Extraction Of Type â…  Collagen From Cortical Bone And Preparation Of A Collagen-Gelatin Scaffold

The regeneration and functiona l recovery of periphera l nerve defects has beenone of the difficult and hot spots in the field of neuroscience research. In mostcases, it is difficult for nerve fibers to be connected by anastomosis after injuries,due to the particularity of the structure and function of periphera l nerve. Walleria ndegeneration taking place in the far end of nerve fibers ma y lead to total or partialloss of sensation, movement or function of limbs. And breakage or defects ofnerve fibers are the severest types of injuries. The most widely used technique forbrid ging defects in periphera l nerve is the use of autologous nerve graft. However,it is limited in applica tion because of the shortage of autologous nerve orsecondary injuries in donor-sites. In recent years, tissue engineering techniqueshave been employed successfully in the repair of periphera l nerve injuries.Preparation of nerve scaffolds (such as artificia l nerve) to repair injuries ofperiphera l nerve pla ys an important role in recent research.Preparation of scaffold materia ls using natural extracellula r matrix (ECM) isthe new direction of the research. ECM has advantages of cellular adhesion,motility, and signa l transduction. Scaffold materia ls made from ECM havefavorable biodegradability and biocompatibility, which beneficia lly inducing thegrowth of nerve fiber. Meanwhile, taking the good biocompatibility and biomecha nica l properties of ECM into considera tion, it would have the potentia l toachieve better results in preparing periphera l nerve tissue engineering materia ls inthe near future.Colla gen belongs to ECM complex, and is the essentia l protein of the body.Colla gen ma inly exists in tissues like bone, cartila ge, skin, and so on. It pla ys animportant role in the control of differentiation, proliferation, maturation andmigration of Schwann cells, as well as regenera tion and growth of axons . Colla genhas advantages of favorable biodegradability, anti-stress capabilities,compressivestrength, low immunogenicity and toxicity, and is easily acquired. Therefore, it iswidely used in fracture healing,cartila ge regeneration, cornea allograft, andpreparation of artificia l-skin.In this study, traditiona l enzymatic method of extracting typeⅠcolla gen fromcortica l bone was improved , and scaffolds were prepared from extracted colla genin order to explore a simple and practica l method to supply basic research andclinica l applica tions with colla gen in high purity but low price.The cortical bone was disintegrated, dehydrated, decalcifica ted and defattedinto bone matrix powder. The osseins were extracte d by using improved pepsindigestion method with a procedure of dissolution, centrifugation, dia lysis andlyophilization, then were characterized by SDS-PAGE and amino-acidcomposition analysis. The biomaterials were made of colla gen sample and gelatinusing freeze-drying method, and the alignment regularities of microscopicchannels and their course directions were observed under the scanning electronicmicroscope. The size of the micropores and the factor of porosity were alsomeasured. The results showed that:1,The cortical bone was disintegrated into bone matrix powder with adia meter of approxima tely 0.6mm~2mm, defatted for 48h and decalcifica ted for84h while changing the decalcified fluid respectively at 12h, 36h, 60h. Theprocedure is easy ,with low cost and effective decalcifica tion.2,The bone matrix powder was dissolved in the mixed solution of glacia l acetic acid and pepsin for 96h and then centrifuged, dia lyzed and lyophilized. Thecolla gen extracted with a high purity was confirmed to be typeⅠcolla gen bySDS-PAGE and amino-acid composition analysis.3,Nerve scaffolds were prepared from colla gen and gelatin obtained from theexperiments. All the scaffolds were circular cylinder with the dia meters of20μm~100μm, the microscopic channels were arranged in parallel ma nners, andthe pore size of the channels were uniform.In conclusion, we successfully obtained typeⅠcolla gen with high purity bythe improved pepsin digestion method. All the scaffolds were circular cylinderwith the dia meters of 20μm~100μm, the microscopic channels were arranged inparallel ma nners, and the pore size of the channels were uniform. The threedimensional structure is close to that of sciatic nerve. This kind of nerve scaffoldspossesses good biocompatibility and favorable bionic effect. As stated above,colla gen extracted from cortica l bone was of high purity that could be applied inpreparation of colla gen products.