Recent advances in composite materials for biomedical purposes

Introduction
The word ""composite"" refers to the combination, on a macroscopic scale, of two or more
materials, different for composition, morphology and general physical properties. In many
cases, and depending on the constituent properties, composites can be designed with a view
to produce materials with properties tailored to fulfill specific chemical, physical or
mechanical requirements. Today composite materials have many different applications such
as aeronautic, automotive, naval, and so on. Consequently, many composite biomaterials
have recently been studied and tested for medical application. Some of them are currently
commercialized for their advantages over traditional materials. Most human tissues such as
bones, tendons, skin, ligaments, teeth, etc., are composites, made up of single constituents
whose amount, distribution, morphology and properties determine the final behavior of the
resulting tissue or organ. Fabricated composites can to some extent, be used to make
prostheses able to mimic these biological tissues, to match their mechanical behavior and to
restore the mechanical functions of the damaged tissue. Different types of composites that are
already in use or are being investigated for various biomedical applications are presented.
In the light of ever-present need for modernization and improvement of biomedical
devices used in treatment of severe wounds and tissue damage, more and more research
around the world is dedicated to investigation of novel composites and nanocomposites for
medical applications, such as wound dressings and tissue implants. Polymers, synthetic or
natural, are prevalent foundation of such materials, because of their similarities to real tissue,
capability to take on many shapes and forms, and almost immeasurable possibilities to tailor
their properties to the unique requirements of target use. Bio composite are widely used
because of their biocompatibility, hydrophilicity and malleability, which allow relatively easy
processing and wide range of applications. Composites comprise of two or more materials at
different composition and produce tailored physical and mechanical properties. In biomedical
applications, implants materials are usually made of metals, which yield optimal interaction
with the hosting tissue. However, metallic implants applied in human body were known to
cause problems like allergies, strain mismatch, and susceptibility to corrosion.
Thus, composites have been introduced for its structure biocompatibility to the human
tissue. Researches focus on developing load-bearing implant and devices from fiber-
reinforced polymer like PEEK-carbon fiber composite and dentistry restoration materials. In

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order to manufacture composite implants at high volume, squeeze forming and injection
molding are considered cost-effective processing.

Fig 1: Levels of hierarchy in bone

Reference: Lakes, R. S. Materials with structural hierarchy, Nature (2014), 361, 511–515

Composite materials are used for various biomedical application such as:
Skins
Tissue Engineering
Scaffolds
Bone regeneration
Cartilages
Heart valves
Dental applications
Membranes
Wound healing
Other such
Composites are of great significance in tissue engineering because they provide a favorable
environment for the growth and differentiation of cells. They play a pivotal role in tissue
engineering through cell seeding and proliferation and formation of new tissue in three-
dimensions, thus showing great promise in tissue engineering research. One of the major

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advantages of natural polymers is their low immunogenic potential and high abundance. This
section deals with some natural polymer composites and their medical applications.

Composite for Dental applications

Reference: J. Sabbagh R.J. McConnell M. Clancy McConnell, Posterior composites: update
on cavities and filling techniques, Journal of Dentistry, published on (2016)

Filling material requirements in all material development, the basic requirements of a dental
filling material should not be forgotten: Every filling material should have good optical
characteristics, and the physical properties should correspond with those of dental hard tissue.
Wear resistance and the effect on the antagonist should be similar to the properties of enamel.
It is also important that the material is easily distinguishable from dental tissue on x-ray. The
material should be easy to handle and easy to polish. Likewise, the material should form a
sufficient bond with dental tissue or at least with a dental adhesive. The material should, of
course, be tasteless and biocompatible. Most of these requirements are recorded in the ISO
standards. However, the range allowed for dental products is very wide and this may lead to
the approval of products, which only narrowly meet the requirements.

Fig 2: Initial situation: Loss of tooth substance due to wear and obvious staining. The patient
was not satisfied with the aesthetics.
Reference: Wilson NH, Lynch CD. The teaching of posterior resin composites: Planning
based on 25 years of research. Journal of Dentistry 2014: 42: page no. 503-516

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Fig 3: Aesthetic improvement after home bleaching and direct composite veneers. The crown
of tooth 43 was left and the discoloured tooth margin was masked using an opaque coloured
hybrid composite.
Reference: Wilson NH, Lynch CD. The teaching of posterior resin composites: Planning
based on 25 years of research. Journal of Dentistry 2014: 42: page no. 503-516

Silorane The name of this material class refers to its chemical composition from Siloxanes
and Oxirans. This product class aims to have lower shrinkage, longer resistance to fading and
less marginal discolouration. The silorane monomer ring differs obviously from the chain-
monomers of hybrid composites. The hydrophobic properties of the material are caused by
siloxanes. Exogenous discolouration and water absorption are reduced. The oxirane rings are
responsible for the physical properties and the low shrinkage. Siloranes are polymerized by a
cationic reaction in contrast to methaycrylates, which crosslink via radicals. The
photoinitiator system is based on three components: light absorbing camphor chinon, an
electron donor (eg amine) and an idonium salt. The camphor chinon is excited and reacts with
the electron donor, which reduces the iodonium salt to an acidic cation in the process. This
starts the opening process of the oxirane ring. The opening of the oxirane rings during the
polymerisation process compensates to some degree for the polymerisation shrinkage. The
fillers in Filtek Silorane®, the only silorane material on the market at the moment, consist of
0.1–2.0 μm quartz particles and radiopaque yttrium fluoride.
A comprehensive study of Filtek Silorane® was carried out by Weinmann et al. It
confirms the low shrinkage (< 1%) and found that the light stability of the silorane was seven
times longer than for methacrylates. The siloranes low shrinkage leads to a lower contraction
stress. The silorane-based filling material was shown to have both low water absorption and

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water solubility. The adhesion of streptococci observed on the surface of silorane restorations
was low, maybe because of its hydrophobic properties.

Fig 4: Tooth-/restoration fracture in tooth 46, after excavation the high degree of tooth
destruction is evident. The available financing is limited, direct adhesive composite build-up.
Reference: Ibarra ET, Lien W, Casey J, Dixon SA, Vandewalle KS. , Physical properties of a
new sonically placed composite resin restorative material. General Dentistry (2015) May-Jun;
63(3): page no. 51-65

Ormocers Ormocers, a word originally derived from organically modified ceramic, were
originally developed for science and technology (e. g. for special surfaces like protective
coatings, non-stick surfaces, anti-static coatings and non-reflective coatings). In contrast to
conventional composites, the ormocer matrix is not only organic but also inorganic.
Therefore monomers are better embedded in the matrix what reduces the release of
monomers. Ormocers basically consist of three components – organic and inorganic portions
and the polysiloxanes. The proportions of those components can effect the mechanical,
thermal and optical qualities of the material: 1. The organic polymers influence the polarity,
the ability to cross link, hardness and optical behaviour. 2. The glass and ceramic components
(inorganic constituents) are responsible for thermal expansion and chemical stability. 3. The
polysiloxanes influence the elasticity, interface properties and processing. The inorganic
components are bound to the organic polymers by multifunctional silane molecules. After
polymerisation the organic portion of the methacrylate groups form a three-dimensional
network. In spite of all efforts to create a better restorative material using ormocers, their

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performance (cervical and occlusal marginal adaptation) was significantly worse when
compared to today’s hybrid composites, after cyclical loading in a laboratory test.
However, no significant differences were found in a five-year clinical comparison. At
the same filler content, ormocers have a reduced polymerisation shrinkage compared to
hybrid composites or at a lower filler content of the ormocer the polymerisation shrinkage is
equal to that of a conventional composite.

