Microporous Structures On Mineralized Collagen Promote Bone Regeneration By Modulating Macrophages Polarization

Background and objectiveThe repair of large-scale and segmental jaw defects caused by tumor,trauma and congenital diseases is a difficult problem to be solved urgently in clinical.In recent years,with the advance in science and technology,bone tissue engineering develops rapidly and reveals good prospects in bone repair.Mineralized Collagen(MC),similar to natural bone in chemical composition and microstructure,is an artificial biomimetic bone material prepared through biomimetic mineralization technology.Studies have confirmed that MC can provide a good environment for bone marrow mesenchymal stem cells and induce stem cells osteogenic differentiation,indicating that it is a promising scaffold for bone regeneration and repair.Traditionally,research about bone regeneration and repair mainly focus on the osteoblastic differentiation of stem cells.However,bone regeneration and repair is a multicellular behavior in vivo.Among these cells,macrophages play a key role in the process of inflammation and tissue reconstruction after biomaterial implantation.The physical and chemical properties of biomaterials can regulate the polarization of macrophages,modulating their performance in promoting inflammation or promoting tissue repair.Previous reports about MC mainly emphasize the promotion role in osteogenic differentiation of stem cells.However,whether macrophages participate in MC-related bone regeneration is still unclear.Our research tried to explore better design for optimize MC scaffold based on macrophages polarization.Considering the effects in facilitating cell infiltration,nutrient/oxygen exchange and angiogenesis,as well as in keeping the mechanical strength of materials and the maintenance of bone repair space,pore size and porosity are the key determinants of a successful scaffold among various factors.By adjusting the pore size and porosity,a scaffold can obtain favorable performances in inducing cell adhesion and migration,promoting angiogenesis and bone regeneration.However,there are few reports about the effect of MC microporous structure on macrophage polarization and bone regeneration.In this study,we designed MC scaffolds with different pore sizes and porosity.We compared the effects of these MC scaffolds on osteogenesis in vitro and in vivo,as well as on regulating macrophages polarization-induced osteogenic differentiation of MC3T3-E1.Furthermore,the mechanism by which mineralized collagen microporous structure regulates M2 macrophage polarization was primarily discussed.This study presented a favourable microporous structure of MC scaffold and provided new ideas and experimental basis for optimizing the MC structure based on macrophages phenotypic transformation.MethodsⅠ Effect of MC microporous structure on osteogenesis in vitro and in vivo1 MCs with different microporous structure were prepared and named MC-A,MC-B,MC-C respectively.The pore size,porosity,hydrophilicity,hardness and compressive strength of the materials were characterized by field emission scanning electron microscopy,porosity measurement instrument,contact angle measurement instrument,shore hardness tester,and electric universal material testing machine.2 CCK-8,AM/PI staining and immunocytochemistry were used to determine the effects of MC micropores on the cell activity and cytoskeleton of mouse osteoblast precursor MC3T3-E1 cells.The ALP activity and the expression of osteogenic related genes ALP,COL-1,OCN and OPN in MC3T3-E1 cells were measured unsing by ALP staining and qRT-PCR.3 The critical mandibular defect model of rats was constructed.The effects of MC microporous structure on the repair of mandibular defects in rats were evaluated using X-ray,H&E staining,Masson staining,immunohistochemical staining.Immunohistochemical staining was also used to detect the infiltration of macrophages around MCs.Ⅱ The effect of macrophage immune microenvironment induced by MC on osteogenic differentiation1 MCs extracts were served as control group for osteogenic induction and named as M-A,M-B and M-C,respectively.Macrophages were seeded on the surface of MC with different microporous structures,culture supernatant after 3 days was collected to prepare conditioned medium,named CM-A,CM-B,and CM-C,respectively.2 The effects of macrophage conditioned medium CM-A,CM-B and CM-C on the activity,cell spreading ability and osteogenic differentiation of MC3T3-E1 cells were investigated by CCK-8,immunocytochemistry,ALP staining,alizarin red staining and qRT-PCR,respectively.Ⅲ Functions of MC microporous structure in macrophages biological behavior and polarization1 Effects of MC microporous structure on biological behaviors of RAW264.7 cells:The morphology and adhesion of macrophages were detected by field emission scanning electron microscopy,cytoskeleton staining,and adhesion spot staining.2 Effects of MC micropore structure on the polarization of RAW264.7 cells:qRT-PCR,Western blot were used to determine the expression of iNOS and ARG-1 gene and protein expression;ELISA was used to measure the secretion levels of cytokines TNF-α,IL-1β,IL-6,IL-10,VEGF and TGF-β.3 Macrophage polarization in rat mandibular defect planted with MC with different micropore structure:One week after implantation,immunohistochemical staining was performed to detect the expression of macrophage marker CD68,Ml macrophage marker iNOS and M2 macrophage marker ARG-1.