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.

Enhancing tendon-bone integration and healing with advanced multi-layer nanofiber-reinforced 3D scaffolds for acellular tendon complexes.

Advancements in tissue engineering are crucial for successfully healing tendon-bone connections, especially in situations like anterior cruciate ligament (ACL) restoration. This study presents a new and innovative three-dimensional scaffold, reinforced with nanofibers, that is specifically intended for acellular tendon complexes. The scaffold consists of a distinct layered arrangement comprising an acellular tendon core, a middle layer of polyurethane/type I collagen (PU/Col I) yarn, and an outside layer of poly (L-lactic acid)/bioactive glass (PLLA/BG) nanofiber membrane. Every layer is designed to fulfill specific yet harmonious purposes. The acellular tendon core is a solid structural base and a favorable environment for tendon cell functions, resulting in considerable tensile strength. The central PU/Col I yarn layer is vital in promoting the tendinogenic differentiation of stem cells derived from tendons and increasing the expression of critical tendinogenic factors. The external PLLA/BG nanofiber membrane fosters the process of bone marrow mesenchymal stem cells differentiating into bone cells and enhances the expression of markers associated with bone formation. Our scaffold's biocompatibility and multi-functional design were confirmed through extensive in vivo evaluations, such as histological staining and biomechanical analyses. These assessments combined showed notable enhancements in ACL repair and healing. This study emphasizes the promise of multi-layered nanofiber scaffolds in orthopedic tissue engineering and also introduces new possibilities for the creation of improved materials for regenerating the tendon-bone interface.

Magnetic Seeding of SPIO-BMSCs Into a Biphasic Scaffold Can Promote Tendon-Bone Healing After Rotator Cuff Repair.

BACKGROUND: The tendon-bone interface (TBI) in the rotator cuff has a poor intrinsic capacity for healing, which increases the risk of retear after rotator cuff repair (RCR). However, facilitating regeneration of the TBI still remains a great clinical challenge. Herein, the authors established a novel strategy based on magnetic seeding to enhance the TBI regeneration. HYPOTHESIS: Magnetic seeding bone marrow mesenchymal stem cells labeled with superparamagnetic iron oxide (SPIO-BMSCs) into a biphasic scaffold can promote tendon-bone healing after RCR. STUDY DESIGN: Controlled laboratory study. METHODS: BMSCs were labeled with SPIOs. Prussian blue staining, CCK-8 tests, Western blot, and quantitative reverse transcription polymerase chain reaction (PCR) were used to determine the optimal effect concentration of SPIOs on cell bioactivities and abilities. Then SPIO-BMSCs were magnetically seeded into a biphasic scaffold under a magnetic field. The seeding efficacy was assessed by a scanning electron microscope, and the potential mechanism in chondrogenic differentiation after seeding SPIO-BMSCs into the scaffold was evaluated by Western blot and PCR. Furthermore, the effect of SPIO-BMSC/biphasic scaffold on tendon-bone healing after RCR using a rat model was examined using histological analysis, enzyme-linked immunosorbent assay, and biomechanical evaluation. RESULTS: BMSCs labeled with 100 µg/mL SPIO had no effect on cell bioactivities and the ability of chondrogenic differentiation. SPIO-BMSCs were magnetically seeded into a biphasic scaffold, which offered a high seeding efficacy to enhance chondrogenic differentiation of SPIO-BMSCs via the CDR1as/miR-7/FGF2 pathway for TBI formation in vitro. Furthermore, in vivo application of the biphasic scaffold with magnetically seeded SPIO-BMSCs showed their regenerative potential, indicating that they could significantly accelerate and promote TBI healing with superior biomechanical properties after RCR in a rat rotator cuff tear model. CONCLUSION: Magnetically seeding SPIO-BMSCs into a biphasic scaffold enhanced seeding efficacy to promote cell distribution and condensation. This construct enhanced the chondrogenesis process via the CDR1as/miR-7/FGF2 pathway and further promoted tendon-bone healing after RCR in a rat rotator cuff tear model. CLINICAL RELEVANCE: This study provides an alternative strategy for improving TBI healing after RCR.