Composite for scaffold regeneration

Reference: Gareth Turnbull, Jon Clarke, Fred Picard, Philip Riches, Luanluan Jia Fengxuan
Han, Bin Li, Wenmiao Shu, 3D bioactive composite scaffolds for bone tissue engineering,
Bioactive Materials (2017) page 1 – 37

In general, the ideal 3D scaffold is composed of a biocompatible, biodegradable material with

similar mechanical properties to the tissue which it is to be implanted in. Scaffolds by design
are not intended to be permanent implants and will ideally facilitate host cells to deposit
extracellular matrix (ECM) and replace the scaffold structure over time. The 3D architecture
of the scaffold should be highly porous with an interconnected structure to allow cell and
nutrient migration. The scaffold surface should also be optimized to facilitate cell attachment,
proliferation and differentiation. Form a surgical point of view; it is also desirable for the
scaffold material to be easily manipulated into different shapes and sizes to allow in-situ
treatment of individual patient bone defects.
Scaffolds and their breakdown products must above all be biocompatible. This
requires scaffold materials to be nontoxic to cells, easily eliminated from the body and to
elicit negligible immune response through their presence .Controlled biodegradability is also
an essential characteristic for a scaffold to achieve; if a scaffold degrades too quickly,
mechanical failure could occur. This is particularly relevant in BTE, as an implanted scaffold
is likely to undergo load bearing and could fracture if unable to provide mechanical support
whilst new bone is forming. Similarly,if a scaffold does not degrade sufficiently quickly an
inflammatory response could be triggered towards the foreign material of the scaffold,
impairing tissue regeneration. Growth factors also have a significant role to play in successful
bone tissue engineering scaffolds. The processes of new bone formation and extracellular
matrix deposition are regulated by a range of growth factors and biomolecules. Bone
morphogenic proteins (BMP) play a critical role in bone and cartilage development, and have
the ability to trigger proliferation and differentiation of osteoprogenitor cells.

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 There are several examples of scaffolds that have successfully incorporated BMPs
resulting in improved bone formation. However, risk of bone formation within soft tissues, or
heterotopic ossification, is also associated with use of BMPs. Vascular endothelial growth
factor (VEGF) has also frequently been included in scaffolds, with desirable properties
including the ability to enhance blood vessel formation and bone formation in vivo.
Transforming growth factor b (TGF b), platelet-derived growth factor (PDGF), insulin-like
growth-factor 1 (IGF-1), and fibroblast growth factors (FGFs) provide further examples of
growth factors that have been utilised in bone and cartilage tissue engineering . The
microarchitecture of scaffolds is also centrally important in encouraging cell viability and
fostering tissue ingrowth.

Fig 5: (A) SEM image showing interconnected porous structure of human trabecular bone (B)
Pores and interconnecting pores demonstrated in hydroxyapatite scaffold. Pores are
circled; arrows indicate interconnecting pores, which allow communication between pores.
Reference: K. Doi, et al., Development of implant/interconnected porous hydroxyapatite
complex as new concept graft material, PLoS One 7 (11) (2015)

An interconnected pore structure, in the absence of an engineered blood

supply, allows inwards diffusion of oxygen and nutrients and outwards diffusion of waste
products from the scaffold. Porosity also supports cell migration into the scaffold and
improves available surface area for cell-scaffold binding and interaction with surrounding
tissues .Individual pore size within the scaffold is an important consideration. It has
previously been shown that scaffold pore density and size significantly impact upon cellular
growth and attachment .As pore size decreases, the surface area of the scaffold increases.
This increases the availability of scaffold ligands for cells to bind to and interact. However, if
pore sizes become too small, cells may struggle to migrate into the scaffold structure.

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Scaffolds must therefore be precisely engineered with parameters favorable to the cells and
tissue that they will be exposed to.

Fig 6: Summary of bioprinting process

Reference: K. Doi, et al., Development of implant/interconnected porous hydroxyapatite
complex as new concept graft material, PLoS One 7 (11) (2015)

Bioceramics, including ceramic composites, amorphous glasses and crystalline ceramics,

show great promise within BTE as mechanically strong materials, with favourable bioactivity
Further material properties can include corrosion resistance, resistance to compression, and a
weakness to shearing and tensile forces, resulting in brittleness Perhaps the most frequently
utilised crystalline bioceramics in BTE are calcium phosphates (CaPs), partly due to their
prevalence in native bone tissue. Hydroxyapatite (HA), tricalcium phosphate (TCP) and a
composite of both substances known as biphasic calcium phosphate (BCP) have all been
adapted in BTE scaffolds.Cell mediated degradation of these ceramics in vivo produces
calcium and phosphate ions, which promote new bone formation through osteoinduction.

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CaPs also share a large degree of similarity in structure and chemical composition to the
mineral content of native bone.
This allows CaP constructs to provide a biocompatible, osteoconductive interface
capable of facilitating integration with host tissue without formation of scar tissue. HA has
excellent properties for BTE, including biocompatibility,controlled degradation and lack of
cytotoxicity. HA can also stimulate endogenous expression of osteogenic growth factors such
as bone morphogenetic protein (BMP) and enhance alkaline phosphatase (ALP) activity in
mesenchymal stem cells (MSCs). This is particularly important as ALP activity is pivotal in
the early mineralization process associated with bone formation.

Fig 7: Micro-CT and histomorphic analysis showing new bone formation in polymer-coated
BG scaffolds implanted in mice for 8 weeks
Reference: F. Westhauser, et al., Three-dimensional polymer coated 45S5-type bioactive
glass scaffolds seeded with human mesenchymal stem cells show bone formation in vivo, J.
Mater. Sci. Mater. Med. 27 (7) (2016) 119.

Hyaluronic acid (HLA) is a natural glycosaminoglycan found widely throughout connective,

epithelial and neural tissues. As one of the chief components of the extracellular matrix, HLA
contributes significantly to cell proliferation and migration. It typically has a very large
molecular weight and has been adapted into attempts at both hard and soft tissue engineering,
particularly as a hydrogel (as discussed in the later hydrogel section).The mechanical
properties of HA can be readily improved through processes such as crosslinking , whilst it is
naturally viscoelastic, biodegradable and biocompatible, making it an ideal material for BTE.
HLA has been implemented in composite scaffolds by several groups.

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Fig 8: Photograph of injectable 3D-formed composite of b-TCP beads and alginate capable of
triggering MSC osteogenic differentiation in vivo. (A) and light microscope photograph
of the composite (B). SEM photographs of the composite (C) and surface of the composite
(D). The composite was composed of b-TCP beads
Reference: Matsuno. T. Matsuno, et al., Preparation of injectable 3D-formed beta-tricalcium
phosphate bead/alginate composite for bone tissue engineering, Dent. Mater. J. 27 (6) (2017)
page no. 827-834

Electron microscopy found the scaffolds to have an interconnected porous morphology. The
addition of GO also resulted in less swelling and ultimately contributed to enhanced
structural integrity of the scaffold. However, introduction of GO also caused a 35% reduction
in porosity of the GOeCSeHLA scaffold. MC3T3 (Osteoblasts) cells
were seen to adhere and proliferate better on SV loaded GOeCSeHLA scaffolds throughout
48 h of in vitro analysis. This was reflected by significantly higher scaffold mineralization
being found in SV loaded scaffolds after 14 days of analysis. Overall, the SV loaded
GOeCSeHLA scaffold appeared to offer a successful option for BTE, with the addition of SV
significantly accelerating bioactivity and osteogenesis.Jing et al. also combined HLA and CS
in a porous scaffold for bone tissue engineering. Mixing CS and HLA together as liquids,
they used a freeze-drying approach to form porous, 3D scaffolds.

