REVIEW ARTICLE
Chitosan Based Systems for Tissue Engineering Part 1: Hard Tissues
H. Çiğdem ARCA*, Sevda ŞENEL*°
Chitosan Based Systems for Tissue Engineering Part 1: Hard Tissues
Summary
Chitosan is a natural polymer with favorable properties such as biocompatibility, biodegradability, non-allergenic and non- toxic which make it a very promising material for scaffold in tissue engineering. It also exerts bioactive properties such as antibacterial and wound-healing. In the first part of this review, a general introduction to tissue engineering and chitosan will be given. Applications of chitosan based systems in hard tissue engineering such as bone, cartilage and periodontal are reviewed with recent and relevant examples, and the factors affecting the formulation properties are discussed. Furthermore, the approaches to enhance the properties of the chitosan-based scaffolds will be mentioned.
Applications of chitosan in soft tissue engineering will be reviewed in the second part to be published in this journal.
Key Words: Chitosan; tissue engineering; bone; cartilage;
periodontal; scaffold Received: 18.09.2009 Revised: 10.11.2009 Accepted: 17.11.2009
Doku Mühendisliği İçin Kitosan İçeren Sistemler Bölüm 1: Sert Dokular
Özet
Doğal bir polimer olan kitosan biyouyumlu, biyoparçalana- bilir, non-allerjenik, non-toksik özellikleri nedeniyle doku iskelesi olarak doku mühendisliğinde ümit vaadeden bir materyal olarak karşımıza çıkmaktadır. Kitosan ayrıca antimikrobiyal ve yara iyileştirici özelliklere de sahiptir. Bu derlemenin ilk kısmında doku mühendisliği ve kitosana genel bir giriş sunulmaktadır. Kitosan bazlı sistemlerin kemik, kıkırdak ve periodontal dokular gibi sert doku mühendisliğinde uygulamaları, özellikle son yıllarda yapılan çalışmalardan örneklerle birlikte sunulacak ve formülasyona etki eden faktörler tartışılacaktır. Ayrıca kitosan bazlı doku iskelelerinin iyileştirilmesi için mevcut yaklaşımlardan bahsedilecektir. Kitosanın yumuşak doku mühendisliğinde uygulamaları ise bu dergide yayınlanacak ikinci bölümde bahsedilecektir.
Anahtar Kelimeler: Kitosan; doku mühendisliği; kemik;
kıkırdak; periodontal; doku iskelesi
* Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100-Ankara, Turkey
° Corresponding author E-mail: ssenel@hacettepe.edu.tr INTRODUCTION
Tissue engineering is an emerging multidisciplinary field involving chemistry, biology, physics, genetics, pharmacy, medicine and engineering. It uses synthetic or naturally derived or engineered biomaterials to replace damaged or defective tissues and organs.
Every year millions of people worldwide suffer from tissue damages and organ failure. The aim of tissue engineering is to improve the life quality of these
millions of people by improving the tissue and organ functions.
Langer and Vacanti (1) have defined tissue engineering as combination of life sciences and engineering principles in order to describe structure- function relationships in mammalian tissues and the development of biological substitutes to restore,
maintain or improve tissue function.
There are 4 approaches for tissue engineering. The first approach includes the application of biomaterials as scaffolds, which are structures prepared to create an artificial cellular environment. (2). The second approach is utilization of cells for creating artificial tissues and organs either with or without polymers (3-5). The third approach includes scaffolds and biosignals (6), which are substances normally present in tissues and capable of stimulating cellular growth, proliferation and cellular differentiation. Finally the fourth approach includes scaffold, biosignals and cells together in order to minimize the differences between the artificial and the cellular environment (7).
The tissue engineering approaches have been extended with the gene-activated matrix (GAM) technology which is a direct gene transfer strategy.
Scaffold and the associated DNA, responsible for the secretion of biological signal molecules by encoding them (8), built up the GAM. After its implantation into tissue defect, granulation tissue fibroblasts migrate into the GAM and promote tissue regeneration by expression of the therapeutic gene (9).
