In the present study, the feasibility of applying LIPUS-BMSC-Exos to promote fibrocartilage regeneration and alleviate fatty infiltration to induce the chondrogenesis of MSCs and was evaluated. We found that LIPUS-BMSC-Exos, the nanocarriers secreted by BMSCs which were preconditioned by LIPUS, could effectively promote BTI healing and ameliorate rotator cuff fatty infiltration through enhancing fibrocartilage regeneration, reducing supraspinatus muscle fat accumulation. We also showed that LIPUS-BMSC-Exos promoted chondrogenetic differentiation and inhibit adipoogenetic differentiation of native BMSCs in vitro. Moreover, we demonstrated that miR-140, a potently pro-chondrogenic miRNA, was extremely highly enriched in LIPUS-BMSC-Exos. To sum up, we showed that LIPUS-BMSC-Exos could promote the shift from adipogenic to chondrogenic differentiation of BMSCs via delivering miR-140, thus enhancing BTI fibrocartilage regeneration and ameliorate rotator cuff fatty infiltration.
Over the past years, the application of MSCs for cartilage tissue repair and regeneration has received considerable attention. 23, 66, 74 In recent years, an increasing number of studies have reported that the paracrine actions are the main mechanism by which MSCs exert their therapeutic effects. 35, 61 Exos, recently identified as an important paracrine factors that mediate the intercellular communication, are critical effectors of MSCs. 33, 52, 57, 61, 70 It has been demonstrated that Exos can transfer proteins, RNA and other bioactive compounds into target cells and create an optimal microenvironment for maintaining cellular dynamic homeostasis, thus completing the transmission of biological information. 29, 59 Due to specific advantages of Exos therapy such as high stability, low immunogenicity, non-tumorigenicity and non-vascular thrombosis, 13, 59 MSCs-Exos therapy has been receiving increasing attention for tissuse regeneration.
As for cartilage regeneration, the role of native MSCs-derived Exos for chondrogenesis is indefinable. Liu et al41 developed an in situ formed acellular hydrogel glue tissue patch combining MSC-Exos for cartilage defect repair and found that the patch can retain MSC-Exos and positively regulate both chondrocytes and BMSCs in vitro and promote cartilage defect repair in vivo. In view of the effect of in situ hydrogel glue in the study, there is not valid evidence to indicate MSC-Exos can effectively induce chondrogenesis alone. Jing et al28 demonstated that small extracellular vesicles (sEVs) derived from native human umbilical cord mesenchymal stem cells (hUCMSCs) do not generate obvious pro-chondrogenesis effects both in vitro and in vivo. However, Zhang et al77 demonstrated the efficacy of human embryonic MSC exosomes in promoting cartilage regeneration, and the utility of MSC exosomes as a ready-to-use and “cell-free” therapeutic alternative to cell-based MSC therapy in vivo. As for BTI healing, Wang et al71 showed that adipose stem cell–derived Exos can decrease fatty infiltration and enhance BTI healing in vivo. In accordance with Wang et al71 research, the present study also revealed improved BTI healing which can be judged by the decrease of fibrous scar tissue formation, augment of fibrocartilaginous enthesis regeneration and better biomechanical properties. Meanwhile, although significant suppression of adipogenesis of native BMSCs were determined by BMSC-Exos, we did not found obvious pro-chongdrogenesis effect of native BMSCs by BMSC-Exos in vitro, which was in consistent with Jing et al28 results. Actually, the results of in vivio and in vitro are not contradictory in the present study. The enhanced BTI healing in the BMSC-Exos group may be ascribed to 2 aspects: First, the anti-inflammatory effects of MSC-Exos such as reduce the infiltration of inflammatory cells into the BTI interface, which can decrease the formation of fibrous scar tissue and enhance the regeneration of the normal tendon-bone insertion site. 31, 39 Second, the angiogenesis effects of MSC-Exos can provide sufficient vascular invasion around the BTI junction, which is essential for promoting BTI healing and possibly contributes to aggregation and chondrogenesis of local stem cells after rotator cuff repair. 26 Combined with all of these results, we can draw conclusions carefully that MSC-Exos may not possess obvious chondrogenetic effect alone,but they can still exhibit a degree of facilitating effect on fibrocartilage regeneration and BTI healing enhancement in vivo by promoting angiogenesis, decreasing infiltration of inflammatory cells and alleviating adipogenesis of MSCs.
