| Background:Rotator cuff(RC)tears are a common bone-tendon interface(BTI)injury to the locomotor system,resulting in neck and shoulder pain,shoulder weakness and impaired motion,and usually require surgical treatment.However,the failure rate after RC injury-repair remains high due to the poor healing ability of the BTI.The lymphatic vessels play a regulatory role in tissue injury-repair and influence tissue healing.However,whether lymphatic vessels are generated and what role the lymphatic vessels system plays in the physiopathological process after BTI injury has not been reported in the literature.Mechanical stimulation has been shown to promote healing after RC injury-repair,but the mechanism remains unclear.In many diseases,the promotion of lymphatic vessels proliferation can enhance tissue healing,and macrophages play an important role in pathological lymphangiogenesis.However,it is still unexplored whether mechanical stimulation promotes lymphangiogenesis through macrophages and thus promotes the healing of BTI.Objectives:The purpose of this study was to investigate the influence of lymphangiogenesis on the healing of the BTI and further examine the impact of mechanical stimulation on lymphatic vessels and their role in the healing of BTI.Methods:(1)To investigate the role of lymphatic vessel generation and healing in RC injury,a mouse RC injury-repair model was constructed and the spatial and temporal distribution of lymphatic vessel generation and changes in the proliferation capacity of lymphatic vessel endothelial cells(LECs)were detected by immunofluorescence.Further administration of the lymphangiogenesis inhibitor SAR131675 intervention,the Evans blue was injected to detect the drainage capacity of the supraspinatus tendon and enzyme-linked immunosorbent assay(ELISA)was used to detect changes in the levels of inflammatory factors at the healing site;the role of lymphangiogenesis in the repair of BTI injury was evaluated by imaging,histology and biomechanics at 2,4 and 8weeks after surgery.(2)To investigate the effect of mechanical stimulation on lymphatic vessels and their role in the healing of BTI,a mouse RC injury-repair model was constructed with treadmill training as an intervention tool.Immunofluorescence was used to detect postoperative lymphangiogenesis,Evans blue injection was used to detect changes in the drainage capacity of the supraspinatus tendon,and ELISA was used to detect changes in the levels of inflammatory factors at the enthesis.Further intervention with the lymphangiogenesis inhibitor SAR131675 was given to detect changes in the drainage capacity of the supraspinatus tendon and the level of inflammatory factors at the enthesis;next,the effect of treadmill training on lymphangiogenesis on the healing of BTI was evaluated from the aspects of imaging,histology and biomechanics at 4 and 8 weeks after surgery.(3)To investigate the role of macrophages in the promotion of lymphatic vascular proliferation by treadmill training,a RC injury-repair model was constructed using Lys M-Cre;R26-td T-DTR mice(macrophage-conjugated diphtheria toxin receptor mice),using treadmill training as an intervention tool;after injection of diphtheria toxin(DT)and PBS,immunofluorescence and flow cytometry were used to detect the clearance efficiency of macrophages in the injury area,and lymphangiogenesis was detected by immunofluorescence.To clarify the effect of mechanical stimulation on vascular endothelial growth factor-c/d(VEGF-C/D)expression in macrophages,transgenic RC injury mice were divided into natural healing and treadmill training groups,and the co-expression of macrophages and VEGF-C/D was detected by immunofluorescence.A mechanically stretched bone marrow-derived macrophages(BMDMs)culture medium-LECs co-culture system was constructed in vitro,and the effect of mechanically stretched BMDMs culture medium on the biological behavior of LECs was tested.LECs proliferation,migration and tubular formation were tested by immunofluorescence,scratch test,Transwell assay and tube formation assay.The expression of VEGF-C/D gene in macrophages was detected by qRT-PCR,and the content of VEGF-C/D protein in macrophage culture medium was detected by ELISA.After further intervention with the VEGF-C/D receptor VEGFR-3 inhibitor SAR131675,the proliferation,migration and tube-forming abilities of LECs were tested by immunofluorescence,scratch test,Transwell assay and tube formation test.Results:(1)The results of immunofluorescence showed that lymphangiogenesis occurred at the healing site of BTI and increased with the time of repair,and the lymphangiogenesis area decreased significantly after 4weeks postoperatively.Proliferative capacity assay revealed that LECs proliferation was observed at both 2 and 4 weeks postoperatively,with a significant increase in LECs proliferation in the injury area at 2 weeks postoperatively and a significant decrease in LECs proliferation in the injury area at 4 weeks postoperatively.After administration of the lymphangiogenesis inhibitor SAR131675,the drainage capacity of the supraspinatus tendon decreased at 2 and 4 weeks after surgery and ELISA results showed that the levels of IL-1β and TNF-α at the BTI increased at2 weeks after surgery;The lymphatic vessel inhibitor group at 4 and 8weeks postoperatively exhibited lower bone morphometric parameters,lower histological scores and poorer biomechanical properties than the control group.(2)The results of immunofluorescence showed that treadmill training significantly promoted the proliferation of lymphatic vessels in the BTI injury area at 2 and 4 weeks after surgery,and the drainage ability test found that the drainage capacity of the supraspinatus tendon was improved at 2 weeks after surgery.In addition,the results of ELISA showed that IL-1β and TNF-α levels decreased at the BTI.After administration of the lymphangiogenesis inhibitor SAR131675,the improvement of the drainage ability of the supraspinatus tendon and the decrease of the level of inflammatory factors at the enthesis promoted by treadmill training disappeared.The results of imaging,histology and biomechanics at 4 and 8 weeks after surgery showed that treadmill training had no effect on the healing of BTI.(3)The results of immunofluorescence and flow cytometry showed that the DT injection could effectively eliminate macrophages in the injured area,and when the macrophages were cleared,the effect of treadmill training on promoting lymphangiogenesis disappeared.In addition,the results of immunofluorescence,qRT-PCR and ELISA showed that treadmill training could promote the expression of VEGF-C in macrophages in the injury area,and mechanical stretching could promote the expression of VEGF-C gene in macrophages and increase the concentration of VEGF-C protein in the culture medium.The results of Ki-67 staining,scratch and Transwell assay,and tube-formation assay showed that mechanically stretched macrophage culture medium could significantly promote the proliferation,migration and tube-formation of LECs;the promotion of proliferation,migration and tube-formation of LECs by mechanically stretched macrophage cultures disappeared after the administration of the VEGFR-3 inhibitor SAR131675 intervention.Conclusion:1)Lymphangiogenesis plays a positive role in RC healing,and targeting the lymphatic drainage at healing site may be a new therapeutic approach to promote RC injury repair;2)Mechanical stimulation enhanced the drainage of the supraspinatus tendon and the clearance of inflammatory factors at the enthesis by promoting lymphatic vessels proliferation in the injury area,and also promotes the healing of the BTI;The possible mechanism is that mechanical stimulation influenced the biological behaviour of LECs by promoting VEGF-C secretion by macrophages that bind to the VEGFR-3receptor on the surface of LECs. |
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