Preparation of chitosan
The purified chitin is subjected to a deacetylation process (Figure 2). To this end, the CI is treated with a concentrated base, most often NaOH,21 and then washed with water and dried. The chitosan thus obtained is in the form of a white powder.13
In the deacetylation process random acetyl groups are removed in favour of the amino group, chitosan is a copolymer ofN– acetyl–D– glucosamine and D–glucosamine units. The presence of the latter groups depends on the degree of deacetylation (DD) that is used to characterise the CS. The more deacetylated the CS is, the fewer N– acetyl–D– glucosamine groups. The value of the DD of chitosan is in the range of 50–95%.17,28
Physicochemical properties
In its structure, chitosan contains functional groups: hydroxyl, amine and acetyl. They have a significant impact on the chemical properties of the polymer and the nature of interactions. The presence of hydrophobic acetyl groups and hydrophilic amine and hydroxyl groups ensures the amphiphilic nature of the polymer.13 The amine groups that are responsible for the cationic nature of the polymer result in a high surface tension of aqueous solutions of this polysaccharide.29
CS is insoluble in water, in popular organic solvents (including methanol, ethanol, isopropanol, toluene), however, it dissolves in dilute aqueous acid solutions, e.g. in hydrochloric acid, acetic acid, formic acid, citric acid.30–32 This property is due to the presence of free amino groups that can protonate at a pH below 6.33 The amine groups become positively changed, breaking the hydrogen bonds and allowing the polymer to dissolve. Above this pH value, the –NH3+ groups deprotonate, lose their charge, and the polymer solubility decreases.16,34 The results of the solubility tests are presented in Table 1.30
The solubility of chitosan also depends on the degree of deacetylation (DD) and the molecular weight of the polysaccharide. The more amino groups there are, i.e. the higher the deacetylation degree, the better the chitosan solubility.23 The higher the molecular weight of the polymer, the more internal hydrogen bonds there are and the more difficult it is to dissolve.17 CS forms water-soluble salts, including formates, acetates, hydrochlorides, citrates.35 To characterise chitosan, its viscosity is often used, which depends on various parameters such as concentration of chitosan in the solution, temperature, degree of deacetylation.36
The cationic nature of chitosan distinguishes it from other natural polysaccharides. Due to its positive charge, chitosan has the ability to form complexes with negatively charged compounds, e.g. polylactide37, poly(glutamic acid)38, DNA39,40, collagen41, alginates42–44, hyaluronic acid45–47, pectin48,49and many others.
The structure of chitosan, which is based on D– glucosamine andN– acetyl–D– glucosamine units, is similar to the structure of naturally occurring glycosaminoglycans (GAG). GAG can build the extracellular matrix and perform various functions in organisms, e.g. they affect adhesion, growth factors and cell receptors, water retention (influencing the resistance of cells to compression). It can be expected that, thanks to its GAG-like structure, chitosan can also interact with animal cells.7,34
Chitosan is readily used in tissue engineering because it is characterised by features desired in biomaterials: biocompatibility, non-toxicity, the ability to biodegrade into non-toxic compounds and bioactivity.50–52 It also demonstrates osteocompatibility (the presence of chitosan does not adversely affect bone regeneration) and osteoconduction (the ability to connect with bone tissue and stimulate its growth).13 CS also has mucoadhesive properties, i.e. it promotes the adhesion of animal mucus cells to the biomaterial. This property is due to the difference in charges between positive chitosan and negative mucus, between which electrostatic interactions are generated.53,54 CS is also credited with the ability to inhibit the growth of neoplastic cells55,56 and promote wound healing.57–60 Chitosan also has antibacterial and antifungal properties (against Gram-positive 61 and Gram-negative bacteria62). These properties are probably due to the interaction between the positively charged polymer and negatively charged lipopolysaccharides or proteins in microbial membranes.59 Interactions with the surface of the bacterial cell reduce the permeability. The antibacterial properties of chitosan may also result from the interaction with the DNA of the bacterial cell, which results in inhibition of RNA synthesis.50,59,63
