The rate of retear after surgical repair remains high. Mesenchymal stem cells (MSCs) have been extensively employed in regenerative medicine for several decades. However, safety and ethical concerns constrain their clinical application. Tendon Stem/Progenitor Cells (TSPCs)-derived exosomes have emerged as promising cell-free therapeutic agents. Therefore, urgent studies are needed to investigate whether TSPC-Exos could enhance tendon-bone healing and elucidate the underlying mechanisms. In this study, TSPC-Exos were found to promote the proliferation, migration, and expression of fibrogenesis markers in BMSCs. Furthermore, TSPC-Exos demonstrated an ability to suppress the polarization of M1 macrophages while promoting M2 macrophage polarization. In a rat model of rotator cuff repair, TSPC-Exos modulated inflammation and improved the histological structure of the tendon-bone interface, the biomechanical properties of the repaired tendon, and the function of the joint. Mechanistically, TSPC-Exos exhibited high expression of miR-21a-5p, which regulated the expression of PDCD4. The PDCD4/AKT/mTOR axis was implicated in the therapeutic effects of TSPC-Exos on proliferation, migration, and fibrogenesis in BMSCs. This study introduces a novel approach utilizing TSPC-Exos therapy as a promising strategy for cell-free therapies, potentially benefiting patients with rotator cuff tear in the future.

Failure to adequately reconstruct the tendon-to-bone interface constitutes the primary etiology underlying rotator cuff retear after surgery. The purpose of this study is to construct a dynamic chondroitin sulfate and chitosan hydrogel scaffold (CHS) with bone morphogenetic protein 2 (BMP2), then seed tendon stem cells (TSCs) on BMP2-CHS for the rotator cuff reconstruction of tendon-to-bone interface. In this dynamic hydrogel system, the scaffold could not only have good biocompatibility and degradability but also significantly promote the proliferation and differentiation of TSCs. The ability of BMP2-CHS combined with TSCs to promote regeneration of tendon-to-bone interface was further verified in the rabbit rotator cuff tear model. The results showed that BMP2-CHS combined with TSCs could induce considerable collagen, fibrocartilage, and bone arrangement and growth at the tendon-to-bone interface and promote the biomechanical properties. Overall, TSCs seeded on CHS with BMP2 can enhance tendon-to-bone healing and provide a new possibility for improving the poor prognosis of rotator cuff surgery.

BACKGROUND: Tendon-to-bone healing is a critical challenge in sports medicine, with its cellular and molecular mechanisms yet to be explored. An efficient murine model could significantly advance our understanding of this process. However, most existing murine animal models face limitations, including a propensity for bleeding, restricted operational space, and a steep learning curve. Thus, the need for a novel and efficient murine animal model to investigate the cellular and molecular mechanisms of tendon-to-bone healing is becoming increasingly evident.
METHODS: In our study, forty-four 9-week-old male C57/BL6 mice underwent transection and reattachment of the Achilles tendon insertion to investigate tendon-to-bone healing. At 2 and 4 weeks postoperatively, mice were killed for histological, Micro-CT, biomechanical, and real-time polymerase chain reaction tests.
RESULTS: Histological staining revealed that the original tissue structure was disrupted and replaced by a fibrovascular scar. Although glycosaminoglycan deposition was present in the cartilage area, the native structure had been destroyed. Biomechanical tests showed that the failure force constituted approximately 44.2% and 77.5% of that in intact tissues, and the ultimate tensile strength increased from 2 to 4 weeks postoperatively. Micro-CT imaging demonstrated a gradual healing process in the bone tunnel from 2 to 4 weeks postoperatively. The expression levels of ACAN, SOX9, Collagen I, and MMPs were detected, with all genes being overexpressed compared to the control group and maintaining high levels at 2 and 4 weeks postoperatively.
CONCLUSIONS: Our results demonstrate that the healing process in our model is aligned with the natural healing process, suggesting the potential for creating a new, efficient, and reproducible mouse animal model to investigate the cellular and molecular mechanisms of tendon-to-bone healing.

