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Prospects of chitosan-based scaffolds for growth factor release in tissue engineering P.R. Sivashankari, M. Prabaharan ∗ Department of Chemistry, Hindustan Institute of Technology and Science, Padur, Chennai 603 103, India

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Article history: Received 25 January 2016 Received in revised form 12 February 2016 Accepted 14 February 2016 Available online xxx Keywords: Chitosan Growth factor Scaffolds Tissue engineering Wound healing

a b s t r a c t Tissue engineering is concerned about the rejuvenation and restoration of diseased and damages tissues/organs using man-made scaffolds that mimic the native environment of the cells. In recent years, a variety of biocompatible and biodegradable natural materials is employed for the fabrication of such scaffolds. Of these natural materials, chitosan is the most preferred one as it imitates the extracellular matrix (ECM) of the cells. Moreover, chitosan-based materials are pro-angiogenic and have antibacterial activity. These materials can be easily fabricated into the desired shape of the scaffolds that are suitable for tissue support and regeneration. Growth factors are small proteins/peptides that support and enhance the growth and differentiation of cells into a specific lineage. It has been observed that scaffolds capable of delivering growth factor promote tissue repair and regeneration at a faster rate when compared to scaffolds without growth factor. The present review focuses on the recent developments on chitosan-based scaffolds for the delivery of growth factors thereby improving and enhancing tissue regeneration. © 2016 Published by Elsevier B.V.

1. Introduction Tissue engineering is the field that deals with restoration of diseased or damaged tissues and organs by controlling the biological microenvironment [1,2]. Tissue engineering consists of a complex cascade of events which includes cell proliferation, differentiation, and synthesis of extracellular matrix [3]. A perfect tissue engineering scaffold should be a template for three-dimensional (3D) growths of tissues by providing porous structure for the growth of tissues, diffusion of oxygen, and delivery of nutrients and it should also mimic the natural microenvironment of the tissue. It should also interact with the surrounding cells and maintain the phenotype of the regenerated tissue. An ideal scaffold should be biocompatible, biodegradable, promote cell adhesion, proliferation and maintains the metabolic activity of the cells [4,5]. Moreover, the scaffolds with suitable pluripotent stem cells, angiogenic potential and prolonged nutrient supply will support the repair and regeneration of various tissues [6]. Chitosan, poly(␤-(1–4)-linked-2-amino-2-deoxy-O-glucose), is a biocompatible and biodegradable natural polymer. It is obtained by the partial deacetylation of chitin that is found in the exoskele-

∗ Corresponding author. Fax: +91 44 2747 4208. E-mail address: [email protected] (M. Prabaharan).

ton of many crustaceans and has many desirable properties in order to use as a biomaterial for tissue engineering and regenerative medicine [7]. Due to the presence of positively charged amino groups, chitosan is mucoadhesive, hemostatic, and capable of binding with cell membranes [8–10]. Chitosan has an ability to make the scaffolds with well interconnected porosity and desired shapes such as hydrogels, sponges, two-dimensional fibers/sheets and 3D porous structures for the improved cell viability by providing the supply of enough oxygen and nutrients [11–13]. Chitosan-based scaffold materials can also exhibit a controlled delivery of loaded therapeutic molecules and growth factors, which makes them a suitable candidate for tissue engineering and regenerative applications. In addition, due to the presence of primary amine and hydroxyl groups, chitosan can be chemically modified to obtain the verities of its derivatives with desired functionalities and properties. During the tissue regeneration, growth factors play a vital role as the essential signaling molecules to initiate the cells for carrying out specific cellular response in the biological environment and proceeding towards the specific lineage [14]. Hence, tissue regeneration can be successfully achieved by controlling the local delivery of growth factors. Different types of growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) have been considered for the

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process of vascularization in bones [15]. It was found that basic fibroblast growth factor (bFGF) promotes angiogenesis, osteogenesis and nerve regeneration [16]. In addition, bFGF and TGF-␤ was found to play an important role in the initiation and progression of tissue repair [17]. In recent years, various types of scaffolds based on chitosan alone or chitosan combined with other biomaterials have been developed for the loading and delivery of growth factors. These scaffolds have shown their ability to accelerate the regeneration of tissues at a higher rate. The aim of this review is to discuss the recent developments of chitosan-based scaffolds designed for the delivery of growth factors in bone, periodontal, nerve, cartilage, and skin tissue engineering and regeneration.

