| Part Ⅰ Compare and analyze the differences of mouse bone marrow derived macrophage induced by LCM and M-CSFObjective:To compare the differences between L929 cell conditioned medium and commercial recombinant macrophage colony stimulating factor(M-CSF)in cultivating bone marrowderived macrophages(BMDM),so as to select the appropriate culture conditions for subsequent experiments.Methods:L929 cells were routinely cultured and the supernatants were obtained.After removing impurities and cell fragments,20%L929 cell supernatants were added to the complete medium to prepare L929 cell conditioned medium(LCM).10ng/mL or 20ng/mL recombinant M-CSF was added to the complete medium to prepare another two groups,respectively.After that,bone marrow cells of C57BL/6 mice were obtained and were cultured in the above mentioned three groups todifferentiate into BMDM.The effects of three groups of culture medium on inducing BMDM were detected by cell morphology observation,flow cytometry,immunofluorescence,CCK-8 cell viability assay kit,qRT-PCR and other experimental techniques.Results:Through microscope observation,the use of complete medium containing 10 ng/mL M-CSF could not differentiate into BMDM well,so it was not used in subsequent experiments.Both 20%LCM and complete medium containing 20 ng/mL M-CSF can induce the differention of BMDM.Most cells of the 20%LCM group are irregular polygonal and star-shaped,while cells of the 20 ng/mL M-CSF group are blunt round,spindle-shaped and plump with good refractive index.Flow cytometry and immunofluorescence indicated that BMDM with purity of about 90%could be induced in these two groups.However,subsequent flow cytometry and qRT-PCR detection revealed that,compared with the 20 ng/mL M-CSF group,the BMDM induced by 20%LCM was less homogeneous with higher levels of CD86 and CD206 expression could be detected.In addition,mRNA expression levels of iNOS,IL-1β,and Arg-1 were also higher.These resutls suggested that the cytokines in the supernatant of L929 cells were complex,which could easily lead to changes in the phenotype of macrophages.Conclusion:Both 20%LCM or the medium containing 20ng/mL M-CSF can induce bone marrow cells into BMDM with high purity.However,BMDM induced by M-CSF has more stable and uniform biological characteristics and is closer to M0 macrophages.For cultivating macrophages,appropriate culture conditions need to be selected according to specific experimental requirements.Part Ⅱ Studies of macrophages efferocytosis and polarization in vitroObjective:To detect the effects of milk fat globule epidermal growth factor 8(MFG-E8)on macrophage efferocytosis and polarization by in vitro experiments.Methods:To induce M1 or M2 polarization,20 ng/mL IFN-γ and 100 ng/mL LPS or 20 ng/mL IL-4 was dissolved into complete medium,respectively.In addition,BMDMs were stimulated by 20 ng/mL MFG-E8.The effects of three different culture conditions on the polarization of BMDMs were detected by flow cytometry,qRT-PCR and immunofluorescence.Mouse bone marrow-derived polymorphonuclears(PMNs)were obtained by Percoll gradient density centrifugation and the phenotypes were identified using flow cytometry.After that,PMNs were starved in serum-free medium for 12h,24h,36h and 48h to induce apoptosis and the apoptotic rates were then quantified by flow cytometry using Annexin V-FITC and PI.The non-stimulated BMDMs was co-cultured with apoptotic PMNs,and the expression level of MFG-E8 in the coculture system was detected.In addition,different doses of MFG-E8 was administered to the co-cultured system.The Efferocytosis index and rate of macrophages were calculated by double fluorescence staining to assess efferocytosis efficiency and determine the optimal concentration of MFG-E8.Next,the polarization and polarization-related bio-markers of BMDM during efferocytosis were detected by ELISA,flow cytometry,and Western-Blot.Results:Microscopic observation,flow cytometry and qRT-PCR confirmed that the use of IFNγ+LPS or IL-4 could induce BMDM into typical M1 or M2 macrophage subtypes,respectively.However,MFG-E8 had no significant effect on the polarization of BMDM cultured alone.Immunofluorescence staining also revealed that the addition of 20 ng/mL MFG-E8 significantly reduced the expression of iNOS in M1 macrophages,but had no significant effect on the expression of CD206 in M2 macrophages.Bone marrow-derived PMNs were successfully obtained by Percoll gradient density centrifugation.After being starved for 48h,PMNs were induced to apoptosis,and the apoptotic rate was already 42.77 ± 4.56%.In addition,we found that BMDMs co-cultured with apoptotic PMNs were able to secrete more MFG-E8 than BMDM cultured alone.Different concentrations of MFG-E8 were added into the co-culture system,and the results showed that with the increase of MFG-E8 concentration,the efferocytosis efficiency of macrophages presented a bell-shaped curve,indicating that there is a threshold concentration of MFG-E8 on macrophage