Circadian Rhythm-Regulated ADSC-Derived sEVs and a Triphasic Microneedle Delivery System to Enhance Tendon-to-Bone Healing.

Modulating the inflammatory microenvironment to reconstruct the fibrocartilaginous layer while promoting tendon repair is crucial for enhancing tendon-to-bone healing in rotator cuff repair (RCR), a persistent challenge in orthopedics. Small extracellular vesicles (sEVs) hold significant potential to modulate inflammation, yet the efficient production of highly bioactive sEVs remains a substantial barrier to their clinical application. Moreover, achieving minimally invasive local delivery of sEVs to the tendon-to-bone interface presents significant technical difficulties. Herein, the circadian rhythm of adipose-derived stem cells is modulated to increase the yield and enhance the inflammatory regulatory capacity of sEVs. Circadian rhythm-regulated sEVs (CR-sEVs) enhance the cyclic adenosine monophosphate signaling pathway in macrophage (Mφ) via platelet factor 4 delivery, thereby inhibiting Mφ M1 polarization. Subsequently, a triphasic microneedle (MN) scaffold with a tip, stem, and base is designed for the local delivery of CR-sEVs (CR-sEVs/MN) at the tendon-to-bone junction, incorporating tendon-derived decellularized extracellular matrix in the base to facilitate tendon repair. CR-sEVs/MN mitigates inflammation, promotes fibrocartilage regeneration, and enhances tendon healing, thereby improving biomechanical strength and shoulder joint function in a rat RCR model. Combining CR-sEVs with this triphasic microneedle delivery system presents a promising strategy for enhancing tendon-to-bone healing in clinical settings.

Exosomes derived from tendon stem/progenitor cells enhance tendon-bone interface healing after rotator cuff repair in a rat model.

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.

Hierarchical Chiral Calcium Silicate Hydrate Films Promote Vascularization for Tendon-to-Bone Healing.

Revascularization after rotator cuff repair is crucial for tendon-to-bone healing. The chirality of materials has been reported to influence their performance in tissue repair. However, data on the use of chiral structures to optimize biomaterials as a revascularization strategy remain scarce. Here, calcium silicate hydrate (CSO) films with hierarchical chirality on the atomic to micrometer scale are developed. Interestingly, levorotatory CSO (L-CSO) films promote the migration and angiogenesis of endothelial cells, whereas dextral and racemic CSO films do not induce the same effects. Molecular analysis demonstrates that L-chirality can be recognized by integrin receptors and leads to the formation of focal adhesion, which activates mechanosensitive ion channel transient receptor potential vanilloid 4 to conduct Ca2+ influx. Consequently, the phosphorylation of serum response factor is biased by Ca2+ influx to promote the vascular endothelial growth factor receptor 2 signaling pathway, resulting in enhanced angiogenesis. After implanted in a rat rotator cuff tear model, L-CSO films strongly enhance vascularization at the enthesis, promoting collagen maturation, increasing bone and fibrocartilage formation, and eventually improving the biomechanical strength. This study reveals the mechanism through which chirality influences angiogenesis in endothelial cells and provides a critical theoretical foundation for the clinical application of chiral biomaterials.

Interposition of acellular amniotic membrane at the tendon to bone interface would be better for healing than overlaying above the tendon to bone junction in the repair of rotator cuff injury.

PURPOSE: The retear rate of rotator cuff (RC) after surgery is high, and the rapid and functional enthesis regeneration remains a challenge. Whether acellular amniotic membrane (AAM) helps to promote the healing of tendon to bone and which treatment is better are both unclear. The study aims to investigate the effect of AAM on the healing of RC and the best treatment for RC repair. METHODS: Thirty-three Sprague Dawley rats underwent RC transection and repair using microsurgical techniques and were randomly divided into the suturing repair only (SRO) group (n = 11), the AAM overlaying (AOL) group (n = 11), and the AAM interposition (AIP) group (n = 11), respectively. Rats were sacrificed at 4 weeks, then examined by subsequent micro-CT, and evaluated by histologic and biomechanical tests. The statistical analyses of one-way ANOVA or Kruskal-Wallis test were performed using with SPSS 23.0. A p < 0.05 was considered a significant difference. RESULTS: AAM being intervened between tendon and bone (AIP group) or overlaid over tendon to bone junction (AOL group) in a rat model, promoted enthesis regeneration, increased new bone and cartilage generation, and improved collagen arrangement and biomechanical properties in comparison with suturing repair only (SRO group) (AOL vs. SRO, p < 0.001, p = 0.004, p = 0.003; AIP vs. SRO, p < 0.001, p < 0.001, p < 0.001). Compared with the AOL group, the AIP group had better results in micro-CT evaluation, histological score, and biomechanical testing (p = 0 0.039, p = 0.011, p = 0.003, respectively). CONCLUSION: In the RC repair model, AAM enhanced regeneration of the tendon to bone junction. This regeneration was more effective when the AAM was intervened at the tendon to bone interface than overlaid above the tendon to bone junction.