Over the last two decades, LIPUS therapy has been proved to be an effective strategy to stimulate tendon-bone junction healing. 8, 43–48, 50, 60 In these studies, an standard 30 mW/cm2 intensity of LIPUS transcutaneous therapy in vivo turned out to enhance BTI healing through promoting angiogenesis, cartilage formation and maturation, endchondral bone formation and increaing the mechanical properties of the healing tissues of the tendon-bone junction. 50, 75 LIUS is also reported to enhance repair of articular cartilage in animal model of cartilage defect. 11, 58 In a rabbit full-thickness osteochondral defects model, Cook et al 11 demonstrate that daily low-intensity pulsed ultrasound had a significant positive effect on the healing of osteochondral defects. Meanwhile, LIPUS can induce chondrogenesis of MSCs in vitro by enhancing the synthesis of matrix proteins such as collagen type II and proteoglycans and expression of chondrogenic markers such as Sox-9 and TIMP-2. 37 Moreover, LIPUS preconditioning of BMSCs in vitro proved to be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo. 12
As valid effect of LIPUS therpy for BTI healing has been comfirmed and the fact that biological functions of the generated Exos were determined by the state of the Exos donor cells, direct use of Exos derived from LIPUS preconditioned MSCs may represent a preferable and promising biotherapy agent for BTI regeneration. Thus, the present study construct Exos derived from BMSCs induced by LIPUS stimulation for fibrocartilage regeneration of BTI healing. What’ more, as fatty infiltration of rotator cuff is the other factor direct associated with unsatisfactory prognosis besides poor BTI healing, we also concern on the effect of LIPUS-BMSC-Exos on fatty infiltration and adipogenetic differentiation of BMSCs. Accordingly, we hypothesized that LIPUS preconditioning might impart Exos derived from MSCs with chondrogenic potential to generate a valid chondrogenic microenvironment. As anticipated, the present results demonstrate that LIPUS-preconditioned BMSCs release Exos with potent chondrogenesis-inducing functions compared to BMSC-Exos both in vivo and in vitro. Meanwhile, LIPUS-BMSC-Exos also exhibit inhibit effect on adipogenesis of BMSCs and fatty infiltration of supraspinatus muscle. These data threw light on the possible mechanism of shift from adipogenetic differentiation to chondrogenetic differentiation of MSCs under the specific chondrogenic microenvironment cultivated by LIPUS-BMSC-Exos.
Exos-derived miRNAs, a class of small (20-24-nucleotide) non-coding RNAs, are one of the most important factors regulating the gene expression of recipient cells through translational repression. 76 Some studies even indicated that Exos mediate intercellular communication mainly through delivering miRNAs. 18, 54 Furthermore, increasing evidence indicates that miRNAs from Exos cargoes are definitely involved in MSCs differentiation processes, such as angiogenesis, osteogenesis, adipogenesis and chondrogenesis. 28, 42, 56, 79 The stepwise chondrogenic differentiation of MSCs is regulated by a series of miRNAs. To clarify the molecular mechanisms of LIPUS-BMSC-Exos on chondrocyte and adipocyte formation, we performed qRT-PCR to detect the key miRNAs that may function. In the chondrogenesis-related miRNA profiles of LIPUS-BMSC-Exos, 14 of 21 (66.67%) miRNAs were significantly up-regulated compared to BMSC-Exos. At the same time, 7 of 11 (63.63%) miRNAs were significantly down-regulated. It is reasonable to assume that LIPUS intervention remodel the miRNAs profiles of BMSCs-derived Exos and the highly enriched chondrogenesis-related miRNA cargoes in KGN-sEV play important roles in regulating transcriptional activity of chondrogenesis-related genes in recipient cells. Strikingly, miR-140, which has been reported to play an important role not only in the stability and maintenance of the cartilage matrix in chondrocytes but also promotes MSCs chondrogenesis, 17 was found to be the most abundant miRNA in LIPUS-BMSC-Exos in the present study. The in vitro investigations revealed that over expressed miR-140 in native BMSCs by miR-140 mimic replicated most of the chondrogenic effect of LIPUS-BMSC-Exos, whereas knockdown of miR-140 nearly abolished the pro-chondrogenic potential of LIPUS-BMSC-Exos, suggesting that miR-140 is one of the critical molecules in LIPUS-BMSC-Exos for stimulating fibrocartilage formation. Interestingly, over expression of miR-140 could suppress adipogenesis of BMSCs. At the same time, most of anti-adipogenesis efficacy by LIPUS-BMSC-Exos could be abolished by miR-140 inhibitor, implying LIPUS treated MSCs-Exos could possibly trigger the stable lineage-specific chondrogenic differentiation other than adipogenetic differentiation of MSCS via transferring miR-140. The possible reason why the regenerated cartilage pellet of the miR-140 mimic group seemed to be inferior compared to LIPUS-BMSC-Exos group might lied in the fact that there are totally 13 miRNAs which were significantly up-regulated in LIPUS-BMSC-Exos, besides miR-140. Whether these miRNAs play essential roles in chondrogenesis of MSCs induced by LIPUS-BMSC-Exos still remains inconclusive. This is also the probable cause why miR-140 inhibitor could not totally reverse the chondrogenetic effect of LIPUS-BMSC-Exos.