Melt Electrowriting Combined with Fused Deposition Modeling Printing for the Fabrication of Three-Dimensional Biomimetic Scaffolds for Osteotendinous Junction Regeneration.

Purpose: This study aims to explore a novel scaffold for osteotendinous junction regeneration and to preliminarily verify its osteogenic and tenogenic abilities in vitro. Methods: A polycaprolactone (PCL) scaffold with aligned and orthogonal fibers was created using melt electrowriting (MEW) and fused deposition modeling (FDM). The scaffold was coated with Type I collagen, and hydroxyapatite was carefully added to separate the regions intended for bone and tendon regeneration, before being rolled into a cylindrical shape. Human adipose-derived stem cells (hADSCs) were seeded to evaluate viability and differentiation. Scaffold characterization was performed with Scanning Electron Microscope (SEM). Osteogenesis was assessed by alkaline phosphatase (ALP) and Alizarin red staining, while immunostaining and transcription-quantitative polymerase chain reaction (RT-qPCR) evaluated osteogenic and tendogenic markers. Results: Scaffolds were developed in four variations: aligned (A), collagen-coated aligned (A+C), orthogonal (O), and mineral-coated orthogonal (O+M). SEM analysis confirmed surface morphology and energy-dispersive X-ray spectroscopy (EDS) verified mineral coating on O+M types. Hydrophilicity and mechanical properties were optimized in modified scaffolds, with A+C showing increased tensile strength and O+M improved in compression. hADSCs demonstrated good viability and morphology across scaffolds, withO+M scaffolds showing higher cell proliferation and osteogenic potential, and A and A+C scaffolds supporting tenogenic differentiation. Conclusion: This study confirms the potential of a novel PCL scaffold with distinct regions for osteogenic and tenogenic differentiation, supporting the regeneration of osteotendinous junctions in vitro.

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.

In Situ Construction of Morphologically Different Hydroxyapatite-Mineralized Structures on a Three-Dimensional Bionic Chitin Scaffold.

Slow healing at the tendon-bone interface is a prominent factor in the failure of tendon repair surgeries. The development of functional biomaterials with 3D gradient structures is urgently needed to improve tendon-bone integration. The crystalline form of hydroxyapatite (HAP) has a crucial impact on cell behavior, which directly influences protein adsorption, such as bone morphogenetic protein 2, the adhesion, proliferation, and osteogenic differentiation with cells. This work aimed to generate gradient mineral structures in situ by stabilizing calcium and phosphate ions using a polymer-induced liquid precursor process. To regulate the crystalline growth of HAP at the interface of ß-chitin, this work made use of the surface properties of the organic matrix found in cuttlefish bone. These techniques allowed us to prepare an organic-inorganic composite gradient scaffold comprising plate-like HAP mineralized in situ on the surface of the scaffold and fibrous HAP in the scaffold's interior. Organic-inorganic composite gradient materials are anticipated for use in tendon-bone healing produced via the in situ construction of gradient-distributed HAP mineralization layers having varying crystalline morphologies on chitin scaffolds that possess a three-dimensional bionic structure.

Advances focusing on the application of decellularization methods in tendon-bone healing.

BACKGROUND: The tendon or ligament is attached to the bone by a triphasic but continuous area of heterogeneous tissue called the tendon-bone interface (TBI). The rapid and functional regeneration of TBI is challenging owing to its complex composition and difficulty in self-healing. The development of new technologies, such as decellularization, has shown promise in the regeneration of TBI. Several ex vivo and in vivo studies have shown that decellularized grafts and decellularized biomaterial scaffolds achieved better efficacy in enhancing TBI healing. However further information on the type of review that is available is needed. AIM OF THE REVIEW: In this review, we discuss the current application of decellularization biomaterials in promoting TBI healing and the possible mechanisms involved. With this work, we would like to reveal how tissues or biomaterials that have been decellularized can improve tendon-bone healing and to provide a theoretical basis for future related studies. KEY SCIENTIFIC CONCEPTS OF THE REVIEW: Decellularization is an emerging technology that utilizes various chemical, enzymatic and/or physical strategies to remove cellular components from tissues while retaining the structure and composition of the extracellular matrix (ECM). After decellularization, the cellular components of the tissue that cause an immune response are removed, while various biologically active biofactors are retained. This review further explores how tissues or biomaterials that have been decellularized improve TBI healing.