UNASSIGNED: To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits.
UNASSIGNED: TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties.
UNASSIGNED: CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05).
UNASSIGNED: TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
UNASSIGNED: 探讨以壳聚糖（chitosan，CS）负载肌腱干细胞（tendon-derived stem cells，TDSCs）构建的可注射型水凝胶（以下简称TDSCs/CS水凝胶）促进兔肩袖腱-骨愈合的效果。.
UNASSIGNED: 取3只成年新西兰大白兔肩袖组织，采用Henderson分步酶消化法分离培养TDSCs，并经多向分化及流式细胞术鉴定。以CS包裹第3代TDSCs构建TDSCs/CS水凝胶，体外培养1～5 d以细胞计数试剂盒8（cell counting kit 8，CCK-8）法检测水凝胶中TDSCs增殖情况，并以单纯TDSCs作为对照，评估TDSCs/CS水凝胶细胞相容性。另取36只成年新西兰大白兔，随机分为3组（ n=12），分别为肩袖修复组（对照组）、肩袖修复+CS水凝胶注射组（CS组）、肩袖修复+DSCs/CS水凝胶注射组（TDSCs/CS组）。3组建立肩袖损伤+单排技术修复模型后，CS组及TDSCs/CS组将对应水凝胶注入腱-骨界面处修复肩袖，对照组不作其他处理。术后观察动物一般情况，于4、8周取材，实时定量PCR（real-time quantitative PCR，qPCR）检测腱-骨界面成肌腱相关基因（腱调蛋白、转录因子），成软骨相关基因（蛋白聚糖、性别决定区Y框蛋白9），成骨相关基因（ALP、人Runt相关转录因子2）表达；另于8周取材行HE、Masson染色观察并行组织学半定量评分，生物力学测试修复肩袖极限载荷和失效部位，评价组织愈合情况和生物力学特性改变。.
UNASSIGNED: CCK-8法检测示共培养后CS水凝胶可促进TDSCs增殖（ P<0.05）。术后各组动物均存活至实验完成。术后4、8周，TSDCs/CS组的成肌腱、成软骨、成骨相关基因相对表达量均高于CS组和对照组（ P<0.05），TSDCs/CS组组内术后8周上述基因相对表达量亦高于术后4周（ P<0.05）。组织学染色示TDSCs/CS组腱-骨界面处有清晰的软骨组织和致密有序胶原形成，半定量分析结果示TDSCs/CS组与对照组相比，细胞数量、胶原纤维分布以及组织学评分增高（ P<0.05），血管数量降低（ P<0.05）；TDSCs/CS组与CS组相比，胶原纤维分布以及组织学评分均增高（ P<0.05），而细胞数量、血管数量差异无统计学意义（ P>0.05）。生物力学检测示所有样本测试过程中均在修复部位失效，TDSCs/CS组极限载荷高于对照组（ P<0.05），但与CS组比较差异无统计学意义（ P>0.05）。.
UNASSIGNED: TDSCs/CS水凝胶可以诱导兔肩袖肌腱和骨之间的软骨再生，促进腱-骨愈合。.

Aging is an independent risk factor for recurrent tearing after surgical repair of rotator cuff ruptures around the tendon-to-bone area. However, aging signature factors and related mechanisms involved in the healing of the rotator cuff are still unknown. We hypothesized that differences in proteins involved in the rotator cuff according to age may affect tendon-to-bone healing. The proteome analysis performed to identify the signature aging proteins of the rotator cuff confirmed the sirtuin signal as an age-specific protein. In particular, the expression of SIRT6 was markedly down-regulated with age. Ingenuity pathway analysis of omics data from age-dependent rat rotator cuffs and linear regression from human rotator cuffs showed SIRT6 to be closely related to the Wnt/β-catenin signal. We confirmed that overexpression of SIRT6 in the rotator cuff and primary tenocyte regulated canonical Wnt signaling by inhibiting the transcriptional expression of sclerostin, a Wnt antagonist. Finally, SIRT6 overexpression promoted tendon-to-bone healing after tenotomy with reconstruction in elderly rats. This approach is considered an effective treatment method for recovery from recurrent rotator cuff tears, which frequently occur in the elderly.