Chitosan degradation
Chitosan is a natural polymer which decomposes under the influence of both chemical and physical factors and enzymes. Chemical factors include depolymerisation under the influence of strong acids, e.g. HCl, HNO2, H3PO4, CH3COOH,64–66 strong oxidants67 or free radicals.68 The products of such reactions are chitosan oligosaccharides with various, low molecular weights.69 Chemical degradation is a fast process, but expensive, poorly controlled (random cutting of the polymer chain70) and highly polluting towards the environment due to the chemical compounds used.55,65,71
Physical methods of CS depolymerisation include, among others, γ radiation, ultrasound. Contrary to chemical depolymerisation, mechanical cuts occur more frequently inside the polymer,72 and the obtained oligosaccharides are characterised by a narrower size distribution.69 Gamma radiation can be used to sterilise chitosan products. It was examined that under the influence of gamma radiation the CS structure changes, there is a decrease in molecular weight already at 15 kGy, which increases with radiation dosage.73 Such changes may affect the properties of the polymer and, consequently, the chitosan-containing medical device. The use of ultrasound is a popular solution due to its low environmental impact. Degradation of chitosan by ultrasound is based on mechanical phenomena and depends on wave intensity, temperature, polymer concentration, ionic forces.74 There are also studies on CS depolymerisation using the cavitation process.69,75,76
The use of enzymes for the depolymerisation of CS is associated with carrying out the reaction in mild conditions and fairly good control of the process. It is also an environmentally friendly method.64,65 The enzyme specific chitosanase can be used in the hydrolysis of β‑1,4‑glycosidic bonds, as well as non-specific chitinases77, lysozym78or cellulases.71 The reduction of the CS molecular weight under the influence of the protease enzyme changed the structure of the oligomer, but did not affect the deacetylation degree.79 Proteases can also be used for the production of monomers (N– acetyl–D– glucosamine units).80 The use of lipase, the enzyme present in the human body, in CS depolymerisation leads to products with different molecular sizes in the 30 – 50 kDa range, without major structural changes to the polymer.81
Metabolism in living organisms
A biodegradable scaffold used in bone regeneration should degrade in the body into known, non-toxic products that can be excreted from the body or incorporated into the body’s natural metabolic cycles. The time of scaffold degradation in the body depends on the place of implanting and the patient (their age, chronic diseases), but on average in bone regeneration it should take from 3 to 9 months.82Hydrolysis of glycosidic bonds in chitosan leads to the production of non-toxic glucosamine oligosaccharides, which can be rapidly cleared from the body in urine.83,84 Chitosan implants do not elicit a long-lasting inflammatory or immune response.52 The inflammation observed after the introduction of chitosan scaffolds depends on the degree of deacetylation of the polysaccharide. Chitosan with a lower DD induced a stronger leukocyte response.85 The rate of degradation of chitosan implemented in the form of microspheres, hydrogels or 3D scaffolds varies. The onset of degradation of chitosan in the form of microspheres was observed 4 weeks after introduction into the body of rats, and after 12 weeks the microspheres disintegrated into small fragments.86 For chitosan hydrogel, strong degradation was observed after 4 weeks.87 In turn, the 3D porous scaffold made of chitosan after 30 days in vivo was completely biodegraded.88 Hydrolysis of bonds in chitosan takes place in the human body under the influence of a non-specific enzyme lysozyme.89 It has been noted that due to the cationic nature of chitosan, it can interact with blood cells. In the presence of chitosan, blood tends to coagulate and form clots on the surface of chitosan.90 After oral administration of a polysaccharide in the organisms of rabbits, a specific response was observed, i.e. an increase in lysozyme secretion by the organism, which may prevent thrombosis.91 In vivo degradation studies of chitosan-poly-L-lactide composites in rats showed that the polysaccharide did not cause an unexpected, negative response, no blood clots were detected after 1 week.92
Cell scaffolding