Ⅳ Mechanism of MC microporous structure regulating RAW264.7 cell polarization1 Transcriptome sequencing technology was applied to obtain differentially expressed gene profiles in RAW264.7 cells treated with three groups of MC.The differentially expressed genes,such as nucleolin(NCL)in RAW264.7 cells,were further verified by qRT-PCR.2 Immunohistochemistry was used to detect the expression and location of NCL in the cells around the MC scaffolds.A NCL inhibitor,AS1411,was used to inhibit the NCL in RAW264.7 cells and a stable cell line of RAW264.7 overexpressing NCL was constructed through infecting the cells with Lv-NCL.The effects of NCL on the expression of polarization markers iNOS and ARG-1 in RAW264.7 cells were detected by qRT-PCR and Western blot.ResultsⅠ Effect of MC microporous structure on osteogenesis in vitro and in vivo1 In this study,three kinds of MCs with different microporous sizes were designed(MC-A,MC-B,MC-C),The mineral phase was hydroxyapatite containing phosphate,and the crystal size was nanometer.The microporous sizes of the scaffolds were 273±13 m,84±3 μm and 9.7±0.2 μm,and the porosity was 80%-90%,70%-80%and 50-60%,respectively.The contact angles of the three scaffolds were all less than 90° with no significant difference,indicating they all possessed hydrophilic surfaces.With the decrease of pore size and porosity,the hardness and compressive strength of the scaffolds increased gradually.2 The three scaffolds could promote the proliferation of MC3T3-E1 cells,with no significant difference among the three groups.ALP staining and quantitative results showed that MC-B group possessed the highest ALP activity.qRT-PCR results suggested that the expression of ALP,COL-1,OCN and OPN in MC-B group was higher than that in the other groups.3 X-ray,H&E staining,Masson staining,immunohistochemistry results indicated that MC-B scaffold significantly promoted new bone formation in vivo.Compared with the control group,a large number of macrophages infiltration was detected in the MC groups.Ⅱ The effect of macrophage immune microenvironment induced by MC during osteogenic differentiationThe macrophage conditioned medium group,especially CM-B group,could significantly promote the proliferation and spreading of MC3T3-E1 cells,as well as enhance the alkaline phosphatase activity,promote the formation of calcium nodules and the expression of osteogenic related genes ALP,COL-1,OCN and RUNX2 of MC3T3-E1 cells compared with the material extract group.Ⅲ The effect of MC microporous structure on the biological behavior and polarization of macrophages1 RAW264.7 cells adhered to the surface of MC-B and MC-C scaffolds around the pores,while macrophages on the surface of MC-A scaffolds scattered inside the pores.Meanwhile,macrophages on the surface of MC-B were spindle shaped(M2 like)with elongated poles,the macrophages on the surface of MC-A were amoeba like(M1 like),while macrophages on MC-C surface were in M0 like resting state.2 The expression of ARG-1,an M2-type marker,was upregulated in the RAW264.7 cells of MC-B group,while iNOS,an M1-type marker,was significantly increased in the cells of MC-A group.The expression levels of the two markers in macrophages of MC-C group were lower than those in the other two groups.ELISA results showed that the expression of VEGF and TGF-β were higher in MC-B group.The secretion of M1 proinflammatory factors TNF-α,IL-6 and INF-y in the MC-A group were significantly higher than that in the other two groups.3 The number of ARG-1 positive cells in MC-B group was significantly higher than that in other groups one week after operation.The number of iNOS positive cells in MC-A group was higher than that in other groups.Ⅳ Mechanism of MC microporous structure regulating macrophage polarization1 The transcriptome sequencing results showed that 458 genes in MC-B group were up-regulated and 527 genes were down regulated compared with the other two groups.GO analysis showed that the genes upregulated in MC-B group involved in nucleosome assembly,chromatin assembly and mRNA binding of macrophages.The expression of NCL,SFPQ,CUL1,STAC2,ECM1,LIF genes was verified by qRT-PCR,which proved that the expression trend of the differentially expressed genes was consistent with the sequencing results.In MC-B group,NCL gene was highly expressed,and NCL protein was highly expressed in bone defect area of rats in MC-B group.2 Following the inhibition of NCL in RAW264.7 cells of MC-B group,M2 polarization marker ARG-1 was significantly downregulated.However,overexpression of NCL in RAW264.7 cells of MC-B group promoted the expression of M2 polarization marker ARG-1.Conclusion1 The MC scaffold with the pore size about 84 μm and the porosity 70%-80%possesses better performance in promoting osteogenesis.2 The MC scaffold with the pore size about 84 μm and the porosity 70%-80%can induce M2 macrophage polarization,and the resulting immune microenvironment further promotes osteogenic differentiation.3 The expression of NCL in macrophages participates in the process of mineralized collagen scaffold induced M2 polarization.