BACKGROUND: Trans-calcaneal suture technique is an economical and effective method for repairing Achilles tendon sleeve avulsion. Whether cancellous bone fixation upon this technique could accelerate tendon-to-bone healing is unknown. The purpose of this study is to compare the effect of cortical versus cancellous bone fixation on tendon-bone healing with a novel rat trans-calcaneal suture model.
METHODS: Trans-calcaneal suture treatment was carried out on the right hindlimb in male Sprague-Dawley rats (N = 80). They were randomly divided into the cortical group (Achilles fixed to the calcaneal cortical bone, n = 40) and the cancellous group (Achilles fixed to the calcaneal cancellous bone, n = 40). Gait analysis and immunohistochemistry were performed 1, 4, 7, and 14 days after the operation. Gross observation, biomechanical analysis, micro-CT, and histological analysis were performed 4 and 8 weeks after surgery. Independent-samples t tests were used for comparison between groups.
RESULTS: At 1, 4, and 7 days, the swing time of the affected limb in the cancellous group decreased, while the duty cycle, the maximum contact area, the print area, and the mean intensity increased significantly. The cross-sectional area of the tendon-bone junction in the cancellous group was smaller, and the failure load and stiffness were higher 4 weeks after the operation. The cancellous group showed more proportion of new bone and a relatively well-organized and dense connective tissue interface with better fibrocartilage-like tissue at 4 weeks after the operation. The ratio of ED2 + macrophages in the cancellous group was significantly higher than in the cortical group on 1, 4, 7, and 14 days. There were no significant differences in gait at 2 weeks, in appearance, biomechanics, new bone formation, and histology at 8 weeks after surgery between the two groups.
CONCLUSIONS: In the new rat trans-calcaneal suture model, cancellous fixation can accelerate tendon-to-bone healing in the early stage, which perhaps is related to the abundant bone marrow tissue in the cancellous bone that modulates the inflammatory processes.

Graft healing after anterior cruciate ligament reconstruction (ACLR) involves slow biological processes, and various types of biological modulations have been explored to promote tendon-to-bone integration. Exosomes have been extensively studied as a promising new cell-free strategy for tissue regeneration, but few studies have reported their potential in tendon-to-bone healing. In this study, a novel type of exosome derived from magnetically actuated (iron oxide nanoparticles (IONPs) combined with a magnetic field) bone mesenchymal stem cells (BMSCs) (IONP-Exos) was developed, and the primary purpose of this study was to determine whether IONP-Exos exert more significant effects on tendon-to-bone healing than normal BMSC-derived exosomes (BMSC-Exos). Here, we isolated and characterized the two types of exosomes, conducted in vitro experiments to measure their effects on fibroblasts (NIH3T3), and performed in vivo experiments to compare the effects on tendon-to-bone integration. Moreover, functional exploration of exosomal miRNAs was further performed by utilizing a series of gain- and loss-of-function experiments. Experimental results showed that both BMSC-Exos and IONP-Exos could be shuttled intercellularly into NIH3T3 fibroblasts and enhanced fibroblast activity, including proliferation, migration, and fibrogenesis. In vivo, we found that IONP-Exos significantly prevented peri-tunnel bone loss, promoted more osseous ingrowth into the tendon graft, increased fibrocartilage formation at the tendon-bone tunnel interface, and induced a higher maximum load to failure than BMSC-Exos. Furthermore, overexpression of miR-21-5p remarkably enhanced fibrogenesis in vitro, and SMAD7 was shown to be involved in the promotive effect of IONP-Exos on tendon-to-bone healing. Our findings may provide new insights into the regulatory roles of IONPs in IONP-Exos communication via stimulating exosomal miR-21-5p secretion and the SMAD7 signaling pathway in the fibrogenic process of tendon-to-bone integration. This work could provide a new strategy to promote tendon-to-bone healing for tissue engineering in the future.

UNASSIGNED: Decreasing the proinflammatory M1 macrophages or shifting the polarization status from M1 to M2 phenotype is thought to be beneficial for tendon-to-bone healing. In anterior cruciate ligament reconstruction (ACLR), using an insertion-preserved hamstring tendon (IP-HT) graft compared with a free hamstring tendon (FHT) graft has been shown to reduce graft necrosis and improve healing. However, the role of macrophage polarization at the tendon-to-bone interface is unclear.
UNASSIGNED: ACLR using IP-HT graft would facilitate the phenotype shift from M1 to M2 macrophages at the tendon-to-bone interface.
UNASSIGNED: Controlled laboratory study.
UNASSIGNED: Unilateral ACLR was performed on 42 healthy New Zealand White rabbits (study group, 21 rabbits with IP-HT graft; control group, 21 rabbits with FHT graft). At days 1, 3, and 7 and weeks 3, 6, 12, and 24 postoperatively, 3 rabbits in each group were sacrificed to investigate and compare the expression of surrogate markers for M1 macrophages (inducible nitric oxide synthase [iNOS] and tumor necrosis factor α [TNF-α]) and M2 macrophages (CD206 and transforming growth factor β [TGF-β]) via immunohistochemical staining and evaluation.
UNASSIGNED: In the control group, the percentage of iNOS- and TNF-α-positive cells from postoperative day 7 and week 3 increased then decreased by week 6; positive expression of CD206 and TGF-β was weaker and peaked at 3 weeks postoperatively. In the study group, high CD206- and TGF-β-positive expression was observed from weeks 3 to 12 and peaked at week 6, and positive expression of iNOS- and TNF-α was weaker and peaked on day 7. At both 7 days and 3 weeks, the percentages of iNOS- and TNF-α-positive cells in the control group were both significantly higher than in the study group (P ≤ .04 for all). At 6 weeks, the percentages of CD206- and TGF-β-positive cells in the study group were both significantly higher than in the control group (P = .02 and P = .04, respectively).
UNASSIGNED: More expression of surrogate markers for M2 macrophages was observed in the tendon-to-bone healing process after ACLR using IP-HT versus FTP graft.
UNASSIGNED: Using IP-HT grafts in ACLR may facilitate postoperative healing by shifting the local status of macrophage polarization at the tendon-to-bone interface.

