Role of low-intensity pulsed ultrasound in regulating macrophage polarization to accelerate tendon-bone interface repair.

Low-intensity pulsed ultrasound (LIPUS) has been proven to accelerate the healing of the tendon-bone interface (TBI), and macrophages are considered to play an important regulatory role. This study was designed to explore the polarization of macrophages during treatment of TBI injury with LIPUS. In a rat model of rotator cuff tear, LIPUS or mock sonication (controls) was administered from 1 week postoperatively. The supraspinatus-supraspinatus tendon-humerus complexes were harvested for further evaluation at different time points for measures such as new bone formation, TBI maturity, ultimate failure load and stiffness, and types of macrophages. In vitro, bone marrow-derived macrophages were cultured, and polarization was identified after stimulation with or without LIPUS (the LIPUS or control groups, respectively). Two weeks posttreatment, the LIPUS group showed higher bone volume/total volume ratios and better TBI maturity scores. Six weeks posttreatment, the failure load of the LIPUS group was significantly higher than that of the control group. LIPUS also accelerated initial inflammatory macrophage accumulation and facilitated anti-inflammatory macrophage polarization (M2) in the late period. In the in vitro macrophage polarization model, the LIPUS group showed a higher proportion of M2 macrophages and mRNA expression of anti-inflammatory genes than the control group, while there was no significant difference in the proinflammatory macrophages between the two groups. Our observations revealed that macrophage polarization may be a potential mechanism of LIPUS treatment for TBI repair.

Mechanical stimulation improves rotator cuff tendon-bone healing via activating IL-4/JAK/STAT signaling pathway mediated macrophage M2 polarization.

Background: It is well known that appropriate mechanical stimulation benefits tendon-bone (T-B) healing, however, the mechanisms behind this are still uncovered completely. Here, we aimed to explore whether the IL-4/JAK/STAT signaling pathway mediated macrophage polarization was involved in mechanical stimulation induced T-B healing. Method: C57BL/6 mice rotator cuff (RC) repair model was established, and the mice were randomly allocated to the following group. 1. Mice were allowed for free cage activities after surgery (FC group); 2. Mice received treadmill running initiated on postoperative day 7 (TR group); 3. Mice only received a local injection of hydrogel containing IL-4 neutralizing antibody without postoperative intervention (FC â€‹+ â€‹AF-404-SP group); 4. Mice received a local injection of hydrogel containing IL-4 neutralizing antibody and postoperative treadmill running (TR â€‹+ â€‹AF-404-SP group). The expression of IL-4 within supraspinatus tendon (SST) enthesis was measured by Enzyme-linked immunosorbent assay (ELISA). In addition, the activation of JAK/STAT signaling pathway in macrophages and identification of macrophage phenotype at the RC insertion site was detected by Flow cytometry and qRT-PCR. T-B healing quality in this RC repair model was evaluated by histological staining, Micro-computed tomography (Micro-CT) scanning, and biomechanical testing. Result: In this study, using the RC repair model, we confirmed that generation of IL-4, activation of the JAK/STAT signaling pathway in macrophages, the ability of macrophages to polarize towards M2 subtype, and T-B healing quality were significantly enhanced in TR group compared to FC group. When comparing FC â€‹+ â€‹AF-404-SP group with TR â€‹+ â€‹AF-404-SP group, it was found that the mechanical stimulation induced this effect was depleted following the blockade of the IL-4/JAK/STAT signaling pathway. Conclusion: Our finding suggested that mechanical stimulation could accelerate T-B healing via activating the IL-4/JAK/STAT signaling pathway that modulates macrophages to polarize towards M2 subtype. The translational potential of this article: This is the first study to reveal a significant role of mechanical stimulation in the IL-4/JAK/STAT signaling pathway activation and macrophage polarization during RC T-B healing, which highlights the IL-4/JAK/STAT signaling pathway as a potential target to mediate macrophage M2 polarization and improves T-B healing for RC repair.

Characterize the microstructure change after tendon enthesis injury using synchrotron radiation µCT.

The microstructure of the bone-tendon interface (BTI) deserves in-depth investigation. In this study, we first aimed to extend the application of synchrotron radiation µCT to characterize the gradient structure of supraspinatus tendon (SST) enthesis, from both tissue morphology to cell distribution. Second, to acquire detailed morphological information of SST enthesis when after injury. Our results showed that in normal enthesis, the phenotype of chondrocyte in BTI was dependent on its distance to subchondral bone. After injury, the fibrocartilage cells were disrupted, as evidenced by reduced lacunae size. Our observation may partly explain the loss of BTI mechanical properties after injury, and we believe the application of synchrotron radiation microcomputed tomography will have promising potential for characterizing the morphology changes in enthesis and for evaluating the therapeutic effects of interventions in preclinical studies.

