Study On The Constructed Cornea Stroma With Collagen-chitosan And Bacterialcellulose

The corneal tissue engineering is a medicinal proposition, which applies biological properties of corneal cells and combines the corneal regeneration engineering. It is one of the focuses of investigation on the present ophthalmology. It has been improved that the regeneration of corneal stroma can not form the three-dimensional structure only depending on the self-propagation of keratocyte. Biological scaffolds need good tissue compatibility, intensity and plasticity. Therefore, the biomaterial with reconstructed corneal stroma has more demands and is harder to found corneal stroma than the corneal epithelium and endothelium in the corneal tissue engineering. Therefore the biomaterials are paid close attention by more people.Biological scaffold used to cultured corneal stroma cell are all imperfect previously. In this study, two biomaterials, namely, the co-mixing membrane of the collagen-chitosan and bacterialcellulose were chosen to fabricate corneal stroma of rabbit and human,and make the investigation on the replantation. Moreover, the effect of PDTC on the proliferation of keratocyte was researched by molecular biology. In this experiment, the newest biomaterial (the co-mixing membrane of the collagen-chitosan) and the nono-biomaterial (the bacterialcellulose) were used as scaffold to fabricate the corneal stroma and observe the biological compatibility respectively. Experiments as follows:1. The construction of biomembrane with the corneal stroma and the collagen-chitosan.It was chosen that the flap-eared white healthy rabbit and healthy human corneas (Derived from adult donators, and the residual materials of the keratoplasty with homogeneity variant cornea). The purity of corneal cells was identified by immunohistochemistry and immunity fluorescence dye after separated in vitro, cultivated, passaged and propagatined. Then corneal cells were seeded onto the co-mixing membrane of the collagen-chitosan and cultivated. The biomembrane was implanted into variant corneal stroma sac when the cells spread all over the co-mixing membrane. The growth of compounds was observed by the corneal confocal microscope and the anterior segment OCT in vivo. The corneal stroma implanted was taken away and detected by immunohistochemistry over four weeks.Results: The keratocyte in the co-mixing membrane of the collagen-chitosan grew into three-dimensional structure. Immunofluorescence displayed that the cells expanded all over the biomembrane. The biomembrane was translucence. The corneas did not have serious inflammatory reaction after transplantation. The fibril organization of biomembrane approached that of the normal corneal stroma in vivo micrography and ex vivo histological inspection. Corneal cells of the acceptor were normal. The boundary between the inserts and the surrounding tissues was clear then became unclear gradually. Vacuolus and Similar corneal cellular structure can be observed after degradation. Histolysis and necrosis of the cornea haven't be observed after transplantation. The biological compatibility was good.2. The construction of corneal stroma and bacterialcellulose compositeThe condition of drawed the materials from cornea is entirely consistent with the group of collagen-chitosan. The methods of identification and culturing are ditto. The 2-3 mm thick bacterialcellulose membrane was cuted into 1.5 cm in diameter, 0.5 mm thick sheet. Then seed corneal stromal cells onto the bacterial cellulose membrane. The cells spread all over the membrane. The sheet was implanted into variant corneal stroma sac. The result was detected by biopsy immunohistochemistry (the same methods as collagen-chitosan group).Results: The bacterialcellulose membrane is stronger and less transparente than collagen-chitosan membrane. The fibril structure of the biomembrane approached that of the normal corneal stroma and degradated gradually. The speed of degradation was slower than collagen-chitosan group. The corneal cells grow well..The morphous and amount of near normal cells did not changed. The compound membrane confluenced with the normal corneal stroma .There were no necrosis and dissolve. The biocompatibility is identical to collagen-chitosan membrane Group.3. The effect of PDTC on corneal stromal cell proliferation.It was observed the effect of PDTC on the proliferation of corneal stromal cells by MTT assay. The effect of PDTC on the corneal stromal cells transcription of TGF-βwas detected by real-time quantitative PCR. The effect of PDTC on corneal stromal cell to express TGF-βwas detected by immunofluorescence assay.Results: PDTC could inhibit the proliferation of corneal stromal cell stimulated by LPS.Conclusion:1. The corneal stromal cells were isolated from rabbit and human and expanded in vitro. The cells were implanted into collagen-chitosan mixed membrane. The cells were growing well monitored under the immunofluorescence and laser confocal microscopy. The engineered human corneal stromal cells and collagen-chitosan biological membrane were reconstructed successfully in vitro. 2. The cells were seeded on the bacterialcellulose biomembrane .The cells growed well and completely filled in the scaffold, possessing the characteristics of stromal cells. The results indicated that the natural biological nanometer material-bacterialcellulose were biologically safe without changing the biological feature of the cells. The corneal stromal biomembrane were successfully reconstructed applying the bacterialcellulose.3. After homogeneous variant corneal transplantation of two kinds of constructed corneal stromal biomembranes, the transplanted materials were detected histologically in vivo and ex vivo. They both showed non-toxic to normal corneal stroma. The necrosis and dissolve haven't been discovered . Both of them showed high histocompatibility and capable of degradation. The collagen-chitosan biomembrane degraded faster. The bacterialcellulose was firstly applied in the corneal engineering and could be an ideal corneal stromal reconstruction biomaterial.4. The histomorphological change in transplanted lagophthalmos were observed by applying cornea confocal microscope Cs-4 and anterior segment OCT. The change were observed in the first, second and fourth week after the transplantation. We confirmed that the two constructed cornea stromal membranes were degraded with no effect on the morphous and amount of normal cornea cells. The cornea confocal microscope Cs-4 and anterior segment OCT can be applied as a new way of observing the reconstructed change in cornea tissue in vivo.5. Apply MTT to detect the affect of PDTC on cornea cells, we found PDTC can obviously degrade the OD value of LPS group. Apply the real-time PCR to detect the affect of PDTC on the transcription of TGF-βin keratocyte though level of mRNA,we found the group with LPS after 24h has five times higher level of mRNA of TGF-βthan the normal group. Group with PDTC alone had no effect on the transcription of mRNA of TGF-βbut reverse the elevated transcription of TGF-βby LPS. It is the first time to confirm that PDTC can inhibit the transcription and expression of TGF-βin cornea stromal cells. It is significant in the tissue engineering of cornea regenerated and the therapy of cornea diseases.In conclusion, we use two brilliant biomaterial to study the construction of corneal stroma. The bacterial cellulose has a better perspective and provide a new way of study the regeneration of cornea. It is feasible and a new field of cornea regeneration and it is a hope for the blind of cornea.