Promote Tendon-bone Healing Research Articles

A novel design magnesium alloy suture anchor promotes fibrocartilaginous enthesis regeneration in rabbit rotator cuff repair

Regarding the current materials used for suture anchors for rotator cuff repair, there are still limitations in terms of degradability, mechanical properties, and bioactivities in clinical applications. Magnesium alloys have preliminarily been shown to promote tendon-bone healing with good prospects for application as anchor materials. However, the design of anchor structures for the degradation characteristics of magnesium alloy materials has not been considered, which is critical for the practical application of magnesium alloy anchors. The mechanism by which magnesium promotes tendon bone healing remains to be clarified. Here, we proposed a novel split hollowed magnesium alloy suture anchors for the repair of rabbit rotator cuff injury. We found that novel split hollowed magnesium alloy anchors structure effectively solved the problem of failure due to degradation of traditional eyelet structure, providing reliable suture fixation. The open architecture facilitates the metabolic resorption of the degradation products of and promotes the ingrowth of bone tissue. Histological staining showed that magnesium anchors have better ability to promote regeneration at the fibrocartilage interface compared to PLLA anchors. The higher expression of fibrocartilage markers (Aggrecan, COL2A1, and Sox9) at the tendon-bone interface in magnesium anchors, which promotes chondrocyte differentiation at the tendon-bone interface and matrix formation, which is more conducive to achieving regeneration and maturation of fibrocartilage enthesis. Hence, this study provides a basis for further research on the clinical application of degradable magnesium alloy suture anchors.

A New Tissue Engineering Strategy to Promote Tendon-bone Healing: Regulation of Osteogenic and Chondrogenic Differentiation of Tendon-derived Stem Cells.

In the field of sports medicine, repair surgery for anterior cruciate ligament (ACL) and rotator cuff (RC) injuries are remarkably common. Despite the availability of relatively effective treatment modalities, outcomes often fall short of expectations. This comprehensive review aims to thoroughly examine current strategies employed to promote tendon-bone healing and analyze pertinent preclinical and clinical research. Amidst ongoing investigations, tendon-derived stem cells (TDSCs), which have comparatively limited prior exploration, have garnered increasing attention in the context of tendon-bone healing, emerging as a promising cell type for regenerative therapies. This review article delves into the potential of combining TDSCs with tissue engineering methods, with ACL reconstruction as the main focus. It comprehensively reviews relevant research on ACL and RC healing to address the issues of graft healing and bone tunnel integration. To optimize tendon-bone healing outcomes, our emphasis lies in not only reconstructing the original microstructure of the tendon-bone interface but also achieving proper bone tunnel integration, encompassing both cartilage and bone formation. In this endeavor, we thoroughly analyze the transcriptional and molecular regulatory variables governing TDSCs differentiation, incorporating a retrospective analysis utilizing single-cell sequencing, with the aim of unearthing relevant signaling pathways and processes. By presenting a novel strategy rooted in TDSCs-driven osteogenic and chondrogenic differentiation for tendon-bone healing, this study paves the way for potential future research avenues and promising therapeutic applications. It is anticipated that the findings herein will contribute to advancing the field of tendon-bone healing and foster the exploration of TDSCs as a viable option for regenerative therapies in the future.

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.

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.

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

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. 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. 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-β 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. 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-β. 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. 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.

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.

Strategies to promote tendon-bone healing after anterior cruciate ligament reconstruction: Present and future.

At present, anterior cruciate ligament (ACL) reconstruction still has a high failure rate. Tendon graft and bone tunnel surface angiogenesis and bony ingrowth are the main physiological processes of tendon-bone healing, and also the main reasons for the postoperative efficacy of ACL reconstruction. Poor tendon-bone healing has been also identified as one of the main causes of unsatisfactory treatment outcomes. The physiological process of tendon-bone healing is complicated because the tendon-bone junction requires the organic fusion of the tendon graft with the bone tissue. The failure of the operation is often caused by tendon dislocation or scar healing. Therefore, it is important to study the possible risk factors for tendon-bone healing and strategies to promote it. This review comprehensively analyzed the risk factors contributing to tendon-bone healing failure after ACL reconstruction. Additionally, we discuss the current strategies used to promote tendon-bone healing following ACL reconstruction.

