| Experiment One:In vitro culture and identification of MSCs and LECsMesenchymal stem cells and limbal epithelial cells were cultured and passaged in DMEM, EpiLife?, D/F12 plus supplements, and low [Ca2+] KSFM, respectively. MSCs were induced to adipogenic and osteogenic differentiation for cell identification, and the induced MSCs were not used in in vivo transplantation. Expression of ABCG2, PCNA, CX43, CK3, and CK12 were detected by RT-PCR and immunocytochemistry.In vitro results showed adherence and extension of primary cells in 24 hours, and confluence within 7 days. MSCs and LECs had fibroblast or epithelium morphology, respectively. Positive stain of Oil red O and Alizarin red reflected the adipogenesis and osteogenesis of MSCs. RT-PCR results showed ABCG2, CX43, CK3, and CK12 expression in cultured LECs. Immunocytochemical results revealed positive stain of ABCG2, PCNA, CK3/12 in cultured LECs.Taken together, cultured MSCs without induction were prepared for later transplantation, and cultured LECs could partly keep progenitor or stem cell property, and exhibited clonal expansion and proliferation ability.Experiment Two: Preparation of DPC, optical and mechanical measurements of DPCWe prepared DPC as follows: fresh porcine corneas were soaked to swell in distilled water at 4oC for 12-18 hours. The anterior lamellar stromal buttons were cut, and immersed in 0.25% trypsin, 40U/ml DNAase and RNAase at 25oC for 30 minutes, and 1M NaOH at 25oC for 2 hours, respectively. After washes in distilled water, incubated DPC with 10% trehalose solution at 37oC for 2 hours, froze at -80oC for 1 hour, and lyophilized in a freeze dryer for 16 hours. Finally, 60Co gamma-irradiation was used for sterilization.Flat pieces of fully hydrated DPC, HAM, and RC were used for optical and mechanical measurements. Refractive indices (RI) of DPC, HAM, and RC were recorded using a Abbe refractometer. Transmittances of the three materials were measured over narrow spectral regions centered at 450,500,550,600 and 650nm on a UV/vis spectrometer. The tensile strength and elastic modulus of DPC, HAM, and RC were determined on an Instron micro tester.The RI of DPC was 1.3313, and that of HAM was measured as 1.3322, while the RI of RC measured was 1.3340. The transmittance value of DPC was 50%, lower than that for HAM, which was measured as 70%. DPC was inferior to RC which measured as 92%. The tensile strength of DPC was 2.4MPa, lower than the tensile strength of HAM measured as 3.6MPa and RC as 4.5MPa. The elastic modulus for DPC was 3.8MPa, and that of HAM was 0.78MPa, both lower than that measured for RC as 5.3MPa.In conclusion, DPC had similar optical and mechanical properties to amniotic membrane.Experiment Three: Mesenchymal stem cell transplantation with DPC carrier to alkali burned cornea in a rabbit modelA disc paper saturated with 1mol/L NaOH was used to damage rabbit corneas, and corneas induced with new vessels were randomly divided into 4 treatment groups of 10 samples each: MSCs-DPC transplantation group, LECs-DPC transplantation group, DPC alone transplantation group, and alkali burn group without transplantation. Cultured MSCs and LECs were labeled with PKH26, seeded onto DPC respectively, and MSCs-DPC construct and LECs-DPC construct were cultured. MSCs or LECs grown on DPC were transplanted. The damaged corneal surfaces were carefully keratectomized under anesthesia. DPC with and without grown cells were sutured onto the corneal surface. The grafted cells on DPC were then covered with soft contact lens. After surgery, 0.3% dexamathazone and 1% cyclosporine were applied in LECs group.Rabbit corneas were examined twice a week for 4 months. The corneal surface was examined and graded for transparency, neovascularization, and corneal survival. In 4 months after transplantation, corneas were collected, fixed, and embeded for sectioning. For hematoxylin and eosin staining, slides were stained with H&E. For masson trichrome staining, slides were stained with Masson Trichrome. For PKH26 tracking, slides were examined. For transdifferentiation examination, slides were incubated with primary antibody of CK3/12, and fluorescein-conjugated secondary antibody for immunofluorescent microscopy. For transmission electron microscopy, ultraslides were stained with 5% uranyl acetate and with Reynold's lead citrate, and examined. Corneas were harvested in the 1st, 2nd, 3rd, 4th, and 5th week after MSCs transplantation. Supernatants of the corneal tissue homogenates were assayed for the content of MIP-2 by Real Time PCR.Clinical general view showed that corneas in MSCs-DPC transplantation group and LECs-DPC transplantation group were clear and blood vessel-free postoperatively. Alkali burned corneas had massive opaque edema and new vessels at 1 month. Inflammation and neovascularization in the corneal surface of MSCs or LECs transplanted group were much less than those in the corneal surface of control groups at 1 month after transplantation. And inflammation and neovascularization were not detected in MSCs transplanted corneas at months 4, at the same time, there showed white scar in alkali burned corneas and opaque residue in DPC alone transplanted corneas.Statistical analyses indicated that MSCs transplanted corneas, and LECs transplanted corneas with administration enjoyed sooner repair of more transparent and less vascular corneas, and longer transparent maintenance.PKH26 expression was detected in the rabbit cornea 2 months after transplantation of MSCs on DPC or LECs on DPC. Whereas cytokeratin 3/12 expression cannot be detected in the PKH26 positive sites of rabbits transplanted with MSCs on DPC. It shows that MSCs survived but did not differentiate into epithelial cells.To confirm the gross observataion in MSCs group, histologicl examination was done. HE and Masson Trichrome results showed that implanted MSCs, together with DPC fibers, could be easily observed in the anterior layer of the cornea 1 month after transplantation, with local inflammatory infiltration and new vessels around the grafted lamellar, and with surface covered by 3-4 cellular layers. By contrast, alkali burned corneas without transplantation in 1 month became thicker; inflammation took place and the neovascularization was massive. In 2-3 months, DPC fibers in MSCs transplanted group were mostly degraded, and new tissues were formed. Decreased inflammation and regressive new vessels were also observed in MSCs grafted corneas. However, the newly generated tissues were still distinguishable from adjacent native stromata. By months 4 of implantation, DPC fibers were completely degraded, and inflammation and new vessels were not detected at all. And the newly formed tissues became more mature and revealed a spatially arranged and highly organized configuration parallel to the corneal surface, similar in appearance to their adjacent tissues, with no clear boundary observable between the engineered and native tissues.On months 4, transmission electron microscopy of MSCs transplanted corneas exhibited formation of desmosomes and hemidesmosomes.Down-regulated expression of MIP-2 was observed in MSCs transplant group, as compared with the alkali burn group.In summary, this study demonstrated that mesenchymal stem cell transplantation using DPC as carrier could repair the alkali burned cornea in a rabbit model. Clinical, statistical, and histological outcomes suggested that MSCs transplantation improved corneal transparency, decreased corneal new vessels, and prolonged maintenance of corneal transparency. MSCs may have anti-inflammatory effect on corneal recovery after alkali burn.
|
PDF Full Text Request