Magnesium Phosphate Cement With Carboxymethyl Chitosan And Sodium Alginate For Promoting Bone Repairand Its Mechanism

Objective:Although human bone has a certain self-healing ability,this ability cannot cope with a wide range of clinical injuries.Therefore,the repair of bone defect is still one of the biggest problems in clinical orthopedics.Bone transplantation is one of the most important treatment methods for bone defect.However,currently,implants used for bone transplantation therapy are greatly limited.Autologous bone is of limited source,and allogeneic bone has risks of immune rejection and transmission of infectious diseases.Therefore,this study aims to develop a kind of bioactive composite magnesium phosphate bone cement（MPC-CMCS/SA）by adding carboxymethyl chitosan and sodium alginate.Through comprehensive evaluation of the material’s physical and chemical properties such as compressive strength,injectable,solidification time,collapsibility resistance and in vitro degradation rate,it was determined whether the material could be used as an early filling material for bone defects and play a certain role in mechanical support.Through the evaluation of physical and chemical properties,select the material group with the most excellent properties.In vitro and in vivo experiments were conducted to evaluate the biocompatibility of the material,explore the optimal addition amount of carboxymethyl chitosan and sodium alginate,and analyze the promoting effect and potential mechanism of the material on bone defect repair.Methods:1.The MPC-CMCS/SA system consists of two phases:solid and liquid.The solid phase was uniformly mixed with reburned magnesium oxide and potassium dihydrogen phosphate at a molar ratio of 1.5:1.Carboxymethyl chitosan and sodium alginate were evenly mixed with 1%,2%,3%and 4%of the mass fraction of solid phase,respectively,and added into deionized water to form a solution.The solid and liquid phases were evenly mixed according to the ratio of solid to liquid 2:1,and transferred to the customized mold.After the composite material was self-cured for24h,the mold was released,and 1000 mesh sand paper was taken to polish the material.After polishing,it was placed at 37℃and 100%humidity for 48h.2.Detect the physical and chemical properties of MPC-CMCS/SA.Universal material testing machine was used to measure its compressive strength;Use p H agent to measure its p H value;The solidification time was measured by vicar instrument.Chemical composition was measured by X-ray diffractometer.Fourier transform infrared spectrometer was used to measure the chemical bond changes.The surface morphology was measured by field scanning electron microscopy.Use thermocouple to measure its exothermic temperature;The release concentration of magnesium ion was measured by inductively coupled plasma atomic emission spectrometry.The physical and chemical properties of MPC-CMCS/SA in each group were evaluated,and the group with the most excellent performance was selected for subsequent in vivo and in vitro experiments.3.The biological properties of MPC-CMCS/SA were detected.The growth of osteoblasts was evaluated by cell proliferation assay.The adhesion of osteoblasts on the surface of MPC-CMCS/SA was evaluated by field scanning electron microscopy（FSEM）and laser confocal microscopy（LSCM）.QPCR and Western blotting were used to evaluate the effect of MPC-CMCS/SA on promoting the expression of bone differentiation.The rat skull defect model was constructed,and micro-CT and histological staining were used to evaluate the effect of MPC-CMCS/SA on promoting bone defect repair in experimental animals.On this basis,the mechanism of MPC-CMCS/SA promoting osteogenic differentiation was explored.Results:1.Injectable results showed that with the increase of CMCS and SA,the injection time of MPC-CMCS/SA composite was prolonged,and the injection time of MPC-CMCS/SA-4 was up to 2.9 minutes.The results showed that the solidification time of MPC-CMCS/SA increased significantly with the increase of CMCS and SA,and the solidification time of MPC-CMCS/SA（4%）reached 12.8 minutes.The results of exothermic temperature showed that with the addition of CMCS and SA,the maximum temperature of hydration reaction decreased significantly,and the maximum temperature of MPC-CMCS/SA（4%）decreased to 39.4℃.The results of compressive strength showed that the compressive strength remained stable for about72 hours after the initial solidification.With the increase of CMCS and SA,the compressive strength increases first and then decreases.The compressive strength of MPC-CMCS/SA（2%）is the highest,about 1.4 times that of MPC.In vitro degradation rate results showed that the addition of CMCS and SA delayed the degradation of MPC-CMCS/SA,and the degradation of MPC-CMCS/SA（2%）was the slowest.The results showed that the collapse-resistance of MPC-CMCS/SA increased first and then decreased with the increase of CMCS and SA,and the collapse-resistance of MPC-CMCS/SA（2%）was the best.XRD analysis results showed that