In general, tissue engineering investigations continue for the repair of many different tissues like as bone (10,11), cartilage (12), cornea (13), skin (14,15), liver (16), nerve (17), adipocyte (18,19), periodontal tissue (7,20), blood vessel (21), skeletal muscle (22,23), abdominal wall (24,25), pulmonary tissue (26,27), vagina (28), urologic tissues (29), heart valves (30,31), myocardial tissue (32) and trachea (33,34).
In the mid-1990’s, the first products of tissue engineering, for wound care and orthopedic applications came on the market. In 2008, this market approached to $1.5 billion, and it is expected to grow at a rate of 16.2% annually between 2008-2013.
In 2013 the expected capacity of the market is $3.2 billion (35). According to 2007 analysis, in the U.S.
alone, the total potential of the market was over $85 billion (36).
Despite the intensive studies performed on tissue
engineering, there are still significant technical challenges needed to be overcome before getting on the market, which are summarized below (37):
– scale up process of cell cultures to produce large amounts of viable cells in sterile condition and without genetic changes,
– the efficient manufacture of biocompatible materials from chemical synthesis or transgenic plants and animals,
– long-term tissue and cell storage in different environmental conditions,
– requirement of potential adverse reaction inhibition,
– requirement of non-immunogenic universal- donor cell lines.
Importance of scaffold in tissue engineering applications
Scaffolds are artificial structures on which cells are seeded or migrated and capable of supporting three- dimensional tissue formation until the cells produce adequate extracellular matrix (ECM) to support the structure mechanically. In general, scaffolds are described as the structures that take part in restoring of organ functionality permanently or temporarily (11).
The ideal properties of a scaffold can be summarized as follows (11,38-42) :
– provides anatomic shape and volume,
– should be compatible with the surrounding biological fluids and tissues, in order to minimize the immunological response,
– scaffold itself and its degradation products should be non-toxic,
– should be prepared by a biodegradable polymer with an adequate degradation rate (if the degradation is faster than the cell proliferation, the scaffold might degrade before the tissue construction. If the degradation of the scaffold is slower than the cell proliferation, cell death can be observed)
– mechanical properties of the scaffold should provide temporary mechanical support at the site
of implantation. It should be capable to carry and deliver cells and/or biosignals,
– should have the favorable surface properties for cell attachment and differentiation,
– should be porous in order to increase the surface area for cell attachment,
– pores of the scaffold should be interconnected for not only cell migration but also for the diffusion of gases, nutrients and metabolic wastes otherwise cell death can be observed.
In addition to all these essential properties, for engineering applications of elastic tissues like blood vessel, skin, heart valves, cartilage, tendon, and bladder, elasticity of scaffolds is regarded as an important designing parameter (43,44). It has been reported in several groups that mechanical stimulation that involves cyclic strain of the cell- scaffold construct or shear stress can influence the quality of the resulting tissue or tissue regeneration (45-47). It is very important to design scaffolds that can maintain their mechanical integrity after in vivo application which might help to convey the mechanical signals to the cells attached onto them.
To achieve this, the designed scaffolds must be elastic and should resist cyclic mechanical strains without any break or any other permanent deformation (48).
The most frequently used polymers for scaffold construction, the details of which are precluded due to the brevity of the current article, are polycaprolactone (49,50), poly(lactic-co-glycolic acid) (51,52), poly(ethylene glycol) (53,54), poly(vinyl alcohol) (55, 56), polyurethane (57,58), alginate (59,60), gelatin (61), collagen (62), silk (63), starch (64) and chitosan (65). In this review, after giving a general introduction to chitosan, its applications in different hard tissues will be discussed.