Comparison of 3 Different Surgical Techniques for Rotator Cuff Repair in a Rabbit Model: Direct Suture, Inlay Suture, and Polyether Ether Ketone (PEEK) Suture Anchor.

BACKGROUND: Rotator cuff tears have been repaired using the transosseous method for decades. The direct suture (DS) technique has been widely used for rotator cuff tears; however, the retear rate is relatively high. Suture anchors are now used frequently for rotator cuff repair (RCR) in accordance with recent developments in materials. However, polyether ether ketone (PEEK) may still cause complications such as the formation of cysts and osteophytes. Some studies have developed the inlay suture (IS) technique for RCR. PURPOSE/HYPOTHESIS: To compare how 3 different surgical techniques-namely, the DS, IS, and PEEK suture anchor (PSA)-affect tendon-bone healing after RCR. We hypothesized that the IS technique would lead to better tendon-to-bone healing and that the repaired structure would be similar to the normal enthesis. STUDY DESIGN: Controlled laboratory study. METHODS: Acute infraspinatus tendon tears were created in 36 six-month-old male rabbits, which were divided into 3 groups based on the technique used for RCR: DS, IS, and PSA. Animals were euthanized at 6 and 12 weeks postoperatively and underwent a histological assessment and imaging. The expression of related proteins was demonstrated by immunohistochemistry and immunofluorescence staining. Mechanical properties were evaluated by biomechanical testing. RESULTS: At 12 weeks, regeneration of the enthesis was observed in the 3 groups. However, the DS group showed a lower type I collagen content than the PSA and IS groups, which was similar to the results for scleraxis. The DS group displayed a significantly inferior type II collagen expression and proteoglycan deposition after safranin O/fast green and sirius red staining. With regard to runt-related transcription factor 2 and alkaline phosphatase, the IS group showed upregulated expression levels compared with the other 2 groups. CONCLUSION: Compared with the DS technique, the PSA and IS techniques contributed to the improved maturation of tendons and fibrocartilage regeneration, while the IS technique particularly promoted osteogenesis at the enthesis. CLINICAL RELEVANCE: The IS and PSA techniques may be more beneficial for tendon-bone healing after RCR.

Healthy Tendon Stem Cell-Derived Exosomes Promote Tendon-To-Bone Healing of Aged Chronic Rotator Cuff Tears by Breaking the Positive-Feedback Cross-Talk between Senescent Tendon Stem Cells and Macrophages through the Modulation of Macrophage Polarization.

The re-tear rate of rotator cuff tears (RCT) after surgical repair is high, especially in aged patients with chronic tears. Senescent tendon stem cells (s-TSCs) generally exist in aged and chronically torn rotator cuff tendons and are closely associated with impaired tendon-to-bone healing results. The present study found a positive feedback cross-talk between s-TSCs and macrophages. The conditioned medium (CM) from s-STCs can promote macrophage polarization mainly toward the M1 phenotype, whose CM reciprocally accelerated further s-TSC senescence. Additional healthy tendon stem-cells derived exosomes (h-TSC-Exos) can break this positive feedback cross-talk by skewing macrophage polarization from the M1 phenotype to the M2 phenotype, attenuating s-TSCs senescence. S-TSC senescence acceleration or attenuation effects induced by M1 or M2 macrophages are associated with the inhibition or activation of the bone morphogenetic protein 4 signaling pathway following RNA sequencing analysis. Using an aged-chronic rotator cuff tear rat model, it is found that h-TSC-Exos can shift the microenvironment in the tendon-to-bone interface from a pro-inflammatory to an anti-inflammatory type at the acute postoperative stage and improve the tendon-to-bone healing results, which are associated with the rejuvenated s-TSCs. Therefore, this study proposed a potential strategy to improve the healing of aged chronic RCT.