Human umbilical cord mesenchymal stem cells overexpressing RUNX1 promote tendon-bone healing by inhibiting osteolysis, enhancing osteogenesis and promoting angiogenesis.

BACKGROUND: Rotator cuff injury (RCI) is a common shoulder injury, which is difficult to be completely repaired by surgery. Hence, new strategies are needed to promote the healing of tendon-bone. OBJECTIVE: We aimed to investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) overexpressing RUNX1 on the tendon-bone healing after RCI, and to further explore its mechanism. METHODS: Lentiviral vector was used to mediate the overexpression of RUNX1. RUNX1-overexpressed UCB-MSCs (referred to as MSC-RUNX1) were co-cultured with osteoclasts, and TRAP staining was performed to observe the formation of osteoclasts. Then MSC-RUNX1 was cultured in osteogenic differentiation medium, Alizarin red staining was conducted to detect osteogenic differentiation. The expression of markers of osteogenesis and osteoclast was detected by RT-qPCR. EA. hy926 cells were co-cultured with MSC-RUNX1. Transwell assay was used to detect the migration, and the expression of angiogenesis related-genes VEGF and TGF-ß was detected by RT-qPCR. The rat rotator cuff reconstruction model was established and MSCs were injected at the tendon-bone junction. Biomechanical test and micro-CT scanning were performed, and HE, Masson and Alcian Blue staining were used for histological evaluation of tendon-bone healing. TUNEL and PCNA immunofluorescence (IF) staining were performed to evaluate apoptosis and proliferation at the tendon-bone healing site. The levels of TNF-α, IL-6 and IL-8 in serum were detected by ELISA. The expression of CD31 and Endomucin that related to angiogenesis was detected by IF. Safranin O-fast and TRAP/CD40L immunohistochemical staining were used to assess the levels of osteoclasts and osteoblasts at the tendon-bone healing site. RESULTS: hUC-MSCs overexpressing RUNX1 inhibited osteoclast formation and promoted osteogenic differentiation. MSC-RUNX1 could promote the migration and tube formation of EA. hy926 cells, and up-regulate the levels of VEGF and TGF-ß. Model mice treated with MSC-RUNX1 partially restored the biomechanical indexes. Treatment of MSC-RUNX1 obviously increased the bone density, accompanied by the formation of new bone. In vivo experiments showed that MSC-RUNX1 treatment could promote tendon-bone healing and inhibit inflammatory response in rats. MSC-RUNX1 treatment also promoted angiogenesis at the tendon-bone healing site, while inhibiting osteoclast formation and promoting osteogenic differentiation. CONCLUSION: hUC-MSCs overexpressing RUNX1 can inhibit the formation of osteoclasts and differentiation of osteoblasts, promote angiogenesis and inhibit inflammation, thereby promoting tendon-bone healing after RCI.

Platelet-rich plasma-derived exosomes promote rotator cuff tendon-bone healing.

BACKGROUND: Rotator cuff tear (RCT) is the most common type of shoulder joint injury, platelet-rich plasma-derived exosomes (PRP-exos) are highly promising in tissue repair and regeneration. The purpose of this study was to determine the function of PRP-exos in rotator cuff tendon-bone healing. METHODS: PRP-exos were isolated from the rabbit whole blood by differential ultracentrifugation and characterized through transmission electron microscopy assay, nanoparticle tracking analysis, and western blotting. Alkaline phosphatase and Von Kossa staining were used to show tendon-derived stem cell (TDSC) differentiation. RT-qPCR and western blotting were performed to detect COL II, SOX-9, and TIMP-1. To determine the therapeutic effects of PRP-exos in vivo. Thirty New Zealand white rabbits were divided into control, model, and PRP-exos groups. The RCT animal model was constructed. The changes in tendon-bone tissue were determined by HE staining. Contents of COL-II, SOX-9, and TIMP-1 were determined by immunohistochemistry staining. RESULTS: PRP-exos were successfully isolated from rabbit blood. PRP-exos promoted TDSC proliferation and differentiation and also induced tendon-specific markers COL II, SOX-9, and TIMP-1 production. In vivo study revealed that PRP-exos promoted early healing of injured tendons. Rabbits treated with PRP-exos had better tissue arrangement in the tear site. Additionally, the contents of COL II, SOX-9, and TIMP-1 were also increased in the RCT rabbit model after PRP-exos treatment. CONCLUSIONS: PRP-exos enhanced tendon-bone healing by promoting TDSC proliferation and differentiation. This finding indicates that PRP-exos can serve as a promising strategy to treat rotator cuff tendon-bone healing.

