Preparation And Research Of Collagen Scaffold For Tissue Engineering

The incidence of fracture and bone defects resulting from trauma, tumors, and infections is increasing, and efforts are needed to facilitate and enhance the fracture repair and reconstruction. The treatment of bone defects in particular is a challenge facing the orthopedic surgeons. However, there are several drawbacks to the commonly used methods, such as autograft, allograft and xenograft. With the advances in tissue engineering, repair using an engeineered construct exhibited more advantages. Collagen, one of the essential constituents of bone, possessed several valuable properties:it acts as a structural support in mature tissues while playing a role of orientation in developing tissues; it has high tension, low malleability, oriented fibrils and controlled cross-linking; it exhibits perfect biocompatibility, minimal or no antigenicity to evoke immune rejection, strong affinity for cells stimulateing their proliferation and differentiation; it degrades with the process being controlled to produce amino-acid and small peptides which can be used as raw materials to reconstruct the cells or excreted as metabolic products.The present study aimed to prepare a novel collagen product from several biomaterials for the use in tissue engineering.1. Preparation of high purity collagen from 4 biomaterials.[Aim] To prepare high purity collagen typeâ… from 4 biomaterials to fulfill the needs in bone tissue engineering. [Methods] Bovine cortical bone, bovine achilles tendon, porcine achilles tendon and porcine skin were splitted into pieces of 0.2-0.5mm. After being immersed in glacial acetic acid, they were extracted with pepsin by a limited enzyme digestion method. Then the crude products were dissolved, centrifuged, dialysed and freeze drying to produce pure collagen typeâ… . The extraction rate was calculated. [Result] Pure collagen typeâ… can be prepared from all four biomaterials, with the purity of the product from bovine cortical bone being the highest while extraction rate from bovine Achilles tendon being the highest. [Conclusions] With the use of limited enzyme digestion method we could obtain high yields of collagen typeâ… from the four biomaterials, suggesting a bright prospect for the application of this method in tissue engineering.2. Analysis and identification of the collagen product[Aim] To analyze and identify the collagen typeâ… prepared from the 4 biomaterials in comparison with Sigma products. [Methods] Collagen typeâ… was extracted from bovine cortical bone, bovine Achilles tendon, porcine Achilles tendon and porcine skin using limited enzyme digestion in acidic conditions, and the final product was identified with use of ultraviolet absorbance, amino-acid analysis, SDS-PAGE electrophoresis with its purity being calculated in comparison with Sigma products. [Result] The collagen typeâ… extracted in the present study showed the maximum wave length of ultraviolet absorbence of 230 nm, and the result of amino-acid analysis and SDS-PAGE electrophoresis were characteristic of those for collagen typeâ… . The purity of the collagen from bovine cortical bone was the highest(96.12%)which was superior to the Sigma product. [Conclusions] High purity collagen was prepared from 4 biomaterials using limited enzyme digestion method, with that from bovine cortical bone being the best.3. Research of biological properties of the tissue engineering collagen scaffold[Aim] To investigate the cell compatibility and biodegradation of the high parity collagen typeâ… used for scaffold in tissue engineering. [Methods] High purity collagen typeâ… which had been extracted and identified as noted above was used to coat a slide glass and then coincubated with marrow stromal cells. Cell adherence, cell toxicity were assessed and cell growth curve was made. In the meantime, high purity collagen typeâ… was used to construct a scaffold that was genipin-crosslinked and implanted in mouse muscle to observe biodegradation. [Results] High purity collagen typeâ… showed strong cell adherence, no cell toxicity, enhanced cell growth, and the genipin-crosslinked collagen began to degrade at 2 weeks after implantation and completely degraded at 4 weeks. [Conclusions] Collagen typeâ… prepared from bovine cortical bone exhibited good biocompatibility and a degradation rate commensurate with new bone formation. The collagen produce thus prepared is well suited for use in bone tissue engineering.As shown in the present study, high purity collagen typeâ… can be prepared from bovine achilles tendon, porcine achilles tendon, porcine skin, and bovine cortical bone in particular. The collagen produce thus obtained has a good biocompatibility and biodegradation making it suitable for use in bone tissue engineering.