Mechanical Stress Affects Tendon-Bone Healing Via Modulating The Polarization Of Local Macrophages

Section Ⅰ Effect of mechanical stress on tendon-bone healing and its modulatory function in the polarization of local macrophagesObjectiveTo investigate the effect of different strengths of mechanical stress on tendon-bone healing and macrophage polarization at the interface after anterior cruciate ligament(ACL)reconstruction.MethodsA rabbit ACL reconstruction model was established.All animals were randomly divided into 3 groups after 2 weeks of postoperative cast immobilization: animals in the no mechanical stress(nMS)group were subjected to prolonged cast immobilization postoperatively,and animals in the moderate mechanical stress(mMS)group and excessive mechanical stress(eMS)group were subjected to a running exercise on the treadmill at a speed of 13 m/min and 20 m/min,respectively.Immunohistochemical staining and qRT-PCR were used to detect macrophage polarization at the tendon-bone interface at 4 weeks postoperatively.Histological analysis,Micro-CT,and biomechanical testing were used at 6and 12 weeks postoperatively to assess tendon-bone healing in each group.ResultsImmunohistochemistry and qRT-PCR indicated that the expression of M2 macrophage markers at the tendon-bone interface significantly increased in the mMS group at 4 weeks postoperatively compared with the other two groups,whereas the expression of M1 macrophage markers significantly increased in the eMS group.The histological staining results suggested that compared with the other two groups,at 6 weeks postoperatively,there was a significant increase in fibrocartilage regeneration at the tendon-bone interface in the mMS group,the average interface width was narrower,and the tendon-bone boundaries were already indistinct.While in the eMS group,there was a greater infiltration of inflammatory cells at the interface,with a lack of fibrocartilage regeneration,the average interface width was wider,and the tendon-bone boundaries were still distinct.At 12 weeks postoperatively,the tendon-bone boundary had disappeared and the interface tissue was more mature and orderly in the mMS group compared with the other two groups.Compared with the nMS group,the tendon-bone boundary was no longer distinct in the eMS group,and more chondrocytes were arranged in the direction of the tendon fibers,resulting in a more mature interface.Micro-CT showed that the peritunnel bone formation and trabecular microstructural parameters of the mMS group were significantly better than those of the other two groups at 6 and 12 weeks postoperatively.At 6 weeks postoperatively,the nMS group had better parameters than the eMS group,but at 12 weeks postoperatively,the eMS group outperformed the nMS group in all parameters except trabecular bone thickness.Biomechanical tests showed that the femur-graft-tibia complex in the mMS group had significantly better maximum failure load and tendon stiffness at 6 and 12 weeks postoperatively than the remaining two groups.Although the nMS group was superior to the eMS group in both indexes at 6 weeks postoperatively,the mechanical properties of complex in the eMS group were significantly stronger than those of the nMS group by 12 weeks postoperatively.ConclusionDelayed loading of moderate mechanical stress after ACL reconstruction promotes M2 polarization of macrophages at the tendon-bone interface,thereby improving tendon-bone healing.In contrast,excessive mechanical stress leads to M1 macrophage aggregation at the interface in early stage,although the long-term healing outcome is superior to that under no stress stimulation,it is still not comparable to that achieved with moderate mechanical stress stimulation.Section Ⅱ Mechanical stress modulates macrophage polarization by affecting paracrine effect of mesenchymal stem cellsObjectiveTo investigate the effect of conditioned medium of bone marrow mesenchymal stem cells(BMSC-CM)with different strengths of mechanical stress intervention on macrophage polarization.MethodsModerate(mMS)or excessive mechanical stress(eMS)was applied to BMSCs.Subsequently,unstressed conditioned medium(CM),moderate mechanical stressed conditioned medium(mMS-CM),and excessive mechanical stressed conditioned medium(eMS-CM)were collected for culturing M1 