Fabrication And Study Of Collagen-Chondroitin Sulfate-Hyaluronic Acid Scaffold Imitating ECM For Dermal Tissue Engineering

Introduction: Patients with full-thickness skin injuries suffer from a substantial loss of dermis, and the regeneration of dermis could not occur spontaneously in adult individual. Due to tissue engineering is now showing promise as a possible method for dermal repair, seeking a suitable scaffold has become more and more important. Desirable properties of the scaffold include growth promotion, biological stability, reasonable tensile strength and ease of handling. Traditionally, dermal tissue engineering studies have used polylactic acid (PLA), polyglycolic acid (PGA), or a copolymer of PGA and PLA (PLGA) serve as scaffold. However, these materials have certain shortcoming in that they are hydrophobic and need pre-wetting before cell seeding. Moreover, their degradation products are acidic and lower the pH around tissue after in vivo implantation, which may cause severe inflammation. Since the first collagen-GAG scaffold was reported by Yannas and Burke, Collagen materials have been extensively used for skin tissue engineering. There are two keys. Firstly, Collagen (Co) is a natural biomaterial and it is the major dermal constituent approximately 60-80% of the dry weight of fat-free skin. Secondly, Co has also many advantageous properties including hemostatic properties, low antigenicity and high growth promotion. However, high degradation rate and deficient mechanical property of Co often fail to meet the requirement of specific application. Recent years, many scholars have been interested in imitating the natural extracellular matrix (ECM) and have been fabricated some scaffolds such as Co-CS, Co-HA and gelatin-CS-HA. These scaffolds had lower degradation rates and higher mechanical strength than collagen alone, but the biocompatibility was not satisfactory to us. The major problem is that they only partial imitate ECM. Fortunately, these experiences lead us to believe that applying Co, CS and HA which are the main natural components of the dermal ECM to fabricate a Co/CS/HA scaffold is feasible and the scaffold might be a better choose for dermal tissue engineering.Objective: The objective of this study was to develop a novel scaffold imitating the ingredients and their ratios of natural dermal matrix and to evaluate its biological activity.Methods: We applied different ratios and different synthetic methods to fabricate nine kinds of cross-linked collagen/chondroitin sulfate/hyaluronic acid (Co/CS/HA) scaffolds for dermal tissue engineering. By comparison of the morphology, mechanical properties and biodegradation rates, we optimized a novel scaffold. This scaffold was fabricated by the procedures which Co, CS and HA were firstly synthesized together in the ratio of 9:1:1 to form a membrane and then cross-linked (CL) with 5 mM of EDC (Co-CS-HA/CL 9:1:1). From the results of comparison, we also found that the ratio of 9:1:1 was better than other ratios. So as follows we evaluated the biocompatibility and the metabolic activity of Co-CS-HA/CL 9:1:1, Co-HA/CS CL 9:1:1 and Co-CS/HA CL 9:1:1 in vitro. These three kinds of scaffolds were used as experimental groups with an open wound without scaffold as control group to evaluate their effects on promoting wound healing in vivo.Results: Morphology observation showed the novel Co-CS-HA/CL 9:1:1 scaffold had uniform and widely interconnected pores with mean diameters of 109±11μm and adequate porosity of about 94%. Mechanical property and biodegradation assessment indicated that it had more degradation-resistant and higher elastic modulus than other scaffolds. Biocompatibility and metabolic activity assay showed it had no apparent cytotoxicity and could more promote cells attachment and proliferation. When scaffolds were seeded with allogenic skin fibroblasts and implanted on the dorsum of SD rats for 6 weeks, the novel Co-CS-HA/CL 9:1:1 skin equivalent could more successfully repair full thickness skin defects in SD rats. The histology was more approximate to normal skin than those of the controls within 6 weeks.Conclusion: The novel Co-CS-HA/CL 9:1:1 tri-copolymer has potential to be used as a scaffold for dermal tissue engineering.