| Magnesium phosphate-based bone cement(MPBC)has been widely used in clinical research of orthopedic implant because that it has superior early strength,curing time,degradation rate and biological activity than calcium phosphate cement.The degradation products of MPBC contain not only calcium and phosphate ions,but also magnesium and citrate ions.Citric acid is usually used as a retarder or dispersant in bone cement to regulate its physical and chemical properties.Although some research progresses have been made on the metabolic pathways and biological effects of MPBC degradation products,such as calcium,magnesium and phosphate,there is a lack of deep understanding on the biological effects of citrate in MPBC degradation products and the regulation of citrate on other degradation products,such as calcium and phosphate.In this paper,the biological effects of MPBC degradation products on bone tissue regeneration were studied,with the emphasis on the role of citrate in bone tissue regeneration,including osteogenic differentiation,osteogenic mineralization and vascularization,as well as the regulation of citrate on calcium and phosphate mediated extracellular mineral deposition,and the inhibitory effects of citrate and its modified polymers on oxidative stress and inflammatory response were studied.Firstly,the citric acid solution was mixed with cement solid powder to prepare citrate modified MPBC.The effect of citrate on the phase structure of MPBC was studied by FT-IR,XRD and XPS.The effects of citrate on osteogenic differentiation and angiogenesis were studied by using murine preosteoblast cell line(MC3T3-E1)and human umbilical vein endothelial cells(HUVECs)models,and the regulation of citrate on the crystal structure of bone apatite was explored in the osteogenic mineralization model of osteoblasts.Finally,the effects of citrate on the angiogenesis and osteogenesis of MPBC were evaluated in the rat skull defect model.The results show that citrate can regulate bone tissue and angiogenesis in different areas of the interface between bone cement and bone tissue,inhibit calcium and phosphorus mediated mineral deposition in the high concentration ion area near the material,promote angiogenesis,and promote osteogenic differentiation and mineralization in the low concentration ion area far away from the material.The relationship between calcium phosphate mediated ATP synthesis and metabolism,ERK1/2 pathway and BMP-2 expression was studied.On this basis,the relationship between citrate mediated ATP synthesis and metabolism and osteogenic differentiation was further investigated.Finally,the effect of citrate on the activity of bone marrow mesenchymal stem cells(BMSCs)in high calcium and high phosphate environment was studied.The results showed that phosphate and citrate were transported to the intracellular environment in different ways to affect the synthesis and secretion of ATP.This biological process is very important for the expression of BMP-2 and the phosphorylation of ERK1/2.However,calcium directly upregulates the expression of BMP-2 and the phosphorylation of ERK1/2.This biological process does not depend on the synthesis and secretion of ATP.In addition,the maintenance of appropriate citrate concentration can inhibit the early mineral deposition,avoid cell death and apoptosis,and is conducive to the osteogenic differentiation and mineralization of cells.DPPH,ABTS,lipid peroxidation and iron chelation were used to evaluate the scavenging effect of citrate on free radicals.The protective effect of citrate on the activity and apoptosis of BMSCs was evaluated by hydrogen peroxide induced oxidative stress model.The anti-inflammatory and anti-oxidation ability of citrate in vivo was evaluated by lipopolysaccharide induced inflammatory response and oxidative stress in rat air pouch model.The above studies showed that citrate showed a stable molecular structure and did not directly react with oxides,and appropriate concentrations of citrate ions exert positive effects on the metabolic activity,membrane morphology and apoptosis of BMSCs under oxidative stress.Citrate exerts significant inhibitory effects on LPS-induced oxidative stress and inflammatory reactions.We performed proteomics assays to identify proteins regulated by citrate in BMSCs and further reveal the anti-oxidant mechanisms of citrate.By applying the i TRAQ quantitative proteomics analysis,we found that,the expression of 171 proteins was regulated by citrate compared to that of the control.A large number of differentially expressed proteins that exert potential anti oxidant effects following citrate treatment was identified.Further functional studies revealed that the nuclear receptor peroxisome proliferator-activated receptor ?(PPAR?),a key protein involved in the transcription factors,contributes to the upregulation of various free radical scavenging proteins and the downregulation of diverse components of the inflammatory responses,revealing the role of citrate in the regulation of redox signal and the role of PPAR ? signal in this process.Finally,we used citric acid modified polyvinyl alcohol(PVA-C)as a model biomaterial to investigate the role of citric acid on the material stimulated anti-oxidant and anti-inflammatory effects.In cellular-based assays,PVA-C extracts showed a protective effect on bone marrow mesenchymal stem cells(BMSCs)under oxidative stress.It could enhance the antiapoptotic ability of stem cells by inhibiting reactive oxygen species.Further studies revealed that PVA-C extracts up-regulated the nuclear receptor peroxisome proliferator-activated receptor ?(PPAR?)and superoxide dismutase [Mn](SOD2).In vivo animal assays,PVA-C extracts showed significant inhibitory effects on the oxidative stress and inflammatory reaction which were induced by lipopolysaccharide(LPS).These findings suggest that the citric acid modified polymer can regulate the redox signaling of stem cells and tissues by the release of citric acid from materials,leading to enhanced oxidative stress-induced degenerative diseases and inflammatory diseases therapy. |
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