| Background:Bone grafting is one of the most common surgical procedures to increase bone regeneration through surgically implanted materials,only behind blood transfusion and tissue transplantation.According to the incomplete statistics,more than two million bone grafts are performed wor-ldwide each year.At present,the materials for bone reconstruction mainly come from autograft and allograft materials.Autologous bone has various advantages,such as bone conduction,inducibility and osteogenesis,so the use of autogenous bone grafts has become the gold standard for bone regeneration.However,because of the limited supply of autologous bone and complications in the donor area,autogenous bone grafts are still very limited in clinical practice.Therefore,in our study,we prepared the novel strontium-containing ?-calcium sulfate hemihydrate promotes by adopting the co-precipitation and hydrothermal techniques,and established the critical tibia bone defect model in SD rats.We further explored the biocompatibility and safety of different content of strontium-containing ?-calcium sulfate hemihydrate.Furthennore,we demonstrated the effects and mechanism of different content of strontium-containing ?-calcium sulfate on bone repairing both in ivitro and in vivo.Objective:1.To explore the effects of the new bone substitute material(strontium-containing a-calcium sulfate hemihydrate)on bone marrow mesenchymal stem cells(BMSC)in viftro.2.The bone defect models were created by using Sprague-Dawley(SD)rats,which were treated with strontium-containing ?-calcium sulfate hemihydrate to investigate the effect of strontium-containing ?-hemihydratc calcium sulfate on bone formation.3.After treatment with strontium-containing ?-hemihydrate calcium sulfate,the tibia was collected from the defect model,the pathology and other basic tests were performed and the bone formation and the related signal pathway were assessed.Methods:1.The cultured BMSC were treated with 0%,5%,10%strontium-containing?-calcium sulfate hemihydrate for 1,3,7 days.The proliferation ability of BMSCs was detected by MTT assay and soft agar cloning methods;cell apoptosis was evaluated by Annexin V/PI double staining;the invasion ability of BMSCs was assessed by Transwells assay;the osteogenic differentiation of cells was analyzed by Alizarin red staining;Runx2,Osterix,ALP,OCN and BSP expressions were measured by qRT-PCR assay;TGF-?,Smad2/3 and ?-catenin expressions were analyzed by western blot assay.2.The general anesthesia was induced by intramuscular injection of 3%pentobarbital sodium at 30mg/kg body weight.The skin over the proximal tibia was incised and periosteum was cleared using a periosteal elevator.A defect(3 mm wide and 5 mm long)was created by using a micro-burr with a 0.8 mm tip,which starts form below the articular surface in the anteromedial cortex at both tibias.SD rats were divided into four groups to fill the defects with different materials.Blank group,no material was implanted in bone defects;calcium sulfate group,calcium sulfate was implanted in bone defects;5%strontium-containing ?-calcium sulfate hemihydrate group,5%strontium-containing ?-calcium sulfate hemihydrate was implanted in bone defects;10%strontium-containing ?-calcium sulfate hemihydrate group,10%strontium-containing ?-calcium sulfate hemihydrate was implanted in bone defects.After implantation of different materials into the defect site of the tibia of SD rats,the bone reconstruction status was observed by nicroCT.Relative bone volume(BV/TV),trabecular bone number(Tb.N),bone density(BMD),trabecular bone thickness(Tb.Th)and trabecular bone separation degree(Tb.Sp)were measured;X-ray was used to examine the reconstruction of tibial defect in SD rats.3.After the bone defect model was intervened by substitute material,the tibial segment of the bone defect was collected,and the effects of different implant materials on the repair of the tibial defect in SD rats were studied by haematoxylin and eosin(HE)staining,masson staining.qRT-PCR assay was used to analyze the nRNA expression levels of Runx2,Ostenix,ALP,OCN,BSP.Western blot assay was performed to detect the protein expression levels of TGF-?,Smad2/3 and ?-catenin.In addition,immunohistochemical(IHC)assays were used to detect the bone repair at the site of bone transplantation.Results:1.The results from MTT and plate cloning assays showed that the effect of each material on cell proliferation was no significant difference at 25%leachate concentration,indicating that the material cytotoxicity is small;flow cytometry results showed that there was no difference in apoptosis level.The results from ELISA and alizarin red staining assays showed that the osteogenic capacity was significantly enhanced with the content of strontium-contained calcium sulphate The qPCR results showed that the 1RNA expression levels of Runx2,Ostenix,ALP,OCN and BSP also were increased with tlhe content of strontium-contained calcium sulphate;The results from western blot assay also showed that the protein expression levels of TGF-? and Smad2/3 were activated by strontium-contained calcium sulphate2.The results of microCT and X-ray showed that the recovery of bone defect in the blank group was worse than that of the rats in the blank group.At the same time.the recoveiry was better with the increase of the content of strontium-contained calcium sulfate,and 10%strontium sulfate-The results showed that the relative bone volume or volume fraction of rats after 5%and 10%strontium sulfate-containing material implantation was significantly better than the blank group and the calcium sulfate group.3.Hematoxylin and eosin staining and Masson staining showed that the location of defect in bone defect group was poorly recovered,the synthesis of collagen fibers was low,and the expression of osteocalcin was low.After implanting,the bone remodeling was significantly improved.At the same time,with the increase of strontium content,the better the condition,the more collagen fibers are synthesized and the more osteocalcin is expressed.Among them,sti*ontium sulfate with 10%strontium content showed the best recovery.Finally,the expression levels of TGF-? and Smad2/3 were up-regulated with the strontium content increasing.Conclusion:1.In vitro,calcium sulfate with 0%-10%strontium cointcent in 25%leaching solution had less cytotoxicity on BMSC and no obvious effect on cell apoptosis.However,with the increase of strontium content,osteogenic capability was enhanced;strontium-containing calcium sulfate can promote osteogenic differentiation through TGF-?/Smad2/3 molecular signaling pathway.2.In vivo,the bone defect of tibia was serious in blank group;while the bone defect of tibia was better in 0%,5%,10%strontium-containing ?-calcium sulfate hemihydirate groups compared with blank group,and the situation of bone repairing was best in SD rats with shin defect treated with 10%strontium-containing?-calcium sulfate hemihydrate.The specific manifestations include increased bone density,increased collagen fiber synthesis at the defect location,unregulated osteocalcin,unregulated TGF-scaffold/smad2/3.3.New materials containing strontium sulfate with 5%and 10%strontium content have a significant recovery and improvement effect on bone defects. |
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