Human Trials on the Prevention of Tunnel Widening by the Emdogain in Anterior Cruciate Ligament Reconstruction.

Background Although anterior cruciate ligament reconstruction (ACLR) is an established procedure, some problems remain, such as bone tunnel widening after ACLR. In animal studies, Emdogain (EMD) prevented tunnel widening by promoting tendon-bone healing. This study aimed to evaluate the effects of EMD on the prevention of tunnel widening after anterior cruciate ligament (ACL) injury in humans. Methods Nineteen patients who underwent ACLR were included. Seven patients in the EMD group were administered EMDs into the femoral tunnel during ACLR, while 12 patients in the control group were not administered EMDs. After surgery, at two and four weeks and three, six, and 12 months, femoral and tibial tunnel widening were evaluated on computed tomography images. Anteroposterior laxity and clinical scores such as the Lysholm score, the International Knee Documentation Committee (IKDC) subjective form, and the Knee Injury and Osteoarthritis Outcome Score (KOOS) were assessed beforesurgery and 12 months postoperatively. Results Tunnel widening on the femoral side was significantly smaller in the EMD group than in the control group at two weeks. However, there was no significant difference between the two groups at 12 months. There were no significant differences in anteroposterior laxity and clinical scores between the groups before and 12 months after surgery. Conclusion EMD administration into the bone tunnel did not prevent tunnel widening at 12 months after ACLR, although tunnel widening of the femoral tunnel was reduced by EMD administration in the early phase.

A retrospective case series of the treatment of spontaneous quadriceps tendon rupture in patients with uremia and secondary hyperparathyroidism.

Spontaneous quadriceps tendon rupture (QTR) is a rare complication of uremia. Secondary hyperparathyroidism (SHPT) is considered the leading cause of QTR in uremia patients. QTR in patients with uremia and SHPT are treated with active surgical repair along with the treatment of SHPT using medication or parathyroidectomy (PTX). The impact of PTX for SHPT on tendon healing remains unclear. The purpose of this study was to introduce surgical procedures for QTR and to determine the functional recovery of the repaired quadriceps tendon (QT) after PTX. Between Jan 2014 and Dec 2018, eight uremia patients underwent PTX after a ruptured QT was repaired by figure-of-eight trans-osseous sutures with an overlapping tightening suture technique. Biochemical indices were measured before and one year after PTX to evaluate the control of SHPT. The changes in bone mineral density (BMD) were determined by comparing x-ray images at pre-PTX and during follow-up. The assessment of the functional recovery of the repaired QT was conducted at the last follow-up using multiple functional parameters. Eight patients (fourteen tendons) were retrospectively evaluated at an average follow-up of 3.46 ± 1.37 years after PTX. ALP and iPTH levels one year after PTX were significantly lower than at pre-PTX (P = 0.017, P < 0.001, respectively). Although there was no statistical differences compared to pre-PTX, serum phosphorus levels decreased and returned to normal one year after PTX (P = 0.101). BMD significantly increased at the last follow-up compared to pre-PTX. The average Lysholm score was 73.5 ± 11.07 and the average Tegner activity score was 2.63 ± 1.06. The active knee ROM after repair averaged an extension of 2.85 ± 3.78° to a flexion angle of 113.21 ± 10.12°. The quadriceps muscle strength was grade IV and the mean Insall-Salvati index was 0.93 ± 0.10 in all of the knees with tendon ruptures. All patients were able to walk without external help. Figure-of-eight trans-osseous sutures with an overlapping tightening suture technique is an economical and effective treatment for spontaneous QTR in patients with uremia and SHPT. PTX may promote tendon-bone healing in patients with uremia and SHPT.

Injectable FHE+BP composites hydrogel with enhanced regenerative capacity of tendon-bone interface for anterior cruciate ligament reconstruction.