Chitosan
Chitosan is a deacetylated derivative of chitin, the most abundant biopolymer in nature after cellulose and found in the shells of crustaceans and walls of fungi. It is a binary heteropolysaccharide which consists of (1-4)-linked 2-acetamide-2-deoxy-b-D-glucopyranose and
2-amino-2-deoxy-b-D-glucopyranose residues (66- 68). Chitosan exhibits a variety of physicochemical and biological properties depending on the molecular weight and deacetylation degree. The degree of acetylation represents the proportion of N-acetyl-D- glucosamine units with respect to the total number of units and can be employed to differentiate between chitin and chitosan. Chitin with a degree of deacetylation of 65-70 % or above is generally known as chitosan. Molecular weight of chitosan may range between 10,000 to 2 million Dalton (67).
Chitosan is degraded in vivo via enzymatic hydrolysis by lysozyme which is normally produced by macrophages. After the dissolution of the polymer in acidic media, the amino groups become protonated and render the molecule positively charged (67).
Due to the cationic nature of chitosan, it interacts with anionic glycosaminoglycans, proteoglycans and other negatively charged molecules. Since great number of cytokines and growth factors are linked to glycosaminoglycans, a scaffold containing chitosan- glycosaminoglycans complex is expected to carry growth factors more efficiently (69).
Chitosan is a promising polymer for tissue engineering due to its favorable properties such as being non toxic, non allergenic, mucoadhesive, biocompatible and biodegradable, and also accelerating cell proliferation. Furthermore, it has structural similarity to glycosaminoglycans which are the major component of the extracellular matrix (70-72). It is a also good candidate for gene delivery because due to its positive charge, it makes complex with negatively charged DNA, and protects it from nuclease degradation (73).
The type of chitosan has significant effects on scaffold properties. Molecular weight of chitosan has been shown to influence swelling and biodegradation properties as well as cell proliferation (74). A decrease in molecular weight resulted in lower water uptake and favored dissolution (75). The degradation rate is inversely related to the degree of crystallinity which is controlled mainly by the degree of deacetylation (DD), hence the higher the deacetylation results in lower degradation rate. As a result, highly
deacetylated form is expected to survive in vivo for months. Depending upon the degradation rate, both the mechanical and solubility properties are affected (76). It was shown that 95% deacetylated chitosan showed better mechanical property than that of 88% deacetylated chitosan of similar molecular weight. On the other hand cytocompatibility and morphology of the different deacetylated chitosan were found to be similar (74).
While chitosan is a promising scaffold material, it has still some limitations. It was reported that the degradation products of chitosan can significantly change the angiogenic behavior of endothelial cells at cellular and molecular levels (77). Moreover chitosan has poor solubility (71), and the mechanical strength of chitosan scaffolds needs be improved. In several investigations, it was shown that this natural polymer is lack of long term stability (78). To achieve the desired mechanical properties of chitosan scaffolds, bioceramics such as hydroxyapatite (10,65,70,79), or calcium phosphate (80); biomaterials like gelatin (72,81), collagen (13,71), alginate (18) or inorganic material such as wollastonite (82,83) can be used.
Application of chitosan in hard tissue engineering In this section, the applications of chitosan as scaffold for tissue engineering will be reviewed for various hard tissues such as bone, cartilage and periodontal tissue.
Bone tissue
In general, bone defects resulting from trauma, tumor, infections, biochemical disorders, or abnormal skeletal development require surgical intervention.
Although the use of bone grafts is an option for bone repair and regeneration, it has serious limitations such as necessity of an extra surgery, morbidity, pain and hypersensitivity at the donor site and limited amount of collection (84). One of the main approaches to overcome these problems is tissue engineering.
Among the various approaches for tissue engineering, as chitosan is generally applied as scaffolds, it will be focused only on scaffolds in this section.
Mechanical properties are critical for the scaffolds of hard tissues like bone and cartilage for transmission
of mechanical force and matrix mineralization formation (70). To meet the desired mechanical and chemical requirements for bone tissue engineering applications, different preparation methods and scaffold structures have been investigated.
Chitosan has been widely used as a scaffold material for bone tissue engineering and the most commonly used preparation methods are lyophilization, bio- mineralization, particle aggregation, electro spinning and gelation. Recent studies on applications of chitosan in bone tissue engineering are summarized in Table 1.