efferocytosis and 500 ng/mL was most effective in the current study.ELISA,flow cytometry and Western-Blot revealed that MFG-E8 at 500ng/mL could promote the macrophages M2 polarization in the co-culture system,and the bio-markers associated with M2 macrophages were also significantly increased.Conclusion:Using of MFG-E8 alone has no significant effect on the polarization of BMDM,but it could partially reverse the expression level of iNOS in M1 BMDM.There is a maximum threshold for the effect of MFG-E8 on efferocytosis.An appropriate concentration of MFG-E8 can enhance the macrophage efferocytosis,promote M2 polarization,increase the expression of anti-inflammatory cytokines,and reduce the expression of pro-inflammatory cytokines.Part Ⅲ MFG-E8 promotes tendon-bone healing by regualting macrophage efferocytosis and M2 polarization after anterior cruciate ligament reconstructionObjective:To investigate the effects of milk fat globule epidermal growth factor 8(MFG-E8)on macrophage efferocytosis,M2 polarization and tendon-bone healing in vivo.Methods:5%GelMA-30 hydrogel was prepared as the carrier of MFG-E8.The biocompatibility,in vitro degradation rate and cumulative drug release rate of the hydrogel were tested.The SD rat anterior cruciate ligament reconstruction(ACLR)model was established,and the control group,hydrogel group and hydrogel+MFG-E8 group were set up.After the experimental animals were sacrificed at the specific time points,the femur-tendon graft-tibia complex was harvested.At 4,8 and 12 weeks post-operation,micro CT,HE staining and biomechanical test were used to evaluate tendon-bone healing.At 5th and 10th day postoperation,the polarization of macrophages and the clearance of apoptotic cells at the tendonbone interface were evaluated by immunohistochemistry,TUNEL staining and Western-Blot,respectively.The expression levels of osteogenic,chondrogenic and adipogenic related genes were evaluated using the qRT-PCR technique at 10th day post-operation.Finally,rat bone marrow mesenchymal stem cells(BMSCs)were isolated and the multipotential capacity of BMSCs was examined using osteogenic,chondrogenic and adipogenic differentiation media.After that,a co-culture system of BMDMs and apoptotic PMNs(with or without 500ng/mL MFG-E8)was established.The supernatants of the two co-culture systems were added to osteogenic differentiation medium,so as to further evaluate the effect of efferocytosis at tendonbone interface on osteogenic differentiation of BMSCs.Results:5%GelMA-30 hydrogel has good biocompatibility.MFG-E8 in the hydrogel exhibited a release behavior with initial bursts of 79.6%from 0 to 11 days,while a dgradation curve similar to the release performance was obtained and 84.7%weight loss of the mixture was reached after 20 days,indicating that the mixture had good degradability.We successfully constructed the rat anterior cruciate ligament reconstruction model.The MFG-E8 and GelMA mixture was fluid-like before UV light irradiation at 37℃,and it could transform to a slightly viscous liquid gel when irradiated with 405 nm UV light for 10s at 37℃,while the injectability was also guaranteed.Micro-CT examination showed that at specific time points,the new bone formation of the hydrogel+MFG-E8 group was significantly increased,and the bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N)and trabecular separation(Tb.Sp)were significantly better than those of the other two groups.HE staining indicated the early appearance of Sharpey fibers in this group.In addition,more osseous ingrowth into the interface,better tendon remodeling and hardly visible gaps were observed in the MFG-E8 group at 12 weeks post-operation.Compared with the other two groups,the maximum failure load and stiffness in the MFG-E8 group were always significantly better.Immunohistochemistry and Western-Blot revealed that the expression levels of iNOS and IL1β(M1-related)at the tendon-bone interface in the hydrogel+MFG-E8 group were decreased,while the expression levels of CD206 and IL-10(M2-related)were increased at 5th and 10th post-operation.TUNEL staining also suggested that more apoptotic cells at the tendon-bone interface in this group were eliminated at an early stage.In subsequent experiments,BMSCs were successfully isolated and were identified by the multi-directional differentiation ability.We found that after the supernatant of co-culture system containing MFG-E8 was added,alizarin red staining indicated that BMSCs exhibited more active osteogenic differentiation and better mineralization ability.Conclusion:In the current study,we found that MFG-E8 could attenuate inflammatory response via enhanced macrophage efferocytosis and M2 polarization,which ultimately resulted in reduced inflammatory bone loss,promote BMSC osteogenesis and ultimately increased peri-tunnel new bone formation and better osteointegration.In this manner,MFG-E8 could act as a new therapeutic strategy for enhancing tendon-bone healing in patients undergoing ACLR. |