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 Stem cell colonisation and proliferation within the scaffold was demonstrated by
DNA assays and confocal imaging. Correia et al.also used a freeze-drying processed to
prepare composite scaffolds of chitosan and HLA, though directed their scaffolds at cartilage
tissue engineering. Within this study incorporation of HLA enhanced cartilage ECM
production, chondrocyte proliferation and cell adhesion to scaffold surfaces. Kim et al.
combined HLA and collagen in a scaffold directed at regenerating cartilage. The hybrid
scaffolds were prepared by adding 0.1, 0.3 or 0.5 wt% collagen to HLA. The HLA was then
crosslinked with ethylene glycol diglycidyl ether, followed by a freeze-drying process.
The degradation time of the hybrid scaffolds in vitro increased with increasing
collagen concentration. In vitro chondrocyte growth on the scaffolds was also improved by
increasing collagen concentration over 2 weeks in culture. Furthermore, glycosaminoglycan
(GAGA) concentration in the hybrid scaffolds was higher than in pure HLA scaffolds. These
composite scaffolds would therefore seem to have potential for in vivo cartilage regeneration.

Composite for heart valve

Hastings, R. N., & Jenson, M. L. (2018). U.S. Patent No. 9,883,941. Washington, DC: U.S.
Patent and Trademark Office
Background. Decellularized extracellular matrix has been suggested as a scaffold for heart
valve tissue engineering or direct implantation. However, cell removal impairs the physical
properties of the valve structure and exposes bare collagen fibers that are highly
thrombogenic. Matrix/polymer hybrid valves with improved biological and mechanical
characteristics may be advantageous. Methods. Porcine aortic valves were decellularized
enzymatically and impregnated with biodegradable poly (hydroxybutyrate) by a stepwise
solvent exchange process. Biocompatibility was tested in vitro using cell proliferation and
coagulation assays. Proinflammatory activity was assessed in vivo by implantation of matrix/
polymer patches in the rabbit aorta. Biomechanic valve properties and fluid dynamics were
tested in a pressure/ flow-controlled pulse duplicating system.

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Fig 9: Patients who underwent aortic valve-sparing procedures experienced fewer major
adverse valve-related events than patients who underwent composite valve graft procedures
with either a bioprosthesis or a mechanical prosthesis.
Reference: Maral Ouzounian, Cedric Manlhiot, Nachum Abraham, MSC, Carolyn David,
RN, Christopher M. Feindel, Valve sparing root replacement compared with composite valve
graft procedures in patients with aortic root dilation, Journal of the American college of
cardiology (2016), vol. 68, page no. 78 – 85

Matrix/ polymer hybrid valves were implanted in pulmonary and aortic position in sheep.
Results. Biocompatibility assays indicated that human blood vessel cells survive and
proliferate on matrix/ polymer hybrid tissue. In vitro activation of cellular and plasmatic
coagulation cascades was lower than with uncoated control tissue. After implantation in the
rabbit aorta, matrix/polymer hybrid patches healed well, with complete endothelialization,
mild leukocyte infiltration, and less calcification than control tissue. Matrix/polymer hybrid
tissue had superior tensile strength and suture retention strength, and hybrid valves showed
good fluid dynamic performance. The two valves in aortic position performed well, with
complete endothelialization and limited inflammatory cell invasion after 12 weeks. Of the
two valves in pulmonary position, one failed. Conclusions. Matrix/polymer hybrid tissue

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valves have good biological and biomechanic characteristics and may provide superior
replacement valves

Fig 10: Gross morphology of the two porcine biomatrix/polymer hybrid valves 12 weeks
after implantation in pulmonary position in sheep. (A) The first valve was completely
degenerated probably due to bacterial endocarditis. (B) The second valve was in excellent
condition, with near-normal gross morphology
Reference: Christof Stamm,Amir Khosravi, Niels Grabow,Kathleen Schmohl, Nadine
Treckmann, Anne Drechsel , Biomatrix/Polymer Composite Material for Heart Valve Tissue
Engineering, Ann Thorac Surg 78 (2015), page no. 2084–93

Autograft and allograft are considered ultimate for bone grafting procedure providing
osteoconductive and osteoinductive growth factors. However, limitations in donor site,
additional surgery, disease transmission and expenditure poses a need to develop alternatives
to autograft and allograft. The repair and replacement of injured or defect bone is a critical
problem in orthopedic treatment throughout worldwide. In recent years, significant
development has been made in organ replacement, surgical reconstruction and the use of
artificial prostheses to treat the loss or failure of an organ or tissue. Due to limited supply of
natural bone for grafting, the need for synthetic bone substitutes which possess same
physiochemical and biological properties as natural bone is ever increasing.
These limitations and concerns created substantial interest in the development of
artificial materials as bone graft substitutes or exenterate. At present days, cell and scaffold
based tissue engineering treatment are being explored for a better treatment of bone related
ailments. Several techniques and materials have been employed for the orthopedic treatment
for the past several decades. Bone is mainly composed of organic and inorganic portion such

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as collagen and nano hydroxyapatite. The prepared composite materials should mimic the
natural function of bone when it is replaced. Moreover, it should replace the function of
extracellular matrix and should not be toxic to surrounding tissue environment, allowing cells
to proliferate and differentiate in a normal pace. In bone tissue engineering, biodegradable
and biocompatible materials introduced at the defective region should mimic all the natural
functions of the normal bone such as porosity, cell proliferation, etc.

Fig.11. Tendon is a complex physiological system. Tendon fascicles represent the basic unit
comprising the ‘‘intrinsic compartment” (tendon cells and a multiscale arrangement of
collagen assemblies). The ‘‘extrinsic tendon compartment” represents synovium-like tissues
that connect to the immune, vascular, and nervous systems. The possible synergism between
the intrinsic and extrinsic compartment, and the role that individual compartments play in the
maintenance of healthy tissue versus the initiation, progression and healing of tendinopathy.
Reference- Snedeker, Jess G., and Jasper Foolen. "Tendon injury and repair–A perspective
on the basic mechanisms of tendon disease and future clinical therapy." Acta biomaterial,
Published- 1 September 2017

Over the last four decades, there is a growing interest in the field of artificial organ material
preparation, transplantation, surgical reconstruction and the use of artificial prostheses to treat
the loss or failure of an organ or tissue. In the recent years, natural polymers are being

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considered by researchers for tissue engineering due to their biocompatibility and
biodegradability nature. The well-known limitations associated with clinical use of autografts
and allografts continue to drive efforts to develop bone graft substitutes using the principles
of biomaterials and tissue engineering. Artificial bone substitutes usually include metals,
ceramics (e.g., hydroxyapatite (HAp) and/ or MWCNT, Chitosan etc.). Artificial bone
substitutes are effective in bone defect repair; however, they are not ideal. Metallic implants
may cause atrophy of surrounding tissue through stress shielding, requiring corrective
procedures. Ceramics can aid in the healing of the bone defect by acting, preferably
temporarily, as a scaffold for bone in-growth. Almost all of the materials have some
drawbacks and their selection usually requires some degree of compromise. Thus, the search
for novel biomaterials as alternatives for pure biomaterials remains an important topic in
medical research. Because of the possibility of combining the advantages of different pure
materials, composite biomaterials are more attractive than pure biomaterials.
Recently, many composite materials have been developed for biomedical applications
and they include ceramics/ceramics composites (for example, HA/plaster of Paris, calcium
phosphate/calcium carbonate), polymer/polymer composites (PLA/collagen), ceramic/metal
composite (HA-coated Ti), and ceramics/polymer composites (HA/CNT, HA/collagen and
HA/chitin). Kim et al developed a biodegradable composite scaffold using chitosan grafted
with functionalized multiwalled carbon nanotube in addition to HA (f-MWCNT-
gchitosan/HAp) scaffolds were prepared for the first time via freezedrying method and
physiochemically characterized as bone graft substitutes. The major objective of this research
work is to develop indigenous technology for substituting the traditionally used metal bone
fracture fixation devices and to develop biodegradable, biocompatible scaffolds for bone
tissue engineering using gelatin, chitosan, hydroxyapatite, MWCNT. These composite
scaffolds from biopolymer and bioactive ceramic materials could provide an optimum
environment for cell growth, and then organized into a real bone.