Features of black phosphorous (BP) nano sheets such as enhancing mineralization and reducing cytotoxicity in bone regeneration field have been reported. Thermo-responsive FHE hydrogel (mainly composed of oxidized hyaluronic acid (OHA), poly-ε-L-lysine (ε-EPL) and F127) also showed a desired outcome in skin regeneration due to its stability and antibacterial benefits. This study investigated the application of BP-FHE hydrogel in anterior cruciate ligament reconstruction (ACLR) both in in vitro and in vivo, and addressed its effects on tendon and bone healing. This BP-FHE hydrogel is expected to bring the benefits of both components (thermo-sensitivity, induced osteogenesis and easy delivery) to optimize the clinical application of ACLR and enhance the recovery. Our in vitro results confirmed the potential role of BP-FHE via significantly increased rBMSC attachment, proliferation and osteogenic differentiation with ARS and PCR analysis. Moreover, In vivo results indicated that BP-FHE hydrogels can successfully optimize the recovery of ACLR through enhancing osteogenesis and improving the integration of tendon and bone interface. Further results of Biomechanical testing and Micro-CT analysis [bone tunnel area (mm2) and bone volume/total volume (%)] demonstrated that BP can indeed accelerate bone ingrowth. Additionally, histological staining (H&E, Masson and Safranin O/fast green) and immunohistochemical analysis (COL I, COL III and BMP-2) strongly supported the ability of BP to promote tendon-bone healing after ACLR in murine animal models.

Strategies for promoting tendon-bone healing: Current status and prospects.

Tendon-bone insertion (TBI) injuries are common, primarily involving the rotator cuff (RC) and anterior cruciate ligament (ACL). At present, repair surgery and reconstructive surgery are the main treatments, and the main factor determining the curative effect of surgery is postoperative tendon-bone healing, which requires the stable combination of the transplanted tendon and the bone tunnel to ensure the stability of the joint. Fibrocartilage and bone formation are the main physiological processes in the bone marrow tract. Therefore, therapeutic measures conducive to these processes are likely to be applied clinically to promote tendon-bone healing. In recent years, biomaterials and compounds, stem cells, cell factors, platelet-rich plasma, exosomes, physical therapy, and other technologies have been widely used in the study of promoting tendon-bone healing. This review provides a comprehensive summary of strategies used to promote tendon-bone healing and analyses relevant preclinical and clinical studies. The potential application value of these strategies in promoting tendon-bone healing was also discussed.

Investigation of the medium-term effect of osteoprotegerin/bone morphogenetic protein 2 combining with collagen sponges on tendon-bone healing in a rabbit.

Osteoprotegerin (OPG) and bone morphogenetic protein-2 (BMP-2) could be administered sequentially to promote tendon-bone healing. There remain several unresolved issues in our previously published study: a) the release kinetics of OPG/BMP-2 from the OPG/BMP-2/collagen sponge (CS) combination in vitro remained unclear; b) the medium-term effect of the OPG/BMP-2/CS combination was not analyzed. Hence, we design this study to address the issues mentioned above. 30 rabbits undergoing anterior cruciate ligament reconstruction (ACLR) with an Achilles tendon autograft randomly received one of the 3 delivery at the femoral and tibial tunnels: OPG/BMP-2, OPG/BMP-2/CS combination, and nothing (blank control). At 8 and 24 weeks post-surgery, the biomechanical tests and histologic analysis were used to evaluate the tendon-bone healing. In mechanical tests, the OPG/BMP-2/CS group showed a higher final failure load and stiffness than the other groups at 8 and 24 weeks. Additionally, the maximum stretching distance showed a decreasing trend. The mechanical failure pattern of samples shifted from a tunnel pull-away to a graft midsubstance rupture after OPG/BMP-2/CS-treated. From histological analysis, the OPG/BMP-2/CS treatment increased the amount of collagen fibers (collagen I and II) and promoted fibrocartilage attachment. CS as a carrier promotes the medium-term effect of OPG and BMP-2 on tendon-bone healing at the tendon-bone interface in a rabbit ACLR model. OPG, BMP-2 and CS were already applied in several clinical practice, but a further study of clinic use of OPG/BMP-2/CS is still needed.

