The Osteogenic Effects And In Vivo Application Of Chitosan/siCkip-1 Biofunctionalized Titanium Implant

With the rapid development in recent years, dental implant has become one of the major prosthodontic approaches for edentulous patients, and the 10-year survival rate could be about 95%. However, patients with systemic bone disease, such as osteoporosis, still suffer from the risk of implant failure, which is due to impaired bone formation and osstointegration. There are many approaches to improve the osstointegration in osteoporotic condition, among which biofunctionalization represents a promising one. It is found that casein kinase-2 interacting protein-1(Ckip-1) is key to bone metabolism by negatively regulating the osteoblast differation and bone formation.The Ckip-1 knockdown by si Ckip-1 notably increases the bone formation in osteoporotic condition. Thus, Ckip-1 si RNA(si Ckip-1) is a good candidate for the implant biofunctionalization to improve the bone formation, thereby to increase the success rate of dental implants.In this study, using chitosan as a gene carrier, we prepared CS / si Ckip-1 complex through electrostatic interactions, and then developed CS/si Ckip-1 biofunctionalized TA Ti implant(TA-CS/si Ckip-1) by coating the CS/si R complex onto the thermal alkali(TA) treatment Ti implants. At first, we observed the implant surface microstructure and analyzed its surface properties; then we evaluated the transfection of si Ckip-1, as well as the cell proliferation and osteogenic differentiation level of the rat primary bone marrow mesenchymal stem cells(r BMMSCs) on the surface of the implant; finally, the in vivo osseointegration in the osteoporostic rat model was assessed. Our study was designed to confirm the feasibility of the application of si Ckip-1 by evaluating its effects comprehensively. The purpose of our study is to provide a basis for surface modification by si RNA in osteoporotic condition and to guide the design of biofunctionalized implants.PartⅠ Preparation and surface analysis of TA-CS/si Ckip-1[Objective] To develop TA-CS/si Ckip-1 and analyze its surface properties, and provide a suitable model for the following experiments.[Methods] The hydrodynamic size and the zeta potential of the CS/si Ckip-1 complex were examined by a Malvern zeta sizer. For TA treatment, the PT samples were soaked in sodium hydroxide aqueous solution at 60 °C. Then the CS/si Ckip-1 complex was loaded onto the TA samples, and TA-CS/si Ckip-1 was developed.The surface morphology of PT, TA and TA-CS/si R substrates were observed by scanning electron microscopy(SEM). The surface of Ti-CS/si Ckip-1 with Cy3 labeled si Ckip-1 was scanned by confocal laser scanning microscopy(CLSM). The surface wettability of the Ti samples was evaluated by using the contact angle system. The protein adsorption of BSA was quantified by the BCA protein quantification kit.[Results] The size of CS/ si Ckip-1 complex varied from 100 to 1000 nm with an average value of ~ 235.9±25.6 nm. The mean zeta potential of CS/ si Ckip-1 complex was about 13.7 ±2.77 m V.TA possessed a microporous/nanofibrous network structure. CS/si Ckip-1complex was evenly covered on the TA and could enter into the microporous and the interfibrous space. The thickness of the CS/ si Ckip-1 complex layer was about 2000 nm.TA treatment could significantly increase the surface wettability. And the contact angle of TA-CS/si Ckip-1 was much lower compared to that of TA but still higher than that of PT. TA-CS/si Ckip-1 resulted in significantly enhanced protein adsorption amounts which were even higher than those induced by PT and TA.[Conclusion] CS/si Ckip-1 complex formulated by electrostatic interaction owned low poly dispersition index and showed positive charge. The complex could cover on the TA implants evenly. TA-CS/si Ckip-1 showed enhanced wettability and protein adsorption ability.Part Ⅱ The regulation of TA-CS/si Ckip-1 on biological function of r BMMSCs[Objective]To evaluate the proliferation and osteogenic differentiation of r BMMSCs on TA-CS/si Ckip-1 implants.[Methods] The r BMMSC cells were insolated using all bone marrow adherence method and seeded on TA-CS/si Ckip-1 with Cy3 labeled si Ckip-1. After culturing for 24 hours, the transfection of si Ckip-1 was observed by confocal laser scanning microscopy. 1, 4 and 7 days after seeding, the cell proliferation was evaluated quantitatively using CCK-8. At predetermined time points, the alkaline phosphatase(ALP) production, collagen secretion and extracellular matrix(ECM) mineralization were measured to evaluate the osteogenic differentiation of MSCs on the Ti samples[Results]The cells at passage 2–4 were used in the experiments. 48 h after the cell seeding, almost all the si Ckip-1 was located surrounding the cell nucleus, indicating a successful cellular internalization of the CS/si Ckip-1 complex.Compared to PT, TA led to significantly higher cell proliferation. No apparent difference in cell proliferation was observed among TA, TA-CS, TA-CS/si NC and TA-CS/si Ckip-1 at all time slots. TA-CS/si Ckip-1 generated much more ALP and collagen product and better ECM mineralization than all the controls.[Conclusion] TA-CS/si Ckip-1 showed good biocompatibility, and si Ckip-1 could enter into the cells and improve the proliferation and osteogenic differentiation of r BMMSCs.Part Ⅲ The effects of TA-CS/si Ckip-1 on osseointegration of osteoporostic rat[Objective]To establish the rat osteoporosis model and evaluate the effect of TA-CS/si Ckip-1 on the osseointegration in osteoporotic condition.[Methods]The female SD rats underwent bilateral ovariectomy(OVX),and after three months the femurs were scanned by micro-CT.The implants were inserted into the femurs of the osteoporotic rats. After 4 and 12 weeks, the osseointegration was evaluated using Micro-CT reconstruction and quantitative analysis, histological observation(Van-Greson staining), EDX scanning of bone-implant interface and biomechanical assessment(pull-out test).[Results]Micro-CT showed that the osteoporotic model was successful established. Micro-CT indicated that the new bone formation around TA-CS/si Ckip-1 was far better than the controls. VG staining showed that the bone on TA-CS/si Ckip-1 was continuous and closely contacted the implant surface. The line-scanning indicated that TA-CS/sickip-1 had the maximum range of newly formed bone around implants, as indexed by the Ca and P rich substance. Bone-implant bonding strength increased with the healing time from 4 to 12 weeks. TA-CS/si Ckip-1 showed obviously higher maximal pull-out force and ultimate shear strength compared to the three controls at both time slots.[Conclusion] In vivo, TA-CS/si Ckip-1 could significantly improve the osseointegration in osteoporotic condition.