| The osteogenesis and angiogenesis of calcium phosphate ceramic can be enhanced by bioactive ions substitution.It is well known that new bone formation is closely bound up to vascularization.We hold the opinion that anyway that can improve the osteogenesis and/or angiogenesis of calcium phosphate ceramic was considered to possess the potential to enhance the osteoinductive property of calcium phosphate ceramic.In this study,bioactive ions(zinc,silicon,strontium and magnesium)with a wide doping range were introduced into calcium phosphate by a doping method using the experimental self-assembly high throughput equipment.The ion doing content range of calcium phosphate powder extracts and scaffolds with the highest in vitro osteogenic and angiogenic activities was first screened out by using proper high throughput screening methods,and the mechanisms of pro-osteogenesis and pro-angiogenesis was summarized.After that,the in vivo osteoinduction and bone defect repairing experiments was conducted to verified the in vitro screening results.Finally,the most effective pro-osteogenesis doping content or content range of a specialized bioactive ion was screened out.This study provides references for improving the osteogenic property and application of calcium phosphate-based materials utilizing ion doping strategy in bone tissue engineering.27 groups of zinc-doped biphasic calcium phosphate(BCP)powders with gradient zinc doping content(0-10 mol.%)but similar compositions were successfully synthesized by a high throughput chemical precipitation method.Zinc tended to enter the β-TCP structure by substituting calcium at the Ca(5)and Ca(4)sites.A burst release of zinc ion in the BCPs into the extracts appeared when the zinc doping content was higher than 6 mol.% and resulted in cell apoptosis.The in vitro osteogenic activity screening results of powder extracts showed that the most effective zinc doping content range and doping content was 2.0-3.0 mol.% and ~2.4 mol.%,respectively.The in vitro osteogenic activity results of the mouse bone marrow stromal cells(m BMSCs)co-cultured with scaffolds showed that the BCP scaffolds substituted with 2.5 mol.% of zinc possessed the highest osteogenic activities.The BCP scaffolds with zinc doping content ≥ 2.5 mol.% can effectively inhibit the differentiation and formation of osteoclasts.Furthermore,the highest new bone formation ratio was found in the BCP scaffolds substituted with 2.5 mol.% of zinc in the in vivo osteoinduction experiment which conducted in the dorsal muscle of beagles.As for the bone defects repaired experiment conducted in the femur bone of rabbits,zinc doping can effectively promote the formation and ingrowth of new bone in BCP scaffolds and the highest new bone formation ratio was also found in the BCP scaffolds substituted with 2.5 mol.% of zinc.The inflammatory reaction and fibrous capsule formation around BCP implanted were reduced by zinc doping,and the introduction of zinc can help the macrophages polarized into M2 phenotype,and consequently improved the osteogenic differentiation of stem cells and accelerated the bone defect regeneration.Hence,an appropriate doping content of zinc can promote the osteogenesis of BCP from many aspects.The phase compositions of strontium-doped calcium phosphate powders prepared by high throughput method were more complicated than zinc-doped calcium phosphate powders,for the doping content range of strontium(0-100 mol.%)was larger.All of the strontium-doped calcium phosphate powder extracts were nontoxicity,and strontium doping can effectively improve the ALP activities and up regulated the osteogenic related genes expression of m BMSCs cultured in the strontium-doped calcium phosphate powder extracts.The screening results showed that the most effective osteogenesis stimulative doping content range of strontium was 30-50 mol.%.Scaffolds with strontium doping content ≥ 20 mol.%can release strontium continuously.Strontium doping can also promote the proliferation and osteogenic differentiation of m BMSCs cultured on the surface of calcium phosphate scaffolds,and the most effective osteogenesis promotional doping content of strontium was further verified to be 30 mol.% according to the ALP activities and the expression of osteogenic related genes.The initial doping content range of silicon in BCP was 0~10 mol.%,and silicon doping was beneficial to the generation of α-TCP,only in the condition of silicon doping content ≤ 4mol.