[An experimental study of mesenchymal stem cells in tissue engineering scaffolds implanted in rabbit corneal lamellae to increase keratoprosthesis biointegration].

OBJECTIVE: To complete a preliminary evaluation of the feasibility of implanting the complex of mouse bone marrow mesenchymal stem cells (BMSC) and a tissue engineering scaffold into rabbit corneal lamellae, based on which a solution may be proposed to consolidate the keratoprosthesis and the recipient surface, and to reduce the risk of complications.

METHODS: This experimental study was composed of two parts. (1) In vitro: some mouse BMSC were marked with red fluorescent proteins (RFP) and integrated with a decellularized pig articular cartilage extracellular matrix (ECM) scaffold. The cell survival was observed under a fluorescence microscope at 4 and 8 weeks. The cell distribution was examined by toluidine blue staining. The pore structure and the cell adhesion were observed under a scanning electron microscope. (2) in vivo: the complex of mouse BMSC and a decellularized scaffold was implanted into the lamellar cornea of 8 rabbit eyes with the fellow eyes as the controls. The eyes were sampled for observation using HE staining under a light microscope at 2, 4 and 8 weeks, respectively. The cell survival was examined under a fluorescence microscope, and the intracorneal cell survival at 8 weeks was observed using in vivo imaging. The conditions of ocular anterior segment of all the experimental animals were recorded.

RESULTS: (1) Under the scanning electron microscope, the ECM scaffolds showed satisfactory porosity required for the adhesion and growth of cells and tissues, and the cell distribution over the cell-scaffold complex can be observed by toluidine blue staining. (2) Under the immunofluorescence microscope, cell proliferation was observed in vitro and in the interlamellar space (the maximum observation time was 8 weeks) after the RFP-marked mouse BMSC were integrated in vitro with ECM scaffolds. (3) Under the light microscope (HE staining), the stromal cells were detected to increase at each timepoint. A small number of monocytes and some mouse BMSC were observed in the superficial layer of corneal stroma, with sparsely and orderly arranged collagenous fibers and no neovascularization. All the epithelial cells appeared as mononuclear, columnar and undamaged, and the shape of ECM scaffolds, which were fused with the collagens, became unclear. (4) By in vivo imaging, it was found that the mouse BMSC survived for 8 weeks after being integrated with scaffolds and implanted into the interlamellar space of rabbit cornea. (5) After the implantation of cell-scaffold complex, severe postoperative inflammatory reactions, obvious conjunctival congestion and neovascularization were not observed. The corneal tissues surrounding the recipient area were transparent. One week later, mild inflammatory reactions were barely observed, and the cornea was transparent enough to observe the scaffold in the stromal layers. Four weeks later, the scaffolds became thinner. Eight weeks later, the scaffolds became extremely thin with normal vascular system in the corneal limbus.

CONCLUSIONS: The ECM scaffold is a solid and biocompatible carrier for the growth and proliferation of BMSC. The mouse BMSC can grow and proliferate in the microenvironment of the interlamellar space of cornea.