Effect of interpolymer complex formation between chondroitin sulfate and chitosan-gelatin hydrogel on physico-chemical and rheological properties
Graphical abstract
Introduction
Tissue engineering attempts to develop biomaterials with similar structural and functional properties as of native extracellular matrix (ECM) to promote the regeneration of new tissues and repair the damaged ones (Hunt, Chen, Van Veen, & Bryan, 2014; Zhang et al., 2018). Natural and synthetic polymers are prevalent compounds for making biologically active artificial biomaterial substitutes (Sherbiny & Yacoub, 2013). Natural polymers such as chitosan, hyaluronic acid, alginate, collagen, fibrin are biomaterials with good cytocompatibility and low immunogenic properties, but their lower mechanical stability and uncontrolled degradation rates are undesirable (Ha, Quan, Vu, & Si, 2013; Hsu, Hung, & Chen, 2016). Synthetic polymers such as polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA) are widely used polymers in biomaterial fabrication due to their good biodegradability and high mechanical properties (Piskin & Hoffman, 1986; Wei, Deng, Guo, & Deng, 2018). Biomaterials should have the following characteristics to facilitate tissue regeneration process: i) providing interconnected porous network system for cells to grow and migrate, and transfer of adequate nutrients, growth factor and gases (Dan et al., 2016); ii) attract excessive amount of white blood cells to participate in wound healing (Reinke & Sorg, 2012); iii) less in vivo toxicity and anti-inflammatory activity (Greaves, Iqbal, Baguneid, & Bayat, 2013); iv) resistant to shear forces (Zheng et al., 2007); v) flexibility (Mao et al., 2002); vi) optimum degradation rate (Sandeep Kumar, Praveen, Raj, Chennazhi, & Jayakumar, 2014); and vii) pathogen free and contact with the tissues for a longer duration (Bello, Falabella, & Eaglstein, 2001).
Hydrogels consisting of natural polymers are the most favorable and extensively studied biomaterials due to their inherent soft-tissue like properties, moderate mechanical stability, tunable surface modification, dual nature (elastic and diffusive properties) of hydrogel, low toxicity, stable crosslinked three-dimensional intact mesh network and large surface area for cellular contact, for their application in both regenerative and reparative process (Chirani et al., 2015; Singh, Patel, & Singh, 2016). The stable polymeric network of hydrogels can absorb and retain excessive exudates in the wound vicinity followed by digestion of eschar and necrotic tissue (Ambrosio, 2014; Madaghiele, Sannino, Ambrosio, & Demitri, 2014). Hydrogels are also widely used as a bone regenerative graft, soft tissue repair, cartilage repair, nerve substitutes and they also enhance the wound healing process (Machado et al., 2007; Silva, Reis, Correlo, & Marques, 2019). The cells, various growth factors and drugs can be delivered by incorporating into the hydrogel (Peter et al., 2010). Controlled delivery of growth factor using hydrogels has shown great therapeutic potential in the clinical applications (Silva, Richard, Bessodes, Scherman, & Merten, 2009; Wang et al., 2017). Growth factors such as vascular endothelial growth factor (VEGFs) and fibroblast growth factor (FGF) play a major role in tissue regeneration through angiogenesis, and bone morphogenesis growth factor (BMPs), transforming growth factor β (TGF-β) and insulin-like growth factors (IGFs) are mostly used osteogenic protein for generation of new bone in bone defects (Brandi & Osdoby, 2006; Martino, Briquez, Maruyama, & Hubbell, 2015). On the other hand, hydrogels possessing self-healing property are highly potential candidates in drug delivery and wound healing (Tu et al., 2019). For example, self-healable chitosan-based hydrogel has been developed to repair the central nerve injury by promoting cell proliferation (Tseng et al., 2015) and chitosan-cellulose nanofiber self-healing hydrogel have been used for neuroregeneration process in zebrafish (Cheng, Huang, Wei, & Hsu, 2019).
Chitosan, a naturally occurring linear polysaccharide structurally analogous to glycosaminoglycans, contains randomly distributed b-(1–4)-linked D-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit). Its biocompatibility, biodegradability, hemostatic activity, acceleration of wound healing, in vivo anti-inflammatory, anti-allergic and osteoconductive properties play a major role in tissue regeneration (Ceccaldi et al., 2017; Khakharia & Khanna, 2012). The presence of abundant functional groups on the chitosan surface is favorable for chemical and physical surface modification and crosslinking (Fu, Yang, & Guo, 2018). The degraded products of chitosan by tissue enzymes are used as an effective agent in tissue regeneration on a wounded skin surface (Vazquez, Ruiz, Zuniga, Koppel, & Olvera, 2015).
Gelatin is a partially denatured biocompatible derivative of collagen. The high-water uptake capacity, variation in physicochemical properties by altering the surface functional sites, cell adhesion, cell attachment and cell migration characteristics of gelatin has been reported earlier (Jaipan, Nguyen, & Narayan, 2017; Li, Zhang, Naoki, & Guoping, 2017). It has been proven that gelatin is an active compound in the wound healing process by preventing the fluid loss due to exudation at the local inflammation generated in the wound site (Nguyen, Nguyen, & Hsieh, 2013; Tanaka, Nagate, & Matsuda, 2005). It is also reported that gelatin forms a polyelectrolytic complex with chitosan which leads to the formation of three dimensional (3D) polymeric scaffolds at an appropriate pH condition (Ng, Yeong, & Naing, 2016a, 2016b; Voron’ko, Derkach, Kuchina, & Sokolan, 2016).