Early treadmill running delays rotator cuff healing via Neuropeptide Y mediated inactivation of the Wnt/ß-catenin signaling.

BACKGROUND: Defining the optimal rehabilitation programs for rotator cuff healing remains a challenge. Early treadmill running may have negative effects on tendon-bone interface (TBI) healing with increased expression of Neuropeptide Y (NPY). However, the underlying mechanism is still unknown. METHODS: The mice were randomly assigned to four groups: control group, treadmill group, treadmill â€‹+ â€‹BIBO3304 group and BIBO3304 group alone. Specifically, the control group was allowed free cage activity without any treatment after surgery. The treadmill group received early treadmill running initiated from postoperative day 2. The treadmill â€‹+ â€‹BIBO3304 group received treadmill running combined with intra-articular injection of BIBO3304 postoperatively. The BIBO3304 group only received type 1 NPY receptor (Y1 receptor, Y1R) antagonist BIBO3304 postoperatively. Healing outcomes of the rotator cuff were evaluated by histological analysis, synchrotron radiation micro-computed tomography (SR-µCT) scanning, and biomechanical testing at 4 and 8 weeks after surgery. The expression of NPY and its Y1 receptor during the treadmill running were tested by immunofluorescence. In addition, the related signaling pathway of Neuropeptide Y among all groups was detected by immunohistochemistry and western-blot. RESULTS: Immunofluorescence results show that early treadmill training could lead to a significant increase in the expression of NPY at the healing site, and Y1R was widely expressed in both normal or injured rotator cuff without statistical difference. At the same time, early treadmill running delayed the healing of rotator cuff, as indicated with unsatisfactory outcomes, including a significantly lower histological score, decreased bone formation and inferior biomechanical properties at postoperative week 4 and 8. Moreover, the use of BIBO3304 could partly alleviate the negative effects of early treadmill running on the healing of rotator cuff and promote the natural healing process of rotator cuff, as evidenced by significant differences observed between the treadmill and treadmill â€‹+ â€‹BIBO3304 groups, as well as observed between the control and BIBO3304 groups. On the other hand, the expressions of Wnt3a and ß-catenin in the treadmill group were significantly lower compared with the other groups, while the expression in the BIBO3304 group was the highest, as evaluated by immunohistochemistry and western-blot. CONCLUSIONS: Early treadmill running increased the expression of NPY at the RC healing site, which might burden the expression of Wnt3a/ß-catenin and delay the healing process, inhibition of Y1 receptor with BIBO3304 could promote bone-tendon healing through the Wnt/ß-catenin signaling.The translational potential of this article: This is the first study to evaluate the specific role of the NPY-Y1R axis and its underlying mechanism by which early treadmill running delays bone-tendon healing. Further, our study may provide references of precise and individualized exercise-based rehabilitation strategies for TBI healing in clinic. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This is the first study to evaluate the specific role of the NPY-Y1R axis and its underlying mechanism by which early treadmill running delays bone-tendon healing. Further, our study may provide references of precise and individualized exercise-based rehabilitation strategies for TBI healing in clinic.

Effect of Exercise Intensity on the Healing of the Bone-Tendon Interface: A Mouse Rotator Cuff Injury Model Study.

BACKGROUND: Injuries at the bone-tendon interface (BTI) are common findings in clinical practice. Rehabilitation procedures after BTI surgery are important but are controversial. PURPOSE: To investigate the effects of different exercise intensities on BTI healing by means of an established mouse rotator cuff injury model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 150 specific pathogen free male C57BL/6 mice, with supraspinatus insertion injury, were randomly assigned to 1 of 5 groups according to postoperative rehabilitation of different exercise intensities: (1) control group, (2) low-intensity exercise group, (3) moderate-intensity exercise group, (4) high-intensity exercise group, and (5) increasing-intensity exercise group (IG). The specimens were harvested 4 or 8 weeks postoperatively for microarchitectural, histological, molecular biological, and mechanical evaluations. RESULTS: Histological test results showed that the degrees of tissue fusion and polysaccharide protein distribution at the healing interface at 4 and 8 weeks after surgery were significantly better in the IG than in the other 4 groups. Synchrotron radiation micro-computed tomography showed that the quantity of subchondral bone at the enthesis (bone volume/total volume fraction, trabecular thickness, trabecular number) was higher and trabecular separation was lower in the IG than in the other 4 groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the healing interface in the IG expressed more transcription factors, such as sox 9, runx 2, and scleraxis, than the interfaces in the other groups. Although no significant difference was seen in the cross-sectional area between the groups at postoperative weeks 4 and 8 (P > .05), the tensile load, ultimate strength, and stiffness of the specimens in the IG were significantly better than those in the other 4 groups (P < .05). CONCLUSION: The rehabilitation program with increasing-intensity exercise was beneficial for BTI healing. CLINICAL RELEVANCE: The results of this study provide evidence supporting the use of a simple and progressive exercise rehabilitation program after rotator cuff surgery.