Chitosan can be used alone in preparation of the scaffold. Malafaya et al. (11) has reported that mechanical stability of the chitosan based scaffold was increased with particle aggregation method and after its in vivo application, enhanced organization of the extracellular matrix (ECM) and neovascularization was observed (Fig. 1).
In general, chitosan has been combined with hydroxyapatite (HA) to form scaffolds in order to improve the mechanical properties. Particle aggregated HA/chitosan scaffold was prepared, and shown that mechanical stability was provided even at high frequencies. However it was found to be cytotoxic to L929 fibroblast cells when compared to chitosan alone (65). The significant increase in compression modulus of the chitosan-HA composite was attributed to the strong interaction between chitosan and HA to form a chitosan–HA complex in bone tissue engineering (65,70,75). In a nanocomposite scaffold, HA nanoparticles were uniformly covered by the organic chitosan network, and the interactions between the network and HA (HA with NH+3) was reported to be similar to those occurring between the components of bone (Ca2+ and PO43-) (70).
Another scaffold composed of chitosan-HA, prepared with the same type of chitosan but by a different preparation method, was shown to have good biocompatibility and besides the increased mechanical properties, cell differentiation to osteoblasts and chondrocytes was also observed (75). In preparation of the scaffold, HA was
Table 1. Recent studies on chitosan based systems for bone tissue engineering
Scaffold content
Pore size Chitosan type
Preparation method
Form
In vitro testing Mechanical Ref
properties Cell culture studies on scaffold PLGA/nHA,
CS/pDNA nanoparticles (encoding BMP-2)
Particle size: 100 nm
Medium MW CS ( DD: 75-85%, Sigma Aldrich, USA)
Electrospinning Membrane (Microfiber)
Increased tensile
strength High cell attachment and high cell viability, increased DNA release when CS nanoparticles before electrospinning;
cytotoxic and high transfection efficiency when CS nanoparticles added after electrospinnig Human marrow stem cells
10
nHA/CS
Pore size: 45-125 μm Medium MW CS 250 kDa, DD: 75%;
High MW CS 400 kDa, DD:83%, (Aldrich, USA)
Lyophilization Sponge
Induced compression
modulus Increased cell attachment, 1.5 times greater cell proliferation, Mouse preosteoblasts (MC3T3-E1) 70
nHA, CS/gelatin Particle size: 17–25 nm
CS (MW 2.0x102 kDa, DD: 85%, Qingdao Medical Institute, China)
Biomineralization
Film NS
Good biocompatibility, increased cell attachment and proliferation, allowed osteogenic differentiation Mesenchymal stem cells
79
CS/poly(butylene succinate), CS nanofibres
Particle size: 500 μm
Medium MW CS ( DD: 85%, France Chitin, France)
Electrospinning Membrane (Microfiber)
Increased tensile strength, no significant difference in tensile stress
NS 85
PLA, CS
microspheres, BMP- 2 derived synthetic peptide
Pore size: 100-300 μm
CS (MW: 2.0x102 kDa, DD: 90%, Beijing Chemical Reagents Company, China)
Lyophilization Sponge
Significantly increased compressive strength and modulus
NS 86
CS, biphasic calcium phosphate Pore size: 100 μm
CS (800 kDa, DD:
>85%, Sigma, USA) Lyophilization Sponge
Porosity: 80 % cell attachment and spreading, more prominent actin cytoskeletons, significantly higher ALP activity and osteocalcin production Mouse mesenchymal stem cells, preosteoblasts
87
CS (drug loaded) Medium MW CS
( DD: 85%, Aldrich) Lyophilization (loaded with su- percritical fluid technology) Sponge
Porosity: 87 %
NS 88
CS/PLGA microspheres Particle size:500- 710μm
CS (DD: 83.3 %, Vanson HaloSources, Inc., USA)
Sintering by heat-
ing Significantly increased compressive modulus and compressive strength; decreased porosity with increasing sintering temperature Porosity: 28-37 %
Significantly increased ALP activity, no significant difference in cell proliferation, significantly higher osteopontin and bone sialoprotein Mesenchymal stem cells
89
CS:Chitosan, CML: water-soluble chitosan derivative, CSC: chondroitin-6-sulfate, DD: degree of deacetylation, DS:
dermatan sulfate, ECM: extracellular matrix, HA: hydroxyapatite, MW: Molecular weight, NS: not studied, PLA:
poly(lactic acid), PLGA: Poly (DL-lactide-co-glycolide)
impregnated in a polyurethane (PU) sponge, then PU-HA sponge was burned in the furnace to remove the organic matrix. Then 3% chitosan gel and HA scaffold was placed in a mold and lyophilized.
