scaffolds can benefit from a comprehensive
evaluation of all aspects of the Diamond Concept including
vascularization instead of only focusing on osteogenic
■ ACKNOWLEDGMENTS
The authors acknowledge the financial support of CIHR and
differentiation and mineralization. NSERC via the Collaborative Health Research Program,
While research examining the combination of the different Canada Research Chair − Tier 1 in Regenerative Medicine
aspects of the Diamond Concept is prominent in vitro, its and Nanomedicine, as well as FRQ-S funding. All schematic
application has been lacking in vivo. As previously discussed, in figures were created using the Biorender software under paid
vivo studies in the literature incorporate 3 main components subscription, and graphs were created using Graphpad Prism.
including imaging, staining, and quantitative assays. Micro-CT
imaging of the regenerated bone should become the gold
standard for imaging methodologies since it provides an
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The Diamond Concept Enigma: Recent Trends of Its Implementation in Cross-Linked Chitosan-Based Scaffolds For Bone Tissue Engineering

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1. INTRODUCTION In terms of bone regeneration, the scaffold needs to have a

Chitosan Citric acid86

ACS Appl. Bio Mater. 2023, 6, 2515−2545

Hardness testing98 1.4% Hardness of material

In simulated body fluid96,117,134,146 7.4% Degradation weight

ACS Appl. Bio Mater. 2023, 6, 2515−2545

Resazurin Assay68,103 2.2%

OPN,65,78,82,102,120,121,124,130,131 BMPs,82,152 ON,70 OSX65

ACS Appl. Bio Mater. 2023, 6, 2515−2545

Subcutaneous Chitosan97 Silicon-substituted calcium phosphate, rhBMP-2 and heparin97

Cranial/mandi- Chitosan77 Lysozyme,141 Hydroxyapatite77

Tibial model Chitosan89 Hydroxyapatite and pyrophosphatase89

Cranial/mandi- Chitosan/collagen112 Hydroxyapatite112

metabolic activity, cytotoxicity, and adhesion. However, the Authors

(222) Lafage-Proust, M.-H.; Roche, B.; Langer, M.; Cleret, D.;

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