Tissue engineering is looking for solutions that could replace the need for autographic or allographic transplants. One of the proposed solutions is the use of scaffolds that mimic the basic roles of tissues and enable their regeneration.93,94
Such scaffolding is usually a three-dimensional, porous structure made of biomaterial. The main task of scaffolding is to support the regenerating tissue by providing a rack on the surface of which cells can settle.8 Scaffolds should imitate the tissue for which they are used to regenerate, its structure and properties.95 The implant must not cause a negative immune response, it should be metabolised by the body, easy to shape and sterilise, and it should be durable so that it can be stored in ex vivo conditions.96
Scaffolds made of chitosan alone
Due to the properties of chitosan, such as non-toxicity, biocompatibility, affinity to animal mucus and biodegradability, it is a polymer very readily used in tissue engineering.4 It supports cell adhesion and proliferation, and thus tissue regeneration. It allows you to easily model the scaffolding to a specific shape and create porous structures with open pores. These features make chitosan a potential candidate for scaffolding material.97
The temperatures used during the production of 3D scaffolding significantly affect, among others, the pore size or mechanical properties related to the compressive modulus.98Chitosan with a higher degree of deacetylation was characterised by higher mechanical resistance, slower degradation time and lower absorbability of the scaffold.99 High DD (DD 88% and 95% tested) supports cell proliferation on the surface of the chitosan scaffold.99 The porosity of the scaffold has a significant influence on its mechanical properties. The more porous the structure, the worse the mechanical properties. The decrease in the porosity from 94.1% to 82.5% was related to an increase in Young’s modulus to 5.2 kPa and 520 kPa, respectively, while the pore size decreased. Cell adhesion was possible on both types of scaffolds, but better conditions were provided by scaffolds with lower porosity, which was associated with an increase in fibre density.100
Due to the properties of chitosan, it is a readily used biomaterial. In vitro and in vivo studies are conducted on the use of chitosan scaffolds in the treatment of damage to various tissues. The interactions and influence of CS on cells are investigated, among others, spinal cord52, bones, cartilage101, tendons102, skin103.
The use of chitosan in the treatment of the spinal cord has been studied. The implants were inserted into the meninges or directly into the spinal cord for 6 or 12 months, respectively. The host cell response (rat) was low, which indicates the inert nature of chitosan and the possibility of using the material in long-term therapies.52 In search of chitosan with the best properties for the osteochondral treatment, porous chitosan scaffolds were prepared. In vivo studies indicate that the cellular response depended on the properties of the chitosan used, and chitosan with a DD of 83%, a relatively low molecular weight of 11.49 KDa and a high content of calcium, remaining due to the lack of demineralisation during the preparation, turned out to be the best for applications. After implant placement, its degradation and shaping of the subchondral bone were observed.101
Active layers can be applied to the scaffolds to improve the interaction at the biomaterial-tissue interface.104 The surface of the chitosan scaffolds was mineralised to obtain apatite layers imitating bone. After modification, the scaffolds are characterised by greater rigidity and smaller pore sizes, yet sufficiently large to allow unhindered migration of bone cells.105 The conductedin vitro studies indicate that the scaffold after apatite modification supports the adhesion and proliferation of cells to a greater extent than the unmodified version. Mineralised scaffolds are better suited for use in bone tissue engineering.106,107
The cultivation of chondrocyte cells is aided by the use of porous chitosan microspheres (porosity ~90%, pore size 5 – 60 μm) with diameters of 180 – 280 μm108 and CS fibres with diameters between 4 and 22 μm. The fibres maintained the viability of chondrocytes (over 85%) and supported the formation of the extracellular matrix (ECM). However, it was concluded that reducing the size and the use of nanofibers could improve interactions with chondrocytes.109
Chitosan as a scaffold surface modifier