2. Delivery of growth factors 2.1. In bone tissue engineering Bone regeneration and repair is an intricate and challenging process, which involves the role of various hormones, growth factors, and cytokines. A complex cascade of molecular events governs the bone regeneration process. Among the various growth factors involved in bone regeneration, bone morphogenetic proteins (BMPs) such as BMP-2, BMP-6 and BMP-7 that belongs to TGF␤ family are the most important and influence bone repair to a great extent. It is proved that BMPs are capable of inducing in vitro osteoblast differentiation, in vivo bone formation and increased alkaline phosphatase (ALP) expression which is the early indicator of cellular differentiation towards osteoblast phenotype [18]. Although BMPs are highly required for bone formation, their structural complicity, systemic side effects, short half-life and rapid clearance from the system are some of the hurdles in current usage of these growth factors. An ideal carrier for the delivery of BMPs should exhibit overall increased total release amount as well as a sustained release for the prolonged time. Moreover, the carrier material should protect the protein from denaturation. In this context, chitosan-based materials can be ideal for the immobilization and delivery of BMP-2 since BMP-2 maintains its intact structure at the isoelectric point between 5 and 6 that is close to the isoelectric point of chitosan [19]. BMP-2-loaded polyelectrolyte complex made up of chitosan and hyaluronic acid exhibited controlled release of the growth factor in pre-osteoblastic cells in vitro [20]. Real-Time PCR analysis revealed the increased expression of pre-osteogenic genes in the cells treated with BMP-2-loaded chitosan-hyaluronic acid microspheres. It is found that negatively charged heparin forms a complex with the basic amino acids of BMP-2 and thus confers good stability and controlled release to the growth factor. Since sulfated chitosan mimics the structure of heparin, it was proved to be a better candidate for the delivery of BMP-2 in vitro. Cao et al. developed gelatin hydrogels loaded with recombinant human BMP-2 (rhBMP-2) encapsulated 2-N, 6-O-sulfated chitosan nanoparticles for bone regeneration [21]. These systems protected and enhanced the bioactivity of encapsulated rhBMP-2 due to the formation of electrostatic assemblies of sulfated chitosan nanoparticles in the hydrogel network. Due to the presence of rhBMP-2, the scaffold systems enhanced the ALP activity and mineralization in cultured human mesenchymal stem cells (hMSCs). The rhBMP-2 loaded scaffolds were also found to exhibit the complete regeneration of critical size defect induced in rabbit femur and an increased bone mineral content and bone mineral density when compared to the control groups. The gelatin hydrogels loaded with sulfated chitosan nanoparticles presented a two-phase release of the encapsulated rhBMP-2 as shown in Fig. 1. First, an initial burst from the partially swollen hydrogels was observed. Thereafter, they showed a sustained release as the hydrogel degraded over a period of time. Niu et al. fabricated chitosan microspheres Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2016.02.043

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loaded with BMP-2 derived synthetic peptide by emulsion method using sodium tripolyphosphate (TPP) as an ionic cross-linking agent [22]. These microspheres were found to be possessed 80% encapsulation efficiency and exhibited slow release up to 7 days at pH 7.4. The peptide released from the microspheres was found to be retained its biological activity in vitro. For in vivo tissue engineering application, 3D scaffolds are advantageous as they possess appreciable mechanical strength and provide a suitable microenvironment for the growth of cells. In this respect, a porous composite scaffold made up of nanohydroxyapatite, collagen and poly (l-lactic acid) (PLA) loaded with growth factor encapsulated chitosan microparticles was developed as a promising material for tissue engineering [23]. In this work, chitosan microspheres loaded with bovine serum albumin (BSA) and BMP-2 were prepared by the emulsion method using TPP as a cross-linking agent. The microspheres were found to be spherical in shape with the sizes in the range of 10–60 ␮m as shown in Fig. 2. It was found that the mechanical property and degradation rate of the 3D composite scaffold was increased due to the presence of encapsulated chitosan microspheres. This composite scaffold was found to mimic the natural bone microenvironment and showed an appreciable in vitro bioactivity. Ferrand et al. developed an improved method for the delivery of BMP-2 for bone regeneration using electrospun poly(␧-caprolactone) nanofibers [24]. In this study, BMP-2 combined with chitosan was coated onto the surface of poly(␧-caprolactone) nanofibers by the layer-bylayer (LBL) method. The nanofibers prepared using this technique were found to mimic the fibrillar nature of the bone matrix. Moreover, they exhibited an improved in vitro and in vivo osteopontin gene expression and calcium phosphate biomineralization. As the small amount of BMP-2 protein is used, LBL method is more economic and leads to reduced side effects due to BMP-2 overdosing. Freeze-dried chitosan scaffolds loaded with BMP-6 were developed by embedding technique [25]. The loading of BMP-6 was determined as 100 ng/3 mg of dry chitosan scaffolds. When compared to the control, BMP-6-loaded scaffolds exhibited an increase in the expression of osteocalcin, alkaline phosphatase as well as mineralization, which infers that BMP-6-loaded chitosan scaffolds support and enhance the osteogenesis in vitro. Macroporous chitosan scaffolds loaded with either BMP-2 or insulin-like growth factor (IGF-1) was studied for their bone healing property in vivo [26]. In this study, chitosan scaffold with varying pore size from 70 to 900 ␮m was prepared by liquid hardening method. The absorption efficiency of BMP-2 and IGF-1 was found to be 87 ± 2% and 90 ± 2%, respectively. In the in vivo rabbit models, chitosan scaffolds loaded with IGF-1 exhibited good osteoblastic differentiation than BMP-2-loaded chitosan scaffolds. Sintered porous scaffolds based on chitosan-poly(lactide-co-glycolide) (PLGA) microspheres loaded with heparin and rhBMP-2 were developed and studied their bone regeneration ability in vivo in a rabbit model [27]. The rhBMP-2 loaded scaffolds showed an improved mechanical strength than growth factor free scaffolds. Moreover, they found to accelerate bone formation more efficiently. A simultaneous or subsequent release of two or more growth factors can accelerate bone regeneration more efficiently when compared to single growth factor release. Yilgor et al. prepared chitosan-poly(ethylene oxide) blended fibers by the wet spinning method using acetic acid as a solvent [28]. These blended fibers showed an improved stability and fiber thickness due to the increased total polymer concentration. It was observed that the concentration of acetic acid had an effect on the surface morphology of the fibers. The fibers prepared in 2% and 5% acetic acid showed smooth and rough surfaces, respectively (Fig. 3). In this study, BMP-2 encapsulated PLGA and BMP-7 encapsulated poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocapsules were loaded within or surface of the chitosan-poly(ethylene oxide)