HA/chitosan/gelatin based scaffolds, prepared by biomineralization method in Ca(NO3)2-Na3PO4 tris buffer solution, were also studied, and increased cell attachment and proliferation was shown besides the osteogenic differentiation.
However, instability of the particulate HA is often encountered when the particles are mixed with saline or patient’s blood and hence migrate from the implanted site into surrounding tissues by causing damage to healthy tissue (94).
Biodegredable and FDA approved poly(lactic acid- glycolic acid) is another polymer used for the formation of composites with chitosan (10,89). It has been shown that the scaffold built by chitosan-PLGA composite is capable of supporting osteoblastic cell attachment and proliferation. In a study on PLGA/nanoHA scaffold with dispersed chitosan nanoparticles, even though all the scaffolds were prepared by electrospinning, addition stage of the chitosan nanoparticles were found to affect the properties of the scaffold. When the chitosan nanoparticles were added to the scaffold before electrospinning, tensile strength, DNA release and cell viability was found to increase significantly, whereas when added after electrospinning, it was found to be cytotoxic (10).
In another study, chitosan-PLGA microspheres were incorporated with sintern into chitosan-PLGA scaffold, and were compared to PLGA scaffolds without chitosan. It was shown that the presence of
chitosan facilitated the maturation of the MC3T3-E1 cells, and higher mineralized matrix formation besides the enhanced osteoblast phenotype expression and differentiation was observed (89).
Beside the different preparation methods and scaffold contents, particle size was also found to affect the scaffold properties. The use of nanoparticles was shown to have advantages over microparticles in bone tissue engineering applications (95). The decrease in size was reported to result in increased cellular adhesion, and also enhanced osteoblast proliferation and differentiation.
Cartilage tissue
Osteochondral defects are lesions of the articular cartilage where the underlying bone tissue is also damaged. Since cartilage is an avascular tissue, it can hardly heal itself (55,91). Currently, osteochondral defects are mostly treated by (i) osteochondral autograft transfer; (ii) filling the lesion with autologous, precultured chondrocytes (autologous chondrocyte transplantation, ACT); or (iii) matrix- induced autologous chondrocyte implantation (65,91).
Although some studies have achieved to repair small cartilage defects, no accepted method for complete repair of osteochondral defects exists (65). Hence, engineered cartilage tissues appear to be promising for the treatment of the osteochondral defects.
Scaffold systems have been developed by different preparation methods using combination of different polymers. Applications of chitosan in cartilage tissue engineering are summarized in Table 2.
Chitosan based hydrogels have been prepared in commonly in presence of polyvinyl alcohol due to Figure 1. The growing connective tissue and increased neo-vascularization in particle-aggregated scaffolds (A) 1 week after; B) 2 weeks after, and C) 12 weeks after implantation (11).
