Characterization and in vivo Evaluation of Amniotic Membrane and Collagen Based Hydrogel on Rat Model

Abstract

Skin burn wounds and injuries continue to be a major problem affecting communities worldwide. Rapid and effective treatment of burnt skin is crucial to fasten wound closure and healing properly. Amniotic grafts are widely used as wound healing biomaterials. Sophisticated processing, high cost, availability, and the need of medics for transplantation limit the application of amnion grafts. This study was aimed to prepare a novel hydrogel from amniotic membrane and collagen extract combined with external epithelial sheath for burn wound healing which overcome the limitations of graft. 2% human amniotic membrane (AM), Collagen and AM-Collagen gels were formulated using different concentrations of carboxymethyl cellulose sodium (CMC-Na) salt. An external thin protective covering known as epithelial sheath (ES) was also prepared using different concentrations of collagen and chitosan. Various physical properties like swelling ratio, water absorption (%), equilibrium water content (%), gel fraction (%) and spreadability of gel formulations were analysed to find out the best concentration of CMC-Na for final wound healing applications. The formulated gels were found at neutral pH, acceptable swelling ratio, good spreadability and acceptable gel fraction (%). But 6% CMC-Na concentration was found to be best for final application. The same process was adopted for epithelial sheaths as well and epithelial sheath containing 10 mg/ml concentration of both collagen and chitosan was found to be optimum for final animal experiment. In vitro cytotoxicity and red blood cell biocompatibility tests of final formulated gels were performed. The gels were observed non-cytotoxic at brine shrimp lethality test and compatible with human red blood cells. Following skin irritation study, second-degree burns were induced on dorsal region of wistar rats; and gels along with ES were applied to observe the wound healing potential at in-vivo study. Wound contraction and re-epithelialization of gel treated skin were macroscopically measured. No erythema and edema were observed in skin irritation experiments confirming the applicability of the gels. AM-Collagen-ES treated group had maximum average body weight, food intake and water intake. At the end of the study (day 16), AM-Collagen-ES treated group was superior in wound contraction (72±3.27%) over other treatment groups and it was statistically more significant (P<0.0000009). While negative control group was healed only 22.67±2.31%, AM-Collagen gel treated group, AM gel treated group, Collagen gel treated group and positive control group were healed 62.5±4.43% (P<0.00001), 52±3.27% (P<0.00004), 36±4% (P<0.001) and 41±3.83% (P<0.0004) respectively. For complete epithelialization, AM-Collagen-ES treated group was required only (16.75±0.96) days while AM-Collagen gel treatment, AM gel treatment, Collagen gel treatment, positive control and negative control group were needed (18.75±0.50), (20.5±1.29), (24.75±0.96), (22.25±0.96) and (28+0.82) days respectively. From the above result, we can demonstrate that a gel consisting a combination of collagen and amnion with the presence of epithelial sheath has promising effect on the wound healing process.