Study On The Bilaver Collagen Based Scaffold For Skin Repair And Appendage Regeneration

Collagen is the major component of extracellular matrix (ECM).The ability of collagen served as a substrate for cell adhesion is based on the RGD peptide and exhibits superior biocompatibility compared with other natural polymers.The use of collagen-based biomaterials in the field of skin tissue engineering has been intensively growing over the past decades. In this paper, the acetic acid and pepsin were used to extract collagen from the bovine tendon in order to study on extraction and purification process of collagen. Experimental results showed that when the temperature of the extraction experiment at 4℃, swelling time of acetic acid was 24hours, the optimum concentration of pepsin was 0.1%. For salt precipitation, the optimum concentration of sodium chloride was 3mol/L, time of 12 hours. The rate of collagen purity achieved above 98%. The extracted protein was confirmed to be collagen by FTIR, UV spectral analysis and amino acids analysis. In addition, the high-purity of the extracted collagen was proved by SDS-PAGE gel electrophoresis experiment. Collagen fiber solution was observed by transmission electron microscopy. According to thermal performance test,the collagen denaturation temperature was around 65℃. Total sugar and heavy metal content were accord with national standard.For mimicking the structures and functions of the human skin, the purpose of this work is to construct a bilayer artificial skin, an infection-preventing asymmetric collagen scaffold. The upper layer is the collagen fiber dense layer containing gelatin microspheres (GMs) loaded with the antibiotic silver sulfadiazine(AgSD). while the collagen fiber dense layer, functions as a temporary epidermis, which can play a role in controlling water loss and inhibiting bacterial entry and soon. The bottom layer is the collagen-chitosan porous sponge. the collagen-chitosan dermal scaffold is equivalent to the dermal extracellular matrix, promoting the regeneration of the damaged dermis in situ.The infection-preventing asymmetric collagen scaffold has good mechanical properties and can control the evaporation of water vapor. Meanwhile it exhibited an obvious inhibition zone against Gram negative and positive bacteria. The collagen scaffold has been designed to deliver AgSD over the wound surface in a uniform and controlled fashion for72hours. This controlled release helped to reduce the cellular toxicity of AgSD. In vitro fibroblast culture demonstrated that the collagen scaffold showed good biocompatibility and bioviability.To achieve the regeneration of skin appendages sweat gland during wound healing, we encorded the targeted gene into the plasmid by gene-engineering to get the pDNA-EGF, pDNA-EDA that could continualy express the targeted protein Hepatocyte Growth Factor (EGF and EDA). After incorporated the seed cells, mesenchymal stem cells(BMSCs), the gene-activated scaffold with BMSCs was fabricated. BDE with BMSCs and EGF,EDA as experiment group was implanted into the SD-Rat full-thickness skin defect model compared with the blank scaffold with BMSCs and the BDE with EGF as control groups. The HE staining shown some mature sweat gland-like structures 8w post-surgery. To verify the structures of sweat gland, immunohistochemical staining (IHC) was carried out. The result shown the sweat gland-like structures 2w,4w and 8w post-surgery was positive for CEA,CK8 and CK14 which demonstrated the sweat gland specificity of these structures. RT-qPCR and Western Blotting shown the expression of CEA, CK8 and CK14 in gene and protein level was higher compared with the control groups.