Low-Intensity Pulsed Ultrasound Promotes Bone-Tendon Interface Repair By Regulating Macrophage Polarization

BackgroundAs a cost-effective treatment,low-Intensity pulsed ultrasound(LIPUS)has been proved to accelerate the healing of tendon-bone interface(TBI),yet the underlying mechanism needs further investigation.Macrophages,with antigen presentation and phagocytosis in the process of inflammation,can rapidly converge to the lesion area and participate in the repair of tissue damage when inflammation occurs,are considered to play an important regulatory role in the whole process of tissue repair and the core of microenvironmental regulation.By clearing macrophages at the bone-tendon interface with liposomal clodronate,we found that promoting effect of LIPUS on the damaged bone-tendon interface was weakened or even disappeared.This study investigated the effects of LIPUS on the bone-tendon interface healing process in a rotator cuff model in rats and described the polarization and spatio-temporal distribution characteristics of macrophages with or without LIPUS treatment.In vitro,the potential molecular therapeutic mechanism of LIPUS based on macrophages was explored by macrophage polarization models.ObjectiveBased on the clear correlation between LIPUS and macrophages in promoting bone-tendon interface healing,we intend to explore how LIPUS can promote bone-tendon interface healing by regulating macrophages.Methods(1)In vivo:A total of 100 mature male Sprague Dawley rats(12 weeks of age)were selected for standard rotator cuff injury repair model.After surgery,they were randomly allocated into two groups:control group(n=50)and LIPUS group(n=50).The density and spatial-temporal distribution of M1 and M2 macrophages at the bone-tendon interface were detected by immunohistochemistry.Micro-CT detected new bone formation and mineralization.The mechanical properties of SSTH-C(supraspinatus-supraspinatus tendon-humerus complexes)were evaluated by biomechanical tests.A histological scoring system was used to assess the maturity of the bone-tendon interface after reconstruction.(2)In vitro:Bone Marrow Derived Macrophages(BMDM)were cultured to establish M1 and M2 macrophage polarization models in vitro.Flow cytometry was used to compare the proportion of M1/M2macrophages successfully induced in the two polarization models with or without LIPUS intervention.q RT-PCR was used to detect the m RNA expression of macrophage phenotype marker and inflammation-related genes in the two polarization models with or without LIPUS intervention.Results(1)In vivo:Immunohistochemical results indicated that F4/80~+cells increased at the early stage of repair,CD163~+macrophages increased at the later stage and that of LIPUS group were significantly more than the control group at the bone-tendon interface at post-treatment 6 weeks.Micro-CT results showed that LIPUS was beneficial to the formation of new bone,and the BV/TV of new bone in the LIPUS group was higher than that in the control group after 2 weeks of treatment.Biomechanical tests suggested that LIPUS could improve the failure load and stiffness of the bone-tendon interface,and both the failure load and stiffness of SSTH complexes in LIPUS group were significantly higher than control group at post-treatment 6 weeks.Histological score system suggested that LIPUS accelerated the remodeling of the bone-tendon interface,and the maturity of the LIPUS group was significantly better than the control group at post-treatment 1-and 2-week time points.(2)In vitro:q RT-PCR and flow cytometry results showed that the m RNA expression of CCR7 in M1 macrophage polarization model and the m RNA expression of CD163 and IL-10 in M2 macrophage polarization model were increased,the proportion of M2 macrophages successfully induced in M2 macrophage polarization model was increased.ConclusionLIPUS accelerated the gathering of F4/80~+macrophages at the early stage of TBI healing and promoted the polarization of M2 macrophages at the later healing stage to advance the BTI healing.