% and calcination temperature < 1100 °C did BCP(HA + β-TCP)generated in silicon-doped calcium phosphate powders.All the silicon doped calcium phosphate powder extracts were nontoxicity,and calcium phosphate powder with an appropriate silicon doping content can effectively improve the ALP activities and up regulated the osteogenic related genes expression of m BMSCs cultured in the silicon-doped calcium phosphate powder extracts.The in vitro osteogenic screening results showed that the most effective silicon doping content range was 2.0-4.0 mol.%,according to osteogenic activities and immune responses of m BMSCs and Raw 264.7,respectively.Moreover,the most effective osteogenesis promotional doping content of silicon was furtherly confirmed to be 4 mol.%,according to screening results of m BMSCs co-cultured with silicon-doped scaffolds.The initial doping content range of magnesium in BCP was 0-100 mol.%,and all of the magnesium-doped calcium phosphate powder extracts were nontoxicity.The phase composition of HA decreased as the doping content of magnesium(≤ 21 mol.%)increased.The extracts from powders whose magnesium doping content in the range of 0-9 mol.% was showed to have high osteogenic activities and the extracts of calcium phosphate powder doped with 3 mol.% of magnesium showed the highest pro-osteogenic properties.Scaffolds with magnesium doping content ≥ 3 mol.% can release magnesium continuously and scaffold substituted with 5 mol.% of magnesium displayed the highest in vitro osteogenic activities.Furthermore,magnesium-doped scaffolds with a doping content ≥ 5 mol.% can promote the proliferation,up regulate the expression of angiogenic related genes and nitric oxide(NO)of human umbilical vein endothelial cells(HUVECs).The in vitro tube formation assay also displayed that the ions microenvironment generated by magnesium-doped(≥ 5 mol.%)scaffolds was beneficial to the migration,rearrangement and vascular-like tube formation of HUVECs.The comparative in vitro and in vivo angiogenic and osteogenic experiments were conducted for the calcium phosphate scaffolds with the optimum doping content of zinc,silicon,magnesium and strontium,respectively.Result showed that,at the osteogenic induction microenvironment,the cells cultured on the surface of zinc-doped BCP scaffolds showed the highest ALP activity,and cells cultured on the surface of magnesium-and strontium-doped scaffolds expressed the highest osteogenic related genes.At the un-osteogenic induction microenvironment,the cells cultured on the surface of zinc-and magnesium-doped scaffolds showed the highest ALP activity,and cells cultured on the surface of strontium-doped scaffolds expressed the highest osteogenic related genes content.Zinc and strontium doping can effectively restrict the formation of osteoclasts.However,silicon doping and magnesium doping made no significantly difference to the osteoclast differentiation.Magnesium-doped scaffold and its extract up-regulated the expression of angiogenic related genes in HUVECs most effectively.The extracts from magnesium-and strontium-doped scaffolds can promote the production of NO in HUVECs.The ions microenvironments generated by silicon-and strontium-doped scaffolds were beneficial to the in vitro tube formation of HUVECs.At the inflammation microenvironment generated by macrophage,zinc-doped scaffold promoted the osteoblast differentiation most effectively because zinc doping can restrict the polarization of M1 phenotype;strontium-doped scaffold up-regulated the angiogenic differentiation most effectively on account of the activation of macrophage by the release of strontium.As for the in vivo ectopic bone formation and bone defect repaired conducted in beagle dorsal muscle and rabbit femur bone,respectively,zinc doping and silicon doping mostly induced the ectopic new bone formation and mature.Furthermore,in the early stage of implantation,zinc and silicon doping can most effectively promote the new bone formation and ingrowth in scaffolds implanted in rabbit femur bone.However,in the late stage of implantation,the scaffolds doped with 30 mol.% of strontium degraded more than any other scaffolds,and the highest degradation rate was accompanied by the highest new bone formation rate.In a word,with an appropriate doping content,zinc,strontium,silicon,and magnesium doping all can improve the osteogenic properties of calcium phosphate.However,the most effective doping content and the mechanism of pro-osteogenic and pro-angiogenic varied with the variety of ions.This study provides theoretical and experimental foundation for the designing and developing of tissue-inductive calcium phosphate by using bioactive ions substitution. |
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