Research progress of traditional Chinese medicine regulating related signaling pathways to promote tendon-bone healing / 中国药房

Tendon-bone healing is a complex biological process. Multiple signaling pathways are involved in tendon-bone healing， including transforming growth factor-β signaling pathway， bone morphogenetic protein signaling pathway， Wnt signaling pathway， fibroblast growth factor signaling pathway and nuclear transcription factor-κB signaling pathway. This paper summarizes the research status of traditional Chinese medicine regulating related signaling pathways to promote tendon-bone healing. It is found that a variety of traditional Chinese medicine monomers or herbal extracts （such as baicalein， icariin， total flavonoids of Drynaria fortunei， parthenolide， total saponins of Panax notoginseng， etc.） and traditional Chinese medicine compounds （such as Taohong siwu decoction， Liuwei dihuang pill， Xujin jiegu liquid， etc.） can promote bone formation， anti-inflammatory， anti-oxidation， by regulating the above signaling pathways， thereby effectively promoting tendon-bone healing.

Engineered exosomes: A promising strategy for tendon-bone healing.

BACKGROUND: Due to the spatiotemporal complexity of the composition, structure, and cell population of the tendon-bone interface (TBI), it is difficult to achieve true healing. Recent research is increasingly focusing on engineered exosomes, which are a promising strategy for TBI regeneration. AIM OF REVIEW: This review discusses the physiological and pathological characteristics of TBI and the application and limitations of natural exosomes in the field of tendon-bone healing. The definition, loading strategies, and spatiotemporal properties of engineered exosomes were elaborated. We also summarize the application and future research directions of engineered exosomes in the field of tendon-bone healing. KEY SCIENTIFIC CONCEPTS OF REVIEW: Engineered exosomes can spatially deliver cargo to targeted sites and temporally realize the sustained release of therapeutic molecules in TBI. This review expounds on the multidifferentiation of engineered exosomes for tendon-bone healing, which effectively improves the biological and biomechanical properties of TBI. Engineered exosomes could be a promising strategy for tendon-bone healing.

Bone marrow mesenchymal stem cell­derived exosomes: A novel therapeutic agent for tendon­bone healing (Review).

In sports medicine, injuries related to the insertion of tendons into bones, including rotator cuff injuries, anterior cruciate ligament injuries and Achilles tendon ruptures, are commonly observed. However, traditional therapies have proven to be insufficient in achieving satisfactory outcomes due to the intricate anatomical structure associated with these injuries. Adult bone marrow mesenchymal stem cells possess self­renewal and multi­directional differentiation potential and can generate various mesenchymal tissues to aid in the recovery of bone, cartilage, adipose tissue and bone marrow hematopoietic tissue. In addition, extracellular vesicles derived from bone marrow mesenchymal stem cells known as exosomes, contain lipids, proteins and nucleic acids that govern the tissue microenvironment, facilitate tissue repair and perform various biological functions. Studies have demonstrated that bone marrow mesenchymal stem cell­derived exosomes can function as natural nanocapsules for drug delivery and can enhance tendon­bone healing strength. The present review discusses the latest research results on the role of exosomes released by bone marrow mesenchymal stem cells in tendon­bone healing and provides valuable information for implementing these techniques in regenerative medicine and sports health.

