| Research backgroundBum, acute trauma and chronic ulcer may lead to full-thickness defect of skin. Skin wound repair and healing is one of the fundamental problems in the field of surgery. Engineering skin substitutes provide a new source of advanced therapies for the treatment of extensive bum and chronic ulcer even scar prevention. Design principles have been suggested by Yanas and Burke at 1980, among which the selection of a dermal substitute is of the most importance. A dermal substitute with good cell compatibility and tissue compatibility can be degraded and absorbed gradually after transplantation, and then a newly formed dermal tissue is developed at the wounded site. Collagen-glycosammoglycan is often used to produce dermal substitutes abroad. Some commercial products like Integra have been developed , but they are always very expensive. Thus, we are about to develop a low costly dermal substitute to meet the biological and clinical requirement of skin reconstruction. Collagen and chitosan are both extensively distributed in nature and are both easy to extract. They can be mixed to fabricate a pliable spongy with tensile strength. We use collagen andchitosan to make spongy membrane as a dermal substitute in order to develop tissue engineering skin. It is very necessary to investigate the biocompatibility and biodegradation of the coUagen-chitosan spongy membrane as an assessment of safety and standardization of a tissue engineering medical product.ObjectiveTo investigate the cell compatibility, tissue compatibility and degradation of collagen-chitosan spongy membrane in vivo. The practicability and reliability of collagen-chitosan spongy membrane are assessed as a dermal substitute to facilitate the development of tissue engineering skin in future.Methods1. Fabrication of collagen-chitosan spongy membraneCollagen type I was harvested from bovine tendon. Purified chitosan was extracted from chitins using sodium hydroxid. Collagen then was mixed with 10% chitosan to fabricate a highly porous membrane by Lyophilization . The membrane was cross linked by 0.25% Glutaraldehyde or by dry heat.2. Investigation of cell compatibility of collagen-chitosan spongy membrane. (l).Cultivation of dermal fibroblast:Dermal fibroblasts were harvested by using enzyme digest technique from child foreskin after circumcision.3-4 generation of cells were used as work cells.(2) Cultivation of dermal fibroblast on collagen-chitosan spongy membrane. The density of cultivated dermal fibroblast was adjusted to 5X 105/cm2,Cell suspension was inoculated on Glutaraldehyde cross-linked collagen chitosan spongy membrane to make a cell-matrix complex. The cell-matrix complex was incubated with DMEM and 10% bovine serum under condition of 37,5%CO2- The medium was changed daily.(3) Cell-matrix complex paraffin section was made after 7 days incubation. Sections were stained by HE and were observed under light microscope.(4)Observation on Cell-matrix complex with confocal microscope. Cell-matrix complex was stained by fluorochrome CFDA-AM (lOug/ml, lOOul) after 7 days incubation, The sample was scanned by confocal microscope to observe cell-growth in the matrix.3 -. Investigation of tissue compatibility of collagen-chitosan spongy membrane.(1) subcutaneous embedding of collagen-chitosan spongy membrane on rabbit.Twelve healthy rabbits were divided into four groups randomly. There are three types of collagen-chitosan membrane: non cross-linked type, dry heat cross-linked type and Glutaraldehyde cross-linked type. Each type of membrane was embedded subcutaneously on dorsal surface of rabbit ear. Four of each type were implanted in every group. Rabbit were breeded separately, general and local changes were recorded.(2)Histological studyHarvests were performed in randomly selected group at 3,7,14 and 28 days after surgery. At harvest, the implantation sites were removed in a full-thickness manner (including both sides of the ear skin and cartilage). Sections were then stained wit...
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