Study On The Extraction And The Biocompatibility Of Collagen From Bovines Tendons

Objectives1.To extract collagen from bovine tendons and assay the characteristics of the collagen.2.To observe the contraction of tissue engineered skins composed of different types of collagen.3.To study the effect of different types of collagen to the adhesion of the keratinocytes, and observe the microstructure between the collagen and the cells by atomic force microscope.4.To examine the synthesis and secretion of collagen by composited chitosan tissue engineered skin by Picric Sirius red Staining.Methods1.We extract the bovine tendon collagen by using acetic acid and ficin and check the biological properties including amino acid and heat denaturation temperature, infrared spectrum (IR). 2.We isolated and cultured kerationcyte and fibroblast from child foreskin. Under the established methods of our lab, we observed the contraction of different tissue engineered skins composed of bovine tendon collagen and rat tail collagen, respectively, and the curve of contraction of different tissue engineered skins was figured. 3.Culture plates were covered by bovine tendon collagen, rat tail collagen and standard typeⅣcollagen, respectively. Then the same passage of kerationcytes was seeded in one density of cells. Un-adherent Cells were counted by viable count at different phases and different types of collagen. the curve of un-adherent cells at different conditions was figured. and we observed the microstructure between the collagen and the cells by atomic force microscope. 4. Tissue skin was stained by picric acid and Sirius red. Synthesis and secretion of TypeⅠand typeⅢcollagen in composited chitosan tissue engineered skin was observed by polarized light microscope and semiquantitative analysis by phtoshop software.Results1. Collagen can be extracted effectively from bovine tendon by acetic acid and ficin. The collagen was identified as TypeⅠcollagen. 2.Less contraction was found in the tissue engineered skin composed of bovine tendon collagen than in that of rat tail collagen with the same concentration. 3.The adhesion ability of typeⅣcollagen to kerationcyte is higher than that of bovine tendon collagen and rat tail collagen in the first one hour, but ?since that, there is no significant distinction among them. 4. There are typeⅠandⅢcollagen both in tissue-engineered skin and in normal skin. The percentage of typeⅠandⅢcollagen in normal skin are 98.51±11.28 and 33.93±3.90, the percentage of typeⅠandⅢcollagen in the tissue engineered skin are 91.42±3.82 and 30.75±2.29,respectively.There is no significant difference in the tissue engineered skin and normal skin(P >0.05). The percentage of typeⅠ,Ⅲcollagen in the tissue engineered skin is detected on 8th , 15th , 30thd , The percentage ofⅠtype collagen are 50.75±4.22,63.57±6.87,91.42±3.82.The percentage of typeⅢcollagen are 16.90±1.64,23.65±4.08,30.75±2.29. There is significant differences among three tissues engineered skin(P<0.01).ConclusionsCollagen can be extracted effectively from bovine tendon by acetic acid and ficin. The contraction of tissue engineered skin composed of bovine tendon collagen is less than that of rat tail collagen. It shows that the bovine tendon collagen can notably decrease the contraction of tissue engineered skin and means lots of help to the stable mechanic property required by the clinic application. Compared with typeⅣcollagen, Collagen from bovine tendon and rat tail have similar adhesion ability to cells. The microstructure between the collagens and the cells was oberved by atomic force microscope. There is no distinguished difference in collagen content and distribution between tissue engineered skin cultured for 1 month and normal skin. Significant difference was found in collagen content and types of tissue engineered skin at varied phases. The distribution of collagen in the tissue engineered skin and normal skin could be observed by the method of Sirius red and polarized light. TypeⅠcollagen shows red or orange and typeⅢcollagen appear green.