Exosomes derived from bone marrow mesenchymal stem cell preconditioned by low-intensity pulsed ultrasound stimulation promote bone-tendon interface fibrocartilage regeneration and ameliorate rotator cuff fatty infiltration.

Background: Fibrovascular scar healing of bone-tendon interface (BTI) instead of functional fibrocartilage regeneration is the main concern associated with unsatisfactory prognosis in rotator cuff repair. Mesenchymal stem cells (MSCs) exosomes have been reported to be a new promising cell-free approach for rotator cuff healing. Whereas, controversies abound in whether exosomes of native MSCs alone can effectively induce chondrogenesis. Purpose: To explore the effect of exosomes derived from low-intensity pulsed ultrasound stimulation (LIPUS)-preconditioned bone marrow mesenchymal stem cells (LIPUS-BMSC-Exos) or un-preconditioned BMSCs (BMSC-Exos) on rotator cuff healing and the underlying mechanism. Methods: C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair were randomly assigned to saline, BMSCs-Exos or LIPUS-BMSC-Exos injection therapy. Histological, immunofluorescent and biomechanical tests were detected to investigate the effect of exosomes injection on BTI healing and muscle fatty infiltration of the repaired rotator cuff. In vitro, native BMSCs were incubated with BMSC-Exos or LIPUS-BMSC-Exos and then chondrogenic/adipogenic differentiation were observed. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the chondrogenesis/adipogenesis-related miRNA profiles of LIPUS-BMSC-Exos and BMSC-Exos. The chondrogenic/adipogenic potential of the key miRNA was verified through function recover test with its mimic and inhibitor. Results: The results indicated that the biomechanical properties of the supraspinatus tendon-humeral junction were significantly improved in the LIPUS-BMSC-Exos group than that of the BMSCs-Exos group. The LIPUS-BMSC-Exos group also exhibited a higher histological score and more newly regenerated fibrocartilage at the repair site at postoperative 2 and 4 weeks and less fatty infiltration at 4 weeks than the BMSCs-Exos group. In vitro, co-culture of BMSCs with LIPUS-BMSC-Exos could significantly promote BMSCs chondrogenic differentiation and inhibit adipogenic differentiation. Subsequently, qRT-PCR revealed significantly higher enrichment of chondrogenic miRNAs and less enrichment of adipogenic miRNAs in LIPUS-BMSC-Exos compared with BMSC-Exos. Moreover, we demonstrated that this chondrogenesis-inducing potential was primarily attributed to miR-140, one of the most abundant miRNAs in LIPUS-BMSC-Exos. Conclusion: LIPUS-preconditioned BMSC-Exos can effectively promote BTI fibrocartilage regeneration and ameliorate supraspinatus fatty infiltration by positive regulation of pro-chondrogenesis and anti-adipogenesis, which was primarily through delivering miR-140. The translational potential of this article: These findings propose an innovative "LIPUS combined Exosomes strategy" for rotator cuff healing which combines both physiotherapeutic and biotherapeutic advantages. This strategy possesses a good translational potential as a local injection of LIPUS preconditioned BMSC-derived Exos during operation can be not only efficient for promoting fibrocartilage regeneration and ameliorating rotator cuff fatty infiltration, but also time-saving, simple and convenient for patients.

Comparative effect of skeletal stem cells versus bone marrow mesenchymal stem cells on rotator cuff tendon-bone healing.

Background: Bone marrow mesenchymal stem cells (BMSCs) have immense potential in applications for the enhancement of tendon-bone (T-B) healing. Recently, it has been well-reported that skeletal stem cells (SSCs) could induce bone and cartilage regeneration. Therefore, SSCs represent a promising choice for cell-based therapies to improve T-B healing. In this study, we aimed to compare the therapeutic potential of SSCs and BMSCs for tendon-bone healing. Methods: SSCs and BMSCs were isolated by flow cytometry, and their proliferation ability was measured by CCK-8 assay. The osteogenic, chondrogenic, and adipogenic gene expression in cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair, and the mice were then randomly allocated to 4 groups: control group (tendon-bone interface without any treatment), hydrogel group (administration of blank hydrogel into the tendon-bone interface), hydrogel + BMSCs group (administration of hydrogel with BMSCs into the tendon-bone interface), and hydrogel + SSCs group (administration of hydrogel with SSCs into the tendon-bone interface). Histological staining, Micro-computed tomography (Micro-CT) scanning, biomechanical testing, and qRT-PCR were performed to assay T-B healing at 4 and 8 weeks after surgery. Results: SSCs showed more cell proportion, exhibited stronger multiplication capacity, and expressed higher osteogenic and chondrogenic markers and lower adipogenic markers than BMSCs. In vivo assay, the SSCs group showed a better-maturated interface which was characterized by richer chondrocytes and more proteoglycan deposition, as well as more newly formed bone at the healing site and increased mechanical properties when compared to other there groups. qRT-PCR analysis revealed that the healing interface in the SSCs group expressed more transcription factors essential for osteogenesis and chondrogenesis than the interfaces in the other groups. Conclusions: Overall, the results demonstrated the superior therapeutic potential of SSCs over BMSCs in tendon-bone healing. The translational potential of this article: This current study provides valuable insights that SSCs may be a more effective cell therapy for enhancing T-B healing compared to BMSCs.