Chitosan is often used as a coating material for implants or scaffolds. Such surface modification significantly improves the surface properties of an implant – its bioactivity, biocompatibility, corrosion resistance, as well as, properties supporting bone regeneration, such as osteoconductivity.110 Chitosan layers are stable, and depending on the concentration of chitosan, its molecular weight, the degree of deacetylation and application technique, they degrade at different rates, even allowing for long-term use.111,112
Surface modification significantly influences the mechanical properties of the scaffolding. Three-dimensional, porous scaffolds made of polycaprolactam (PCL) were covered with layers of chitosan at various concentrations, from 1% to 3%. As the concentration of chitosan in the layer increases, the compression strength and the compression modulus factor increase, while scaffolding crystallinity decreases. The increase in the first two is likely related to the increase in the amount of amine groups that support PCL and improve the strength of the scaffold. The decrease in crystallinity is a result of the formation of hydrogen bonds between PCL and chitosan. Among the examined, the best osteoblast differentiation was provided by the PCL scaffold covered with a 2.5% chitosan layer.113
It is commonly known that the hydrophilicity/hydrophobicity of the scaffold affects cell adhesion and proliferation. Polylactide (PLA) is a polymer readily used in tissue engineering due to its biocompatibility and bioresorbability. It is however, a hydrophobic polymer that does not promote cell adhesion. In order to increase the hydrophilicity of the polymer, the PLA surface can be modified by applying a layer of chitosan.114 Modification of poly-L­- lactide surface with chitosan also improves mechanical properties of the scaffold, as well as improves adhesion, differentiation, activity, and morphology of chondrocyte cells115 and mouse bone marrow stromal cells (mBMSCs) compared to the unmodified structure, which accelerates the bone regeneration process.51,116The introduction of the PLLA scaffold with a CS layer into skull defects of rats and the examination of bone changes after 12 weeks showed that the modified scaffolds support greater growth of bone tissue with higher density compared to the unmodified ones. This confirms the suspicions that chitosan-coated polylactide scaffolds are sustainable for bone regeneration,116 and modifying the scaffold surface with chitosan significantly improves osteoblast adhesion and proliferation.117
Chitosan surfaces exhibit antimicrobial properties, highly desirable in biomedical applications. The antibacterial nature of CS was tested on Gram-negative strains of E. coli 118,119 and Gram positive Staphylococcus aureus .120 For both groups, the use of chitosan layers reduced bacterial settling.
In addition to applying layers of chitosan itself, layers of mixtures of chitosan and other compounds are also used to improve certain properties. The use of a chitosan layer with ZnO particles on a titanium implant increases the compatibility of such an implant and its corrosion resistance, which results from the closure of pores on the implant surface. The introduction of the oxide improves the antibacterial properties of the layer compared to the layer made of chitosan itself.119 Antibiotics can also be incorporated into the chitosan layer. The compounds bind through weak intermolecular interactions, which allows easy release of the antibiotic and, consequently, the fight against bacteria.121
Complexes with other natural polymers
Due to the cationic nature of the polymer, CS has the ability to spontaneously form stable polyelectrolyte complexes (PEC) with negatively charged structures such as natural polymers. In solution, permanent electrostatic interactions are formed between the cationic amino group of chitosan and the negatively charged group of the polyanion. Complexes are formed in stages, first the primary complex is formed, with chains of polyions randomly arranged in space. Then the chains arrange themselves in orderly structures, which is associated with the formation of hydrogen bonds, van der Waalss forces and others. Concurrently, aggregation into various structures may take place. The wide range of properties, as well as the non-toxicity, biodegradability and biocompatibility of PEC ensure a wide application of the complexes, especially in tissue engineering.122,123 Examples of PEC are shown in Table 2. PEC formation can be investigated, for example, by FTIR (N–H, C=O bond shift study), DSC differential scanning calorimetry (melting point shift) or by examining changes in zeta potential.