Combination of autologous osteochondral and periosteum transplantation effectively promotes fibrocartilage regeneration at the tendon-bone junction of the rotator cuff in rabbits.

Rotator cuff tendon-bone healing often leads to scarring and low biomechanical strength, resulting in a tendency to re-tear. This study examined whether combining autologous osteochondral transplantation and periosteum transplantation increases fibrocartilage transition zone regeneration and improves biomechanical fixation. A total of 48 New Zealand white rabbits were divided into the periosteum, autologous osteochondral, combination of autologous osteochondral and periosteum, and control groups. The supraspinatus tendon was cut from the greater tuberosity and repaired by different transplants. A total of 12 rabbits were used for histological examination (haematoxylin and eosin staining, Masson's staining and Safranin-O staining) at 4, 8 and 12weeks after the repair, and 36 rabbits were used for biomechanical tests (maximal failure load and stiffness). At 4weeks following the operation, each group had a large tendon-bone gap with a small number of disordered collagen fibres. At 8weeks, the tendon-bone gap was smaller than that before the operation, and the tendon-bone gap in each experimental group was smaller with neater and denser collagen fibres and chondrocytes than in the control group, with the osteochondral combined periosteum group having the best results. At 12weeks, the typical tendon-bone transitional structure was observed in the osteochondral combined periosteum group, and more collagen fibres and chondrocytes were generated in each group. The osteochondral combined periosteum group had the largest staining area and the largest amount of cartilage. The maximum tensile strength and stiffness of each group increased over time. There was no significant difference in each group's maximum tensile strength and stiffness at 4weeks after the operation. However, the maximum tensile strength and stiffness of the osteochondral combined periosteum group at 8 and 12weeks after operation were significantly higher than those of other groups (P < 0.05). Histological and biomechanical results show that autologous osteochondral transplantation combined with periosteum transplantation can effectively promote the regeneration of fibrous cartilage in the tendon-bone junction of the rotator cuff. It is concluded that this technique is a new treatment method to promote tendon-bone healing in the rotator cuff.

Regulating Macrophages through Immunomodulatory Biomaterials Is a Promising Strategy for Promoting Tendon-Bone Healing.

The tendon-to-bone interface is a special structure connecting the tendon and bone and is crucial for mechanical load transfer between dissimilar tissues. After an injury, fibrous scar tissues replace the native tendon-to-bone interface, creating a weak spot that needs to endure extra loading, significantly decreasing the mechanical properties of the motor system. Macrophages play a critical role in tendon-bone healing and can be divided into various phenotypes, according to their inducing stimuli and function. During the early stages of tendon-bone healing, M1 macrophages are predominant, while during the later stages, M2 macrophages replace the M1 macrophages. The two macrophage phenotypes play a significant, yet distinct, role in tendon-bone healing. Growing evidence shows that regulating the macrophage phenotypes is able to promote tendon-bone healing. This review aims to summarize the impact of different macrophages on tendon-bone healing and the current immunomodulatory biomaterials for regulating macrophages, which are used to promote tendon-bone healing. Although macrophages are a promising target for tendon-bone healing, the challenges and limitations of macrophages in tendon-bone healing research are discussed, along with directions for further research.

Synergistic Effect of Reverse Drilling and Bone Dust on Femoral Tendon-Bone Healing After Anterior Cruciate Ligament Reconstruction in a Rabbit Model

