The Experimental Study Of Hydroxyapatite Coating Combined With Mesenchymal Stem Cells To Increase Keratoprosthesis Biointegration

Objective:Preliminary assessment on safety and effectiveness of surface modified materials (surface coating of hydroxyapatite recombination with MSCs in Titanium and PMMA which is the most widely used artificial cornea materials) in vitro and in vivo. To consolidate of the interface and reduce complications such as corneal melting related KPro by surface modification.Methods:The experiment included three parts:Part I.Surface coating of HA in TI and PMMA respectively by MPS and SBF was analysed by SEM and EDX. The strength of the coating and the stability in balanced salt solution were also carried on. Part Ⅱ①Mouse RFP-MSCs seeded in ECM were ovserved in fluorescence microscope in2w,4w and8w, and also examined by SEM and toluidine blue staining.②Compared two material (HA-TI and HA-PMMA) by MTT cell growth curve, respectively seed on rabbit MSCs and human MSCs. Observation of cells and ECM were carried by SEM. Rabbit MSCs and human MSCs were induced into bone and cartilage in vitro, respectively identified with alizarin red and alcian blue staining.Part Ⅲ:①Mouse RFP MSCs-ECM were implanted in6eyes of rabbit corneal lamellar pocket (no-cells ECM implantation as control). The survive cells were observed in live imaging and fluorescence microscope. ECM frozen section were examined under light microscope (HE stain, toluidine blue staining).②Rabbit MSCs on two materials nearly induced into cartilage in vitro, were implanted into12rabbit corneal lamellar pocket.Clinical observation and pathology (HE staining, Masson staining and TEM scanning) were carried in2w,4w and8w.③Rabbit MSCs compound two kinds materials were implanted under the6rabbits back muscles. The organization around the materials were examined by HE and Masson staining.Conclusion:①Two methods could achieve uniform continuous coating which was analysed with composition of hydroxyapatite by EDX. Coating with MPS of titanium was more compact and stable which was test in simulated body fluids; Coating for PMMA with SBF was less in strength test and a small amount of calcium salt deposition was observed in the simulated body fluids.②M ouse RFP-MSCs in ECM showed a trend of increasing over time and red marked cells attached net-like were observed by SEM scanning. MTT analysis in three groups show significant difference between HA-PMMA and control group (P<0.01), HA-PMMA and HA-PMMA (P<0.01). Cell number in HA-TI was significantly higher than HA-PMMA by SEM scanning.③M TT analysis showed no obvious difference among three groups in human MSCs in vitro.④Rabbit M SCsand human M SCs could be induced as bone and cartilage in HA coating material surface which were identified by alizarin red and alcian blue staining.⑤Mouse RFP-MSCs implanted into rabbit cornea lamellar pocket could be observed in living imaging (8weeks) and cells counting show a trend of growth in fluorescence microscope. Clinical observation and pathological results showed mild inflammation and no neovascularization.