Experimental Study On Taking Acellular Small Intestinal Submucosa As Substitute For Dermis

Objective: To explore the feasibility of taking acellular small intestinal submucosa as substitute for dermis.Methods: 1. We obtained jejunum from healthy susscrota domestica,removed its serous tunic, muscular layer and mucous membrane and prepared acellular small intestinal submucosa by using trypsin, EDTA and NaCL associated cell-free method. Examination and detection would be conducted to test its physical and chemical performances, histologic structure and microbiology.2. We took acellular small intestinal submucosa as dermis frame and cultivated it together with human epidermal cells to form composite skin. The growth of cell and the structure of composite skin would be comprehended through MTT assay, historical anatomy observation, immunohistochemical detection and scanning electron microscope observation.3. Wound with full-thickness cutaneous deficiency was created on the back of SD rats. Acellular small intestinal submucosa, together with rats'autologous split thickness skin, was used as substitute to repair the wound in the experiment group while grafted sheer autologous split thickness skin was adopted for repair in the control group. The healing effectiveness of wound and related histological changes were observed.Results: 1. Acellular small intestinal submucosa was about 0.1cm thick. It was a semitransparent and white membrane structure which was soft in texture and fine in elasticity. Acellular small intestinal submucosa was a three-dimensional reticular spatial structure with many pores, and no cell was found from inside. Microbiological test showed no microbiological pollution.2. Human epidermal cells could stick to and grow on acellular small intestinal submucosa. MTT assay indicated that A570 value at each fixed time points were statistically insignificant in both groups either used combined cultivation or sheer human epidermal cells cultivation (P＞0.05),which proved that acellular small intestinal submucosa had neither auxo-action nor toxic action on human epidermal cells proliferation, but a good compatibility with these cells. Histological anatomy observation through HE stain and Masson stain showed acellular small intestinal submucosa had a collagen fibrous and reticular structure, with no cell inside but a layer of cells on its surface. Keratin immunohistochemical detection proved the layer of cell on the surface of acellular small intestinal submucosa to be epidermal cells. Observation through scanning electron microscope revealed the confluence growth of epidermal cells on the surface of acellular small intestinal submucosa and its underneath reticular structure through intercellular spaces.3. Dynamic observation conducted 2, 4 and 6 weeks after experiment indicated a satisfactory survival rate of free skin graft in both experiment group and control group after grafting. However, the survival rate of free skin graft in both groups at certain fixed time points was statistically insignificant. Four and six weeks after experiment, the contract rate of skingrafting area in composite skin group decreased remarkably, compared with that of the control group which used sheer autologous split thickness skin, and therefore had statistical significance (P＜0.05). Histological anatomy observation of the composite skin group through HE stain and Masson stain showed that 2 weeks after experiment, the stain degrees of the subepidermic dermis tissue and aceltular small intestinal submucosa differed, as evidenced by two layers with different alignment of collagen. Neogenetic small vessels were observed to grow into acellular small intestinal submucosa, with enormous inflammatory cell infiltrate. There were also fibroblasts migrating inside acellular small intestinal submucosa from the wound. Four weeks after experiment, under the epidermis of the composite skin, we could still observe collagen fibrils with different alignment, enormous inflammatory cell infiltrate, enormous migration of fibroblasts and a great many new vessels.Six weeks after experiment, there was no evident distinction between the two collagen fibrils structures underneath the composite skin, namely dermis structure and acellular small intestinal submucosa, where structures were replaced by collagen of homogeneity; inflammatory cell infiltrate reduced remarkably, while large quantities of new vessels and fibroblasts could still be seen.Conclusions: 1.Cells with antigenic component can be completely removed by using trypsin, EDTA and NaCL associated cell-free method to prepare acellular small intestinal submucosa. The obtained SIS is free from microbiological pollution, with fine elasticity and certain mechanical strength. It retains three-dimensional reticular spatial structure. In addition, it has low cost, wide resources and is easy to prepare, all of which suggest that it is an ideal substitute for dermis.2. Human epidermal cells can not only stick to and grow on acellular small intestinal submucosa, but also become combined with it to form composite skin, which proves that acellular small intestinal submucosa has no cytotoxicity but fine cellular compatibility, and therefore can be cultivated in vitro with human epidermal cells to form composite skin with double-layer structure.3. As a substitute for dermis to repair wound, Acellular small intestinal submucosa has high survival rate after grafting and can improve the healing effectiveness of the raw surface.