The Role Of Macrophages In The Ossification Via Pre-induced Cartilage Templates

Part I Culture and identification of pre-induced cartilage templates in vitroObjective:To isolate and identify mouse bone marrow mesenchymal stem cells and prepare pre-induced cartilage templates in vitro.Materials and Methods:Primary bone marrow-derived mesenchymal stem cells(BMSCs)from C57BL/6 mice were isolated via whole bone marrow adherence method and cultured to the third generation.To identify the BMSCs,flow cytometry(FCM)was used to detect the characteristic surface markers of stem cells.The osteogenic and adipogenic differentiation were conducted using the corresponding differentiation medium and observed by alizarin red and oil red O staining.Then the third-generation BMSCs were seeded into commercial gelatin sponge(Spongostan?R)(10~6 cells/per sponge)and cultured in one week's proliferation medium and four weeks'chondrogenic medium.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the characteristic markers of hypertrophic chondrocytes and their typical morphology were observed by Safranin O staining.Results:FCM results showed that isolated BMSCs characteristically expressed high level of CD44,CD29 and Sca-1,and hardly express hematopoietic cell markers CD31and CD34.The alizarin red staining showed dark red calcified nodules and oil red O staining showed red intracellular lipid droplet formation.The cartilage templates were translucent and have a certain thickness and curvature.They showed a high expression of hypertrophic chondrocytes characteristic markers:collagen II(COLII),aggrecan(Acan)and Y chromosome sex determining gene-related high-mobility box transcription factor 9(SRY-related high mobility group-box gene 9,Sox9).Safranine O staining showed typical lacuna of chondrocyte morphology.Conclusion:Stable mouse bone marrow mesenchymal stem cells were isolated in the study,and can be pre-induced into"cartilage templates"in vitro.Part II Effects of eliminating monocytes/macrophages on cartilage templates'subcutaneous osteogenesis and angiogenesisObjective:To detect the effect of eliminating recipient mice's monocytes/macrophages on the subcutaneous osteogenesis and angiogenesis of the cartilage templates and to determine the involvement of macrophages in the process.Materials and Methods:Monocytes from peripheral blood of mice were eliminated by intravenous injection of clodronate liposomes(one time/three days)to reduce the recruited macrophages to the subcutaneous implantation site.The elimination was identified by FCM using phosphate buffered saline(PBS)liposomes as control.Cartilage templates of the same batch were subcutaneously implanted into the above two groups of differently treated mice.The harvested implants were subjected to micro-computed tomography(?-CT)and immunohistochemistry(IHC)staining to compare the subcutaneous osteogenesis and angiogenesis of the two groups from imaging and histological perspectives.Results:The FCM results showed that the number of CD3~-CD45RA~-F4/80~+CD11b~+monocytes in the mice treated with clodronate liposomes was significantly reduced.The number of recruited macrophages would surely decrease.?-CT results showed that bone volume/total volume(BV/TV),trabecular number(Tb.N)and bone mineral density(BMD)were lower in the clodronate group than that of PBS group,indicating a decrease in osteogenic mass.And structural model index(SMI),trabecular bone pattern factor(Tb.Pf)and the degree of anisotropy(DA)was higher in the clodronate group,indicating that the trabecular bone was looser.The IHC staining also showed that the clodronate treatment of the mice resulted in a decrease expression levels of the osteogenesis marker osteocalcin(OCN)and the vascular endothelial marker CD31.Conclusion:Reducing the number of monocytes/macrophages in mice inhibited the subcutaneous osteogenesis and angiogenesis of cartilage templates.Subcutaneous ossificationviacartilagetemplatesrequirestheinvolvementof monocytes/macrophages.Part III Changes in M2/M1 phenotype of recruited macrophages after cartilagetemplates implantation in normal miceObjective:To backtrack the influence of cartilage templates on recruited macrophages during the process subcutaneous osteogenesis.Materials and Methods:Cartilage templates and empty gelatin sponge were implanted subcutaneously into normal mice.The implants were harvested on the third and seventh day and digested with 3g/ml collagenase and trypsin(0.5 ml:0.5 ml)to single cell suspension.FCM was performed by adding PE fluorescently labeled anti-F4/80 flow antibody,APC fluorescently labeled anti-CD86 flow antibody and PerCP/Cy5.5fluorescently labeled anti-CD206 flow antibody.The ratio of type II macrophages(M2)to type I macrophages(M1)was compared between the two groups to examine the macrophages'phenotypic changes during the process.Results:On the third day(early inflammatory response)and the seventh day(the initiation of osteogenesis process),the M2/M1 ratio of macrophages in the cartilage templates were both significantly higher than that in the empty gelatin sponge.The proportion of M2/M1 on the seventh day was also significantly higher than that on the third day,suggesting that the proportion of M2 phenotype in macrophages increased after cartilage templates implantation.Conclusion:After the cartilage templates were implanted subcutaneously,at the early stage of the immune response and the initiation of osteogenesis,the cartilage templates could promote the recruited macrophage polarize to type II phenotype that supports tissue repair,suggesting they have a favorable immunomodulatory capacity.Part IV In vitro assay of the interplay between cartilage templates andmacrophagesObjective:To investigate the effects of cartilage templates on macrophage polarization and the involved mechanism and to adversely detect polarized macrophages on the expressions of osteogenesis and angiogenesis markers of cartilage templates.Materials and Methods:RAW264.7 cells were co-cultured with cartilage templates via transwell.Firstly,RAW264.7 cells were seeded on the lower chamber of transwell with a density of 5?10~5 cells/well,and the upper chamber was added with sterile gelatin sponge or 5?10~5 third-generation BMSCs or one cartilage template.After co-culture,FCM was used to detect the macrophage polarization and western blot(WB)was used to observe the phosphorylation of Signal transducer and activator of transcription(STAT)-1/3/6.Polarized M1 or M2 was obtained via cell culture medium supplemented with 1?g/ml of lipopolysaccharides(LPS)and 10 ng/ml of interferon?(IFN-?)or 10ng/ml of interleukin-4(IL-4)for 24h.Non-treated RAW264.7 cells were considered to be M0 macrophages as a control.Then one cartilage template was placed in the upper chamber of transwell and the lower chamber was seeded with newly polarized M0,M1or M2 cells(5?10~5 cells/well).After 48 hours'co-culture,qRT-PCR was used to detect the expression of osteogenic and angiogenic markers of the cartilage template.Results:FCM results showed that both BMSCs and cartilage templates promoted the polarization of macrophages to M2 and inhibited their M1 polarization,while cartilage templates were more effective than BMSCs.Western blot results suggested that the mechanism might be that cartilage templates made a higher degree of STAT3phosphorylation and a lower degree of STAT1 phosphorylation than BMSCs.qRT-PCR results showed that both M1 and M2 can enhance the expressions of osteopontin(OPN),runt-related transcription factor 2(Runx2),type I collagen(COLI)and vascular endothelial growth factor(VEGF)to varying degrees.Conclusion:In vitro co-culture assay further confirmed that the cartilage templates promoted M2 polarization of macrophages and inhibited M1 polarization,while the macrophages of both phenotypes could promote the osteogenesis and angiogenesis of cartilage templates to varying degrees.