UNASSIGNED: There is growing interest in various biological supplements to improve tendon healing in patients after arthroscopic rotator cuff repair. The ideal biological supplement to strengthen rotator cuff remains unknown.
UNASSIGNED: To assess the safety and efficacy of autologous cultured dermal fibroblast (ADF) injection on tendon-to-bone healing in patients after arthroscopic rotator cuff repair.
UNASSIGNED: Case series; Level of evidence, 4.
UNASSIGNED: Included were 6 patients who underwent arthroscopic rotator cuff repair between June 2018 and March 2020; all patients had a full-thickness rotator cuff tear (>2 cm) involving the supraspinatus and infraspinatus tendons. The patients were injected with ADF between the repaired tendon and footprint during arthroscopic rotator cuff repair using the suture bridge technique. The safety of ADFs and the procedure was evaluated at 5 weeks postoperatively, and the anatomical healing of the repaired tendon was accessed at 6 months postoperatively using magnetic resonance imaging and at 12 months using ultrasonography. Outcomes including shoulder range of motion (ROM), visual analog scale (VAS) for pain, and functional scores were measured at 6 and 12 months postoperatively.
UNASSIGNED: Adverse reactions to ADF injection were not observed at 6 months after surgery. VAS and functional scores at 6 and 12 months postoperatively were significantly improved compared with preoperative scores (all P < .05). However, there was no significant difference on any ROM variable between preoperative and postoperative measurements at 6 and 12 months (all P > .05). No healing failure was found at 6 and 12 months postoperatively.
UNASSIGNED: There was no adverse reaction to ADF injection, and all patients had successful healing after rotator cuff repair. A simple and easily accessible ADF injection may be a novel treatment option for increasing the healing capacity of torn rotator cuff tendons. Further clinical research is needed to verify the study results.

The healing failure rate after rotator cuff repair is considerably high.
To evaluate the effect of a porous suture containing transforming growth factor beta 1 (TGF-β1) on the sustained release of TGF-β1 and rotator cuff healing in a rat model.
Controlled laboratory study.
A porous suture was developed, and its tensile strength was measured. TGF-β1 was delivered using the porous suture, and a TGF-β1 release test and human fibroblast proliferation assay were performed. For the animal experiment, 30 rats were randomly allocated into 3 groups (n = 10 each). A bilateral supraspinatus tendon tear was made in all the rats, and repair was performed. Group 1 received repair only; group 2, repair and a single injection of TGF-β1; and group 3, repair using the porous suture containing TGF-β1. Eight weeks after repair, biomechanical and histological analyses were performed.
The porous suture was successfully developed with mechanical properties compatible with the conventional suture, and the sustained release of TGF-β1 from the porous suture was confirmed. In addition, the cell proliferation assay confirmed the biological safety of the porous suture. In the animal experiment, group 3 biomechanically exhibited the largest cross-sectional area and the highest ultimate failure load and ultimate stress (all P < .05). Histological examination revealed that group 3 showed significantly better collagen fiber density and tendon-to-bone maturation than did groups 1 and 2 (all P < .05).
The porous suture containing TGF-β1 could sustainedly and safely release TGF-β1, and its use during rotator cuff repair could improve rotator cuff healing, as assessed on the basis of the biomechanical and histological changes in the rat model in this study. Considering the effectiveness, safety, and convenience of the porous suture without extra effort in surgery, the findings of the present study will have a far-reaching effect on the treatment of rotator cuff tears.
The porous suture containing TGF-β1 might improve healing after rotator cuff repair.