Tendon stem cells seeded on dynamic chondroitin sulfate and chitosan hydrogel scaffold with BMP2 enhance tendon-to-bone healing.

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.

[Study on injectable chitosan hydrogel with tendon-derived stem cells for enhancing rotator cuff tendon-to-bone healing].

Objective: 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. Methods: 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. Results: 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). Conclusion: TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.

A novel and efficient murine model for investigating tendon-to-bone healing.

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.

Macroporous Granular Hydrogels Functionalized with Aligned Architecture and Small Extracellular Vesicles Stimulate Osteoporotic Tendon-To-Bone Healing.

Osteoporotic tendon-to-bone healing (TBH) after rotator cuff repair (RCR) is a significant orthopedic challenge. Considering the aligned architecture of the tendon, inflammatory microenvironment at the injury site, and the need for endogenous cell/tissue infiltration, there is an imminent need for an ideal scaffold to promote TBH that has aligned architecture, ability to modulate inflammation, and macroporous structure. Herein, a novel macroporous hydrogel comprising sodium alginate/hyaluronic acid/small extracellular vesicles from adipose-derived stem cells (sEVs) (MHA-sEVs) with aligned architecture and immunomodulatory ability is fabricated. When implanted subcutaneously, MHA-sEVs significantly improve cell infiltration and tissue integration through its macroporous structure. When applied to the osteoporotic RCR model, MHA-sEVs promote TBH by improving tendon repair through macroporous aligned architecture while enhancing bone regeneration by modulating inflammation. Notably, the biomechanical strength of MHA-sEVs is approximately two times higher than the control group, indicating great potential in reducing postoperative retear rates. Further cell-hydrogel interaction studies reveal that the alignment of microfiber gels in MHA-sEVs induces tenogenic differentiation of tendon-derived stem cells, while sEVs improve mitochondrial dysfunction in M1 macrophages (Mφ) and inhibit Mφ polarization toward M1 via nuclear factor-kappaB (NF-κb) signaling pathway. Taken together, MHA-sEVs provide a promising strategy for future clinical application in promoting osteoporotic TBH.

Sirtuin 6 Overexpression Improves Rotator Cuff Tendon-to-Bone Healing in the Aged.

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/ß-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.

Microfracture Lateral to the Greater Tuberosity of the Humerus Enhances Tendon-to-Bone Healing in a Rat Rotator Cuff Model.

BACKGROUND: Microfracture at the rotator cuff insertion is an established surgical marrow-stimulation technique for enhancing rotator cuff healing. However, the effect of lateralized or medialized microfracture on the insertion is unknown. PURPOSE: To compare the biomechanical and histologic effects of microfracture at 3 different regions for rotator cuff repair in a rat model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 72 Sprague-Dawley rats with bilateral supraspinatus tendon insertion detachment were allocated into 4 groups with 4 different interventions: no microfracture at the humeral head as a control group (Con), traditional microfracture at the footprint area (MFA), and medialized microfracture to the footprint area (MMFA) on the articular surface of the humerus or lateralized microfracture to the footprint area at the greater tuberosity (LMFA). All underwent immediate repair. Tendon-to-bone healing was assessed by biomechanical and histologic tests 4 and 8 weeks postoperation. RESULTS: At 4 weeks, the LMFA group showed a significantly superior failure load compared with the other groups (all P < .05). The LMFA and MFA groups showed significantly superior stiffness compared with the Con and MMFA groups (all P < .01). At 8 weeks, superior failure load and stiffness were observed in the LMFA group compared with the control group (all P < .05). Histologic examination revealed that the LMFA group had superior collagen composition and tendon-to-bone maturation at the interface at 4 and 8 weeks compared with the Con group (all P < .05). CONCLUSION: Lateralized microfracture at the greater tuberosity improved the histologic quality of repair tissue and biomechanical strength at the tendon-to-bone insertion after rotator cuff repair in a rat model. CLINICAL RELEVANCE: Microfracture lateral to the footprint area might be a better way to enhance rotator cuff healing clinically.

Enhancement of Tendon-to-Bone Healing: Choose a Monophasic or Hierarchical Scaffold?