[Research progress on bioactive strategies for promoting tendon graft healing after anterior cruciate ligament reconstruction].

Objective: To review the bioactive strategies that enhance tendon graft healing after anterior cruciate ligament reconstruction (ACLR), and to provide insights for improving the therapeutic outcomes of ACLR. Methods: The domestic and foreign literature related to the bioactive strategies for promoting the healing of tendon grafts after ACLR was extensively reviewed and summarized. Results: At present, there are several kinds of bioactive materials related to tendon graft healing after ACLR: growth factors, cells, biodegradable implants/tissue derivatives. By constructing a complex interface simulating the matrix, environment, and regulatory factors required for the growth of native anterior cruciate ligament (ACL), the growth of transplanted tendons is regulated at different levels, thus promoting the healing of tendon grafts. Although the effectiveness of ACLR has been significantly improved in most studies, most of them are still limited to the early stage of animal experiments, and there is still a long way to go from the real clinical promotion. In addition, limited by the current preparation technology, the bionics of the interface still stays at the micron and millimeter level, and tends to be morphological bionics, and the research on the signal mechanism pathway is still insufficient. Conclusion: With the further study of ACL anatomy, development, and the improvement of preparation technology, the research of bioactive strategies to promote the healing of tendon grafts after ACLR is expected to be further promoted.

Cartilage fragments combined with BMSCs-Derived exosomes can promote tendon-bone healing after ACL reconstruction.

Anterior cruciate ligament reconstruction (ACLR) often fails due to the inability of tendon-bone integration to regenerate normal tissues and formation of fibrous scar tissues in the tendon-bone interface. Cartilage fragments and exosomes derived from bone mesenchymal stromal cells (BMSCs-Exos) can enhance enthesis healing. Nevertheless, the effects on the tendon-bone healing of ACLR remain unknown. This study found that BMSCs-Exos can promote the proliferation of chondrocytes in cartilage fragments, and activated the expression of chondro-related genes SOX9 and Aggrecan. The optimal effect concentration was 1012 events/uL. Besides, BMSCs-Exos could significantly upregulated the expression of BMP7 and Smad5 in cartilage fragments, and further enhanced the expression of chondrogenic genes. Moreover, this study established a rat model of ACLR and implanted the BMSCs-Exos/cartilage fragment complex into the femoral bone tunnel. Results demonstrated that the mean diameters of the femoral bone tunnels were significantly smaller in the BE-CF group than those in the CF group (p = 0.038) and control group (p = 0.007) at 8 weeks after surgery. Besides, more new bone formation was observed in the femoral tunnels in the BE-CF group, as demonstrated by a larger BV/TV ratio based on the reconstructed CT scans. Histological results also revealed the regeneration of tendon-bone structures, especially fibrocartilage. Thus, these findings provide a promising result that BMSCs-Exos/cartilage fragment complex can prevent the enlargement of bone tunnel and promote tendon-bone healing after ACLR, which may have resulted from the regulation of the BMP7/Smad5 signaling axis.

Slit Guidance Ligand 3 (SLIT3) Loaded in Hydrogel Microparticles Enhances the Tendon-Bone Healing through Promotion of Type-H Vessel Formation: An Experimental Study in Mice.