Our findings broaden the understanding of the regenerative potential of LIPUS-treated-Exos on chondrogenesis of MSCs, and suggest the application of LIPUS-BMSC-Exos as an off-the-shelf bio-tool for inducing the stable differentiation of MSCs, enhancing BTI healing and ameliorate fatty infiltration of the rotator cuff. In contrast to cumbersome postoperative LIPUS therapy or expensive inducing hormonal cocktails preconditioning which containing transforming growth factor β (TGF-β), insulin-like growth factor (IGF), dexamethasone, insulin, transferrin, selenium, and ascorbic acid, 40 an local injection of LIPUS preconditioned MSC-derived Exos during operation is absolutely economical, simple, and convenient. Moreover, contrast to classical MSC-derived Exos therapy, the LIPUS preconditioned MSC-derived Exos demonstrate substantially superior pro-chondrogenesis efficacy on BTI fibrocartilage regeneration and also better anti-adipogenesis effect on prevent supraspinatus fatty infiltration.
This study has some limitations. First, although we continued to observe the generation of fibrocartilage tissue at the BTI junction which were proved to be most difficult part to be regenerated, the biological and biomechanical properties of BTI junction is also determined by the other 2 zones of BTI, namely the subchondral bone and the tendon. It is still unclear whether LIPUS-BMSC-Exos can make a significant difference on the endochondral calcification, ossification, tendon collagen fiber formation, peritendinous fibrosis and tendon adhesion processes. Therefore, regeneration of the 3 zones of the native enthesis (tendon, fibrocartilage, bone) likely requires more than just simple application of LIPUS-BMSC-Exos at the surgical repair site. Second, in many cases, LIPUS treatment entails 20 min of daily stimulation at intensity of 30 mW/cm2, with a frequency of 1.5 MHz in pulsed-wave mode (0.2-s burst sine waves repeated at 1.0 kHz). 8, 24, 39, 67 Most studies, including this one, have only studied a single LIPUS dose. Different dosages or use of different LIPUS stimulation parameters may reveal different effects of LIPUS on MSC-Exos components and function, thus leading to different outcomes of rotator cuff healing in this specific model. Third, it is noteworthy that inhibition of miR-140 did not thoroughly block the beneficial effects of LIPUS-BMSC-Exos on chondrogenetic differentiation in vitro, indicating the implication of other miRNA or proteins that might influence the regulatory effects of the LIPUS-BMSC-Exos through some unknown mechanisms. Finally, although there are many advantages of mice as a model for acute supraspinatus tendon injury repair, 36 mice are quadrupedal animals and joint motion and loading on the healing site are difficult to control owing to the inability to reliably immobilize the extremity or restrict weight bearing. 72 As mechanical loading is critical to the processes of fibrocartilage formation, mineral accumulation and collagen metabolism that are closely involved in a healing enthesis, it is not suitable to simply translate our findings into rotator cuff tear repair in humans. Meanwhile, The pathophysiological process of the acute rotator cuff injury model is different from that of chronic rotator cuff injury, and the role of LIPUS-BMSC-Exos in chronic rotator cuff injury is unclear and needs further exploration.
In conclusion, our results demonstrate that Exos derived from LIPUS-preconditioned BMSCs are able to promote BTI fibrocartilage regeneration and ameliorate supraspinatus fatty infiltration in a murine rotator cuff repair model. The potential mechanism may be the positive regulation of pro-chondrogenetic and anti-adipogenetic of MSCs differentiation primarily through delivering miR-140. LIPUS-BMSC-Exos may become an innovative “Exos based strategy” for inducing chondrogenic differentiation of native MSCs, thereby proposing a new therapeutic method to BTI regeneration and rotator cuff healing in the near future.