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Fig 12: Cumulative incidence rate of cardiac related mortality
Reference: Tanjina I, Khandaker S S, Shanta B, Papia H, Sunzida H R, Preparation of Carbon
Nanotube Reinforced Gelatin-ChitosanHydroxyapatite, Biocomposite for Bone Tissue
Engineering (2018), vol 3, page no. 13-18

Cytotoxicity analysis of composites The results of cytotoxicity analysis are shown and the
data are presented. It was observed that with increase of content of MWCNT, cytotoxicity is
increased. Survival of cells in the composite samples were >90 to 80% in the sample A and
sample B.Conclusion To prepare a bionanocomposite for bone tissue engineering as an
artificial bone scaffold, we have successfully incorporated MWCNTs in gelatin-chitosan-
hydroxyapatite composite and found that the mechanical and thermal performances of
MWCNTs- gelatin-chitosan-hydroxyapatite nano-composites were satisfactory.
Addition of functionalized CNTs helps boosting the conducting efficiency nano-
composites. The experimental results of this research were confirmed through different
analytical tests. Therefore, total elemental modeling will be part of the future work. Op Acc J
Bio Eng & App Copyrights@ Tanjina I, et al. 7/7 Citation: Tanjina I, Khandaker S S, Shanta

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B, Papia H, Sunzida H R, et al. Preparation of Carbon Nanotube Reinforced Gelatin-Chitosan
Hydroxyapatite Biocomposite for Bone Tissue Engineering. Op Acc J Bio Eng & App 1(3)-
2018. OAJBEA.MS.ID.000112. The study paves the way for the development of a nano-
composite for biomedical application especially in case of bone replacement aspects, since
MWCNT is non-toxic within certain range of content in the composite. Increasing the loading
of MWCNTs to gelatin-chitosan-hydroxyapatite nano-composites increases the
morphological, physico-mechanical, thermal properties of the nano-composites. Compressive
strength of the samples of having 0.1%, 0.15%, 0.25% MWCNT are 0.311, 0.554, 0.609 MPa
respectively, which shows a considerable increase in mechanical property.

Fig 13: FT-IR spectra of composite samples ((a) sample A, (b) sample B, (c) sample C, (d)
Gelatin-Chitosan (97.5:2.5), (e)GelatinChitosan-Hydroxyapatite
Reference: Tanjina I, Khandaker S S, Shanta B, Papia H, Sunzida H R, Preparation of Carbon
Nanotube Reinforced Gelatin-ChitosanHydroxyapatite, Biocomposite for Bone Tissue
Engineering (2018), vol 3, page no. 13-18

Analyzing the same samples with thermogravimetric test, again it showed that the composite
having highest amount CNT (0.25%), was most thermally stable, as highest amount residue (
27.28%) obtained after the decomposition temperature. The morphological studies were
carried out by SEM. The prepared scaffolds have interconnected porous microstructures,
which is convenient for bone growth in the scaffold. It was also observed that 0.1%
MWCNT-composite showed least cytotoxicity and that of 0.25% CNT showed highest toxic
effects on cells. It is recommended that from the above results, (0.1%-0.15%) MWCNT-
gelatinchitosan-hydroxyapatite composite to use as artificial bone scaffold for enhanced
physic-mechanical properties.

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Fig 14: SEM images of a) gelatin-chitosan composite, b) gelatin-chitosan-hydroxyapatite, c)
sample A and d) sample C
Reference: Tanjina I, Khandaker S S, Shanta B, Papia H, Sunzida H R, Preparation of Carbon
Nanotube Reinforced Gelatin-ChitosanHydroxyapatite, Biocomposite for Bone Tissue
Engineering (2018), vol 3, page no. 13-18

Characterization of composites FT-IR analysis The dried sample was embedded in KBR
pellets analyzed by an ATR-FTIR (Attenuated Total Reflectance/ Fourier Transforms
Infrared) spectrophotometer (Model-01831, SHIMADZU Corp, Japan) .The composites were
analyzed by ATR-FTIR. The spectra were recorded in both cases in the absorption band
mode in the range of 4000-400 cm−1. Surface morphology A Scanning Electron Microscopy
(SEM) was used to determine surface morphology of the composites.
SEM analysis has been carried out using a JEOL JSM-6490LA machine. The JSM-
6490LA is a high performance scanning electron microscope with an embedded energy
dispersive X-ray analyser (EDS) which allows for seamless observation. As the surface
morphology of the samples were needed to be analyzed, the non-conducting samples were set
on a carbon tape, made conducting by platinum coating and then placed for analysis at 20kV.
Thermogravimetric analysis Thermogravimetric analysis of the composite samples was done
using TG/DTA EXTAR 6000 STATION, Seiko Instrument Inc. Japan. The TG/DTA module
uses a horizontal system balance mechanism. TGA measuring range: ±200mg (0.2µg), DTA
measuring range: ±1000µV (0.06µV), at ≤ 1000m/min. Thermo gravimetric analysis (TGA)
were performed of each sample using a Perkin-Elmer SetUp (TAQ-500) and a heating rate of
200C/min under a nitrogen atmosphere. Differential scanning calorimetry (DSC) analysis
DSC tests were carried out. The sealed Aluminium pan was put on the calorimeter along with

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an empty sealed pan. Change of heat per gram of sample was recorded at a constant
temperature for 60minutes with a computerized system in dry nitrogen environment.
Compressive strength analysis Compressive strength is the strength of materials, the capacity
of a material or structure to withstand loads tending reduce size.

Fig 15: Cytotoxicity analysis of composite A, B and C

Reference: Tanjina I, Khandaker S S, Shanta B, Papia H, Sunzida H R, Preparation of Carbon
Nanotube Reinforced Gelatin-ChitosanHydroxyapatite, Biocomposite for Bone Tissue
Engineering (2018), vol 3, page no. 13-18

It can be measured by plotting applied force against deformantion in a testing machine. some
materials fracture at their compressive strength limit, others deform irreversibly, so a given
amount deformation may be considered as limit for compressive load. Compressive strength
was measured with Universal Testing Machine (Hounsfield, Model H50 Ks 0404, and UK).
Cytotoxicity analysis Cytotoxic analysis was examined in Centre of Advanced Research in
Sciences. In brief, HeLa, a human cervical carcinoma cell line was maintained in DMEM
(Dulbecco’s Modified Eagle’s medium) containing 1% penicillin-streptomycin (1:1) and
0.2% gentamycin and 10% fetal bovine serum (FBS).
Cells (4×10^4 /400µl) were seeded onto 24-well plates and incubated at 37⁰C+5%
CO2. Next day 100µl of sample (autoclaved previously) was added at each well. Cytotoxicity
was examined under an inverted light microscope after 24h of incubation. Duplication wells
were used for each sample. The measurement was carried out in autoclave (DAC-45, Human
lab, Korea), Biological Bio safety Cabinet (model: NU-400E, Nuaire, USA), CO2 Incubator
(Nuaire, USA), Trinocular microscope with camera (Olympus, Japan). Results and

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Discussion Cytotoxic analysis of MWCNT dispersion .The physically functionalized
MWCNT dispersion (1%) was prepared by 40h sonication with surfactant Sodium Dodecyl.

