L-Arginine intercedes bio-crosslinking of a collagen-chitosan 3D-hybrid scaffold for tissue engineering and regeneration: in silico, in vitro, and in vivo studies

Three-dimensional (3D) collagen (COL) scaffolds have recently emerged as functional biomaterials in tissue engineering and wound healing. The limitation includes toxicity of synthetic crosslinkers, weak mechanical stability, low biocompatibility and fast biodegradation. Bovine collagen incorporated with chitosan (COLCS) was cross-linked with amino acids L-arginine (ARG), glutamic acid (GLU) and lysine (LYS) or without amino acids and was fabricated by the freeze-drying method. Morphological characteristics of 3D porous scaffolds observed by SEM showed a uniform interconnected porous structure with pore size ranging from 51 of 450 mm. Intermolecular interactions between the bio-polymers were examined by FT-IR. Thermal properties using DSC and TGA, swelling ability in PBS (pH 7.4) and biodegradability by collagenase digestion of the scaffolds were assessed. The amino acid cross-linked scaffolds have greatly improved thermal stability and reduced biodegradation. In vitro fibroblast cultures on the 3D scaffolds proved cyto-compatibility and enhanced proliferative ability. Fluorescence microscopy and SEM were conducted to examine cell morphology and its attachment on the scaffolds. Cell viability was also confirmed by using flow cytometry. The overall result authenticates that the ARG cross-linked scaffold (COL-CS-A) augmented cellular growth compare to other cross-linked scaffolds followed by uncross-linked scaffolds (control). In silico studies also proved the fact that ARG can act as a suitable cross-linker for collagen and chitosan complex systems. Moreover, the in vivo wound healing studies in a rat model revealed that the COL-CS-A dressing promotes the healing and accelerates the re-epithelialization and collagen deposition. Overall, the findings confirm that the COL-CS-A hybrid scaffold was an excellent candidate for tissue engineering and also for regenerative medicine.