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Fig. 1. Schematic representation of rhBMP-2 release mechanism from gelatin hydrogels loaded with rhBMP-2 encapsulated sulfated chitosan nanoparticles [21].

Fig. 2. TEM images of chitosan microsphere loaded with (A) BSA and (B) BMP-2 [23].

Fig. 3. SEM images of chitosan-poly(ethylene oxide) fibers prepared in (A) 2% acetic acid and (B) 5% acetic acid [28].

blended fibers. Sustained release of growth factors was found from the nanocapsules loaded within the fibers rather than those loaded on the surface of the fibers. It was observed that the sequential delivery of BMP-2 followed by BMP-7 from the fibers, which facilitated the differentiation of hMSCs more effectively. Chitosan-collagen scaffold prepared by the freeze-drying method was studied as a potential material for the dual delivery of BMP-7 and PDGF-B [29]. Although BMP-7-loaded scaffolds exhibited good in vitro osteoblast differentiation and in vivo bone formation, the

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simultaneous delivery of BMP-7 and PDGF-B from the scaffolds exhibited an efficient bone healing activity in vivo when compared to scaffolds delivering a single growth factor (either BMP-7 or PDGF-B). In the similar line, dual growth factor expressing chitosancollagen scaffolds loaded with BMP-2 and VEGF coding adenovirus were also developed for the increased bone regeneration in and around dental implant [30]. It was found that the ALP expression of the scaffolds loaded with BMP-2 was higher than that of the scaffolds loaded with VEGF. In vivo experiments showed that the

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Fig. 4. Schematic representation for immobilization of growth factors onto chitosan hydrogel [57].

O

Chitosan-NH-CO-CH2(CH2)4CH2-CO-O N Activated chitosan

H2N-RGD

Chitosan-NH-CO-CH2(CH2)4CH2-CONH-RGD RGD immobilized chitosan

O

H2N-EGF

Chitosan-NH-CO-CH2(CH2)4CH2-CONH-EGF EGF immobilized chitosan

Fig. 5. Immobilization of RGD and EGF onto chitosan scaffolds [61].

percentage bone defect filling and new bone-to-implant contact in the scaffolds loaded with only BMP-2 and both BMP-2 and VEGF was improved. Similar like BMPs, the growth factors VEGF and PDGF also involved in the regeneration of bone defects. VEGF is an angiogenesis inducer which plays an important role in postnatal neovascularization. It prolongs cell survival, osteoblast proliferation, differentiation and migration, thereby promotes bone formation by acting in combination with other osteogenic proteins. PDGF-B is an important growth factor for the bone healing process where it is capable of mediating mesenchymal cell proliferation, differentiation as well as chemotaxis. Therefore, delivery of VEGF and PDGF at the site of bone injury can accelerate bone healing process more efficiently. Recently, brushite-chitosan sponges loaded with VEGF encapsulated alginate microspheres and PDGF were prepared and their release mechanism was studied [31]. It was observed that PDGF released more rapidly than VEGF from the scaffold and both the growth factors were located around the site where the scaffold was implanted with only slight systemic exposure. The in vivo studies with rabbit femur proved that this hybrid scaffold is capable of providing sustained release and localization of both the growth factors. A composite scaffold fabricated using the mixture of alginate, chitosan and PLA and tested for its efficiency in sustained release of VEGF for neovascularization during bone healing [32]. To avoid the burst release, VEGF was encapsulated within alginate microspheres and loaded into the composite scaffold. This