The formation of complexes is influenced by the concentration of polymers, the mass ratio of polyelectrolytes, the order of polymer addition, the degree of CS deacetylation, the pH value of the solution, presence of other ions and molecules in the solution. As the concentration of polymers in the solution increases, the size of the particles formed increases. In turn, the mass ratio of the polymers used affects the size, charge and solubility of resulting complexes.124,125 The molecular weight of chitosan and the degree of deacetylation affect the size of the complex. The larger the polymer, the thinner the packing of the compound and the larger the size of the PEC.39 Other molecules or ions than the polymers forming the complex may be present in the solution. They affect the ease of formation of complexes, their stability and properties. An example of such a compound is sodium lauryl sulphate surfactant, the use of which increases the surface roughness and the thickness of the chitosan/alginate (CS/Alg).125,126
The size of the nanoparticles of the chitosan CS/Pc with pectin complex varied depending on the order of addition of polymers. At low polymer concentrations, larger particles were formed when chitosan was added first, the inverse was true at high concentrations. The size of the complexes ranged from 460 nm (low polymer concentration) to 1110 nm (high polymer concentration). The zeta potential depends on the mass ratio of the polymers used, for complexes with different mass ratios it was in the range between 40 and 60 mV, and the advantage of chitosan in the complex increases the value of the potential, i.e. the stability of the complex. CS/Pc nanocomplexes are characterised by low stability compared to other polyelectrolyte complexes, they lose their stability after 14 days, and they degrade after 30 days. The stability is independent of particle size, but is dependent on the pH of the solution (stable at pH 3.5 – 6.0).124
The introduction of a polyanion into a chitosan scaffold has many advantages. The electrostatic interaction between the polymers increases the stability of the scaffolds obtained.127Modification with alginate improves the mechanical properties of the scaffold, the compressive modulus and yield strength after the modification are, respectively, 8.16 MPa and 0.46 MPa, for the chitosan only scaffold these values are about three times lower. Also, the preparation of the scaffold from the complex is simpler, since the environment is not necessarily acidic, it can just as well be alkaline or neutral. The chitosan/alginate complex is non-toxic, has anti-inflammatory effects,128 in many cases supports adhesion, as well as osteoblast proliferation and angiogenesis.129 However, some sources report the CS/Alg surface as unfavourable for cell adhesion.43
Composites with chitosan
Often, implants made of purely natural or synthetic polymers do not have the mechanical properties desired in tissue engineering, for instance, they are not able to carry the required loads.13 To change the properties of the scaffold, a new compound can be introduced into the starting composition to obtain a composite. Examples of complexes are presented in Table 3. The use of the composite influences morphology, mechanical properties, porosity, swelling of the scaffolds as well as biomineralisation and degradation processes. Chitosan is a modifier commonly used in composites, a building material popular in the animal world, composites of which support bone regeneration processes such as adhesion and proliferation of bone cells, osteocalcin secretion or biomineralisation.138–140 On the other hand, nanoparticles are readily introduced into chitosan scaffolds, thanks to which biomineralisation takes place more efficiently and the degradation rate decreases.
The morphology of the scaffolds significantly depends on the concentrations of the polymers used. For the poly-L-lactide + chitosan (PLLA+CS) scaffold, the higher the concentration of chitosan in the starting solution, the more chitosan was deposited on the pore surface, making the pores surface more jagged compared to polylactide systems alone, which is desirable due to an increase in the surface of the implant.139
The use of the PLLA+CS composite significantly affects the rate of scaffold degradation in comparison to the polylactide scaffold. This is explained by the fact that when PLLA hydrolyses into lactic acid, it lowers the pH of the environment, which causes the dissolution of the CS. In this way, along with the depletion of chitosan, the pores of the scaffold increase, which increases its surface contact with water. Also, the hydrophilic properties of chitosan accelerate the diffusion of water into the interior, making hydrolysis of the hydrophobic polylactide faster.92
