The Dynamic Effect Of Macrophages On Fate Determination Of Mesenchymal Stem Cells In Injury-induced Heterotopic Ossification

【Background】Heterotopic ossification(HO)is a paathological process forming the mature bone tissue outside the normal bone and generally divided into two forms: acquired and hereditary HO.Hereditary HO is seen in rare genetic disorders such as Fibrodysplasia ossificans progressiva(FOP)and Progressive osseous heteroplasia(POH).Traumatic HO is commonly occurred after the soft tissue injury,brain injury and surgical injury.In current studies,traumatic HO has a variety of pathogenesis,complicated and varied pathological process,unclear mechanism,extensive involvement of the population,threatening human life and health.Mesenchymal stem cells(MSCs)have strong self-renewal ability and multiple differentiation potential,that is,under certain conditions,MSCs can differentiate into bone cells,chondrocytes,adipocytes and other cells.When the muscle and Achilles tendon are severely injured,the MSCs at the injured site abnormally differentiate into chondrocytes and osteoblasts under the stimulation of inflammatory response,and form ectopic bone through endochondral ossification.Through single cell sequencing,we found that MSCs in the damaged area of HO model mice began to proliferate and subsequently occupied the potential to differentiate into chondrocytes and osteocytes,but the mechanism is still unclear.Macrophages play an important role in the development of HO.The previous single cell sequencing showed that macrophages increased in the injured tibial muscle of HO model mice.Therefore,we focused on macrophages and MSCs and proposed a scientific hypothesis: in HO,macrophages may affect the proliferation of MSCs and affect the bone-cartilage differentiation of MSCs.To verify this hypothesis,we used two types of injury-induced HO model mice,inhibited macrophages of HO model mice by injecting clodronate liposomes into the tail vein,analyzed the proliferation of MSCs in HO site by flow cytometry,and observed the differentiation of MSCs in HO site into cartilage and osteogenesis by immunofluorescence staining and histological staining,and concluded: In traumatic HO model mice,macrophages are inhibited during HO formation,and the proliferation of MSCs at the damaged site is significantly reduced,and the differentiation ability of MSCs to chondrocytes and osteoblasts is weakened,and the volume of HO formation is ultimately reduced.This not only explores a new way for the prevention and treatment of HO,but also has important value and significance for the research and clinical application of adult stem cells in vivo.【Research methods】1.The tibial muscle of NSE-BMP4 transgenic mice was mechanically damaged and the Achilles tendon HO model of C57BL/6 mice was created through Achilles tendon rupture.First,clodronate liposomes and control liposomes were injected into the tail vein of HO model mice 1 day in advance,and then the left hindlimb muscle and Achilles tendon of HO model mice were damaged.The clodronate liposomes and control liposomes were injected continuously into NSE-BMP4 mice 1 day after mechanical injury and C57BL/6 mice 3 days after Achilles tendon rupture.The muscle and Achilles tendon tissues of mice were taken from the injured site,digested by the corresponding collagenase,and the tissues were digested and sifted into a single-cell suspension.Flow cytometry was used to detect the injury of NSE-BMP4 mice and the inhibition of macrophages in C57BL/6 mice at 1 or 3 day after the Achilles tendon rupture,according to cell surface Marker and intracellular cell cycle staining.Proliferation of MSCs at injured sites in mice.2.HO model mice were given clodronate liposomes and control liposomes,and the tissue at the injury site of mice was fixed and dehydrated,then tissue slices were taken and stained with immunofluorescence.Marker F4/80 on the surface of macrophages was used to detect the clearance efficiency of macrophages at the injury site of mice.Chondrocyte surface marker SOX9 and osteoblast surface marker RUNX2 were co-stained to explore whether removal of macrophages in mice would affect the differentiation of MSCs from HO site to cartilage.HE staining,Safranin Ostaining,and Masson staining were used to detect the ossification of the injured sites in mice after the removal of macrophages.3.HO model mice were injected with liposome and clodronate liposomes through caudal vein to clear macrophages involved in HO formation.Mature ectopic ossification was formed 14 dpi in NSE-BMP4 mice,and the difference of HO volume was detected by Micro-CT.【Research results】1.The macrophages in HO model mice were cleared,and the number of MSCs in the injury site and in G2/M proliferation stage were significantly reduced.2.When macrophages in HO model mice were cleared,the differentiation ability of MSCs in the injury site to cartilage and osteoblast was reduced.3.The removal of macrophages in HO model mice can significantly reduce the volume of HO.【Conclusion】1.Inhibition of macrophages can reduce the proliferation of MSCs at the injury site in injury-induced HO model mice.2.Inhibition of macrophages can reduce the chondrogenic differentiation of MSCs at the injury site in injury-induced HO model mice.3.Inhibition of macrophages can reduce HO volume in injury-induced HO model mice.