Composite for collagen

Reference: T. Arahira, M. Todo, Effects of proliferation and differentiation of mesenchymal
stem cells on compressive mechanical behavior of collagen/ b-TCP composite scaffold, J.
Mechanical behavior Biomedical Material Vol (3) (2014) page no. 218 - 222
With calcium phosphate (CaP) based bioceramic components Hydroxyapatite. HA is the most
commonly used calcium phosphate with the molar ratio of Ca/P ¼ 1.67. Moreover, the
biology bone is mainly constructed by the inorganic HA and organic collage components.
In addition, these two components possess great biocompatibility, osteoconductivity
and bone-bonding ability. Therefore, the collagen/HA. Composite biomaterial scaffolds have
been extensively investigated and used for bone tissue engineering scaffolds. The mechanical
strength of pure collagen scaffolds is extraordinarily low, which immensely limits their wider
applications in tissue regeneration. The compressive modulus of collagen scaffold can be
apparently improved by incorporating HA, and the degree of increase is largely related to the
concentration of collagen, the amount of HA, the composite methods and the crosslinking
methods.

Fig 16: X-ray pictures of the bone healing process of rabbit distal femoral condyles after
surgery. (a) Treated with Cerasorb Ortho Foam. (b) The control group with an empty defect.
The red circle indicates drilled holes. Scale bars equal to 1 mm.

20
Reference: T. Arahira, M. Todo, Effects of proliferation and differentiation of mesenchymal
stem cells on compressive mechanical behavior of collagen/ b-TCP composite scaffold, J.
Mechanical behavior Biomedical Material Vol (3) (2014) page 218.

As b-tricalcium phosphate [b-Ca3(PO4)2, b-TCP]. As another representative calcium

phosphate bioceramics, b- TCP has been widely used as bone substitutes for many years due
to its good osteoconductivity, biocompatibility and biodegradability both in vitro and in vivo.
The Ca/P ratio of b-TCP is 1.5. It can not only improve cellular adhesion but also accelerate
their differentiation and proliferation ability. Some scholars have prepared b-TCP based
scaffolds for bone regeneration. Compared with HA, b-TCP has a faster degradation rate and
can be completely replaced by newly formed bone tissues.

Fig 17: Fluorescence microscopy images of the bone defect of SD rats after implantation. (a,
b): 4 weeks, (c, d): 8 weeks. At week 4, newly formed woven bone had been found beside
CM scaffold while no newly formed bone was found in COL scaffold. Larger amount of
newly formed bone can be observed in CM scaffold than that in COL scaffold. TC:
tetracycline, XO: xylenol orange. WB: woven bone. BT: bone tissue, FT: fibrous tissue

21
Reference: S.K. Misra, D. Mohn, T.J. Brunner, W.J. Stark, S.E. Philip, I. Roy, V. Salih, J.C.
Knowles, A.R. Boccaccini, Comparison of nanoscale and microscale bioactive glass on the
properties of P(3HB)/Bioglass composites, Biomaterials 29 (12) (2017) page 1750- 1761

Skin:
Hayashida, K., Saijo, H., & Fujioka, M., Peroneal perforator‐based peroneus longus tendon
and sural neurofasciocutaneous composite flap transfer for a large soft‐tissue defect of the
forearm: A case report. Microsurgery, (2018), 38(1), 85-88

Wound dressing involves the restoration of the continuity of a living tissue and an integrated
response of several cells to injury to promote cellular colonization and remodeling of the
matrix. Therefore, inflammation and neovascularization are required for healing. Material for
wound dressing usually contains an inner gel or scaffold layer that is in direct contact with
the wound site and an outer elastic covering layer that fixes the device to the tissue. Thus far,
scaffolds consist of HA, chitosan, and collagen alone or in combination have been found to
be promising for application in wound healing. A collagen/chitosan sponge on which human
fibroblasts cultured for 1month produced differentiated connective tissue. An in vivo study
showed that a tissue engineering construct of collagen/chitosan, human skin fibroblasts, and
keratinocytes transplanted in the back of nude mice enhanced nerve regeneration, suggesting
that it is a suitable construct for skin substitution. Studies have shown that BC-based
coverings reduce wound pain, accelerate re-epithelization, and decrease wound infection rates
and scarring. Some BC-based wound dressings. Legeza et al. produced a BC-based wound
dressing containing superoxide dismutase (an antioxidant) or poviargol (an antibiotic) for
treating third-degree burns. Metal nanoparticles in biopolymer-based matrix play an
important role in the repair and regeneration of skin wounds. Generally, silver nanoparticles
(AgNPs) prevent or control excessive proliferation of bacteria, which decreases
inflammation. In addition, zinc is essential in protein synthesis and T-cell development and
acts as an antioxidant and calcium is vital for the normal hemostasis of mammalian skin and
modulates keratinocyte proliferation and differentiation.
Based on the characteristics of the metal particles, Kumar et al. synthesized a ˇ-
chitin/AgNPs composite scaffold with a better blood-clotting ability for application in wound
dressing. In addition, alginate-based sponges containing AgNPs have antimicrobial and anti-
inflammatory properties. In addition, chitosan/zinc complex has improved antibacterial and
wound-healing properties. Ninan et al. studied the role of pectin/carboxymethyl

22
cellulose/microfibrillated cellulose scaffolds in the proliferation of NIH3T3 (mouse
embryonic fibroblast cell line) and showed that calcium concentration in the medium greatly
affected cell viability . Although metals are essential for wound healing, the toxicity of metal-
incorporated scaffolds should be controlled.

Vascular graft
Liu, R. H., Ong, C. S., Fukunishi, T., Ong, K., & Hibino, N. (2018). Review of Vascular
Graft Studies in Large Animal Models. Tissue Engineering Part B: Reviews, 24(2), 133-143

New blood vessel formation is a critical requirement in many vascular diseases and in tissue
engineering. Recent efforts involving biopolymers have led to the successful delivery of
growth factors and small molecules in vivo. BC tubes (diameter, 1 mm; length, 5 mm; and
wall thickness, 0.7 mm) prepared by Klemm group were used to replace a part of the carotid
artery in a rat model. After 4 weeks, the BC/carotid artery complex was covered with
connective tissue, indicating that BC can be used as a replacement blood vessel.
Brown et al. developed small tubes made of BC/fibrin composites treated with
glutaraldehyde to crosslink the polymers and to better match the mechanical properties with
those of native small-diameter blood vessels. BC-based blood vessels were found to be stable
vascular conduits and were biocompatible. Morphological analysis showed that structural
modifications occurred in the contact region between BC-based blood vessels and
surrounding media. Presence of fibroblasts in this region suggested the occurrence of
integration without degradation (vitalization). Andrade et al. coated BC with tripeptide
RGD to induce endothelialization and to improve the hemocompatibility of BC. They
cultured human microvascular endothelial cells on RGD-coated BC and observed that the
endothelial cells formed a confluent layer, which inhibited the adhesion of platelets.
Based on the data validation in vivo test of alginate, 3D alginatebased cardiac patch has been
developed. However, a preformed 3D alginate-based cardiac patch has not yet been tested in
clinical practice despite the availability of promising in vitro results.