The Enhancement Effect of Acetylcholine and Pyridostigmine on Bone-Tendon Interface Healing in a Murine Rotator Cuff Model.

BACKGROUND: How to improve rotator cuff healing remains a challenge. Little is known about the effect of the parasympathetic transmitter acetylcholine (ACh) and the acetylcholinesterase inhibitor pyridostigmine (PYR), both of which have anti-inflammatory properties, in the healing process of rotator cuff injury. HYPOTHESIS: ACh and PYR could enhance bone-tendon interface healing in a murine model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 160 C57BL/6 mice underwent unilateral rotator cuff repair surgery. Fibrin gel (FG) was used as a drug carrier. The mice were randomly assigned to 4 groups with 40 mice per group: FG group (received FG alone), 10-5 M ACh group (received FG containing 10-5 M ACh), 10-6 M ACh group (received FG containing 10-6 M ACh), and PYR group (received FG containing 25 µg of PYR). Ten mice in each group were euthanized at 2, 4, 8, and 12 weeks postoperatively. Histologic, immunohistochemical, and biomechanical evaluations were performed for analysis. RESULTS: Histologically, fibrocartilage-like tissue was shown at the repaired site. The proteoglycan content of the 10-5 M ACh group was significantly increased compared with the FG group at 4 weeks. M2 macrophages were identified at the repaired site for all groups at 2 and 4 weeks. At 8 weeks, M2 macrophages withdrew back to the tendon in the FG group, but a number of M2 macrophages were retained at the repaired sites in the ACh and PYR groups. Biomechanically, failure load and stiffness of the ACh and PYR groups were significantly higher than those of the FG group at 4 weeks. The stiffness of the ACh and PYR groups was significantly increased compared with the FG group at 8 weeks (P < .001 for all). At 12 weeks, most of the healing properties of the ACh and PYR groups were not significantly different compared with the FG group. CONCLUSION: ACh and PYR enhanced the early stage of bone-tendon insertion healing after rotator cuff repair. CLINICAL RELEVANCE: These findings imply that ACh and PYR could serve as potential therapeutic strategies for rotator cuff healing.

Acceleration of Bone-Tendon Interface Healing by Low-Intensity Pulsed Ultrasound Is Mediated by Macrophages.

OBJECTIVE: Low-intensity pulsed ultrasound (LIPUS) has been proven to facilitate bone-tendon interface (BTI) healing and regulate some inflammatory cytokines. However, the role of macrophages, a key type of inflammatory cell, during treatment remains unknown. This study aimed to investigate the role of macrophages in the treatment of BTI injury with LIPUS in a rotator cuff tear animal model. METHODS: In this experimental and comparative study, a total of 160 C57BL/6 mature male mice that underwent supraspinatus tendon detachment and repair were randomly assigned to 4 groups: daily ultrasonic treatment and liposomal clodronate (LIPUS+LC), daily ultrasonic treatment and liposomes (LIPUS), daily mock sonication and liposomal clodronate (LC), and daily mock sonication and liposomes (control [CTL]). LIPUS treatment was initiated immediately postoperatively and continued daily until the end of the experimental period. RESULTS: The failure load and stiffness of the supraspinatus tendon-humerus junction were significantly higher in the LIPUS group than in the other groups at postoperative weeks 2 and 4, whereas those in the LIPUS+LC and LC groups were lower than those in the CTL group at postoperative week 4. The LIPUS, LIPUS+LC, and LC groups exhibited significantly more fibrocartilage than the CTL group at 2 weeks. Only the LIPUS group had more fibrocartilage than the CTL group at 4 weeks. Micro-computed tomography results indicated that LIPUS treatment could improve the bone quality of the attachment site after both 2 and 4 weeks. When macrophages were depleted by LC, the bone quality-promoting effect of LIPUS treatment was significantly reduced. CONCLUSION: The enhancement of BTI healing by LIPUS might be mediated by macrophages. IMPACT: In our study, LIPUS treatment appeared to accelerate BTI healing, which was associated with macrophages based on our murine rotator cuff repair model. The expressions of macrophage under LIPUS treatment may offer a potential mechanism to explain BTI healing and the effects of LIPUS on BTI healing.