BACKGROUND: To enhance the healing of tendon to bone, various biomimetically hierarchical scaffolds have been proposed. However, the fabrication of such scaffolds is complicated. Furthermore, the most significant result after a routine repair is loss of the transition zone between the tendon and bone, whose main components are similar to fibrocartilage. PURPOSE: To compare tendon-to-bone healing results in a rabbit model using a monophasic graft (decellularized fibrocartilage graft; DFCG) and hierarchical graft (decellularized tendon-to-bone complex; DTBC) that contain the native hierarchical enthesis. STUDY DESIGN: Controlled laboratory study. METHODS: DFCG and DTBC were harvested from allogenic rabbits. A rabbit model of a chronic rotator cuff tear was established, and 3 groups were assessed: direct repair or repair with DFCG or DTBC fixed between the tendon and bone. Hierarchical evaluations of the repaired tendon-to-bone interface were performed with regard to the tendon zone, transition zone, and bone zone using histological staining and micro-computed tomography scanning. Biomechanical analysis was performed to evaluate the general healing strength. RESULTS: The healing results in the tendon zone exhibited no significant difference among the 3 groups at any time point. In the transition zone, the grade in the direct repair group was significantly lower than that in the DFCG and DTBC groups at 4 weeks, and the grade in the DFCG group was significantly lower than that in the DTBC group at this time point. However, any significant difference between the DFCG group and DTBC group could no longer be detected at 8 and 16 weeks, which was inconsistent with the results of the biomechanical analysis. Micro-computed tomography analysis showed no significant difference among the 3 groups with regard to bone mineral density at 16 weeks. CONCLUSION: A monophasic DFCG was able to achieve enhanced tendon-to-bone healing similar to that with hierarchical DTBC over the long term, with regard to both histological and biomechanical properties. CLINICAL RELEVANCE: Fabrication of a monophasic scaffold instead of a hierarchical scaffold to promote regeneration and remodeling of a transition zone, which was mainly composed of fibrocartilaginous matrix between the tendon and bone, may be sufficient to enhance tendon-to-bone healing.

Three-Dimensional Bioprinting of a Structure-, Composition-, and Mechanics-Graded Biomimetic Scaffold Coated with Specific Decellularized Extracellular Matrix to Improve the Tendon-to-Bone Healing.

Healing of a damaged tendon-to-bone enthesis occurs through the formation of fibrovascular scar tissue with greatly compromised histological and biomechanical properties instead of the regeneration of a new enthesis due to the lack of graded tissue-engineering zones in the interface during the healing process. In the present study, a structure-, composition-, and mechanics-graded biomimetic scaffold (GBS) coated with specific decellularized extracellular matrix (dECM) (GBS-E) aimed to enhance its cellular differentiation inducibilities was fabricated using a three-dimensional (3-D) bioprinting technique. In vitro cellular differentiation studies showed that from the tendon-engineering zone to the bone-engineering zone in the GBS, the tenogenic differentiation inducibility decreased in correspondence with an increase in the osteogenic differentiation inducibility. The chondrogenic differentiation inducibility peaked in the middle, which was in consistent with the graded cellular phenotypes observed in a native tendon-to-bone enthesis, while specific dECM coating from the tendon-engineering zone to the bone-engineering zone (tendon-, cartilage-, and bone-derived dECM, respectively) further enhanced its cellular differentiation inducibilities (GBS-E). In a rabbit rotator cuff tear model, histological analysis showed that the GBS-E group exhibited well-graded tendon-to-bone differentiated properties in the repaired interface that was similar to a native tendon-to-bone enthesis at 16 weeks. Moreover, the biomechanical properties in the GBS-E group were also significantly higher than those in other groups at 16 weeks. Therefore, our findings suggested a promising tissue-engineering strategy for the regeneration of a complex enthesis using a three-dimensional bioprinting technique.

Cryopreserved Adipose-Derived Stem Cell Sheets: An Off-the-Shelf Scaffold for Augmenting Tendon-to-Bone Healing in a Rabbit Model of Chronic Rotator Cuff Tear.