23
Fig 18: (A) Illustration of the biopolymer composites for wound dressing, (B) chitosan/PGA
polyemetrolyte complex, (C) chitin and nanosilver complex, (D) RGDg-PLLA scaffold, (E)
EGF/gelatin microshere
Reference: Sung-Bin Park, Eugene Lih, Kwang-Sook Park, Yoon Ki Joung, Dong Keun Han,
Biopolymer-based functional composites for medical applications, Progress in Polymer
Science 68 (2017) page no. 77–105

In this study, the cellular morphogenesis of human hepatocarcinoma cells is simply

modulating through pore architecture change by gelation temperature (-20 and -80 ◦C).
Synteyer et al. investigated the macroporous alginate scaffolds without any implanted cells as
biocompatible matrices to support the liver regeneration. Alginate scaffolds fully support the

24
survival of the remaining liver cells by providing a replacement environment, thus reducing
cell necrosis, improving hepatic synthetic functions, and improving regeneration.
Recently, Lee et al. described that the encapsulated human hepatocellular carcinoma
(HepG2) cells in the chitosan-alginate fibers were more viable than cells encapsulated in pure
alginate fibers, suggesting that incorporated chitosan provides a better environment for
HepG2 cells than alginate alone. In addition, the adhesion of HepG2 cells on the chitosan-
alginate fiber is much better than that on the alginate fiber. The chitosan-alginate fibers
showed a cell viability that was five times larger than that of the alginate fibers after 7 days.
Furthermore, HepG2 cell proliferation was observed in the chitosan-alginate fibers over the
course of 7 days. These results demonstrate that the chitosan in the fiber plays a key role in
providing a proper environment for encapsulated HepG2 cells, and the chitosan-alginate may
be used as a good scaffold material for liver regeneration. Hepatocyte pre-culturing may offer
opportunities for cell analysis, therapeutic gene manipulation, or simply to bridge the
availability of highly differentiated hepatocytes from a donor liver if the recipient is
temporary not available. Bierwolf et al. demonstrated successful liver repopulation with pre-
cultured human hepatocytes in alginate scaffold. The human hepatocyte spheroids obtained
after 7 days pre-culture and in vitro lentiviral gene transfer are able to repopulate a recipient
liver. Results show that hepatocyte pre-culture system may offer new opportunities to bridge
the availability of highly differentiated hepatocytes from a donor liver. In the future, there is a
possibility that the pre-cultured hepatocytes obtained from a patient can directly transplant.
Pancreatic islet transplantation has potential as a treatment for patients with insulin-
dependent diabetes, but unfortunately, currently available procedures suffer from both low
efficacy and limited reproducibility.
Mechanical and structural properties of fibrin matrix were similar to normal pancreas.
In addition, fibrin matrix shows no cytotoxicity and can enhance differentiation of human
endometrial stem cell to beta cells. Shalaly et al. demonstrated functionalized 3D silk
matrices for generation of insulin-secreting islet-like clusters from mouse and human primary
cells. Significantly, more clusters were formed on silk foam with the cell-binding motif RGD
compared to wild type silk. The obtained clusters formed on silk matrix maintain functional
insulin release upon glucose stimulation in vitro. Moreover, in vivo imaging of transplanted
clusters showed engraftment with increasing vessel formation during time. The size of the
clusters increased over time without cell toxicity. Acute kidney injury may lead to chronic
kidney disease, which can progress toward end-stage renal disease with a mortality rate.
Current treatment options are limited to dialysis and kidney transplantation. However, there

25
are some limitations such as donor organ short age, graft failure and numerous complications
Accordingly, many researchers develop alternative therapies for kidney diseases through
tissue engineering with scaffolds and cells. The kidney has complex 3D tubular structure with
many types of cell populations. This complex structure is difficult to reproduce by traditional
scaffold based tissue engineering techniques.
Therefore, decellularization/recellularization and 3D bioprinting approaches were
attracted to prepare kidney scaffold similar to natural organ. Recently, Lih et al. developed
the kidney-derived ECM incorporated PLGA scaffolds as a cell supporting material by an ice
particle leaching method for kidney tissue regeneration. ECM is composed of natural
polymers such as collagen, laminin, and fibrinogen. The functional ECM proteins
incorporated PLGA scaffolds had a beneficial effect on kidney tissue regeneration.
Interestingly, after grafting the scaffold into a partially nephrectomized mouse kidney,
tubular and glomerular structures were observed within the regenerated tissue and hostgraft
interfaces, and especially neovascularity was found in the kidney ECM incorporated PLGA
scaffold. In organ regeneration research, researchers should found out how to successfully
reproduce the complex structure and tissues of organs. Biopolymer composites in medical
implants. Stent Coronary artery disease is a cardiovascular illness in which an artery is
clogged or narrowed because of hardening due to the deposition of cholesterol, fats, and other
components in the blood. Stents are implantable medical devices that are placed in the
coronary artery by using an inflatable balloon on a catheter to recover the shape of narrowed
or diseased arteries. The 2 most important functions of a stent are to restore normal blood
flow and to prevent restenosis . Biodegradable polymeric stents are less obtrusive than
metallic stents, which can cause arterial vessel rupture. Presence of anti-restenosis drugs
within a stent is another desirable feature. A biodegradable polymeric stent loaded with anti-
restenosis drugs minimizes systemic toxicity, facilitates the healing of vessels injured due to
stent implantation, and prevents restenosis .
The first biodegradable polymeric stent was developed by Stack et al. by using poly-l-
lactic acid. Degradation period of this stent was 9 months. After stent implantation into
animals, minimal thrombosis, moderate neointimal growth, and very limited inflammatory
response were observed. However, further clinical tests on this device are limited. Kum et al.
recently developed oligolactide-grafted magnesium hydroxide/PLA composites that induced
a lower degree of inflammation after mechanical reinforcement. PHB-based biodegradable
stents were developed and implanted into the iliac arteries of New Zealand white rabbits for
30 weeks. Upon implantation, PHB induced inflammatory reactions, stent thrombosis, and

26
lumen narrowing. Another everolimus-eluting bioabsorbable stent composed of PLA
(ABSORB II, Abbott Vascular) is commercilizedmore than 60 countries around the world
including Korea, United States, Japan, and China, and data collected thus far are promising.
The ABSORB II stent showed similar 1-year composite secondary clinical outcomes to the
everolimus-eluting metallic stent (Xience, Abbott Vascular). The ABSORB II stent showed
similar postprocedure area stenosis, minimum lumen area, and eccentricity index to second-
generation drug-eluting stent. In addition, exercise performance and angina status by Seattle
Angina Questionnaires were similar.