The use of composites has a significant influence on mechanical properties. In order to strengthen the macrospheres made of chitosan, montmorillonite (MMT) and/or hydroxyapatite (HAP) were introduced into the system. Composite scaffoldings were characterised by much greater compressive strength. The introduction of MMT and HAP also reduced the swelling of scaffolds, which is probably related to the hydrophobicity of the additives and the binding of hydrophilic amino and hydroxyl CS groups by hydroxyapatite. Interactions with bone cells and cell proliferation supported all systems.141
The chitosan/collagen polyelectrolyte complex (CS/Coll) was enriched with MMT and modified organomontmorillonite (OMMT). The scaffolds made of PEC had weakly connected large pores (about 1 mm). The introduction of MMT and OMMT made it possible to obtain a structure with well connected pores of smaller diameters (200 – 400 μm for CS/Coll+MMT and 50 – 200 μm for CS/Coll+OMMT), which are better suited for seeding cells. Both composites were characterised by lower swelling ratio, degradation rates and greater compressive strength, and they supported biomineralisation better than the CS/Coll complex.142
The introduction of nanomaterials into scaffolds made of chitosan or its complexes affects the properties of the structures. The use of a chitosan-chitin composite with the addition of ZrO2nanoparticles (CS+CI+nZrO2) enables the production of non-toxic scaffolds with a pore size of 150 – 200 μm (larger in the CS+CI system) and improved bioactivity.143 Porous scaffolds made of a combination of chitosan, gelatine and hydroxyapatite (Cs+Ge+nHAP composite) with an average pore size of 100 – 180 μm support the adhesion and development of bone cells. Hydroxyapatite occurs naturally in human bones, while gelatine and chitosan mimic the extracellular matrix (ECM) well. The introduction of nHAP significantly affects the properties of the system, increases the susceptibility to mineralisation of the scaffold, and as a result, significantly improves cytocompatibility, reduces swelling and the rate of degradation. Composite scaffolds are well suited for bone tissue engineering.144 In the same way, the properties of the Cs+Ge scaffolds change after the introduction of SiO2nanoparticles (Cs+Ge+nSiO­2composite).145
Cellular response to chitosan materials
After a scaffold is implanted, the body reacts, which ultimately leads to tissue reconstruction. The damage is immediately recognised and triggers appropriate interactions with blood, clot formation, cellular response, and eventually tissue reconstruction.157 A thin layer of water forms on the scaffold surface, and proteins adhere to it. The bone cells then recognise these proteins and settle on the implant. Cell growth and integration enables tissue to regenerate on the scaffold surface.158,159 Depending on the surface chemistry and implant topography, cells may interact with the material with different intensity.
Cell adhesion
The term “adhesion” describes two phenomena that occur during the settlement of scaffolding. It concerns the short-term generation of interactions between the cell membrane and the material (ionic forces, van der Walls interactions) and long-term cell attachment.159 Biomaterials may affect cell adhesion, be osteoconductive or osteoinductive. Osteoconduction is the ability of a material to connect to bone tissue, provide adequate support for cells and influence the direction of the regeneration process. The osteoconductive material also positively influences the processes of cell proliferation and ECM formation. Osteoinduction involves triggering a series of processes and reactions that lead to bone regeneration through the use of biomolecular signalling devices. One such process is, for example, cell differentiation.160–162
Implants are recognised by the body as foreign bodies and trigger an immune response. The body’s response mechanism after implantation (Figure 3) begins with the formation of a thin layer of water on the surface of the biomaterial within a few nanoseconds. Then, adhesion proteins are deposited, these are peptides with a specific structure, containing the RGD group – the arginine-glycine-aspartic acid combination.163,164 Proteins are deposited on the implant surface within seconds to hours. Depending on the surface properties of the biomaterial, proteins settle in different amounts, densities and conformations.165 The cells of the tissue then recognise the attached proteins, and cell-protein interactions are formed, which last from a few minutes to several days. Cell growth and integration, the final step, enables tissue formation on the scaffold surface.157,158