Table 2. Recent studies on chitosan based systems for cartilage tissue engineering
Scaffold content
Pore Size Chitosan type
Preparation method
Form
In vitro testing Mechanical Ref
properties Cell culture studies on scaffold CSParticle size: 375-
485μm
Pore size: 240-290 μm
Medium MW CS (DD: 85%, Sigma- Aldrich)
Particle
aggregation Mechanically stable for low frequencies Porosity: 26-30 % Interconnectivity:
96-94 %
NS
11
CS/Pluronic hydrogel Pore size: 10-100 µm
High MW CS (DD: 75-85%, Sigma–Aldrich, USA)
Gelation Gel
Increased storage and loss moduli with the increasing cross- linking
sufficient mechanical strength to retain the structure and shape, interconnected pores
Improved cell attachment, significant cell proliferation, increased GAG content Chondrocytes
12
CML hydrogel CS (MW: 6.2 x 105 Da, DD:
78%, Haidebei Company, China)
Gelation
Gel NS
Effective interaction between cells and scaffold,
Chondrocyte
42
PVA/NOCC CS (The Standards and Industrial Research Institute, Malaysia)
Gelation Gel
No significant difference in stress relaxation functions Porosity: 43.3%
NS
55
HA/CS Particle size: 410- 460 μm
Pore size: 225-290 μm
Medium MW CS (DD: 85%, Sigma–
Aldrich)
Particle
aggregation Mechanically stable even for high frequencies Porosity: 28-34 % Interconnectivity:
92-96 %
Decrease in cytotoxicity with higher concentration of glycine
significant increase in cell viability with sintered HA, no significant decrease in cell viability with increasing glutaraldehyde concentration,
reduced cell attachment in the absence of Ca2+
L929 fibroblast cell line
65
HA/CS Pore size: 50-500 µm
Medium MW CS (DD: 85%, Aldrich, Germany)
Sintering by heating then lyophilization Bilayered sponge
High compression modulus, highly interconnected pores Porosity of HA: 60%
Porosity of CS: 75%
Adequate cell attachment, proliferation and differentiation to osteoblasts and chondrocytes, increased ALP activity, no cytotoxic effect
Goat bone marrow stromal cells, L929 fibroblast cell line
75
CS-graft-glycolic acid/phloretic acid
Low MW CS (DD: 85%, Aldrich)
Gelation Gel
Increase in storage modulus, highly elastic
Predominantly living cell (>90%) existence, uniform cell distribution, cell differentiation Chondrocytes
90
Poly (DL-lactide)/
CSPore size: 3-200 µm
CS (MW: 1.41 ± 0.19 x 106 Da, DD:
84%, Fluka)
Solvent extraction, phase separation, freeze drying Sponge
Interconnected with irregular shapes, increased tensile strength and young’s modulus,
Porosity: 83.7-85.3%
Significant cell proliferation, increased GAG and type II collagen production
Chondrocytes
91
CSC/DS/CS Pore size : 100–200 µm
CS (MW: 2.0 x 106
Da, DD: 85%) Lyophilization Sponge
Porosity: 88-91% No significant change in cell attachment and proliferation, significantly increased GAG and collagen production
Chondrocytes
92
Gelatin/CS/
hyaluronan, PLGA microspheres Pore size: 200 µm Particle size: 5-40 µm
CS (MW: 6.2 x 105 Da, DD: 85%, Qingdao Haidebei Bioengineering Co. Ltd., China)
Lyophilization Sponge
Significantly increased compressive modulus,
interconnected pores Porosity: 83-95%
formation of larger aggregates, no significant difference in cell proliferation and ECM secretion compared to control groups Chondrocytes
93
CS:Chitosan, CML: water-soluble chitosan derivative, CSC: chondroitin-6-sulfate, DD: degree of deacetylation, DS:
dermatan sulfate, ECM: extracellular matrix, HA: hydroxyapatite, GAG: glycosaminoglycans, MW: Molecular weight, NS: not studied, NOCC: N,O-carboxymethylated chitosan, PLA: poly(lactic acid), PLGA: Poly (DL-lactide-co-glycolide)
its excellent weight bearing properties, low friction coefficient and biocompatibility (55, 96).
Chitosan based thermosensitive hydrogels have also been studied for the repair of cartilage in order to increase cell seeding efficiency, and to prevent necrosis of seeded cells in the core of scaffold (12).