The chitosan-gelatin polymeric porous network could be a perfect biomaterial due to its flexibility, slow in vivo degradation, biocompatibility, enhanced blood clotting activity, water retaining capacity and good supporting material for cell proliferation and differentiation (Choi, Kim, Lim, & Choi, 2018; Re et al., 2019). However, some of the properties of chitosan-gelatin hydrogels still need more attention to meet all the requirements of a tissue engineering graft. Some of the issues yet to be addressed are small pore size and fewer pores, lack of protein absorption sites, decrease in water absorption and high biodegradability when adding other polymeric material to improve mechanical strength and elasticity (Gautam, Chou, Dinda, Potdar, & Mishra, 2014; Lee, Yoo, & Atala, 2017).
Chondroitin Sulfate (CS) is a water-soluble glycosaminoglycan consisting of a repeating unit of D-glucuronic acid and N-acetylgalactosamine (Djerbal, Jacob, & Kwok, 2017). Owing to its polyanionic nature, it enhances the surface adsorption of protein and growth factors and it also plays a vital role in the regulation of growth factors (Xu & Yonese, 2007; Zhou et al., 2017). Having advantageous properties such as anti-oxidant nature, anti-inflammatory activity (Zhou et al., 2017), anti-apoptotic activity (Charbonneau, Liberelle, Hebert, Crescenzo, & Lerouge, 2011) and good mechanical strength (Farrugia, Lord, Whitelock, & Melrose, 2018) makes it an attractive component of biomaterials. Chitosan and chondroitin sulfate composite biomaterials have been studied in bone and cartilage regeneration (Gacanin et al., 2017; Shen et al., 2015). However, highly water-soluble nature and less cell adhesion properties are abating the use of CS in biomaterials (Kastana et al., 2019; Kaur, Rana, Jain, & Tiwary, 2010).
Apart from cytocompatibility, cell differentiation and proliferation, the biomaterials should have optimal polymer composition and physiological functional characteristics to become a feasible engineered substitute. Considering all the above aspects, we have fabricated a 3D-polymeric composite chitosan-gelatin-chondroitin sulfate (C-GE-CS) hydrogel as a substitute for chitosan/gelatin engineered hydrogel. In this study, we have fabricated the hydrogel with chitosan, gelatin and the physiochemical characteristics were investigated by varying the CS concentration. We are the first to formulate the CS incorporated chitosan-gelatin hydrogel to the best of our knowledge.
Section snippets
Materials
Chitosan (C) [Extra Pure, Mw 290–310 kDa, 90 % DA] was purchased from Sisco Research Laboratories (SRL) Private Limited, Mumbai-India. Gelatin (GE), chondroitin sulfate A salt (CS), glycerol phosphate disodium salt (β-GP), sodium hydrogen carbonate (SHC), acetic acid (AA), hematoxylin, bovine serum albumin (BSA), vascular endothelial growth factor A (VEGF165), fetal bovine serum (FBS), dulbecco’s modified eagle medium (DMEM), 1X penicillin and streptomycin antibiotic solution were purchased
Fabrication of composite hydrogels
Different compositions of hydrogels were fabricated as mentioned in Table 1 along with the examined viscosity of initial hydrogel sol phase, gelation time, the stability of hydrogel thin layer and physical parameters (length, width, and thickness) of hydrogels. The formation of intact hydrogels and thin layer (<2 ± 0.1 mm thickness) stability were determined. It was noticed that a thin layer of hydrogel containing 0.5 % CS was not stable, had typically lower mechanical strength and are
Conclusions
The obtained porous C-GE-CS hydrogels (pore size: 5–150 μm) could be a potential biomaterial for tissue engineering applications such as nutrients, oxygen and waste exudate permeation (15–40 μm) and internalization of several cell types: for endothelial cells (<80 μm), vascular muscle cells (63–150 μm), fibroblasts (38–150 μm) (Saraiva, Miguel, Ribeiro, Coutinho, & Correia, 2015). The prepared hydrogels expressed certain affinity towards growth factor/protein and the prolonged controlled
Funding sources
Science and Engineering Research Board (File No. EMR/2016/002447), Department of Science and Technology, Government of India.
CRediT authorship contribution statement
Ponsubha S: Visualization, Investigation, Methodology, Project administration, Formal analysis, Writing - original draft. Amit Kumar Jaiswal: Conceptualization, Supervision, Funding acquisition, Project administration, Validation, Writing - review & editing.
Declaration of competing interest
There are no conflicts to declare.
Acknowledgements
This research was supported by funding from the Science and Engineering Research Board (File No. EMR/2016/002447), Department of Science and Technology, Government of India. We thank N. Arunai Nambiraj for his help in mechanical testing and Dhivyaa Anandan for proofreading the manuscript. We are also thankful to Satheesh M. Further, we also thank School of Biosciences and Technology (SBST), VIT for providing DST-FIST cum VIT Funded Scanning Electron Microscope Lab Facility and Centre for
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