Poor tendon-bone interface (TBI) integration is one of the major causes contributing to unsatisfactory healing quality in patients after anterior cruciate ligament (ACL) reconstruction. Type H vessels have been recently found to closely modulate bone formation via regulation of the osteo-angiogenic crosstalk, so the strategies favoring type H vessel formation may be promising therapeutic approaches for improved graft osteointegration. In this study, we reported for the first time the treatment outcome of slit guidance ligand 3 (slit3), a novel proangiogenic factor favoring type H vessel formation, in TBI healing in mice with ACL reconstruction. The mice (n = 87) were divided into three groups for various treatments: hydrogel microparticles (HMP, control group), slit3@HMP, and slit3 neutralizing antibody@HMP (slit3-AB@HMP). Histological analysis, gait performance, radiographic measurement, and biomechanical testing were performed to assess the TBI healing quality. Increased bony ingrowth and reduced fibrous scar tissue was formed at the TBI in the slit3@HMP group when compared to the HMP group. Meanwhile, the slit3-AB@HMP inhibited the osseous ingrowth and increased fibrous scar tissue formation relative to the HMP group. Compared to the HMP group, the slit3@HMP favored type H vessel formation at the TBI while the slit3-AB@HMP impeded it. According to micro-CT assessment, compared to the HMP group, the slit3@HMP significantly increased the peri-tunnel bone mass while the slit3-AB@HMP significantly reduced the peri-tunnel bone mass. The mice in the slit3@HMP group showed the best gait performance in terms of stance time, stride length, paw print area, and stance pressure. Dynamic laxity measurement and tensile testing showed the slit3@HMP group exhibited significantly reduced laxity displacement and improved failure load and stiffness relative to the other two groups. Collectively, the injection of slit3 could be used to enhance tendon-bone integration, which may be ascribed to modulation of angiogenesis-osteogenesis crosstalk coupled by type H vessels.

[Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction].

Objective: To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods: After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 µmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results: Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion: After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.

Cocktail-like gradient gelatin/hyaluronic acid bioimplant for enhancing tendon-bone healing in fatty-infiltrated rotator cuff injury models.

The regeneration of enthesis tissue (native tendon-bone interface) at the post-surgically repaired rotator cuff remains a challenge for clinicians, especially with the emergence of degenerative affection such as fatty infiltration that exacerbate poor tendon-bone healing. In this study, we proposed a cocktail-like hydrogel with a four-layer structure (BMSCs+gNC@GH) for enhancing fatty infiltrated tendon-bone healing. As collagen and hyaluronic acid are the main biomacromolecules that constitute the extracellular matrix of enthesis tissue, this hydrogel was composed of UV-curable gelatin/hyaluronic acid (GelMA/HAMA) dual network gel (GH) with nanoclay (NC) and stem cells loaded. The results showed that NC exhibited a cocktail-like gradient distribution in GH, which effectively mimicked the structure of native enthesis and supported the long-term culture and encapsulation of BMSCs. What's more, the gradient variation of NC provided a biological signal for promoting gradient osteogenic differentiation of cells. Based on the in vivo results, BMSCs+gNC@GH effectively promoted fibrocartilage layer regeneration at the tendon-bone interface and inhibited fatty infiltration. Therefore, BMSCs+gNC@GH group exhibited better biomechanical properties. Thus, this cocktail-like implant may be a promising tissue-engineered scaffold for tendon-bone healing, and it provides a new idea for the development of scaffolds with the function of inhibiting degeneration.

Therapeutic Potential of Exosomes in Tendon and Tendon-Bone Healing: A Systematic Review of Preclinical Studies.

Exosomes have been proven to play a positive role in tendon and tendon-bone healing. Here, we systematically review the literature to evaluate the efficacy of exosomes in tendon and tendon-bone healing. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a systematic and comprehensive review of the literature was performed on 21 January 2023. The electronic databases searched included Medline (through PubMed), Web of Science, Embase, Scopus, Cochrane Library and Ovid. In the end, a total of 1794 articles were systematically reviewed. Furthermore, a "snowball" search was also carried out. Finally, forty-six studies were included for analysis, with the total sample size being 1481 rats, 416 mice, 330 rabbits, 48 dogs, and 12 sheep. In these studies, exosomes promoted tendon and tendon-bone healing and displayed improved histological, biomechanical and morphological outcomes. Some studies also suggested the mechanism of exosomes in promoting tendon and tendon-bone healing, mainly through the following aspects: (1) suppressing inflammatory response and regulating macrophage polarization; (2) regulating gene expression, reshaping cell microenvironment and reconstructing extracellular matrix; (3) promoting angiogenesis. The risk of bias in the included studies was low on the whole. This systematic review provides evidence of the positive effect of exosomes on tendon and tendon-bone healing in preclinical studies. The unclear-to-low risk of bias highlights the significance of standardization of outcome reporting. It should be noted that the most suitable source, isolation methods, concentration and administration frequency of exosomes are still unknown. Additionally, few studies have used large animals as subjects. Further studies may be required on comparing the safety and efficacy of different treatment parameters in large animal models, which would be conducive to the design of clinical trials.

