| 1. Background and objectiveUsing tissue engineering cornea as the equivalent of the cornea is a good way to overcoming the deficiency of the donor and postoperative rejection in keratoplasty. The research includes the culturing of the seeding cells, the preparing of the scaffold, the technology of constructing the cell-scaffold complex and the application on the clinical. The ideal scaffold matching the human cornea has not been obtained. The scaffolds in previous research have the drawbacks respectively, such as the cell compatibility, strength, ratio of the degradation, immunogenicity and transparence. To expand the resource of the scaffold of the tissue engineering cornea, this study prepared the acellular porcine corneal stroma with basement membrane (APCS-BM). The methods applied to remove the cells did not impair the native basement membrane of the cornea as far as possible to improve the cell compatibility of the material. This study investigated APCS-BM on histological structure, the cell compatibility and immunogenicity and observed the effect of repairing the rabbit's defect cornea with APCS-BM. 2. Method2.1 The preparation of APCS-BM and the histological observationThe anterior lamellar corneal stroma buttons(1/2 thickness of the cornea)of the fresh porcine cornea were harvested. The epithelium cells were removed with 2.5 mM ethylenediaminetetraacetate (EDTA). The stromal cells were extracted by means of freezing, thawing and the digestion of DNAase & RNase. Lastly, the materials were lyophilized and sterilized by Cobalt-60. The histological structure and ultrastructure of the sample were observed with the hematoxylin and eosin (H&E) section and scanning electron microscopy respectively. The collagen IV and laminin on the surface of APCS were investigated by the immunofluorescence.2.2 The investigation of the cell compatibility of APCS-BMThe 3rd passage epithelium cells and stromal cells of the rabbit's cornea were trypsinized and seeded onto the surface adjacent to the epithelium side and the middle stromal side of APCS respectively. After culture for 10d and 5d respectively, the cell-scaffold constructs were fixed in 4% paraformaldehyde, paraffin embedded, then sectioned and followed by staining with H&E. The 3rd passage fibroblasts of human dermis were seeded on the middle stromal side of APCS. After culture for 3d, the cell-APCS constructs were turnovered and the 3rd passage epithelium cells of human skin were seeded onto the surface adjacent to the epithelium side of APCS. After culture for another 10d, the cell-scaffold constructs were fixed in 4% paraformaldehyde, paraffin embedded, then sectioned and followed by staining with H&E.2.3 The investigation of the immunogenicity of APCS-BMIn the experiment of the ectopic transplantation of APCS-BM, the fresh porcine coenral stroma (control group) and APCS-BM was transplanted into the rat's back subcutaneously. In the experiment of the entopic transplantation of APCS-BM, the fresh porcine corneal stroma (control group) and APCS-BM was transplanted into the rat's corneal stromal sac. The clinic and histologic observation were performed. The leukomonocytes with CD4 molecule were counted by immunofluorescence and the result was analyzed by statistics.2.4 The lamellar keratoplasty on the rabbit's cornea with a graft of APCS-BMThe 7.5mm diameter APCS-BM was fixed on the 7 mm diameter recipient bed with suture in 6 rabbits respectively. The clinic and histologic observation were performed by slit lamp, light microscope and transmission electron microscope.2.5 The lamellar keratoplasty on the rabbit's cornea with a graft of APCS-BM combined with the skin fibroblastsThe fibroblasts of the rabbit's skin labelled by PKH26 were seeded onto APCS-BM, then the complex was transplanted to the rabbit's eye. The clinic and histologic observation were performed by slit lamp, light microscope and fluorescence microscope.3. ResultAPCS-BM prepared was white and endured to suture. The image of H&E showed that the surface of the anterior stroma was consecutive and the collagens in the stroma arranged meshyly. The epithelium and keratocytes were not found in APCS. SEM revealed that there were not cells on the surface of APCS. The surface adjacent to the epithelium side presented a felt-like ultrastructure with various pores, grooves and edges. The surface of the opposite side showed a quite different meshy structure. Immunofluorescence displayed the positive staining for the collagen IV and laminin on the surface adjacent to the epithelium side. The negative staining was observed on the surface of the opposite side. The results of cell culturing in vitro showed 2-3 layers corneal epithelium adhered tightly to APCS-BM 10d after culture and the stromal cells grew into the APCS-BM 5d after culture. In the complex of the epithelium, fibroblast of skin and APCS-BM, The HE section also showed 3-4 layers epithelium adhered tightly to APCS-BM and the fibroblast grew into the APCS-BM.In the experiment of the ectopic transplantation of APCS-BM on the rat, the cuts of skin healed. The skin did not ulcerate and the grafts were not discarded 2 weeks post operation in two groups. The image of HE showed neutrophil and leukomonocytes in the group of APCS-BM were fewer than in fresh porcine cornea. The count of leukomonocytes positive to CD4 indicated the cells in the group of APCS-BM were fewer than in fresh porcine cornea. The result had a difference in statistic. In the experiment of the entopic transplantation of APCS-BM on the rat, the cuts of cornea did not split and the grafts were not discarded 2 weeks post operation in two groups. The edema of the cornea and new vessels in the group of APCS-BM were fewer than in the group of the fresh cornea. The count of leukomonocytes positive to CD4 had a difference in statistic in two groups. The cells in the group of APCS-BM were fewer than in fresh porcine cornea.After the lamellar keratoplasty on the rabbit's cornea with a graft of APCS-BM, the observation of clinic showed the grafts were integrated completely with the receptive cornea. The epithelium covered the scaffold surface completely within 2 weeks after graft. The cornea became transparent gradually. In group 1, the image of HE section showed the epithelium on APCS-BM stratified similar to the normal corneal epithelium 4 weeks after graft. The keratocytes of the recipient grew into APCS-BM. The transmission electron microscope indicated microvillus of the epithelium cell surface, desmosome and hemidesmosome structure formed within 4 weeks. The keratocyte in the scaffold was active and secreted new extracellular matrix.After the lamellar keratoplasty on the rabbit's cornea with a graft of APCS-BM combined with the skin fibroblasts, the epithelium covered the scaffold surface completely within 10-12 d after graft. The defects of corneal stroma were repaired. The H&E section showed the epithelium on APCS-BM stratified and has hyperplasia in some areas 4 weeks postoperation. A lot of fibroblast-like cells could been seen in the graft. The fluorescence microscope showed the cells positive to fluorescence presented in graft until the 5th month post operation.4. ConclusionThe experimental results mentioned above suggest APCS-BM prepared by the means of digestion of EDTA, freezing and thawing and lyophillization is a kind of acellular biologic derivation material. The surface of the material preserves the basement membrane and the stromal collagen is meshy. It has good compatibility with cells. The basement membrane on the surface is beneficial to the growth and adherence of the epithelium. The meshy structure in middle stroma is fit for invading of the stromal cells. It has low immunogenicity. The local immune response induced by it is more lighter than the fresh cornea. Its strength can be tolerant to suture. It can repair the defect of the cornea, integrate with the host cornea and become transparent after lamellar keratoplasty. It can be used for a kind of scaffold of tissue engineering cornea.
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