Preparation Of Cobalt-doped Calcium Phosphate Cement And Study On Its Induced Angiogenesis Of Bone Tissue

Purposes:Hypoxia-inducible factor(HIF-la)is a core transcription factor that regulates tissue and cell adaptation to hypoxic conditions.Divalent cobalt ions as a hypoxia inducer can significantly promote the expression of HIF-1α.In this study,the cobalt of trace element was added into the calcium phosphate cement materials and further mimicked the hypoxia state of cells,leading to spontaneous production of angiogenic factors,such as VEGF-A,etc.Ultimately,we could develop the biodegradable bone repair materials with long induced angiogenesis activity.Besides,the materials had the potential to stimulate the rapid and full angiogenesis surrounding them,which was thus conducive to the new bone formation,alteration and maturity and finally accelerated the procedure of bone defects repair.Methods:1.Injectability,setting time,degradation performance and cobalt ion sustained release effect in vitro of the materials were characterized.2.Evaluations of cytotoxicity,cell adhesion,cell morphology and cytoskeleton were carried out.3.Quantitative real-time PCR was used to detect the expression of HIF-la of MC3T3-E1 and the VEGF-A was detected by ELISA kits.In vitro angiogenesis assay of HUVECs cultured on BD Matrigel TM in the presence of different bone cement extracts was conducted to explore the effects on the angiogenesis of different bone cement extracts.4.To assess the ability of induced angiogenesis and osteogenesis by doped cobolt Calcium phosphate bone cement,the technology of histopathology and immunohistochemistry were performed.Results:1.Although the early injectability of all bone cements was not significantly affected,injectability of them gradually decreased when duration of the injection was more than 3 minutes with the increase of cobalt carbonate content.The initial setting time of bone cement gradually shortened while the final setting time had no significant change with the increase of cobalt carbonate content.The influence of different cobalt carbonate contents on degradation rate of bone cements were as follows:0%>1%>5%>10%.Cobalt-doped bone cements could slowy release cobalt ions,and the sustained-release time almost reached 56 days.2.The cytotoxicity of bone cements with different content of cobalt carbonate(0%,1%,5%and 10%)was respectively as follows:100.4%,123.2%,96.7%and 83.1%.The cells were able to adhere,grow and proliferate on all cements and the ability of promoting cell proliferation was 1%>0%>5%>10%.In addition,the morphology of the cells grown on these cements in which the content of cobalt carbonate was 0%,1%or 5%had no obvious changes.But the morphology of cells grown on the cements in which the content of cobalt carbonate was 10%was significantly different from the other groups.What’s more,the cytoplasm was atrophying and the cell synapses were obviously reduced and shorter.3.Doped cobolt calcium phosphate bone cement could mimic the hypoxia status of cells,and the transcription level of HIF-1a was inhibited by cobalt ions released from bone cements.Besides,the expression level of VEGE-A in these cells was increased with the increase in the content of cobalt carbonate.The junction,circle and total length of the HUVECs cultured with the cobalt-doped calcium phosphate bone cement extracts were significantly higher than those in the HUVECs cultured with control medium.That was to say the effects of inducing angiogenesis were arranged as follows 0%,1%,5%and 10%.4.The immunohistochemistry of subcutaneous tissue section with 1%content of cobalt carbonate showed a large number of capillaries.5%group showed the highest vascular density,but the diameter of vessels was slightly less than the former.The other groups were hardly to find blood vessels.The immunohistochemistry of bone tissue section showed the same results with the the immunohistochemistry of subcutaneous tissue section.2 months after surgery,the blank control and 0%groups indicated without obvious capillaries while the 1%and 5%groups showed obvious capillaries and the 5%group with the highest density.At 3 months postoperatively,the angiogenesis was degenerated.The 1%and 5%groups had the new bones growth.What’s more,there were Hover’s tubes in the 5%group.Bone tissue HE staining showed that the effects of osteogenesis were arranged as follows:control<0%<1%<5%.The results of bone tissue masson staining were follows:the bone collagen of blank control and 0%groups were sparse while the the bone collagen of 1%and 5%groups filled the bone defects fully.Besides,the collagen of 5%group was more dense,mature and obvious.Conclusions:1.The incorporation of cobalt carbonate into conventional calcium phosphate cement to improve the biological function of existing biological materials was of feasibility.2.A small amount of cobalt carbonate(≤1%)could be more conducive to cell proliferation and adhesion and improve the biocompatibility.However,when the cobalt carbonate was more than 10%,it would aggravate the cement of cytotoxicity and further inhibitted cell proliferation and adhesion.3.The cobalt-doped calcium phosphate cement could obviously promote the release of VEGF-A by vascular endothelial cells,which consequently significantly induced the proliferation,migration and differentiation of cells.4.In vitro,the coated cobalt phosphate cement significantly increased local capillary regeneration and synthesis of collagen and then accelerated the formation of callus,alteration and remodeling.