The Research Of The Effect And Mechanism In Zinc Doped Calcium Silicate Coating Integrating With Host Bone

Objectives:Although the success rate of implant placement is high at present,the problem of implant failure is still an urgent problem to be solved currently.Previous studies have confirmed that Zn/Ca ratio of 0.5 zinc-doped calcium silicate ceramic coating has a good role in promoting pre-osteoblast adhesion,proliferation,osteogenesis differentiation,and formating effective integration on the host bone interface suggesting that the coating play an important role in the biological effects of bone integration.Osteoporosis is a common underlying disease in clinical practice and is one of the important factors result in the failure of bone implant embedding.It is very important to explore the zinc-doped calcium silicate ceramic coating with good bioactivity and biocompatibility for bone integration and to study its role and mechanism in bone integration of osteoporosis model.To further explore its role as bone implant coating applied to orthopedics and dental clinical lay the experimental basis.Methods:?1?To build and optimize the zinc-doped calcium silicate ceramic coating.The Zn-Ca ratio of 0.1,0.3,0.5 zinc-doped calcium silicate ceramic powders were prepared by sol-gel method and the ratio of zinc-calcium ions was adjusted.Then,the samples were sprayed with atmospheric plasma spraying system?APS?under the specific parameters.The zinc-doped calcium silicate ceramic coating was obtained to detect the coating,s surface morphology,internal microstructure,chemical stability and the bonding strength with the titanium base material.?2?To study the biological effects of zinc-doped calcium silicate ceramic coating on pre-osteoblast MC3T3-E1 and osteoclast RAW264.3.We cultured the pre-osteoblasts?MC3T3-E1?and osteoclasts?RAW264.7?on the surface of zinc-doped calcium silicate ceramics with vrious Zn/Ca ratios,studied their The biological characteristics to explore the optimum Zn/Ca ratio in the zinc doped calcium silicate coating,and weather the Zn ions which release from the coating con promot osteogenic differentiation.?3?To construct the model of osteoporotic rabbit and study the effect of zinc-doped calcium silicate ceramic coated titanium rod on normal New Zealand rabbits and osteoporosis model.The bone formation and growth around the implant were measured by micro-CT scanning and hard tissue staining.The differences of Interface integration and the the bone trabecular microstructure between the control group and the experimental group was compared.?4?To observe the biological effects of zinc-doped calcium silicate ceramic coating on BMSCs with different Zn/Ca ratios and study the mechanism of zinc-doped calcium silicate ceramic coating promoting osseointegration.We cultured and identified BMSCs extracted from SD rats,then used chemical method,ELISA,q RT-PCR and other methods to explore the effects of BMSCs on titanium alloy sheet,HA?hydroxyapatite?ceramic coating,CaSiO₃ ceramic coating Titanium,Zn/Ca ratio of 0.1,0.3 zinc-doped calcium silicate ceramic coated titanium surface on proliferation,osteogenesis differentiation,osteogenic indicators such as ALP,COL-I,OCN and RUNX2.Moreover,the mechanism of zinc-doped calcium silicate ceramic coating promoting osteogenic differentiation and bone regeneration is not known.In order to further study the potential molecular mechanisms,we explored potential target genes associated with signal transduction of BMSCs osteogenesis differentiation on different ratio coatings.Relevant studies have shown that TGF-?pathway plays an important role in the differentiation of BMSCs into osteogenic and cartilage-forming pedigrees.TGF-?plays an important role in the formation of bone formation by activating their receptors to induce phosphorylation of transcription factors in a group of cells,called Smads.Smad2/3 as a substrate for TGF-?receptors.The activated Smad complex can then be moved into the nucleus to trigger transcription of a set of target genes.We investigated the expression of TGF-?1,Smad2and Smad3,and the gene expression of classical MAPK signaling pathway by q RT-PCR and Western-Blot,including IGF-I,ERK 1/2 and PKC-?.TGF-?inhibitor SB431542 will also be used to study the direct effect of TGF-?signaling on different coatings during BMSCs differentiation.Results:?1?Zinc-doped calcium silicate ceramic coatings with Zn/Ca ratios of 0.1,0.3 and 0.5were prepared respectively.The surface morphology of the three coatings was rough,the internal microstructures were typical ceramics crystal structure and titanium bond strength were better than ceramic coating without Zn ions,hydrophilic were similar to Ca Si O₃ceramic coating which better than pure titanium sheet.All of these three coatings can slowly release Zn ions,the Zn ion concentration increased with the Zn/Ca ratio increasing,and gradually became saturated after 7 days.?2?Zinc-doped calcium silicate ceramic coating with Zn/Ca ratio of 0.3 showed the highest cell viability in the pre-osteoblast MC3T3-E1 cells and significantly promoted osteogenic differentiation,upregulated the expression of ALP,COL 1 and OCN,which showed the lowest cell viability for osteoclast RAW 264.7 cells and significantly inhibited the expression of osteoclast gene OAR,TRAP and HYA1.Therefore,the coating also has the effect of promoting osteoblasts and inhibiting osteoclasts,to form a dynamic balance between osteoblasts/osteoclasts the role of promoting bone integration has the best effect.?3?The osteoporotic rabbit model was successfully constructed,and the bone mass and bone mineral density of the model were significantly lower than those of normal New Zealand rabbits.The zinc-doped calcium silicate ceramic coating showed good biocompatibility and bone formation after implantation of normal mature New Zealand rabbit femur.Micro-CT scan results show that the zinc-doped calcium silicate ceramic coating has a better effect on osteoblastic growth and bone formation than the uncoated titanium rods,HA ceramic coatings and CaSiO3 ceramic coatings Zn/Ca ratio of 0.3 was the best ratio affect on the zinc-doped calcium silicate ceramic coating.The results of histopathological analysis showed that compared with other coatings,the osteoporosis model Can better promote the formation of new bone interface bone,which Zn/Ca ratio of 0.3 zinc-doped calcium silicate ceramic coating the best results.?4?The zinc-doped calcium silicate ceramic coating can significantly promote the proliferation and osteogenic differentiation of BMSCs in vitro,significantly increase the expression of ALP,Col-1,OCN and RUNX-2 mRNA and the activity of ALP,COL-I,OCN secretion and calcium deposition increased with a clear dose or time-dependent trend.Zn/Ca ratio of 0.3 zinc-doped calcium silicate ceramic coating can better promote activity of BMSCs.It was confirmed that TGF-?/Smad signaling pathway plays an important role in the process of bone integrationg of the zinc-doped calcium silicate ceramic coating and the MAPK/ERK signaling pathway doesn't work in the process.Conclusion:Zn/Ca ratios of 0.1,0.3,and 0.5,respectively,have a relatively coarse surface morphology,the internal microstructure of typical ceramic crystals,excellent hydrophilicity,and slow release as a biological Active ion Zn ion.Zn/Ca ratio of 0.3zinc-doped calcium silicate ceramic coating,which have shown the best results in promoting the biological activity of osteoblasts MC3T3-E1 cells and osteogenic differentiation ability,or inhibiting the biological activity of osteoclast RAW 264.7 cells and osteoclast gene expression.and regulation of osteoblasts/osteoclasts dynamic balance.After implanting a successfully constructed osteoporotic rabbit model,the coating also showed optimal osteoblast formation,bone growth,and bone integration.At the same time,the coating in vitro also showed the best resultsin promoting the proliferation of BMSCs,osteogenic differentiation and mineralization process.We confirmed that TGF-?/Smad signaling pathway to play the corresponding cellular biological activity,The MAPK/ERK signal path was not activated during this process.