Biocompatible Study Of Hydroxyapatite Modified Titanium For Keratoprosthesis (in Vitro And In Vivo)

Objectives.Preparation of bioactive hydroxyapatite coating on pure titanium for keratoprosthesis by a acid-alkali chemical treatment. To evaluate in vitro and in vivo biocompatible of hydroxyapatite modified titanium for keratoprosthesis.Methods.1. The specimens (pure titanium skirt for keratoprosthesis) were divided into group A and group B. Group A were first polished, and then treated in boiling 0.6mol/L NaOH solution at 160 ℃ for 24 h; Group B were first etched with mixture of H₂SO₄ and HCl for 1h, and then treated in boiling 0.6mol/L NaOH solution at 160℃ for 24 h; After being washed, the specimens of the two groups were immersed in eight different supersaturated calcification solutions(SCS1-SCS8) for 24h. The composition, the surface morphology and the cross-section were analyzed by means of X-ray diffraction (XRD) and scanning electron microscopy (SEM). The surface roughness before deposition was investigated by Talysurf 5P-120 topographer.2. We used bioactive hydroxyapatite to modify titanium surfaces. Fourth to sixth passage fibroblasts of rabbit cornea were seeded on hydroxyapatite modified titanium surfaces, pure titanium surfaces and glass surfaces. Cell adhension, proliferation and morphology were detected at 3 hours, 24 hours, 48 hours, and 72hours using a acridine orange stain. Further studies of cell morphology were performed using scanning electron microscopy.MTT assay were used to examine cytotoxicity of test materials.3. A total of 15 New Zealand white rabbits and 15 alkali burned rabbit corneas were respectively divided into three groups. Skirt of HA-Ti and Ti were inserted into the corneal stroma of rabbits for a 8 and 16 week period.The third group did not insert skirt as surgery control. Corneal oedema and neovascularisation were evaluated. The interfacial biointegration of skirt/cornea were examined under light microscopy by HE . The extracellular matrix deposited on the surface of skirt were examined by SEM.Results.1. Dense hydroxyapatite coating was deposited on the specimens immersed in the solution of SCSI, and was composed of two sublayers, i.e. an outside loose crystal HA sublayer and an inside dense HA sublayer.A scratching test indicated that the inside sublayer was strongly bonded to the pure titanium substrate. The thickness of the HA coating was about 30μm. The shape of titanium skirt were damaged by polishing, but can be well maintained by the two-step acid-alkali chemical treatment. And compared to group A, the HA crystals of group B growed larger and more sufficient.2. MTT assay indicated that hydroxyapatite modified titanium and pure titanium did not hinder the proliferation of rabbit corneal fibroblasts. Cell counts were significantly greater on hydroxyapatite modified titanium surfaces at each time point(p<0.05). At 3 hours, cell spreading was greater on hydroxyapatite-coated titanium and glass than that on the pure titanium. At 48 hours, compared with pure titanium and glass, the cells on hydroxyapatite modified titanium surfaces had greater spreading area and longer stress fibers. At 72 hours, hydroxyapatite modified titanium surfaces were totally covered by collogen.3. Corneal oedema and neovascularisation were found in all of the skirt inserted eyes in healthy animals, but the corneal oedema subsided within 4 weeks. The number of corneal fibroblasts increased significantly in HA-Ti skirt inserted eyes compared with Ti skirt inserted eyes. The extracellular matrix deposited on the surface of HA-Ti skirt were more dense and tight than that of Ti. Compared with healthy host tissue, skirt/cornea healing after alkali burn were impaired, with evidence of epithelial downgrowth in 17% Ti skirt inserted eyes, and there were more inflammatory cells and corneal fibroblasts in alkali burn cornea/skirt interface than that of heathy cornea.Conclusions.1. By a two-step acid-alkali chemical treatment and a proper solution, a hydroxyapatite coating can be quickly deposited on titanium skirt of keratoprosthesis, which is exempt from the procedure of polishing.2. Hydroxyapatite modified titanium and pure titanium had no cytotoxicity and were safe implantation. Hydroxyapatite modified titanium promoteed superior rabbit corneal fibroblast adhension and proliferation in comparison with pure titanium. Hydroxyapatite coationg improved the bioactivity of titanium.3. Hydroxyapatite modified titanium skirt for keratoprosthesis promoted the interfacial biointegration of skirt andhost cornea, no matter in healthy cornea or diseased cornea (alkali burn cornea). In alkali burn cornea, the biointegration of Kpro skirt were reduced but not prevented.