In addition to the surface properties, the scaffold topography and porosity have a significant impact on cell adhesion. The rough structure increases the contact area between the implant and the surrounding tissue and allows cells to settle. Osteoblast differentiation is supported in particular by nanosized roughness.104,166The scaffold should be characterised by open porosity, which enables cell migration and colonisation of pores. Open porosity supports the process of vascularisation of the resulting tissue.160Cell adhesion is significantly affected by the surface energy of the biomaterial. High surface energy enables very good wettability and adhesion. Osteoblasts are more likely to settle, differentiate faster and multiply on a surface with higher surface energy.167–169
Interaction with groups in chitosan
The biomaterial can interact with the body’s cells in different ways. Significant surface features that influence this are surface properties such as hydrophilicity, the presence of functional groups or surface charge.104,130
Functional groups on the surface of the biomaterial significantly affect the adhesion of bone cells. The binding of osteoblasts to surfaces with different functional groups was investigated. The hydroxyl–OH and amino–NH2 groups present in chitosan supported the adhesion and differentiation of stem cells into osteoblasts better than the carboxyl –COOH and methyl–CH3groups.170 Stem cell subsidence was most strongly supported by the amino group. Depending on the functional groups, the shape of the cells may also change. Flattened cells were preferred for the –NH2 group, and spherical cells for –COOH.171 The surfaces with the amino group also promoted and maintained the process of osteogenesis, both under normal and osteogenesis-promoting conditions.172
The wettability of the biomaterial affects the adhesion of cells. Good wettability facilitates the process of proteins settling on the surface of the material and, as a result, cell adhesion. A greater number of adhesive proteins, and a smaller number of hydrophobic proteins that do not participate in the process, were deposited on the hydrophilic surfaces.163,173
Proteins settle differently on the positively charged surface than on the negatively charged one. Amino groups of the CS promote the deposition of negatively charged proteins and cell membranes, proliferation and differentiation, which results from electrostatic interactions.159
Chitosan with a higher degree of deacetylation provides better conditions for osteoblast subsidence, but to a lesser extent supports the secretion of osteoprotegerin compared to CS with a lower degree of deacetylation.161,174,175
Secreted cellular metabolites
There are several types of cells in bone: osteoblasts, osteocytes, osteoclasts, and there are also bone lining cells.176Osteoblasts are mature, metabolically active bone-building cells that are found at the surface of the bone. The task of osteoblasts is to secrete compounds that make up the intercellular substance.177,178 The most numerous cells in bone tissue (over 90%179) are bone cells – osteocytes. They arise from transformed osteoblasts trapped in the bone matrix, in the bone cavities. Osteocytes form a communication network of bone cells, thanks to which they regulate bone tissue homeostasis. Signals are sent via cleft junctions, chemical secretion, or direct dendrite association.180–182 The bone lining cells are osteoblasts that have not undergone apoptosis or developed into osteocytes. They perform functions related to bone remodelling.176,183 The last group of cells are multinucleated osteoclasts, whose task is to maintain the calcium balance in the tissue and to secrete compounds that break down the bone.184–187
The living cells of the body conduct metabolic processes to produce the energy necessary for life from carbohydrates, proteins and fats. For this, the energy carrier adenosine-5′-triphosphate (ATP), produced in various processes, is most often used. Glucose is the main source of energy in mesenchymal stem cells, which is converted into pyruvic acid and ATP in the process of glycolysis. Osteoblasts obtain ATP through glycolysis and the Krebs cycle, with predominance of the former process. The Krebs cycle is used mainly in the period of higher energy demand, during bone formation. In addition to glucose, osteoblasts obtain ATP from the transformation of amino acids (in particular glutamine) and fats. Osteocyte metabolism is not a well understood process. Osteoclasts generate energy in the processes of glycolysis and the Krebs cycle.188