Barrier membrane
Reference: Sung-Bin Park, Eugene Lih, Kwang-Sook Park, Yoon Ki Joung, Dong Keun Han,
Biopolymer-based functional composites for medical applications, Progress in Polymer
Science 68 (2017) page no. 77–105

Bioresorbable barrier membranes have been developed to prevent the second surgical
removal. Many types of barrier membranes have been used for guided bone regeneration
(GBR) and for preventing postoperative abdominal adhesion. GBR membranes
prevent the in-growth of soft tissue into the bone defect and maintain the defect space during
bone regeneration. Moreover, growth factors incorporated in these membranes provide a
more suitable environment for bone regeneration. Clinically, collagen and expanded
polytetrafluoroethylene (ePTFE) membranes have been widely used for GBR. Types I and III
collagens derived from porcine, bovine, and human have been mainly used for producing
GBR membranes. PLA is combined with poly-glycolic acid, a copolymer, to appropriately
degrade GBR membranes. Silk membranes are still not used commercially in GBR.
However, several recent studies have reported the potential application of silk membranes in
GBR. Kim et al. reported that silk fibroin membranes successfully enhanced new bone
generation in a rat calvarial defect model without any adverse inflammatory reactions.
However, in clinical use, the abovementioned biopolymer GBR membranes generally
indicated less stiffness and space-maintaining ability.
However, many surgeons have reported that SeprafilmTM is breakable and sticky. In
vivo animal studies have shown that N,O-carboxymethyl chitosan is safe and efficacious as
an antiadhesion barrier. A modified chitosan–dextran membrane was used to prevent
peritoneal adhesions in a rat model and was found to significantly reduce abdominal

27
adhesions without adversely affecting wound healing Chang et al. also developed an
antiadhesion membrane made of chitosan and alginate by performing electrospinning.

Fig 19: (A) Illustration of the biopolymer composites for bone and cartilage regeneration: (B)
MC3T3-E1 preosteoblast cells seeded on porous BC scaffolds; Cells nuclei are stained with
DAPI (blue) and actin cytoskeleton is stained with rhodamine-phalloidin (red) (C)
PHB/Bioglass composites (D) PGA/Hap composites by biomineralization (E) BC/HAp
composite
Reference: Sung-Bin Park, Eugene Lih, Kwang-Sook Park, Yoon Ki Joung, Dong Keun Han,
Biopolymer-based functional composites for medical applications, Progress in Polymer
Science 68 (2017) page no. 77–105

It use of biopolymers as drug delivery carrier has been interested due to their bioactivity,
biocompatibility, and biodegradability. In particular, the functional groups on biopolymer
backbone help to design a positively or negatively charged carrier, to induce biological

28
interaction with target organs and cells, and to characterize size, shape, hydrophobicity,
degradation rate, and stimuli-responsibility through chemical modification. Chitosan,
which is originally positive-charged, has been utilized over a wide range of therapeutic
approaches via oral, nasal, intravenous, and ocular routes because of its high affinity to cell
membrane . Khangtragool et al. studied the efficiency of chitosan as an ocular drug delivery
vehicle for topically applied vancomycin in rabbit eyes, and Mahmoud et al. similarly
reported the potential of chitosan nanoparticles contained econazole nitrate as a hydrophobic
model drug which is used for the treatment of many infections. The various researches for
chitosan derivatives were also tried to control degradation rate and drug release profile. For
examples, the release rate of paclitaxel and rutin from chitosan-PCL-PEG micelles was not
only controlled with different crosslinking density but also the degradation rate of high-
crosslinked micelles was prolonged. Moreover, the chemically modified chitosan was
investigated for tumortherapy to require effectively loading poorly water-soluble anticancer
drugs and delivery of a high local drug concentration to targeted tumor. Li et al. suggested
chitosan and inorganic phosphate nanocolloids as an anticancer drug carrier to overcome the
problem of low water solubility and severe toxicity of camtothecin. HA-conjugated chitosan
nanoparticles were developed to deliver oxaliplatin to colorectal tumors and showed targeting
potential to colonic tumors with low systemic toxicity.

Composite for bone regeneration

Reference: Dawei Zhang, Xiaowei Wu, Jingdi Chen, Kaili Lin, The development of collagen
based composite scaffolds for bone regeneration, Bioactive Materials Vol 3 (2018) 129 – 138

Bone is a composite material with an organic phase and an inorganic mineral phase. Major
concerns associated with polymeric scaffolds for bone tissue engineering are low mechanical
strength and shape retention failure. For these reasons, polymer/bioactive ceramic composite
scaffolds have been developed for applications in bone tissue engineering. These scaffolds
exhibit good bioactivity and flexibility; in addition, their microstructure can be controlled to
fit bone defects. Further, these scaffolds have excellent mechanical properties and
osteoconductivity .The inorganic phase can be added to different polymer matrices in the
form of micron-sized or nanoscale particles or fibers.Recently, Misra et al. successfully
prepared PHB and nanoparticulate bioactive glass composites by using solvent casting.
Addition of nanoparticles exerted a significant stiffening effect on the composite
modulus.Young’s modulus increased 1.5 times after theaddition of nanoparticles. In addition,

29
a preliminary cell proliferation study of nanoparticle-containing composites by using
osteoblastlike cells showed good cytocompatibility. Furthermore, an in vitro
degradation study involving simulated body fluids showed high level of bioactivity. Stoica-
Guzun et al. used calcium chloride (CaCl2) and sodium carbonate (Na2CO3) as starting
reactants to promote the deposition of calcium carbonate on BC membranes.
BC nanofibers mimic collagen nanofibers to induce the deposition of Ca-P minerals through
biomineralization.

Fig 21: Swelling behavior of different scaffolds cross-linked by tannic acid based on
chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition after
immersion in PBS for 2, 24, 48, and 72 h
Reference: Beata Kaczmarek, Alina Sionkowskaa, Anna Maria Osyczka, Scaffolds based on
chitosan and collagen with glycosaminoglycans crosslinked by tannic acid,Polymer Testing
65 (2018), page no.163-168

Zhang et al. performed phosphorylation to introduce phosphate groups into the hydroxyl
groups of BC and to promote the growth of calcium phosphate. Wan et al. demonstrated that
phosphorylation effectively triggered hydroxyapatite (HAp) formation on BC. Scaffolds
cultured with cells displayed the formation of bone-like tissue, which was determined in vitro
by using microcomputed tomography and in vivo by using a cranial defect model. Alternative
strategies for improving the compressive strength of silk scaffolds involve addition of HAp or
bioglass particles or in situ crystallization of calcium phosphate on the scaffold surface.
Alginate-based materials and composites have been studied in orthopedic research for

30
promoting osteogenesis, improving osteogenic differentiation, and delivering cells and
growth factors to bone defect sites Cartilage. Cartilage is a tissue with remarkable functions
that are partly attributed to the composition and structure of the highly hydrated ECM.
Polymeric scaffolds used for cartilage regeneration are fabricated from flexible biomaterials
that can withstand compression while providing a supporting environment for chondrocytes.
The most popular polymeric scaffolds currently in clinical use for cartilage regeneration are
collagen and HA, which are natural components of a mature hyaline cartilage and are capable
of integrating into endogenous cartilage material. Several studies have also been performed
on the use of alginate as scaffolds. Alginate implants are capable of supporting chondrocyte
viability and producing a cartilage-like ECM as early as 4 weeks after implantation.
However, alginate implants do not have sufficient mechanical properties compared with
native cartilage tissue.

