The Experimental Study Of Modified Titanium Skirt For Keratoprosthesis

ObjectivesThe research is engaged in developing an improved titanium skirt for keratoprosthesis,the aim of this study was to evaluate biocompatible of keratoprosthesis of novel design.And also to assess the long term clinical outcomes implanted to rabbit corneas(including compalications),to investigate the effect of MMP-2 and TIMP-2 for the skirt inserted eyes in rabbit,and the expression of MMP-2 and TIMP-2 during the course of cornea with HA/SB-Ti skirt and SB-Ti skirt,in order to explore the potential roles of MMP-2 and TIMP-2 during the course of the skirt inserted eyes in rabbit.Methods1.The pure titanium skirt for keratoprosthesis with three hands were first Sandblasted,and then bioactive hydroxyapatite coated on Sandblasted titanium for keratoprosthesis by a acid-alkali chemical treatment.The improved artificial cornea consists of a skirt and a transparent center of polymethyl methacrylate PMMA.The composition,the surface morphology and the cross-section were analyzed by means of X-ray diffraction(XRD) and scanning electron microscopy (SEM).2.A total of 18 New Zealand white rabbits and 18 alkali burned rabbit corneas were respectively divided into three groups.Skirt of HA/SB-Ti and SB-Ti were inserted into the corneal stroma of rabbits for a 1 and 3 month 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 and TEM.The extracellular matrix deposited on the surface of skirt was examined by SEM.3.The transparent center was implanted to rabbit corneas after 3 months,and then assesses the long term clinical outcomes and compalications implanted to rabbit comeas.To investigate the effect of MMP-2 and TIMP-2 for the skirt inserted eyes in rabbit,the corneas were immunohistochemically analyzed with antibodies directed against MMP-2 and TIMP-2,and the expression of MMP-2 and TIMP-2 during the course of cornea with HA/SB-Ti skirt and SB-Ti skirt,and pathohistology and computerized corneal analysis were studied on month 1 and 3. The expression of MMP-2 and TIMP-2mRNAlevel was determined by real time-polymerase chainreaction,and its protein level was determined by western blot.Results1.Dense hydroxyapatite coating was deposited on the Sandblasted specimens by an acid-alkali chemical treatment,and was composed of two sublayers,i.e.an outside loose crystal HA sublayer and an inside dense HA sublayer.The thickness of the HA coating was about 30μm.The shape of titanium skirt was well maintained by the two-step acid-alkali chemical treatment.And compared to group SB-Ti,the HA crystals of group HA/SB-Ti growed larger and more sufficient.The skirt surfaces expanded and keratoprosthesis became firm when keratoprosthesis of novel design consist of three hands.The radius of transparent center of novel design is 8.874 mm;thickness of transparent center is 3.8 mm.2.Corneal oedema and neovascularisation were found in all of the skirt inserted eyes in healthy animals,but the corneal oedema subsided within 1 week.The skirt have been retained,now up to a period of 5 months,with no severe complications such as cataract,retrocorneal membrane formation and retinal detachment,but there was one case of corneal dissolving being found on the 1 month.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 was more dense and tight than that of Ti. Compared with healthy host tissue,skirt/cornea healing after alkali burn was impaired;there were more inflammatory cells and corneal fibroblasts in alkali burn cornea/skirt interface than that of heathy cornea.The investigation of the effects of skirt surface on the interaction of group HA/SB-Ti and SB-Ti,The results showed:HA/SB-Ti surface markedly influenced the corneal fibroblasts attachment pattern and orientation in relation to the rim of the skirt,cells attaching to it perpendicularly or at an angle.The observation of the tissue sections of HA/SB-Ti skirt inserted eyes revealed that there were cells and collagen-like fibres perpendicular to or at an angle to the rough interface and that dramatic changes occured in the cellular morphology within the interface,from spindle shape on the surface to round or ovoid shape with a large cellular body and abundant cellular plasm,which suggested that the cellular function was extremely active,in accordance with the results of TEM.3.The keratoprosthesis of novel design have been retained during the experiment without severe complications such as cataract,retinal detachment and extrusion.Compared with HA/SB-Ti skirt inserted eyes from epithelium healing, corneal inflammation,neovascularization and scarring were all markedly improved in group E after injure,MMP-2 and TIMP-2 immunoreactivities were expressed in the normal corneas,predominantly in the corneal epithelium.After injury,immunoreactivities of both MMP-2 and TIMP-2 were increased notably in the healing corneal epithelium,infiltrating inflammatory cells,stromal fibroblasts and ingrowing vascular endothelial cells.The expression of MMP-2 was lower in the corneal tissue sections of HA/SB-Ti skirt inserted eyes than that in the tissue sections of SB-Ti skirt inserted eyes(P＜0.01).The Western bolt revealed no significant differences of MMP-2mRNA between group 3 month and 2 week(t test of Ct,P＞0.05);MMP-2 immunoreactivities were absent or lowly expressed predominantly in the corneal epithelium of normal corneas.The expression of MMP-2,TIMP-2 mRNA level was paralled that of protein level.Conclusions1.The titanium rough surface produced by sandblast(SB),then by a two-step acid-alkali chemical treatment and a proper solution,a hydroxyapatite coating can be quickly deposited on Sandblasted titanium skirt of keratoprosthesis, which is exempt from the procedure of polishing.The skirt surfaces expanded and keratoprosthesis became firm when keratoprosthesis of novel design consist of three hands. 2.Hydroxyapatite modified Sandblasted titanium skirt for keratoprosthesis promoted superior rabbit corneal fibroblast adhension and proliferation in comparison with Sandblasted titanium,and it could promote the interfacial biointegration of skirt and host cornea,no matter in healthy comea or diseased cornea(alkali burn cornea).Hydroxyapatite coationg improved the bioactivity of titanium.3.By Immunohistochemitry and analysis system of pathologicpicture,the relative studies showed the different expressions of MMP-2 and TIMP-2 in different period and they have simultaneous incidence with corneal dissolving. The studies of MMP-2,TIMP-2 can provide a new way to prevent the incidence of corneal dissolving after surgery for keratoprosthesis,.The exciting and inhibiting mechanism of MMP-2 and TIMP-2 as well as how to balance those in order to prevent corneal dissolving perforation are still needed to be studied.