Background: Anterior cruciate ligament (ACL) injuries and bone tunnel enlargement (BTE) after ACL reconstruction (ACLR) remain frequent issues. Bone dust (BD) produced by tunnel preparation with osteogenic ability and reverse drilling (RD), an easy compaction technique, make it accessible to enhance tendon-bone healing in the clinic. Hypothesis: RD and BD synergistically promote tendon-bone healing by improving peritunnel bone and preventing BTE in femurs. Study Design: Controlled laboratory study. Methods: In total, 96 New Zealand White rabbits underwent ACLR. The semitendinosus tendon was freed before medial parapatellar arthrotomy. After the native ACL was transected, bone tunnels were prepared through the footprint of the native ACL. All animals were randomly assigned to 1 of 4 groups according to different tunnel preparation methods: group 1 (irrigation after extraction drilling [ED]; control group), group 2 (irrigation after RD), group 3 (no irrigation after ED), and group 4 (no irrigation after RD). BD was harvested by irrigating tunnels and was characterized by morphology and size. The specimens underwent microarchitectural, histological, and biomechanical evaluations at 4, 8, and 12 weeks postoperatively. Results: Micro–computed tomography demonstrated more peritunnel bone and less BTE in the femurs of group 4 compared with the other groups. Histologically, BD possessed osteogenic activity in bone tunnels postoperatively. Meanwhile, group 4 regenerated a higher amount of the tendon-bone interface and more peritunnel bone than group 1. Biomechanically, group 4 showed higher failure loads and stiffness than group 1. However, peritunnel bone loss, active osteoclasts, and significant BTE were found in the femurs of group 1 and group 3 at 12 weeks postoperatively, while no strong correlation was found between BTE and inflammatory cytokines. Scanning electron microscopy and particle size analysis suggested that BD produced by ED and RD had no difference in size. Conclusion: Tendon-bone healing was facilitated by the synergistic effect of RD and BD in femurs. Clinical Relevance: This study provides a more accessible and effective surgical strategy to promote tendon-bone healing after ACLR by increasing peritunnel bone and preventing BTE in femurs.

Trend of Bioactive Molecules and Biomaterial Coating in Promoting Tendon—Bone Healing

The tendon-bone junction (TBJ) is a graded structure consisting of tendons, nonmineralised, and mineralised fibrocartilage and bone. Given the complex gradient of the TBJ structure, TBJ healing is particularly challenging. Injuries to the TBJ such as anterior cruciate ligament (ACL) tears and rotator cuff injuries are common and serious sports injuries, affecting more than 250,000 patients annually in the United States, particularly people older than 50 years. ACL reconstruction and rotator cuff repair are the commonly performed TBJ repair surgeries. However, the re-tear rate is high post-operation. In recent years, studies on improving TBJ healing have focused on promoting tendon-bone integration at tendon sites. This process includes the use of periosteum, hydrogels, scaffolds, growth factors, stem cells or other reconstruction materials that promote bone growth or ligament attachment. In this study, we will highlight the utilisation of the unique properties of biomaterial coating in promoting tendon-bone healing and discuss recent advances in understanding their role in TBJ healing. Furthermore, we aim to provide a systematic and comprehensive review of approaches to promoting TBJ healing.

BMSC-derived exosomes promote tendon-bone healing after anterior cruciate ligament reconstruction by regulating M1/M2 macrophage polarization in rats

BackgroundRecent studies have shown that bone marrow stromal cell-derived exosomes (BMSC-Exos) can be used for tissue repair. However, whether the BMSC-Exos can promote tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) is still unclear. In this study, we observed in vivo and in vitro the effect of rat BMSC-Exos on tendon-bone healing after ACLR and its possible mechanism.MethodsHighly expressed miRNAs in rat BMSC-Exos were selected by bioinformatics and verified in vitro. The effect of overexpressed miRNA in BMSC-Exos on M2 macrophage polarization was observed. A rat model of ACLR was established. The experimental components were divided into three groups: the control group, the BMSC-Exos group, and the BMSC-Exos with miR-23a-3p overexpression (BMSC-Exos mimic) group. Biomechanical tests, micro-CT, and histological staining were performed for analysis.ResultsBioinformatics analysis showed that miR-23a-3p was highly expressed in rat BMSC-Exos and could target interferon regulatory factor 1 (IRF1, a crucial regulator in M1 macrophage polarization). In vitro, compared with the control group or the BMSC-Exos group, the BMSC-Exos mimic more significantly promoted the polarization of macrophages from M1 to M2. In vivo, at 2 weeks, the number of M2 macrophages in the early local stage of ACLR was significantly increased in the BMSC-Exos mimic group; at 4 and 8 weeks, compared with the control group or the BMSC-Exos group, the bone tunnels of the tibia and femur sides of the rats in the BMSC-Exos mimic group were significantly smaller, the interface between the graft and the bone was narrowed, the bone volume/total volume ratio (BV/TV) increased, the collagen type II alpha 1 level increased, and the mechanical strength increased.ConclusionsBMSC-Exos promoted M1 macrophage to M2 macrophage polarization via miR-23a-3p, reduced the early inflammatory reaction at the tendon-bone interface, and promoted early healing after ACLR.

