Preliminary Study On The Effect Of TRPV5 Pathway On Osteoclast Bone Resorption In Osteoclast And Coral Tissue Engineered Bone Culture System

Background Bone transplantation is one of the most common surgical operations.Many patients have a large range of bone defects due to various factors such as trauma,tumor and congenital malformation,so bone transplantation is needed to fill the corresponding bone defects.The gold standard for the selection of fillings is autologous bone.However,,the clinical application of autologous bone graft is limited due to the limited supply,secondary injury and increased risk of infection,so corresponding bone replacement materials are needed to fill in.Coral tissue engineering bone retains the loose and porous structure of natural coral bone similar to human bone and is a good bone substitute material.Coral tissue engeering bone(CTEB)also known as Coralline Hydroxyapatite(CHA).CTEB is the product of natural coral bone after "hydrothermal replacement",which converts the calcium carbonate into hydroxyapatite in a certain proportion.While maintaining the pore structure similar to that of human cancellous bone,CTEB also increases its mechanical strength,as well as its osteoconductivity and biocompatibility.However,the degradation and absorption rate of CETB in vivo is not well matched with the growth rate of new bone,and the degradation and absorption rate is slow,which limits its clinical application.Therefore,it is very important to reveal the mechanism of coral tissue engineered bone degradation to further promote its application.Osteoclast(OC)is the only known cell with Osteoclast effect in human body.TRPV5 is an epithelial Ca2+ channel protein,expressed on the brushy border membrane of osteoclasts,and is crucial for Osteoclast differentiation,calcium ion homeostasis and bone resorption.This study intends to take TRPV5 as the entry point to study the degradation mechanism of coral tissue engineered bone,provide a theoretical basis for the construction of an ideal degradation and absorption rate,and further solve the problem of the source of bone replacement materials.Objective The in vitro degradation model of coral tissue engineered bone was established by co-culture of coral tissue engineered bone and osteoclasts,and the ion channel mechanism of coral tissue engineered bone degradation was partially revealed by taking the TRPV5 channel as the entry point.Methods We induced mouse mononuclear macrophage RAW264.7 into osteoclast by RANKL and M-SCF and identified it by anti-tartaric acid phosphatase(TRAP)staining.Select the best passage method of RAW264.7 by cell morphology and positive number of TRAP staining.Coral tissue engineered bone slices were prepared and co-cultured with osteoclasts to construct a coral tissue engineered bone co-culture system.The growth of osteoclasts on coral tissue engineered bone was observed by cellular immunoluminescence.The expression of TRPV5 on the induced osteoclasts was determined by laser confocal analysis.The osteoclasts and coral tissue engineered bone culture system were treated with different concentrations of Ru R(0,,50,500,5000,nmol/L)and at different times(1,2,3 d).The changes of the bone resorption lacunae area were observed by light microscope and electron microscope.Results TRAP staining showed that RAW264.7 can be induced to differentiate into mature osteoclasts.RAW264.7 cells passaged by cold stimulation have less morphological changes and can differentiate more osteoclasts;scanning electron microscopy results show that osteoclasts are in Coral tissue engineered bones grow well;a co-culture system of osteoclasts and coral tissue engineered bones can be established;CCK8 results show that after 24 h,48h,72 h culture,the OD values measured by the CHA co-culture group are all greater than those of the control group, with significant differences(P<0.01);The results of Annexin V showed that the apoptotic rate was higher in the co-culture group with coral tissue engineered bone(CHA)(P<0.01);the results of Fluo-4AM calcium ion probe showed that the cells in the CHA co-culture group The internal calcium ion concentration is higher(P<0.05);WB results show that TRPV5 on OC is significantly suppressed after treatment with50,100,and 5000 nmol/ml Ru R for 2 days;laser confocal results show that after 500,5000 nmol/ml Ru R After 2 days of treatment,the F-actin ring on OC increased significantly(P<0.05),while the F-actin ring of osteoclasts treated with 50nmol/ml Ru R for 2 days was not significantly different from the control group;Ru R had an effect on osteoclast bone The inhibitory effect of absorption capacity increases with the increase of its concentration(0,50,100,5000 nmol/ml).Conclusions Compared with the traditional trypsin digestion and passage method,RAW264.7 cells are more suitable for passage by cold stimulation.RAW264.7 cells passaged by cold stimulation have less morphological changes and induce more osteoclasts;Osteoclasts can grow well on coral tissue-engineered bone slices.TRPV5 can be successfully expressed on the OC of the co-culture system.Osteoclasts and bone slices can be used to establish coral tissue engineering bone degradation models in vitro;under in vitro culture conditions,coral tissues Engineering bone can promote the proliferation and apoptosis of RAW264.7 cells and osteoclasts;Ru R can promote the increase of the F-actin ring of osteoclasts;when TRPV5 is inhibited,osteoclasts degrade coral tissue engineering bone resorption significantly,TRPV5 It has the theoretical basis for regulating the bone degradation rate of coral tissue engineering.