or M2 macrophages induced by bone marrowderived macrophages(BMDMs),respectively.The effect of different BMSC-CM on the polarization of BMDMs was determined by flow cytometry,qRT-PCR,and Western blot.ResultsFlow cytometry showed that incubation of M1 macrophages with mMS-CM significantly decreased their CD86 expression while increasing CD163 expression,whereas eMS-CM significantly decreased CD163 expression while increasing CD86 expression in M2 macrophages.qRT-PCR showed that,compared with the other two CMs,the mMS-CM significantly decreased the expression of inflammatory factors(IL-1β,TNF-α)while increased the expression of anti-inflammatory factors(IL-10,Arg-1)in M1 macrophages.Meanwhile,eMS-CM significantly down-regulated the expression of anti-inflammatory factors and up-regulated the expression of inflammatory factors in M2 macrophages.Western blot suggested that mMS-CM significantly reduced the protein expression of iNOS in M1 macrophages while increasing the protein expression of Arg-1 compared with the other two CMs.In contrast,eMS-CM significantly decreased Arg-1 expression in M2 macrophages while increasing iNOS expression.ConclusionCompared with unstressed BMSC-CM,BMSC-CM with moderate mechanical stress intervention has stronger anti-inflammatory properties and significantly promotes the polarization of M1 macrophages to M2,while BMSC-CM with excessive mechanical stress intervention has stronger pro-inflammatory properties and significantly promotes the polarization of M2 macrophages to M1.Section Ⅲ Mesenchymal stem cells under different strengths of mechanical stress regulate macrophage polarization through the p38 MAPK and PI3K/Akt signaling pathwaysObjectiveTo investigate the mechanism by which conditioned medium of bone marrow mesenchymal stem cells(BMSC-CM)modulates macrophage polarization after different strengths of mechanical stress intervention.MethodsModerate(mMS)or excessive mechanical stress(eMS)was applied to BMSCs.Subsequently,unstressed conditioned medium(CM),moderate mechanical stressed conditioned medium(mMS-CM),and excessive mechanical stressed conditioned medium(eMS-CM)were collected for culturing M1 or M2 macrophages induced by bone marrowderived macrophages(BMDMs),respectively.Macrophages cultured with different BMSCCM were collected,and total RNA was extracted and analyzed by RNA sequencing(RNAseq)to screen for differentially expressed genes and enriched signaling pathways.After blocking the relevant signaling pathways using specific inhibitors,the downstream signaling pathways as well as effector proteins were detected by qRT-PCR and Western blot to verify the interaction between the relevant signaling pathways.ResultsTwenty-two signaling pathways co-enriched among the three comparison groups(M1 vs.M1 + CM;M1 vs.M1 + mMS-CM;M1 + CM vs.M1 + mMS-CM)were screened by RNA-seq,among which the p38 MAPK/NF-κB and PI3K/Akt/STAT6 pathways,associated with macrophage polarization and inflammatory response,were selected for validation.Western blot showed that both pathways were activated in M1 or M2 macrophages cultured with BMSC-CM under distinct conditions.Inhibition of Akt activity in M1 macrophages significantly attenuated the anti-inflammatory effect of mMS-CM,whereas inhibition of p38 activity in M2 macrophages significantly attenuated the pro-inflammatory effect of eMSCM.The phenomenon of differential regulation of macrophages by different BMSC-CMs was associated with negative crosstalk between PI3K/Akt and p38 MAPK pathways.In M1 macrophages,mMS-CM increased Akt phosphorylation while inhibiting p38 phosphorylation and led to STAT6 activation and NF-κB inhibition,and the inflammatory response attenuated,thus contributing to the M2 transition of macrophages.Significantly increased levels of p38 phosphorylation in eMS-CM cultured M2 macrophages inversely inhibited Akt phosphorylation,which led to STAT6 inhibition and NF-κB activation,as well as enhanced inflammatory response,which in turn contributed to the M1 transition of macrophages.ConclusionBMSC-CM stimulated with different strengths of mechanical stress can regulate the activity of NF-κB and STAT6 by modulating p38 MAPK and PI3K/Akt signaling pathways,thus managing the intensity of the inflammatory response to achieve the regulation of macrophage polarization.