Electrospun hydroxyapatite loaded L-polylactic acid aligned nanofibrous membrane patch for rotator cuff repair

Rotator cuff repair remains a challenge clinically due to the high retear rate after surgical intervention. There is a significant need to develop functional biomaterials facilitating tendon-to-bone integration. In this study, hydroxyapatite (HA) incorporated polylactic acid (PLLA) aligned nanofibrous membranes were fabricated by electrospinning as a low-cost sustainable rotator cuff patch. The morphology, physical, mechanical and in vitro cell assays of the nanofibrous membranes were characterized. The results showed that the nanofibrous membrane maintained a rough surface and weakened hydrophobicity. It has excellent cytocompatibility, and the cells were oriented along the direction of fiber arrangement. What's more, the PLLA-HA nanofibrous membrane could increase the alkaline phosphatase (ALP) expression in rat bone marrow mesenchymal stem cells (BMSCs), indicating that the electrospinning PLLA-HA nanofibrous membrane can better induce the bone formation of rat BMSCs cells. When the mass ratio of PLLA to HA exceeds 3: 1, with the increase of the HA content, the patch showed rising induction ability. The results suggested that electrospinning PLLA-HA nanofibrous membranes are an ideal patch for promoting tendon-bone healing and reducing the secondary tear rate. Furthermore, the use of biodegradable polymers and low-cost preparation methods presented the possibility for commercial production of these nanofibrous membranes.

Exosomes derived from magnetically actuated bone mesenchymal stem cells promote tendon-bone healing through the miR-21-5p/SMAD7 pathway.

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.

MFG-E8 promotes tendon-bone healing by regualting macrophage efferocytosis and M2 polarization after anterior cruciate ligament reconstruction.

BackgroundScar tissue formation at the tendon-bone interface caused by excessive inflammation leads to insufficient healing strength, while the phagocytic clearance of dying cells (efferocytosis) has profound consequences on macrophage polarisation and the inflammatory response. Modulating the inflammatory microenvironment may have satisfactory curative effects in patients with anterior cruciate ligament reconstruction (ACLR).MethodsBone marrow-derived macrophages (BMDMs) and polymorphonuclear leukocytes (PMNs) were harvested from bone marrow. The effects of milk fat globulin protein E8 (MFG-E8) on macrophage polarisation were compared with those of M1 and M2 macrophages induced by conventional methods. The BMDMs and apoptotic PMNs co-culture system was used to assess the efficiency of efferocytosis. The biological functions of MFG-E8 in tendon-bone healing by regulating macrophage efferocytosis and polarisation were further investigated using a rat ACLR model.ResultsBMDMs and PMNs were successfully isolated. Compared to conventional induction methods, MFG-E8 alone did not significantly induce macrophage M1 or M2 polarisation, but it could partially reverse the expression of inducible nitric oxide synthase (iNOS) in M1 macrophages. In vitro studies revealed that appropriate dosing of MFG-E8 could significantly promote the efficiency of macrophage efferocytosis and subsequently increase M2 polarisation. More importantly, significantly increased peri-tunnel new bone formation, tighter connected interface and better mechanical properties were observed after ACLR when treated with MFG-E8 in vivo. We further demonstrated that MFG-E8 remarkably facilitated the clearance of apoptotic cells and increased the number of M2 macrophages at the interface between the tendon graft and bone tunnel in the early postoperative stage.ConclusionMFG-E8 promoted tendon-bone healing histologically and biomechanically, probably by the regulation of inflammatory processes via macrophage efferocytosis and M2 polarisation.The translational potential of this article: Regulation of macrophage efferocytosis and M2 polarization by MFG-E8 is expected to be a therapeutic strategy for promoting tendon-bone healing in patients undergoing ACLR.