Periosteum progenitors could stimulate bone regeneration in aged murine bone defect model.

Periosteal stem cells are critical for bone regeneration, while the numbers will decrease with age. This study focused on whether Prx1+ cell, a kind of periosteal stem cell, could stimulate bone regeneration in aged mice. Four weeks and 12 months old Prx1CreER-GFP; Rosa26tdTomato mice were used to reveal the degree of Prx1+ cells participating in the femoral fracture healing procedure. One week, 8 weeks, 12 and 24 months old Prx1CreER-GFP mice were used to analyse the real-time distribution of Prx1+ cells. Twelve months old C57BL/6 male mice (n = 96) were used to create the bone defect model and, respectively, received hydrogel, hydrogel with Prx1- mesenchymal stem cells and hydrogel with Prx1+ cells. H&E staining, Synchrotron radiation-microcomputed tomography and mechanical test were used to analyse the healing results. The results showed that tdTomato+ cells were involved in bone regeneration, especially in young mice. At the same time, GFP+ cells decreased significantly with age. The Prx1+ cells group could significantly improve bone regeneration in the murine bone defect model via directly differentiating into osteoblasts and had better osteogenic differentiation ability than Prx1- mesenchymal stem cells. Our finding revealed that the quantity of Prx1+ cells might account for decreased bone regeneration ability in aged mice, and transplantation of Prx1+ cells could improve bone regeneration at the bone defect site.

Structure and ingredient-based biomimetic scaffolds combining with autologous bone marrow-derived mesenchymal stem cell sheets for bone-tendon healing.

Tendon attaches to bone across a robust fibrocartilaginous tissue termed the bone-tendon interface (BTI), commonly injured in the field of sports medicine and orthopedics with poor prognosis. So far, there is still a lack of effective clinical interventions to achieve functional healing post BTI injury. However, tissue-engineering may be a promising treatment strategy. In this study, a gradient book-type triphasic (bone-fibrocartilage-tendon) scaffold is fabricated based on the heterogeneous structure and ingredient of BTI. After decellularization, the scaffold exhibits no residual cells, while the characteristic extracellular matrix of the original bone, fibrocartilage and tendon is well preserved. Meanwhile, the bone, fibrocartilage and tendon regions of the acellular scaffold are superior in osteogenic, chondrogenic and tenogenic inducibility, respectively. Furthermore, autologous bone marrow mesenchymal stem cell (BMSC) sheets (CS) combined with the acellular scaffolds is transplanted into the lesion site of a rabbit BTI injury model to investigate the therapeutic effects. Our results show that the CS modified scaffold not only successfully achieves triple biomimetic of BTI in structure, ingredient and cell distribution, but also effectively accelerates bone-tendon (B-T) healing. In general, this work demonstrates book-type acellular triphasic scaffold combined with autologous BMSCs sheets is a promising graft for repairing BTI injury.

-Book-shaped decellularized tendon matrix scaffold combined with bone marrow mesenchymal stem cells-sheets for repair of achilles tendon defect in rabbit.

Tissue-engineering approaches have great potential to improve the treatment of tendon injuries which are major musculoskeletal disorders. The purpose of this study was to assess the tissue engineering potential of a novel multilayered decellularized tendon "book" scaffold with bone marrow mesenchymal stem cells (BMSCs) sheets for repair of an Achilles tendon defect in a rabbit model. In this study, we developed a novel book-shaped decellularized scaffold derived from the extracellular matrix of tendon tissues from New Zealand white rabbits. Hematoxylin and eosin (H&E) staining, 4', 6-diamidino-2-phenylindole (DAPI) staining, DNA quantitation, and scanning electron microscopy (SEM) confirmed the efficiency of decellularization. After culturing BMSCs on decellularized scaffolds, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, SEM, quantitative real time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis demonstrated that decellularized scaffolds have the capacity to yield homogeneous distribution and alignment of BMSCs, as well as support their differentiation into tendon. Tenomodulin and Alpha-1 collagen type I are important indicators for evaluating tenogenic differentiation of BMSCs. When decellularized "book" scaffolds with BMSCs sheets were used to repair a 1 mm Achilles tendon defect, histomorphological analysis, immunohistochemical assessment, and biomechanical testing showed that the book-shaped decellularized tendon matrix scaffold and BMSCs sheets could promote the regeneration of type I collagen at the wound site during healing, and improve the mechanical properties of the repaired tendon. Therefore, the results of this study suggest that the novel decellularized "book" tendon scaffolds combined with BMSCs sheets have therapeutic effects on improving the healing quality of the Achilles tendon. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-11, 2019.