characteristic peaks of KMg PO₄·6H₂O and unreacted Mg O could be observed in both MPC group and MPC-CMCS/SA group.Ftir analysis results show that compared with MPC,MPC-CMCS and MPC-SA,the characteristic peak of MPC-CMCS/SA shifts,the vibration frequency decreases,and the peak shifts to a longer wavelength direction.Scanning electron microscopy（SEM）results showed that MPC was mainly composed of KMg PO₄·6H₂O crystal,with high surface brittleness and many cracks.With the increase of CMCS and SA,the surface cracks of MPC-CMCS/SA decrease first and then increase.SEM images of MPC-CMCS/SA（2%）showed that the cracks between KMg PO₄·6H₂O crystals were filled and the surface was smoother after adding CMCS and SA.2.As MPC-CMCS/SA（2%）showed relatively best physical and chemical properties,it was selected as a further experiment with MPC group as the control.The morphology and adhesion of MC3T3-E1 cells were detected by fluorescence imaging and scanning electron microscopy.The results showed that MC3T3-E1 cells adhered and diffused on the two groups of samples,and the cells in MPC-CMCS/SA（2%）group formed more pseudopodia and diffused in a wider range.In addition,fluorescence images showed that the number of cells in the MPC-CMCS/SA（2%）group was higher than that in the MPC group,and the indexes of cell adhesion Actin in the MPC-CMCS/SA（2%）group was higher than this in the MPC group.ALP activity is an important indicator of osteoblast activity during osteogenic differentiation.ALP results showed that ALP activity increased rapidly in all MC3T3-E1 cells from day 7 to day 14.On day 14,ALP activity increased almost linearly with the increase of CMCS/SA.QPCR results showed that the expression levels of osteogenic genes were also increased in the cells cultured on the MPC/CMCS/SA group compared to the cells grown on the MPC group at 7 and 14days.Western blotting results showed that the effect of MPC-CMCS/SA on the expression of osteogenic related proteins was consistent with the gene expression data.In animal experiments,micro-CT 3D reconstruction results of bone defects showed that after 1 month of healing,new bone was implanted in the central region and edge of the sample in the MPC-CMCS/SA group,while almost no new bone was formed in the MPC group,mainly at the edge.Three months after implantation,the degradation rate of the MPC-CMCS/SA sample was faster than that of MPC,resulting in thicker and more continuous new bone formation.In addition,the results of new bone structure parameters showed that the bone volume fraction BV/TV of THE MPC-CMCS/SA group was slightly higher than that of the MPC group at 1month.At 3 months,BV/TV was significantly increased in MPC-CMCS/SA（2%）group.Tb.N and Tb.Th were consistent with BV/TV,while Tb.Sp was opposite.Histological results included HE,von Kossa and Masson staining.HE staining revealed a mild to moderate inflammatory response in both groups after one month,with a small number of macrophages and foreign body giant cells around the samples.In addition,minimal bone formation was observed at the margins of both groups of samples,with higher bone formation in the MPC-CMCS/SA group than in the MPC group.Von Kossa and Masson staining were consistent with HE staining.Three months after implantation,cement residue in the MPC-CMCS/SA group was less than that in the MPC group.Both groups showed more bone formation than the one-month slice.Bone formation mainly occurred in the MPC-CMCS/SA group,and bone ingrowth mainly occurred in the dural side of the MPC group,with more fibrous tissue in the implant center.Focal adhesion kinase（FAK）plays an important role in adhesion dependent signal transduction and osteogenesis.MPC-CMCS/SA（1%）and MPC-CMCS/SA（2%）promoted FAK phosphorylation and inhibited the phosphorylation ofβ-catenin,a downstream effector of FAK,in MC3T3-E1 cells at24 h.When FAK inhibitors were added to the MPC-CMCS/SA（2%）group,FAK phosphorylation was inhibited andβ-catenin phosphorylation was enhanced.Conclusion:In this paper,a kind of composite magnesium phosphate bone cement（MPC-CMCS/SA）with good physical and chemical properties and biological activity was successfully constructed by adding carboxymethyl chitosan and sodium alginate.The experimental results showed that the addition of carboxymethyl chitosan and sodium alginate improved the compressive strength of MPC,delayed the degradation rate in vitro,prolonged the solidification time,and reduced the exothermic temperature.The results of in vitro and in vivo experiments showed that the addition of carboxymethyl chitosan and sodium alginate improved the biocompatibility of MPC,increased the adhesion and proliferation of cells on the material,promoted osteogenic differentiation and new bone formation,and was beneficial to the repair of bone defects.On this basis,we found that MPC-CMCS/SA regulates the process of osteogenic differentiation through the Integrin-Fak-Wnt signaling pathway.In this study,MPC-CMCS/SA is considered as a bioactive bone repair material with good clinical application prospect.