A Combined Treatment of BMP2 and Soluble VEGFR1 for the Enhancement of Tendon-Bone Healing by Regulating Injury-Activated Skeletal Stem Cell Lineage.

BACKGROUND: Bone morphogenetic protein 2 (BMP2) is an appealing osteogenic and chondrogenic growth factor for promoting tendon-bone healing. Recently, it has been reported that soluble vascular endothelial growth factor (VEGF) receptor 1 (sVEGFR1) (a VEGF receptor antagonist) could enhance BMP2-induced bone repair and cartilage regeneration; thus, their combined application may represent a promising treatment to improve tendon-bone healing. Moreover, BMP2 could stimulate skeletal stem cell (SSC) expansion and formation, which is responsible for wounded tendon-bone interface repair. However, whether the codelivery of BMP2 and sVEGFR1 increases tendon enthesis injury-activated SSCs better than does BMP2 alone needs further research. PURPOSE: To study the effect of BMP2 combined with sVEGFR1 on tendon-bone healing and injury-activated SSC lineage. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 128 C57BL/6 mice that underwent unilateral supraspinatus tendon detachment and repair were randomly assigned to 4 groups: (1) untreated control group; (2) hydrogel group, which received a local injection of the blank hydrogel at the injured site; (3) BMP2 group, which received an injection of hydrogel with BMP2; and (4) BMP2 with sVEGFR1 group, which received an injection of hydrogel with BMP2 and sVEGFR1. Histology, micro-computed tomography, and biomechanical tests were conducted to evaluate tendon-bone healing at 4 and 8 weeks after surgery. In addition, flow cytometry was performed to detect the proportion of SSCs and their downstream differentiated subtypes, including bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors within supraspinatus tendon enthesis at 1 week postoperatively. RESULTS: The repaired interface in BMP2 with sVEGFR1 group showed a significantly improved collagen fiber continuity, increased fibrocartilage, greater newly formed bone, and elevated mechanical properties compared with the other 3 groups. There were more SSCs; bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors in the BMP2 with sVEGFR1 group than that in the other groups. CONCLUSION: Our study suggests that the combined delivery of BMP2 and sVEGFR1 could promote tendon-bone healing and stimulate the expansion of SSCs and their downstream progeny within the injured tendon-bone interface. CLINICAL RELEVANCE: Combining BMP2 with sVEGFR1 may be a good clinical treatment for wounded tendon enthesis healing.

Intermittent fasting promotes repair of rotator cuff injury in the early postoperative period by regulating the gut microbiota.

Background: The repair of rotator cuff injury is affected by lifestyle and metabolic factors. Intermittent fasting (IF) can promote repair of damaged tissue by regulating intestinal flora, which provides an idea of therapy for rotator cuff injury. The aim of this study was to investigate the effects of fasting on rotator cuff repair after injury, and the role of intestinal flora or a single strain in this process. Methods: Mice underwent rotator cuff injury were treated with intermittent fasting or fed ad libitum. Fasting began one month before surgery and continued until euthanasia. Fresh feces were collected at 2 weeks before surgery, on the day of surgery, and 2, 4, 8 weeks postoperatively for 16S rRNA microbiome sequencing. Supraspinatus tendon-humerus â€‹(SSTH) complex was collected at 2, 4 and 8 weeks after surgery. Live parabacteroides distasonis (Parabacteroides distasonis) was used for repair of rotator cuff injury, with equal amount of pasteurized P. distasonis (KPD) or sterile anaerobic phosphate buffer saline (PBS) as control. Biomechanical, radiological, histological analysis were used to assess the effect of rotator cuff repair. Results: Biomechanical, radiological and histological analysis indicated that intermittent fasting significantly promoted the repair of rotator cuff injury in the early postoperative period (P ＜ 0.05), but significantly inhibited the repair of rotator cuff injury at 4 weeks postoperatively (P ＜ 0.05). 16S rRNA Microbiome sequencing result showed that P. distasonis was the species with the most obvious changes in intestinal flora of mice after fasting. The results of tensile test, X-ray analysis and histological analysis indicated that the live P. distasonis (LPD) significantly impaired the biomechanical properties, bone regeneration and fibrocartilage regeneration of enthesis postoperatively (P ＜ 0.05). Conclusion: Intermittent fasting promoted repair of rotator cuff injury in the early postoperative period by regulating the gut microbiota, in which P. distasonis played an important role. The translational potential of this article: Intermittent fasting (IF) may be a beneficial lifestyle for the repair of rotator cuff injury in the early postoperative period in clinical, and the influence of a certain strain on the repair of rotator cuff injury may also provide an idea for the treatment of rotator cuff injury in the future.