These injectable systems can form a gel at the applied site at the body temperature before becoming rigid (97). Among the advantages of thermosensitive gels are no need for surgery, high cell seeding efficiency, good transport and easily corporation with therapeutics drugs. On the other hand, these systems can be inadequate for mechanical support and can have stability problems (12,98), though it was shown that chitosan-pluronic (CP) graft copolymer thermosensitive hydrogel system has adequate mechanical strength to retain the structure and shape.
The storage and loss moduli of the 20 % CP hydrogel was found to be higher than that of 16 % CP hydrogel (Fig. 2). These results indicated that introducing the chitosan to the formulation increases the mechanical strength and stability of the CP hydrogel (12).
Chitosan-PDLL (poly(DL-lactide)) (91) and gelatin- chitosan-hyaluronan with PLGA microspheres (93) based scaffolds have also been studied for cartilage tissue engineering. Chitosan-PDLL sponge was prepared by solvent extraction at ambient temperature for 2 days, phase separation and lyophilization at -75ºC (99). It was reported that the
concentration of sodium hydroxyde in the extraction solution, the composition ratio of components and the freezing temperature were the determining parameters for its morphology. Gelatin-chitosan- hyaluronan sponges were prepared by freeze drying.
Both of the sponge forms were found to be highly interconnected and their porosity values were larger than 83%. After chondrocytes seeding, significant cell proliferation was observed on the chitosan-PDLL scaffolds whereas no significant proliferation was observed on the gelatin-chitosan-hyaluronan sponge until the second week. After the second week, high cell activity was observed.
Periodontal tissue
Periodontal diseases (e.g. periodontitis, gingivitis) can lead to destruction of periodontal tissues like gingival, alveolar bone, periodontal ligament (PDL), and cementum (100). Periodontitis, one of the most common infections in humans, affecting in its most severe form, approximately 10% of the population can lead to tooth loss (101). There are various methods for the treatment such as autogenous, allogenic bone grafts implantation (7) however with many limitations. Low number of cells at periodontium can be the main limitation. Since the defect hardly has the optimal conditions for the migration, proliferation, differentiation or protein synthesis of cells, the treatment which depends on the cells reside of the periodontium is questionable (102).
Therefore tissue engineering approach is promising for periodontal tissue.
Figure 2. The storage modulus (G’) and loss modulus (G’’) of the 16 % (A) and 20 % (B) chitosan-pluronic hydrogel in phosphate-buffered saline buffer (the applied frequency: 0.1 Hz) (12).
There are many studies in which chitosan based scaffolds have been investigated for periodontal tissue
engineering. Applications of chitosan in periodontal tissue regeneration are summarized in Table 3.
Table 3. Recent studies on chitosan based systems for periodontal tissue engineering Scaffold
content /
Pore Size Chitosan type
Preparation method /
Form
In vitro testing
In vivo testing Ref Mechanical
properties Cell culture studies on scaffold CS/collagen,
CS/pDNA nanoparticles (encoding PDGF), Particle size 30-40 nm
Pore size: 200-300 μm
CS (DD:>
85%, Sigma, USA)
Lyophilization Sponge with nanoparticles
High degree of interconnectivity, Porosity: > 90%
Improved growth rate and proliferation, formation of periodontal tissue-like structure,
PDL cells
NS 9
CS/coral (calcium carbonate), pDNA (encoding PDGFB)
Pore size: 200-300 μm
CS
(DD:> 85%) Lyophilization Sponge
NS
Significant cell proliferation, no cytotoxicity, significantly increased mRNA expression levels of PDGFB
HPLCs
Athymic mice
Sampling: Day 2, Week 4 (Applied with HPLC cells)
No inflammation, increased expression of PDGFB, cell proliferation, new vascular tissue growth
7
HA beads (bFGF)- CS
Particle size: 40 μm
Pore size: 20-100 μm
CS (DD: >85%, Sigma- Aldrich)
Lyophilization Sponge
Interconnected
structure significant increase in the cementoblast and PDL cell counts,
well organized F-actin meshwork,
higher mineralization, significantly higher alkaline phosphatese activity
PDL cells, cementoblasts
NS 102
