| Objective:Osteolysis characterized by bone destruction,is a kind of orthopaedic disease associated with overactive bone resorption,such as osteoporosis,rheumatoid arthritis,etc.But the mechanism of its occurrence and progress is not yet clear.Biomedical material Calcium Phosphate ceramics(CaP)are of a similar composition with bone mineral salt components for bone more approachable and can be widely used in bone repair.Previous studies have shown that small-particle size calcium phosphate ceramics have better therapeutic effect on bone repair.We have three kinds of calcium phosphate ceramics with nano structure preparation into powder,including the hydroxyapatite(HA),Biphasic calcium phosphate(BCP)and beta tricalcium phosphate(?-TCP),inject skull location,study three kinds of injectable calcium phosphate ceramics of osteoclast inhibitory effect and the function of osteoblast proliferation,explore the molecular mechanism of inhibiting osteoclast,and build osteolysis in mice model,through the local injections of HA,BCP and?-TCP,evaluate its potential therapeutic effect on osteolysis.Methods:In the first part,the particle sizes of three kinds of injectable calcium phosphate ceramics(HA,BCP(HA/?-TCP?30/70),and ?-TCP)were observed by transmission electron microscopy(TEM),and the XRD was used to detect the structure changes.The inhibitory effect of three kinds of calcium phosphate ceramic particles(HA,BCP,?-TCP)on osteoclast was investigated.The effect of three kinds of calcium phosphate ceramic particles on BMM proliferation was determined by MTT assay.TRAP staining was used to observe the three kinds of calcium phosphate ceramic particles on osteoclast differentiation,the growth state,morphology and size of mature osteoclasts.To observe the bone resorption of osteoclasts,the fluorescence staining of F-actin(actin ring)was used to study the structural integrity of actin ring.Next,PCR was used to detect the expression of osteoclast expressional genes(Acp5,Ctsk,Fos,Nfatcl,Oscar).BMM were extracted to observe the signaling network of three kinds of calcium phosphate ceramic particles on osteoclast differentiation.Regarding to osteoclast differentiation related network access,first,we used PCR to detect osteoclast related genes in treated with three kinds of calcium phosphate ceramics,next luciferase report gene and Western Blot were clarified,to observe the key mechanism of inhibiting osteoclast differentiation.In the second part,the effects of HA,BCP(HA/?-TCP?30/70)and ?-TCP on osteoblast differentiation were studied.Bone marrow mesenchymal cells(BMMC)were treated with hydroxyapatite,biphasic calcium phosphate,and?-tricalcium phosphate.The number of osteogenic calcification nodules of osteoblasts were observed by alizarin red S staining.ALP quantitative analysis reflects the degree of differentiation and maturation of osteoblasts.The expressions of osteogenic specific genes Alp,Collal,Opn and Runx2 in each group were detected by PCR experiment to explore the induction properties of three kinds of calcium phosphate ceramic particles(HA,BCP,?-TCP)on osteoblasts.In the third part,the lps-induced osteolysis model in mice was constructed and tissue sections were prepared.LPS was injected around the middle suture of the skull of 9 weeks to 12 weeks old male mice,and osteolysis model was established.Micro-CT and histological staining were performed to analyze the severity degree of osteolysis and the protective effect of hydroxyapatite,biphasic calcium phosphate(HA/?-TCP?30/70)and ?-tricalcium phosphate on osteolysis.Results:(1)Particle sizes of HA,BCP,and ?-TCP were at the nanometer level,and X-ray diffraction analysis confirmed that BCP was composed of HA and ?-TCP,and that BCP had great biocompatibility.We cultured Bone Marrow Macrophage(BMM)in medium containing HA,BCP,?-TCP,the results showed that HA,BCP,?-TCP is beneficial to the growth and proliferation of macrophages,and at the same time the research proved that HA,BCP,?-TCP can inhibit osteoclast differentiation.In HA,BCP,?-TCP groups,osteoclasts had small specific surface area,and the number was far less than the RANKL group.F-actin ring was lower than RANKL group,indicating negative bone resorption.In terms of cell mechanism studies,PCR results showed that the activity of NF-?B and NFATc1 was indeed affected after the treatment of HA,BCP and?-TCP,and the activity of-NF-?B and NFATc1 was inhibited by HA,BCP and?-TCP.Secondly,the results of WB and luciferase reporter genes further confirmed the above results.It was found that HA,BCP,?-TCP inhibited the activity of NF-? and NFATc1,thus restrained the differentiation and f-unction of osteoclasts.(2)HA,BCP,?-TCP were used to treat bone marrow mesenchymal cells.After 7,14,and 21 days,we found that HA,BCP,and?-TCP had bone induction and mineralized osteogenesis trend,and could promote the high expression of osteoblast-related genes,and the BCP group had the best effect.(3)HE staining,TRAP staining and immunohistochemical(OPN)staining results showed that lps-induced skull erosion was relatively serious,LPS group had obvious bone defects,the porosity and porosity around the middle suture were statistically significant,and the edges were rough.Compared with the LPS group,HA?BCP and ?-TCP had protective effects on lps-induced osteolysis and can prevent bone loss.The porosity and porosity of the LPS group were far less than that of the LPS group,while the BCP group was close to that of the Control group,both of which had statistical significance.Conclusion:(1)HA?BCP and ?-TCP of three kinds of calcium phosphate ceramics can inhibit osteoclast differentiation and bone resorption function.(2)three kinds of calcium phosphate ceramics(HA,BCP,?-TCP)can inhibit the NF-jB and NFATc1 activity to play for the purpose of osteoclast differentiation and function of interference.(3)Hydroxyapatite,biphasic calcium phosphate and ?-tricalcium phosphate can induce the differentiation of bone marrow mesenchymal cells into osteoblasts.(4)Three kinds of calcium phosphate ceramics(HA,BCP,?-TCP)particles can protect bone mass and prevent bone loss by inhibiting osteoclast differentiation and inducing osteoblasts.(5)Among the studies on the effects of three kinds of calcium phosphate ceramic particles on osteoblasts and osteoclasts,BCP has the best effect,followed by ?-TCP,and HA had less significant effect than BCP and ?-TCP. |
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