Bone marrow mesenchymal stem cell-derived exosomal microRNAs target PI3K/Akt signaling pathway to promote the activation of fibroblasts.

BACKGROUND: Fibroblast plays a major role in tendon-bone healing. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) can activate fibroblasts and promote tendon-bone healing via the contained microRNAs (miRNAs). However, the underlying mechanism is not comprehensively understood. Herein, this study aimed to identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets, and to verify their effects as well as mechanisms on fibroblasts. AIM: To identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets and verify their effects as well as mechanisms on fibroblasts. METHODS: BMSC-derived exosomal miRNAs data (GSE71241, GSE153752, and GSE85341) were downloaded from the Gene Expression Omnibus (GEO) database. The candidate miRNAs were obtained by the intersection of three data sets. TargetScan was used to predict potential target genes for the candidate miRNAs. Functional and pathway analyses were conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively, by processing data with the Metascape. Highly interconnected genes in the protein-protein interaction (PPI) network were analyzed using Cytoscape software. Bromodeoxyuridine, wound healing assay, collagen contraction assay and the expression of COL I and α-smooth muscle actin positive were applied to investigate the cell proliferation, migration and collagen synthesis. Quantitative real-time reverse transcription polymerase chain reaction was applied to determine the cell fibroblastic, tenogenic, and chondrogenic potential. RESULTS: Bioinformatics analyses found two BMSC-derived exosomal miRNAs, has-miR-144-3p and has-miR-23b-3p, were overlapped in three GSE datasets. PPI network analysis and functional enrichment analyses in the GO and KEGG databases indicated that both miRNAs regulated the PI3K/Akt signaling pathway by targeting phosphatase and tensin homolog (PTEN). In vitro experiments confirmed that miR-144-3p and miR-23b-3p stimulated proliferation, migration and collagen synthesis of NIH3T3 fibroblasts. Interfering with PTEN affected the phosphorylation of Akt and thus activated fibroblasts. Inhibition of PTEN also promoted the fibroblastic, tenogenic, and chondrogenic potential of NIH3T3 fibroblasts. CONCLUSION: BMSC-derived exosomes promote fibroblast activation possibly through the PTEN and PI3K/Akt signaling pathways, which may serve as potential targets to further promote tendon-bone healing.

Mesenchymal stem cells and macrophages and their interactions in tendon-bone healing.

Tendon-bone insertion injuries (TBI), such as anterior cruciate ligament (ACL) and rotator cuff injuries, are common degenerative or traumatic pathologies with a negative impact on the patient's daily life, and they cause huge economic losses every year. The healing process after an injury is complex and is dependent on the surrounding environment. Macrophages accumulate during the entire process of tendon and bone healing and their phenotypes progressively transform as they regenerate. As the "sensor and switch of the immune system", mesenchymal stem cells (MSCs) respond to the inflammatory environment and exert immunomodulatory effects during the tendon-bone healing process. When exposed to appropriate stimuli, they can differentiate into different tissues, including chondrocytes, osteocytes, and epithelial cells, promoting reconstruction of the complex transitional structure of the enthesis. It is well known that MSCs and macrophages communicate with each other during tissue repair. In this review, we discuss the roles of macrophages and MSCs in TBI injury and healing. Reciprocal interactions between MSCs and macrophages and some biological processes utilizing their mutual relations in tendon-bone healing are also described. Additionally, we discuss the limitations in our understanding of tendon-bone healing and propose feasible ways to exploit MSC-macrophage interplay to develop an effective therapeutic strategy for TBI injuries. The Translational potential of this article: This paper reviewed the important functions of macrophages and mesenchymal stem cells in tendon-bone healing and described the reciprocal interactions between them during the healing process. By managing macrophage phenotypes, mesenchymal stem cells and the interactions between them, some possible novel therapies for tendon-bone injury may be proposed to promote tendon-bone healing after restoration surgery.