Xenogeneic Transplantation Of Acellular Conjunctiva Matrix For Rabbit Conjunctival Defect

PurposeTo explore the preparation of acellular conjunctiva matrix(ACM) and observe the performance of of acellular conjunctival transplantation on rabbits to evaluate its feasibility. Methods1. The preparation of ACM.Porcine conjunctivas(3 hours after slaughter, it only contains a little under the conjunctival tissue) were first treated with 0.2% glutaraldehyde for 5 min, then followed by 24 hours of 1%TritonX-100 and 2 hours of 0.25% trypsin / 0.02% EDTA respectively, at 37?. Based on the results of hematoxylin and eosin(HE) and DAPI staining, well decellularized conjunctivas were picked out. Finally, the acellular conjunctivas were air-dried and ultraviolet-sterilized in clean bench for 8 hours to be prepared for use. 2. The correlative examinations of ACM.Detection of light transmittance of acellular conjunctival stroma by visible spectrophotometer, Bacteria and fungi culture line detection, Immunohistochemistry method was used to detect the antigen, Cell toxicity was detected by MTT assay. 3. The application of heterogeneous ACM in repairing conjunctiva defects on rabbits.(1) The creation of conjunctiva defect models in rabbits.Free remove about 20 mm × 8mm of bulbar conjunctiva, fascia and also the corresponding palpebral conjunctiva over the noses of the rabbits.(2) Animal model of transplanted materials20 rabbits of conjunctiva defects were divided into 4 random groups, 5 in each. Group A underwent ACM transplantation,group B underwent amnion transplantation, group C took transplantation of heterogenous conjunctiva without decellularization,while group D was given no treatment. Using slit lamp, the conjunctival healing, conjunctival sac stenosis, conjunctival adhesion and vascular conditions were observed at different times after operation. And the performance of conjunctiva transplantation was evaluated by fluorescein staining, HE staining, and imprint cytology. Results 1. Compared with single use of TritonX-100 or trypsin/EDTAcan effectively remove the conjunctival epithelium and stromal cells. 2. The optics transmittance of ACM proved to be good without antigen or cytotoxicity. The collagen fibers were well structured, and free of cellular components. 3. Acellular porcine conjunctival transplantation group(group A) had good graft survival. 1 week after the operation,fluorescein sodium staining was positive at the defect of eyelid conjunctiva, but the area where the graft covered was smooth and non staining. Neovascularization was found at corneal limbus,and HE staining showed that a single layer of epithelial cells were covering the graft surface with a large number of inflammatory cells cells moving into the acellular conjunctival stroma. 2 weeks after, fluorescein staining went all negative, and a lot of blood vessels had grown in the the graft showing a pink appearance. 3-4 layers of cells were observed at conjunctival epithelium of the graft by HE staining, and the number of inflammatory cells dropped drastically. Imprint cytology results had shown the existence of goblet cells and epithelial cells. Immunofluorescence staining was performed 6 weeks after transplantation, and the positive expression of the epithelial cells proved the success of conjunctival epithelialization. 1 weeks after operation, the amniotic membrane transplantation group(group B) underwent dissolution and absorption of the amniotic membrane. 2 weeks after, the formation of conjunctival scar was found in Group B. In the heterogenou conjunctival transplantation group(group C), the conjunctiva allograft dissolved. Conjunctival scar formation was found in the conjunctival defect group(D group) without treatment. Conclusions 1.The combined treatment of TritonX-100 and trypsin /EDTA can effectively remove conjunctival epithelial and stromal cells. 2.The heterogeneous conjuntiva matrix has no cytotoxicity, low immunogenicity, and with rapid vascularization speed. It can be used as a good conjunctival graft.3. Heterogenenous acellular conjunctival can be used as a bridge to repair the conjunctival epithelium and repair the conjunctival defects.