CS/taurine Chitosan-H (DD: 80%, MW:
1400kDa)
Solvent casting
Film NS NS
Beagle dogs Cellular activity observed both in the mitochondria of fibroblasts and macrophages
104
CS/collagen/
demineralized bone matrix
Protasan UP CL213, (MW:
252.000Da;
DD: 84%, Pronova)
Gelation
Gel
Membrane NS NS
Human
Sampling: 0,3,6 months Statistically significant bone fills compared to Day 0
105
CS/ bovine type I collagen/
Plasmid and virus encoding TGF-ß1 gene
Pore size: 200 μm
Chitosan (DD: 85%, Sigma, USA)
Lyophilization Sponge
Interconnected structure Porosity:80%
Better proliferation
HPLCs
Athymic mice Sampling:week 2
higher transfer efficiency 107
bFGF: basic fibroblast growth factor, CS: chitosan, DD: degree of deacetylation, HA: hydroxyapatite, HPLCs: human periodontal ligament cells, MW: molecular weight, NS: not studied, PDGF: platelet derived growth factor, PDL: periodontal ligament
Effect of chitosan on osteoblast and fibroblast cell attachment was studied in vitro (103). Mouse MC3T3-E1 osteoblasts and 3T3 fibroblasts were grown in the presence of serum on acid soluble and water soluble chitosans. Cell attachment and immunofluorescent analysis at various time points were done to analyze initial phenotypic profiles. Our results suggested that chitosan supports the initial attachment and spreading of osteoblasts preferentially over fibroblasts, and that manipulation of the biopolymer can alter the level of attachment and spreading.
A chitosan film was prepared incorporated with an amino acid, taurine, which is considered to be beneficial for regulating the inflammation process.
The synergistic effects of taurine and chitosan in the experimental defects at the vestibular bone of maxillary canine teeth in dogs has been investigated (104). Cellular activity was observed both in the mitochondria of fibroblasts and macrophages. These ultrastructural alterations were thought to be the sign of the disturbed balance between the generated oxidants and antioxidant defense mechanisms.
Taurine appeared to enhance the acceleration effect of chitosan on wound healing at early periods. This effect can be considered beneficial in tissue repair in destructive diseases like periodontitis.
In another study, the effect of chitosan gel combined with demineralized bone matrix or collagenous membrane on periodontal regeneration has been investigated in twenty chronic periodontitis patients (105). Significant bone healing was observed when compared with baseline indicating that chitosan gel alone or its combination with demineralized bone matrix/collagenous membrane is promising for periodontal regeneration.
The effects of many growth factors on periodontal tissue cells have been evaluated for their implication in periodontal tissue engineering using chitosan scaffolds (106). Porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-b1 (TGF-b1) (107). Results indicated that the pore diameter of the gene combined scaffolds was lower than that of pure
chitosan/collagen scaffold. After implanted in vivo, EGFP-transfected HPLCs not only were found to proliferate but also recruit surrounding tissue to grow in the scaffold, demonstrating the potential of chitosan/
collagen scaffold combined TGF-b1 as a good substrate candidate in periodontal tissue engineering.
It was reported that with bFGF loaded HA beads- chitosan scaffolds a significant increase was observed in the number of cementoblasts which are the main cells of cementum production as well as in the number of PDL cells which are the primary fibroblastic cells (102) .
Chitosan/coral sponge with platelet-derived growth factor B (PDGFB) encoding pDNA was prepared to construct periodontal tissue. Increased expression of PDGFB and significant cell proliferation was observed in vitro, and increased expression of PDGFB and new vascular tissue growth were observed in vivo (7).
Conclusion
In the light of the recent studies reviewed in this article, it is obvious that chitosan is a promising candidate as a supporting material for hard tissue engineering applications owing to its porous structure, gel forming properties, ease of chemical modification, and high affinity to in vivo macromolecules. Yet, more in vivo studies are needed to bring the chitosan- based products on the market in the near future.
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