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scaffold system exhibited initial 13% release of VEGF within first 24 h followed by sustained release up to 5 weeks. Angiogenic gene-activated porous chitosan-hydroxyapatite hybrid scaffolds with pore size about 150–400 ␮m were prepared and analyzed for their potential to induce neovascularization in ectopic bone formation [33]. The attachment, proliferation, and differentiation of human osteoblasts (hOBs) on gene-activated scaffolds were evaluated in vitro and in vivo. The results showed that hOBs cultured on gene-activated scaffolds secreted VEGF, besides maintaining its characteristic phenotype with specific ECM production. In vivo results revealed that gene-activated chitosanhydroxyapatite scaffolds were tissue biocompatible, and provided a suitable environment for neovessel development by employing host endothelial cells into the newly forming ectopic bone-like tissue. Yu et al. developed 2-N, 6-O-sulfated chitosan-coated hierarchical scaffold composed of PLGA microspheres for VEGF delivery [34]. Due to the porous structure and high affinity between VEGF and sulfated chitosan, this scaffold showed an excellent entrapment and sustained release of VEGF. The VEGF-loaded scaffold also demonstrated an improved angiogenesis due to the presence of sulfated chitosan. Recently, hydroxyapatite–chitosan–polyvinyl alcohol composite scaffolds with interconnected porous structures have been considered for craniofacial tissue engineering due to their biocompatibility and sustained release of a pro-neurogenic factor [35]. Release studies showed that these composite scaffolds presented a sustained release of retinoic acid over a

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period of ten days. Moreover, it was found that hydroxyapatite/chitosan/polyvinyl alcohol composite scaffolds provide a better environment for cell attachment and spreading. 2.2. In periodontal tissue engineering Periodontal tissue regeneration is a complex process where the damaged soft and hard periodontal tissues will be cured. Recent advances in tissue engineering considered chitosan-based materials and growth factors to facilitate the regeneration of periodontal tissues. Zhang et al. prepared porous chitosan/collagen scaffolds by a freeze-drying process, and loaded these scaffolds with plasmid and adenoviral vector encoding TGF-␤1 [36]. The pore diameter of the gene-combined scaffolds was found to be lesser than that of control chitosan-collagen scaffold. The scaffold containing adenoviral vector encoding TGF-␤1 showed the highest proliferation rate, and the expression of type I and type III collagen up-regulated in adenoviral vector encoding TGF-␤1 scaffold. This study revealed the potential of chitosan/collagen scaffold loaded with the adenoviral vector encoding TGF-␤1 as a scaffold for periodontal tissue engineering. Peng et al. developed porous chitosan/collagen composite scaffold loaded with PDGF as a gene-activated matrix [37]. The plasmid DNA entrapped in the scaffolds demonstrated a controlled and steady release over 6 weeks and effectively protected by chitosan nanoparticles. MTT assay showed that human periodontal ligament cells (HPDLCs) cultured into the chitosan/collagen composite scaffold achieved high proliferation. Luciferase reporter gene assay showed that this scaffold could express 1.073104 LU/mg proteins after 1 week and 8.973103 LU/mg protein after 2 weeks. These results confirmed that gene-activated matrix based on chitosan/collagen composite scaffold have the potential to be used as periodontal tissue engineering scaffolds. Chitosan scaffolds containing dexamethasone and bFGF were developed for periodontal bone regeneration [38]. Release studies showed that dexamethasone-loaded chitosan scaffolds released total dexamethasone during for 5-days period at a constant rate after the initial burst. However, bFGF release from all scaffolds was found to be completed in 20 h. To increase the release period of bFGF, composite scaffolds were also fabricated using chitosan and hydroxyapatite beads with average particle size of 40 ␮m. Due to the electrostatic interactions between hydroxyapatite and bFGF, controlled release of bFGF up to 7 days was obtained. In the similar line, Akman et al. fabricated a hybrid scaffold based on chitosan and hydroxyapatite loaded with bFGF and assessed its efficacy in the regeneration of periodontal tissues [39]. This growth factor loaded scaffold exhibited initial burst release of bFGF followed by sustained release for up to 168 h. The MTT assay revealed that bFGF-loaded hydroxyapatite–chitosan scaffolds can be used for supporting the cellular structure, proliferation, and mineralization. A thermo-sensitive hydrogel was prepared from chitosan and quaternized chitosan containing ␣, ␤-glycerophosphate for periodontal tissue regeneration [40]. This thermo-sensitive hydrogel was found to be non-toxic and promoted alkaline phosphatase (ALP) activity in HPDLCs in vitro. Moreover, the hydrogel loaded with bFGF had more effect on periodontal regeneration in dogs. Chitosan scaffolds containing BMP-6-loaded alginate microspheres were also developed for periodontal tissue regeneration [41]. Due to the presence of BMP-6-loaded alginate microspheres, the chitosan scaffolds had well interconnected pores and presented a controlled delivery of loaded BMP-6. These results confirmed that controlled release of BMP-6 from the scaffolds had a considerable effect on osteogenic differentiation. Recently, nanocapsules based on PLGA and PHBV containing BMP-4, PDGF, and IGF-I were loaded into chitosan scaffolds for the regeneration of periodontal tissues [42]. The morphology of these scaffolds was found to be a sponge-like open porous Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2016.02.043