Fig 22:Liquid uptake of PBS solution to the scaffolds cross-linked by tannic acid based on
chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition after
immersion in PBS for 2, 24, 48, and 72 h.
Reference: Beata Kaczmarek, Alina Sionkowskaa, Anna Maria Osyczka, Scaffolds based on
chitosan and collagen with glycosaminoglycans crosslinked by tannic acid,Polymer Testing
65 (2018), page no.163-168

Collagen has been investigated for some time, with early in vivo studies indicating
that direct implantation of passaged chondrocytes seeded into collagen scaffolds improves the

31
healing of chondral defects in a canine model. However, mechanical strength of the
regenerated articular cartilage remains to be investigated. Although collagen scaffolds with
premature degradation do not provide the necessary structural integrity for ECM deposition
during chondrocyte cultivation, atelocollagen scaffolds support the formation of cartilage in
vitro with enough elasticity and stiffness to be used in vivo. Several studies have shown that.
Azuma et al. stated that the mechanical properties of BC/poly (dimethyl acrylamide) double
network gel are similar to those of native cartilage. However, in vivo tests that can confirm
the biocompatibility of BC-based cartilage replacements have not yet been performed.
Bone is consisted of bone matrix, cells and bioactive factors, and bone matrix is the
combination of inorganic minerals and organic polymers. Type I collagen fibril made of five
triple-helical collagen chains is the main organic polymer in bone matrix. It plays an
important role in the bone formation and remodeling process. Moreover, collagen is one of
the most commonly used scaffold materials for bone tissue engineering due to its excellent
biocompatibility and biodegradability. However, the low mechanical strength and
osteoinductivity of collagen limit its wider applications in bone regeneration field.
By incorporating different biomaterials, the properties such as porosity, structural stability,
osteoinductivity, osteogenicity of collagen matrixes can be largely improved. This review
summarizes and categorizes different kinds of biomaterials including bioceramic, carbon and
polymer materials used as components to fabricate collagen based composite scaffolds for
bone regeneration.

Scaffold
Reference: Beata Kaczmarek, Alina Sionkowskaa, Anna Maria Osyczka, Scaffolds based on
chitosan and collagen with glycosaminoglycans crosslinked by tannic acid,Polymer Testing
65 (2018), page no.163-168

Scaffolds based on chitosan, collagen, and glycosaminoglycans-enriched ones, cross-linked

by tannic acid can be obtained with the use of the freeze-drying method. Composites were
characterized by different analyses, SEM images, porosity and density measurements,
swelling, liquid uptake, mechanical tests in wet conditions, and enzymatic degradation by
collagenase and hyaluronidase. In addition, the viability of human osteosarcoma
SaOS-2 cells was examined on the obtained scaffolds. The results showed that the scaffolds
based on chitosan, collagen, and glycosaminoglycans cross-linked by tannic acid are not
cytotoxic. Scaffolds are stable in aqueous environment and show high swelling behavior.

32
Each material porosity is above 90% which is appropriate for the tissue engineering
applications. The mechanical parameters of the scaffolds decrease with increasing immersion
time in PBS. SEM images showed the homogeneous scaffold structure with interconnected
pores. Due to their stability and biocompatibility, the scaffolds presented here may be easily
operated to fit such small bone defects.

Glycosaminoglycans (GAGs) are polyanionic linear polysaccharides with a repeating

disaccharide unit’s structure. GAGs are components of various structural and connective
tissues such as skin, cartilage, and cornea. Each tissue produces GAGs with specific
polymeric chains binded with proteins, enzymes, or ions. The connective tissues of
marine organisms are rich in sulfated glycosaminoglycans. The isolation of GAGs from
marine organisms, e.g. the Aetobatus narinari, Cyclopterus lumpus, Anodonta cygnea, or
Salmo salar has already been reported. Glycosaminoglycans can be identified by the
spectrophotometric assay in the absorption spectrum of 1,9-dimethylmethylene blue (DMB) .
The DMB complex can be stabilized by the use of formate buffer. Another dye type for
GAGs identification is Alcian blue . However, the DMB use is a more sensitive method than
Alcian blue use. The obtainment of scaffolds based on collagen with glycosaminoglycans has
already been reported. Such materials present appropriate biocompatibility and are not
cytotoxic. GAGs are also added to chitosan-based materials. The obtained composites were
characterized as biocompatible and bioresorbable. Moreover, the appropriate biological
properties of chitosan/collagen scaffolds with GAGs were reported previously. However,
materials based on chitosan, or their mixture, exhibit low stability in aqueous environment
and need to be cross-linked to improve their properties. Tannic acid is a glucose and gallic
acid polyphenol. It can be applied to scaffolds as a cross-linker due to the hydrogen and
electrostatic interactions formation.
Tannic acid addition to the polymeric scaffolds results in the obtainment of materials
biocompatible and stable in aqueous conditions. The aim of the study was to obtain scaffolds
based on chitosan and collagen, glycosaminoglycans-enriched ones, cross-linked by tannic
acid. The novel materials were characterized by physicochemical methods as well as an in
vitro experiment. Such scaffolds can find a potential clinical application to fill small bone or
cartilage defects. Chitosan and collagen solutions were mixed in the 50/50 wt ratio. During
stirring, 1 and 5 wt.% of the isolated glycosaminoglycans solution was added. Then, tannic
acid was added as a cross-linker in the 20% weight ratio based on the previous studies. The

33
obtained solutions were frozen and lyophilizated at -55 °C and 5Pa (ALPHA 1- 2LD plus,
CHRIST, Germany) for 24 h. As a result, 3D porous structures (scaffolds) were obtained.

Fig 23: Compressive modulus (Emod) of scaffolds cross-linked by tannic acid based on
chitosan, collagen, and their mixture with 1 and 5% glycosaminoglycans addition,
crosslinked by tannic acid.
Reference: Beata Kaczmarek, Alina Sionkowskaa, Anna Maria Osyczka, Scaffolds based on
chitosan and collagen with glycosaminoglycans crosslinked by tannic acid,Polymer Testing
65 (2018), page no.163-168

Fig 24: Maximum compressive force (Fmax) of scaffolds based on chitosan, collagen, and
their mixture with 1 and 5% glycosaminoglycans addition, cross-linked by tannic acid.

34
Reference: Beata Kaczmarek, Alina Sionkowskaa, Anna Maria Osyczka, Scaffolds based on
chitosan and collagen with glycosaminoglycans crosslinked by tannic acid,Polymer Testing
65 (2018), page no.163-168

The scaffolds immersion before measurement allows imitating the conditions inside the body.
All the cross-linked scaffolds were in the cylindrical shape before and after immersion which
allowed for the mechanical testing. Initial measurements showed the highest mechanical
parameters of the scaffolds based on chitosan and chitosan/collagen mixture. However, after
48 and 72 h, swelling was advanced and the compressive modulus and maximum
compressive force decreased. After immersion for a period longer than 72 h, mechanical
parameters did not change significantly (results not showed). The use of the chitosan and
collagen mixture improves the material stability which was observed as the lowest difference
before the first and the last measurement. The amount of glycosaminoglycans did not
significantly influence the scaffolds mechanical properties. The percentage of weight loss as
a result of enzymatic scaffolds degradation by collagenase is listed in Table 2. The results
showed the weight change in the range of 3.5–6.8%. The highest weight loss was noticed for
the collagen scaffold with 5% glycosaminoglycans addition The addition of chitosan to
collagen improves the material stability against collagenase. The results of percentage weight
loss after the enzymatic degradation by hyaluronidase. The enzymatic degradation by
hyaluronidase resulted in scaffolds weight changes higher than in the case of collagenase.
The changes are in the range of 10.5% for collagen, 25% for chitosan/collagen, and 41% for
chitosan. All the scaffolds are sensitive to the enzymatic degradation by hyaluronidase;
however, the presence of chitosan improves this sensitivity. The content of
glycosaminoglycans does not significantly influence the obtained results.

35