| Objectives:Failure of bone implant is a serious clinical problem.Early studies have shown that the zinc-modified calcium silicate(Ca2ZnSi2O7)ceramic coating with Zn-Ca molar ratio of 0.5 possesses better effect of promoting pre-osteoblast adhesion,proliferation and differentiation than calcium silicate(CaSiO3)ceramic coating and forming an effective integration with the host bone,which reveals CaSiO3 ceramic coating modified with zinc ion plays an important role on biological effects of pre-osteoblasts.Bone marrow mesenchymal stem cells(BMSCs)are the main source of differentiation of osteoblasts in vivo.It is essential to explore the best molar ratio of Ca2ZnSi2O7 ceramic coating which has best biological activity for BMSCs,and study the biological effect and the mechanism of action of the extract of Ca2ZnSi2O7 ceramic coating to BMSCs.Also it is particularly important basis for experiments in vivo and makeling further exploration of applications in clinical Orthopaedics as a novel bone implant materialMethods:(1)The extraction,purification and identification of BMSCs.BMSCs was extracted by whole bone marrow adherent culture method from SD rats,and was cultured to the third generation.the positive rates of CD29,CD90,CD45,CD31 were detected via flow cytometry Osteogenic and adipogenic differentiations were performed by Alizarin Red and Oil Red O staining.Vimentin staining was observed after immunofluorescence staining.(2)Construction and optimization of Ca2ZnSi2O7 ceramic coating.Sol-gel method was used as well as moderating of the Zn-Ca molar ratio to build Ca2ZnSi2O7 ceramic powder with Zn-Ca molar ratio of 0.1,0.3,0.5 respectively.Atmospheric plasma spray system(APS)was used to get Ca2ZnSi2O7 ceramic coating via plasma spraying according to specific parameters.Testing surface morphology,bonding strength with a titanium and chemical stability of the coating.(3)To observe the biological effects of Ca2 Zn Si2O7 ceramic coating with different Zn-Ca molar ratio for BMSCs.Ion concentration of the coating leaching solution was detecte.The adherion and proliferation of BMSC were tested by CCK-8 kit and observed by inverted microscope cultured on the surface of coating or in the extract solution.The expressions of ALP,BGP,COL-I were detected by ELISA,when BMSCs was cultured on the coating material or in leach liquor which were in bone-forming agent.Calcium nodule formation were observed after Alizarin red staining.ALP secretion was observed after ALP staining.To explore the Zn-Ca molar ratio of Ca2 Zn Si2O7 ceramics which has the best biological activity on BMSCs.(4)To research signal transduction pathway about TGF-?/Smad,MAPK/ERK and PKC which were mediated by TGF-?1 and IGF-1.Using Ca2ZnSi2O7 ceramic coating with optimum Zn-Ca molar ratio as experimental group,BMSCs were cultured in each group extracts containing osteogenic agent.The mRNA expressions of osteogenic marker gene ALP,BGP,COL-I,TGF-?1,Smad2,Smad3 in TGF-?/Smad signaling pathway,and IGF-I,ERK1,REK2,PKC-? in MAPK/ERK,PKC signal transduction pathway were detected using qRT-PCR method.To explore the effect of TGF-?/Smad,MAPK/ERK and PKC signal transduction pathway when biological activity of BMSCs was promoted by Ca2ZnSi2O7 ceramic coating.Results:(1)Using the whole bone marrow adherent method,a large number of high purity SD rat BMSCs were successfully seperated and cultured,which have good potential of differentiation.(2)Successful preparating of Ca2ZnSi2O7 ceramic coating with Zn-Ca molar ratio of 0.1,0.3,0.5,each of the coating surface were relatively rough,and bond strength with titanium were significantly stronger than the CaSiO3 ceramic coating,but no obvious difference between three groups.When each material was soaked in DMEM/F12 medium for 1d,Ca,Si ion concentration was highest,and Zn ion concentration was equal to ordinary culture medium CaSiO3 coating release,while Ca,Si ion concentration released from Ca2ZnSi2O7 ceramic coating decreased with the increasing of Zn-Ca molar ratio,meanwhile Zn ion concentration increases.(3)BMSCs on Ca2ZnSi2O7 ceramic coating surface and in its extraction have better abilitiy of adhesion and proliferation than uncoated titanium and CaSiO3 ceramic coatings,Ca2ZnSi2O7 ceramic coating with Zn-Ca molar ratio of 0.3 was the best coating significantly.Under the condition of adding osteogenic inducers,BMSCs cultured on Ca2ZnSi2O7 ceramic coating surface and in its extraction have better ability to osteogenetic differentiate,generate ALP,COL-I,BGP,and form calcium nodule than the uncoated titanium plate and CaSiO3 ceramic coatings.The Ca2ZnSi2O7 ceramic coating is the best one significantly,whose Zn-Ca molar ratio is 0.3.(4)ALP,BGP,COL-I,TGF-?1,Smad2,Smad3 m RNA expressions of BMSCs in each group cultured in extracts containing osteogenesis were increased over time,the Ca2ZnSi2O7 ceramic coating whose Zn-Ca molar ratio is 0.3 had highest expression,the subsequent was CaSiO3 group.IGF-I,ERK1,ERK2,PKC mRNA expression moderately elevated with the extension of time,no significant difference between groups.IGF-I mRNA expression was increased markedly when BMSCs were cultured in extracts of Ca2ZnSi2O7 ceramic coating with 0.3 Zn-Ca molar ratio for 21 d.Conclusion:Ca2ZnSi2O7 ceramic coating with different Zn-Ca molar ratio has a rough surface morphology,abundant pore and bioactive chemical components,also can release the biological activity of ion,whether BMSCs are cultured on its surface directly or in its leaching solution,all showed better ability to promote BMSCs adhesion,proliferation and osteogenesis compare with the uncoated titanium and CaSiO3 ceramic coating.The Ca2ZnSi2O7 ceramic coating with 0.3 Zn-Ca molar ratio have the most positive impact on the biology of BMSCs.Therefore,this coating possess superior biocompatibility and bioactivity on BMSCs,which significantly improve the adhesion,proliferation and osteogenetic differentiation,Zn ions released on suitable concentration plays a vital role,good chemical stability and high bonding strength with titanium,coupled with excellent antibacterial property,this material as a new type of bone implant has good prospects.Ca2ZnSi2O7 ceramic coating could promote the osteogenetic differentiation of BMSCs through the TGF-?/Smad signaling pathway,meanwhile the MAPK/ERK,PKC signaling pathways have not been activated.IGF-I may also play a role in later period of the osteogenetic differentiation of BMSCs. |
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