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microstructure with pore size about 100–200 ␮m. The in vitro release studies showed the highest proliferation of hMSCs due to the sudden release of BMP-4 and PDGF from the scaffolds. Zhang et al. impregnated PLGA/polyethylene glycol microspheres containing VEGF into collagen-chitosan scaffolds loaded with human adipose-derived stem cells (hASCs) [43]. In vitro testing revealed that the collagen-chitosan scaffold provided a supportive environment for hASC integration and proliferation. In addition, these scaffolds provided a promising, clinically translatable platform for engineering vascularized soft tissue flap. The engineered adipose tissue with a vascular pedicle could be transferred as a vascularized soft tissue pedicle flap to a recipient site for the repair of soft-tissue defects. 2.3. In nerve tissue engineering Brain is the most difficult organ to regenerate after an injury due to its complex architecture and poor ability to regenerate on its own. Therefore, neural stem cell delivery to the site of damage is an essential step for the regeneration of brain cells. In this respect, brain-derived neurotropic factor (BDNF) plays a vital role in the differentiation of the neurons since it is capable of promoting the differentiation of neuronal stem cells into neurons both in vitro as well as in vivo [44]. Shia et al. investigated the efficiency of BDNF mixed chitosan porous scaffolds loaded with human umbilical cord mesenchymal stromal cells (hUCMSCs) in the treatment of traumatic brain injury [45]. Here, chitosan scaffolds prepared by the freeze-drying method were loaded with BDNF using genipin as a natural cross-linker. It was found that the release of BDNF from the scaffolds enhanced the neuronal differentiation of hUCMSCs. The chitosan-based scaffolds loaded with FGF-2 were also found to be highly efficient in neural stem cell growth and survival when compared to control. Skop et al. prepared genipin cross-linked chitosan-heparin microspheres loaded with FGF-2 by coaxial air flow technique for brain nerve regeneration [46]. Due to the binding of FGF-2 with heparin, the biological activity of FGF-2 was found to be retained. Unlike other tissues in the body, peripheral nerve regeneration requires autografts. Due to the limited availability of autografts, the repair of peripheral nerve injury is quite slow and incomplete. In this context, chitosan-based nerve conduits have been considered as potential alternative to the autografts [47,48]. However, due to their brittleness, rigidness, and uncontrollable degradation, their use in nerve tissue engineering is limited. To avoid these drawbacks, nerve conduits were developed using chitosan combined with gelatin. These hybrid nerve conduits were found to have a controlled degradation and improved mechanical properties [49,50]. Chitosan-gelatin conduits loaded with Schwann cells and TGF-␤ exhibited good recovery rate in the in vivo animal models and the overall outcome of these growth factor-loaded chitosangelatin nerve conduits were found to be similar to autografts [51]. The scaffolds loaded with nerve growth factor (NGF) can improve the neurite outgrowth and promote peripheral nerve regeneration both in vitro and in vivo [52]. Zeng et al. developed NGF-loaded chitosan microspheres encapsulated with chitosan-collagen fibers and studied their ability to promote peripheral nerve regeneration. In this study, NGF-loaded chitosan microspheres were prepared by emulsion-ionic cross-linking method and chitosan-collagen scaffolds were prepared by the freeze-drying method [53]. These NGF-loaded chitosan microspheres were evenly distributed in the micro channels of the chitosan-collagen scaffold and exhibited sustained NGF release for up to 28 days. In vivo studies proved that scaffolds with NGF-loaded chitosan microspheres exhibited increased nerve regeneration than the control. A hybrid scaffold based on alginate-chitosan-gelatin loaded with NGF was prepared by particulate leaching method and tested

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for its efficiency in differentiating induced pluripotent stem cells (iPS) towards neuron lineage [54]. This hybrid scaffold exhibited good cytocompatibility for the iPS cells and increase in NGF concentration induced iPS cells differentiation towards neuronal cells. Mottaghitalab et al. proved that NGF-coated chitosan/poly(vinyl alcohol) electrospun nanofibers exhibit an increased surface to volume ratio and provide a favorable environment for the growth and proliferation of cell lines U373-MG (Glioblastoma-astrocytoma) and SKNMC (Human neuroblastomas) [55]. Chitosan-based hydrogels tagged with growth factor were developed for neural stem cell differentiation [56,57]. In this work, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-␥ (IFN-␥), PDGF-AA, or BMP-2 were immobilized to a thiolated methacrylamide chitosan via a streptavidin linker as shown in Fig. 4 to specify neural stem/progenitor cells (NSPCs) differentiation into neurons, oligodendrocytes, or astrocytes, respectively. The bioactivity of immobilized growth factors was found to be maintained up to 28 days. Immunohistochemical staining of in vivo test samples confirmed that growth factorimmobilized scaffolds exhibited an improved neuronal progenitor cell differentiation. Recently, Barough et al. investigated the differentiation ability of human endometrial stem cells cultured on PLA-chitosan nanofibrous scaffold into neuroglial cells in response to conditioned medium of BE(2)-C human neuroblastoma cells and growth factors, FGF2 and PDGF-AA [58]. The results showed that human endometrial stem cells can attach, grow and differentiate on the PLA-chitosan scaffold. These results revealed that of these scaffolds have the potential to differentiate in neuronal and glial cells in the presence of neuroblastoma conditioned medium on PLA-chitosan scaffold.