The extracellular matrix is mostly produced by osteoblasts. Osteocytes contribute to a lesser extent to the production of matrix, which is due to their structure – mature osteocytes do not have many organelles responsible for secretion.182 ECM consists mainly of collagen (90%) and non-collagen proteins (10%). The vast majority of collagen is type I collagen, however, type III and V are also found in the ECM. The non-collagenous organic part consists of proteoglycans, osteocalcin (also known as bone gamma-carboxyglutamic acid-containing protein (BGLAP)), glycoproteins and Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs).189–191Proteoglycans are proteins with which saccharide glycosaminoglycans (GAGs) are linked. They are mainly secreted by osteoblasts, e.g. biglikan192 or decorin.193Osteocalcin is secreted mainly by mature osteoblasts, but also by osteocytes.194 SIBLINGs are small hydrophilic proteins that contain the same set of amino acids (Arg-Gly-Asp) found in the bone matrix. These compounds are mainly produced by osteocytes (Matrix Extracellular Phosphoglycoprotein195), but to a lesser extent also by osteoblasts (osteopontin, Dentin Matrix Protein-1196,197).198
Osteoclasts are capable of secreting H+–adenosine triphosphate responsible for the dissolution and cathepsin K protease responsible for dissolving the demineralised organic part.199,200
Chitosan has a positive effect on the metabolic behaviour of cells. In vitro studies of MCF-7 cells on chitosan scaffolds were performed. The metabolism of cells in such a system is very similar to that in tissue, which proves the positive effect of the chitosan matrix on the vital processes of cells.201 Similarly, for dental pulp stromal cells, CS has a positive effect on metabolism and proliferation.202
CONCLUSION
Chitosan, a chitin derivative, is a unique polysaccharide. It is obtained in the process of deacetylation of chitin, most often derived from the shells of sea crustaceans. It is a readily used compound in tissue engineering due to its similarity to the glycosaminoglycans present in the body, biocompatibility, bioactivity, non-toxicity, antibacterial properties and non-toxic degradation products that may occur under the influence of human enzymes. Amine groups of chitosan improve the surface properties and provide better adhesion of bone cells. CS can be used in the production of cell scaffolds for bone regeneration as a direct substrate or component of a composite. The cationic nature of the polysaccharide also enables the formation of polyelectrolyte complexes with other natural polymers. It can also be used for surface modification to improve the properties of the scaffold.
AUTHOR INFORMATION
Corresponding Author
*Email:agnieszka.gajadhur@pw.edu.pl
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding Sources
This scientific research was financed from the National Centre for Research and Development as a research project “Lider 11” (LIDER/4/0010/L-11/19/NCBR/2020) titled “Porous, biodegradable implants for the regeneration of spongy bone”.
Notes
The authors declare no competing financial interest. Thanks to Karolina Kalbarczyk for help with graphics.
ABBREVIATIONS
Alg, alginate;
ATP, adenosine triphosphate;
BCNs, bacterial cellulose nanocrystals;
BGLAP, bone gamma-carboxyglutamic acid-containing protein;
CHAP, carbonated hydroxyapatite;
CI, chitin;
Coll, collagen;
CS, chitosan;
CSA, chondroitin sulfate;
DSC, differential scanning calorimetry;
ECM, extracellular matrix;
FTIR, fourier transform infrared spectroscopy;
GAGs, glycosaminoglycans;
Ge, gelatin;
HA, hyaluronic acid
HAP, hydroxyapatite;
MMT, montmorillonite;
nBG, nanobioglass;
nHAP, nano hydroxyapatite;
nPP, nano-pearl powder;
nSiO2, nano-silica;
OMMT, organomontmorillonite;
Pc, pectin;
PCL, polycaprolactone
PCS, phosphorylated chitosan;
PEC, polyelectrolyte complex;
PGS, polyglycerol sebacate;
PLLA, poly-L-lactide;
PVA, poly(vinyl alcohol);
SIBLINGs, Small Integrin-Binding LIgand N-linked Glycoproteins;
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Figure LegendsFigure 1. Chitin (a) and chitosan (b). Figure 2. Production of chitosan. Figure 3. Cell adhesion to biomaterial. After biomaterial implementation thin water layer is formed on the surface, followed by protein adhesion. Cells recognise proteins and adhere to the biomaterial, allowing the tissue to regenerate.Table LegendsTable 1. Chitosan solubility. Table 2. Examples of polyelectrolite complexes with chitosan in tissue engineering Table 3. Examples of chitosan composites in bone tissue engineering