N-methacrylate glycol chitosan is a water soluble polymer and can be readily injected into the site of defect and subsequently photo-polymerized by visible or UV light. This polymeric system has been considered as a potential tissue engineering material due to its non-toxicity and desired biological properties [64]. Sukarto et al. developed ASCs mixed N-methacrylate glycol chitosan hydrogel loaded with BMP-6 and TGF-␤3 microspheres for the regeneration of chondral defects [65]. Here, the effects of growth factors released from the hydrogel on ASC chondrogenesis were examined. The results showed that there was an increased expression of GAG and collagen type II protein in the N-methacrylate glycol chitosan hydrogel during the controlled and local release of growth factors. End-point Real-Time PCR analysis demonstrated that there was a rapid stimulation and improvement of ASC chondrogenesis in the growth factors-loaded hydrogel. It is known that IGF-1 supports the formation of normal cartilage by promoting the growth and differentiation of articular cartilage. Therefore, IGF-1-loaded sintered chitosan microparticles were developed for chondral regeneration [66]. In this work, IGF-1loaded microparticles were sintered in an oven at 60 ◦ C for 3 days in a mold and the resulting scaffold was tested for its release profile. It was found that the scaffolds with the high amount of IGF-1 presented a better release profile for chondral regeneration. Recently, BSA-loaded porous chitosan-alginate hybrid scaffolds cross-liked with TPP and formaldehyde were developed for better cartilage tissue engineering applications [67]. The maximum BSA release was observed from the hybrid scaffolds cross-linked with formaldehyde.

2.4. In cartilage tissue engineering

Chitosan has an inherent ability to promote wound healing as it can activate platelets when it is in contact with blood [68–70]. In recent years, chitosan-based scaffolds encapsulated with growth factors have been considered as the highly preferred biomaterial for efficient and faster wound healing process [71]. Mizuno et al. studied the wound healing ability of hydroxypropyl chitosan scaffolds loaded with bFGF in diabetic mice [72]. The results showed that these scaffolds had a significant effect of wound healing when compared to the scaffolds free from bFGF. Kweon et al. developed an efficient wound healing material based on chitosan-heparin complex [73]. Due to the presence of heparin, this complex can bind with growth factor easily. To assess the wound healing ability, chitosan-heparin complex was applied in the wound of rat model and after 15 days gross and histologic examination was conducted. The results showed that the wound treated with chitosan–heparin complex was cured more rapidly when compared to that treated with control. Recently, bFGF encapsulated gelatin microparticles loaded into chitosan scaffolds have been employed in aged mice to treat pressure ulcers [74]. The bFGF-loaded chitosan scaffolds accelerated the healing of pressure ulcers and exhibited improved angiogenesis. To develop a scaffold with controlled drug release ability for skin tissue engineering, collagen-chitosan-chondroitin sulfate hybrid scaffolds encapsulated with bFGF-loaded PLGA microspheres were developed by Cao et al. [75]. Due to the higher swelling and degradation rate, these scaffolds showed a higher diffusion rate and release of encapsulated bFGF. The cell proliferation studies demonstrated that the collagen-chitosan-chondroitin sulfate hybrid scaffolds had a good biocompatibility and capability to endorse fibroblast cell proliferation and skin tissue regeneration. Bilayer porous collagen-chitosan-silicone membrane loaded with the plasmid encoding VEGF-165 and N, N, N-trimethyl chitosan chloride (TMC) was developed as a dermal equivalent for healing full thickness wounds in porcine models [76]. The experimental results showed that animals treated with VEGF-

In general, cartilage has poor regeneration ability due to the lack of vasculature and low cell content. In recent years, various types of biomaterials have been considered as scaffolds for the effective cartilage repair [59]. Among these, glycosaminoglycans (GAGs) will be more appropriate for the regeneration of cartilage tissues as they play an important role in the process of chondrogenesis [60]. Since chitosan resembles GAGs structurally and has very good wound healing property, it is considered as a potential candidate for the cartilage tissue engineering. Tigli et al. prepared porous chitosan scaffolds covalently immobilized with Arg-GlyAsp (RGD) and epithelial growth factor (EGF) as shown in Fig. 5 and studied their chondrogenic activity [61]. MTT assay revealed that only chitosan-EGF scaffolds showed a substantial increase in cell proliferation. Biochemical analysis showed that GAG and deoxyribonucleic acid (DNA) content of the chondrocyte-seeded scaffolds increases with time. These results indicate that chitosan-EGF scaffolds have the potential for reticular cartilage regeneration. It is known that TGF-␤ has an important role in cartilage tissue engineering where it induces differentiation of mesenchymal stem cells into chondrogenic lineage. However, the short life-time of TGF-␤ limits its use in cartilage tissue engineering. This drawback can be overcome by using the scaffolds that are capable of delivering the TGF-␤ more specifically to the site of cartilage damage. It was observed that chitosan-poly(␧-caprolactone) microspheres retained the stability of encapsulated TGF-␤1 and exhibited the controlled release of TGF-␤1 for the effective cartilage tissue repair [62]. Kim et al. developed a hydrogel by conjugating TGF-␤1 with type II collagen mixed photo-polymerizable chitosan for cartilage regeneration [63]. This hydrogel presented a controlled release of TGF-␤1 due to the chemical conjugation between TGF-␤1 and hydrogel. This hydrogel showed an improved the cellular aggregation and deposition of cartilaginous ECM because of the presence of type II collagen. Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2016.02.043

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2.5. In wound healing and skin tissue engineering

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165/TMC-loaded chitosan-collagen dermal equivalents exhibited neovasculature and faster regeneration of the dermis when compared to scaffolds without growth factor. Similarly, VEGF encoding plasmid DNA-loaded collagen-sulfonated carboxymethyl chitosan porous scaffold was also prepared [77]. Due the presence of VEGF, this scaffold showed an increased angiogenesis for the rapid wound healing. Han et al. constructed hUCMSCs implanted collagenchitosan laser drilling acellular dermal matrix composite scaffold for wound healing application [78]. The collagen-chitosan laser acellular dermal matrix composite had a uniform microporous structure and a higher degree of vascularization in the skin defect wounds on the back of miniature pigs. These results proved that this composite scaffold can be used to grow hUCMSC-derived fibroblasts in vitro and to develop stem cell-derived tissue-engineered dermis. Jiang et al. prepared TGF-␤3-loaded chitosan microspheres by cross-linking emulsion method for the delivery of TGF-␤3 [79]. In this study, synovial cells were cultured and then seeded into the TGF-␤3-loaded scaffold to produce tissue engineering synovial sheath. These scaffolds had good structure and compatibility with cells and a potential to be used for tissue engineered synovial sheath formation. Recently, Periayah et al. investigated platelet morphology changes and the expression level of TGF-␤1 and PDGF-AB in the oligo-chitosan in von Willebrand disease [80]. The results showed that the oligo-chitosan showed dramatic changes in the platelet’s behaviors. The platelet aggregation had occurred depending on the severity of von Willebrand disease. The oligo-chitosan showed an elevated expression level of TGF-␤1 and PDGF-AB. This observation suggested that oligo-chitosan stimulates these mediators from the activated platelets to the early stage of restoring the damaged cells and tissues.

3. Concluding remarks Chitosan and its derivates have received much attention as biomaterials for the fabrication of scaffolds for tissue engineering due to their non-toxicity, biodegradability, and biocompatibility. Chitosan-based scaffolds have the capability to immobilize the growth factors and release them in a sustained manner. In addition, they can retain the bioactivity of immobilized growth factors for longer time period thereby enhance the process of tissue regeneration. The hybrid scaffolds based on chitosan blended with other biomaterials such as collagen, hyaluronic acid, gelatin, hydroxyapatite, PLA, and PLGA are found to have an improved mechanical strength, ability for controlled delivery of growth factor, and adequate bioactivity for bone repair. These hybrid scaffolds have good osteoinductive and osteoconductive effects and proved to deliver a combination of growth factors sequentially and at different rates. Chitosan-based hybrid scaffolds encapsulated with the growth factors such as TGF-␤1, bFGF, and PDGF have the potential to be used for periodontal tissue engineering due to their ability to support the cellular structure, proliferation, and mineralization. The growth factors such as BDNF, FGF-2 and NGF have shown an improved neural cell growth and survival and hence chitosan-based scaffolds loaded with these growth factors have a large potential to be used for different types of nerve regeneration. Since chitosan and its composites encapsulated with TGF-␤ have an ability to promote chondrogenesis by the increased cell proliferation, these materials can be effectively used as scaffolds for cartilage repair. For the efficient wound healing and skin tissue regeneration, different types of growth factors such as bFGF, VEGF, and TGF-␤1, and TGF-␤3 have been loaded with chitosan-based scaffolds. These scaffolds exhibited an increased angiogenesis and neovasculature for the rapid skin tissue regeneration. Although chitosan-based scaffolds provided an exciting opportunity for the delivery of growth factors, Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2016.02.043

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some of the practical problems need to be addressed. As the growth factors loaded in the scaffolds are peptides/proteins, the structure and stability of these molecules should be intact to maintain its activity in vitro and in vivo. Moreover, sustained release of these growth factors from the scaffolds should be more controlled and evaluated. This would help in managing the delivery of exact dosage of growth factors for the regeneration of specific tissues. The scaffolds made by chitosan grafted/blended with stimuli-responsive polymers may have the potential to deliver the growth factors from the scaffolds at the time of requisite. Hence, more research on the development of chitosan-based stimuli-responsive scaffolds would be worthwhile for tissue engineering application. In addition, further studies and complete evaluation as tissue engineering materials would be highly required for the most of chitosan-based scaffolds for their real-time application. Acknowledgments The authors thank DST-Nano Mission (SR/NM/NS-1260/2013), Department of Science and Technology, Government of India for financial support. References [1] K.J. Rambhia, P.X. Ma, J. Controlled Release 219 (2015) 119–128. [2] D. Archana, L. Upadhyay, R.P. Tewari, J. Dutta, Y.B. Huang, P.K. Dutta, Ind. J. Biotechnol. 12 (4) (2013) 475–482. [3] M.S. Zafar, Z. Khurshid, K. Almas, Tissue Eng. Regen. Med. 12 (6) (2015) 387–397. [4] W. Shao, J. He, F. Sang, B. Ding, L. Chen, S. Cui, K. Li, Q. Han, W. Tan, Mater. Sci. Eng. C 58 (2016) 342–351. [5] N. Saxena, A. Jain, D. Archana, H. Kumar, P.K. Dutta, Asian Chitin J. 8 (1) (2012) 13–18. [6] Y. Chen, D. Zeng, L. Ding, X.L. Li, X.T. Liu, W.J. Li, T. Wei, S. Yan, J.H. Xie, L. Wei, Q.S. Zheng, BMC Cell Biol. 16 (1) (2015) 22. [7] A. Semwal, R. Singh, P.K. Dutta, J. Chitin Chitosan Sci. 1 (2013) 87–102. [8] R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biotechnol. Adv. 29 (2011) 322–337. [9] S. Dhivya, S. Saravanan, T.P. Sastry, N. Selvamurugan, J. Nanobiotech. 13 (40) (2015) 1–13. [10] S. Saravanan, S. Vimalraj, M. Vairamani, N. Selvamurugan, J. Biomed. Nanotechnol. 11 (2015) 1124–1138. [11] M. Prabaharan, M.A. Rodriguez-Perez, J.A. de Saja, J.F. Mano, J. Biomed. Mater. Res. B Appl. Biomater. 81B (2) (2007) 427–434. [12] S.C. Jouault, Funct. Marine Biomater: Prop. Appl. (2015) 69–90. [13] P.K. Dutta, K. Rinki, J. Dutta, Chitosan: a promising biomaterial for tissue engineering scaffolds, in: R. Jayakumar, M. Prabaharan, R.A.A. Muzzarelli (Eds.), Chitosan for Biomaterials II, Springer-Verlag, Berlin, Heidelberg, New York, 2011, pp. 45–80. [14] K. Lee, E.A. Silva, D.J. Mooney, J. R. Soc. Interface 8 (2011) 153–170. [15] A. Busilacchi, A. Gigante, M. Mattioli-Belmonte, S. Manzotti, R.A.A. Muzzarelli, Carbohydr. Polym. 98 (2013) 665–676. [16] H.Y. Cheng, M.F. Long, S.H. Wen, K.P. Li, Int. J. Pharm. 376 (2009) 69–75. [17] A.M. Rajam, P. Jithendra, C. Rose, A.B. Mandal, J. Bioact. Compat. Polym. 27 (2012) 265–277. [18] D. Chen, M. Zhao, G.R. Mundy, Growth Fact 22 (4) (2004) 233–241. [19] H.C. Yang, H.H. Jen, L.H. Chuang, W.D. Ming, H.L. Tuan, Dental Mater. 22 (2006) 622–629. [20] S.D. Nath, C. Abueva, B. Kim, B.T. Lee, Carbohydr. Polym. 115 (2015) 160–169. [21] L. Cao, J.A. Werkmeister, J. Wang, V. Glattauer, K.M. McLean, C. Liu, Biomaterials 35 (2014) 2730–2742. [22] X. Niu, Q. Feng, M. Wang, X. Guo, Q. Zheng, J. Microencapsulation 26 (2009) 297–305. [23] X. Niu, Q. Feng, M. Wang, X. Guo, Q. Zheng, J. Controlled Release 134 (2009) 111–117. [24] A. Ferrand, S. Eap, L. Richert, S. Lemoine, D. Kalaskar, S. Demoustier-Champagne, H. Atmani, Y. Mely, F. Fioretti, G. Schlatter, L. Kuhn, G. Ladam, N. Benkirane-Jessel, Macromol. Biosci. 14 (2014) 45–55. [25] A.C. Akman, R.S. Tigli, M. Gumusderelioglu, R.M. Nohutcu, Artif. Organs 34 (2009) 65–74. [26] S.K. Nandi, B. Kundu, D. Basu, Mater. Sci. Eng. C 33 (2013) 1267–1275. [27] T. Jiang, S.P. Nukavarapu, M. Deng, E. Jabbarzadeh, M.D. Kofron, S.B. Doty, W.I. Abdel-Fattah, C.T. Laurencin, Acta Biomater. 6 (2010) 3457–3470. [28] P. Yilgor, K. Tuzlakoglu, R.L. Reis, N. Hasirci, V. Hasirci, Biomaterials 30 (2009) 3551–3559. [29] Y. Zhang, B. Shi, C. Li, Y. Wang, Y. Chen, W. Zhang, T. Luo, X. Cheng, J. Controlled Release 136 (2009) 172–178. [30] T. Luo, W. Zhang, B. Shi, X. Cheng, Y. Zhang, Clin. Oral Implants Res. 23 (2012) 467–474.

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