BACKGROUND: Adipose-derived stem cell (ADSC) sheets have been shown to promote tendon-to-bone healing. However, conventional laboratory preparation methods for ADSC sheets are time-consuming and risky, which precludes their diverse clinical applications. PURPOSE: To explore the utility of off-the-shelf cryopreserved ADSC sheets (c-ADSC sheets) for rotator cuff tendon-to-bone healing. STUDY DESIGN: Controlled laboratory study. METHODS: The ADSC sheets were cryopreserved and thawed for live/dead double staining, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, scanning electron microscopy observation, and biomechanical testing. Clone formation, proliferative capacity, and multilineage differentiation of ADSCs within the c-ADSC sheets were assayed to explore the effect of cryopreservation on stem cell properties. A total of 67 rabbits were randomly divided into 4 groups: normal group (without supraspinatus tendon tears; n = 7), control group (repair alone; n = 20), fresh ADSC (f-ADSC) sheet group (repair; n = 20), and c-ADSC sheet group (repair; n = 20). Rabbit bilateral supraspinatus tendon tears were induced to establish a chronic rotator cuff tear model. Gross observation, micro-computed tomography analysis, histological or immunohistochemical tests, and biomechanical tests were conducted at 6 and 12 weeks after repair. RESULTS: No significant impairment was seen in the cell viability, morphology, and mechanical properties of c-ADSC sheets when compared with f-ADSC sheets. The stem cell properties of ADSC sheets also were preserved by cryopreservation. At 6 and 12 weeks after the repair, the f-ADSC and c-ADSC sheet groups showed superior bone regeneration, higher histological scores, larger fibrocartilage areas, more mature collagen, and better biomechanical results compared with the control group. No obvious difference was seen between the f-ADSC and c-ADSC sheet groups in terms of bone regeneration, histological score, fibrocartilage formation, and biomechanical tests. CONCLUSION: c-ADSC sheets, an off-the-shelf scaffold with a high potential for clinical translational application, can effectively promote rotator cuff tendon-to-bone healing. CLINICAL RELEVANCE: Programmed cryopreservation of ADSC sheets is an efficient off-the-shelf scaffold for rotator cuff tendon-to-bone healing.

Comparison of cortical versus cancellous bone fixation in tendon-to-bone healing with a rat trans-calcaneal suture model for Achilles tendon sleeve avulsion.

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. CONCLUSION: 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.

Effects of a graphene oxide-alginate sheet scaffold on rotator cuff tendon healing in a rat model.

BACKGROUND: Natural polymer scaffolds used to promote rotator cuff healing have limitations in terms of their mechanical and biochemical properties. This animal study aimed to investigate the effects of combined graphene oxide (GO) and alginate scaffold and the toxicity of GO on rotator cuff healing in a rat model. METHODS: First, the mechanical properties of a GO/alginate scaffold and a pure alginate scaffold were compared. The in vitro cytotoxicity of and proliferation of human tenocytes with the GO/alginate scaffold were evaluated by CCK-8 assay. For the in vivo experiment, 20 male rats were randomly divided into two groups (n = 10 each), and supraspinatus repair was performed: group 1 underwent supraspinatus repair alone, and group 2 underwent supraspinatus repair with the GO/alginate scaffold. Biomechanical and histological analyses were performed to evaluate the quality of tendon-to-bone healing 8 weeks after rotator cuff repair. RESULTS: The GO/alginate scaffold exhibited an increased maximum load (p = .001) and tensile strength (p = .001). In the cytotoxicity test, the cell survival rate with the GO/alginate scaffold was 102.08%. The proliferation rate of human tenocytes was no significant difference between the GO/alginate and alginate groups for 1, 3, 5, and 7 days. Biomechanically, group 2 exhibited a significantly greater ultimate failure load (p < .001), ultimate stress (p < .001), and stiffness (p < .001) than group 1. The histological analysis revealed that the tendon-to-bone interface in group 2 showed more collagen fibers bridging, tendon-to-bone integration, longitudinally oriented collagen fibers, and fibrocartilage formation than in group 1. CONCLUSION: A small amount of GO added to alginate improved the mechanical properties of the scaffold without evidence of cytotoxicity. At 8 weeks after rotator cuff repair, the GO/alginate scaffold improved tendon-to-bone healing without causing any signs of toxicity in a rat model.

Effect of secretory leucocyte protease inhibitor on early tendon-to-bone healing after anterior cruciate ligament reconstruction in a rat model.

AIMS: To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction. METHODS: In vitro: the mobility of the rat bone mesenchymal stem cells (BMSCs) treated with SLPI was evaluated by scratch assay. Then the expression levels of osteogenic differentiation-related genes were analyzed by real-time quantitative PCR (qPCR) to determine the osteogenic effect of SLPI on BMSCs. In vivo: a rat model of ACL reconstruction was used to verify the effect of SLPI on tendon-to-bone healing. All the animals of the SLPI group and the negative control (NC) group were euthanized for histological evaluation, micro-CT scanning, and biomechanical testing. RESULTS: SLPI improved the migration ability of BMSCs and upregulated the expression of genes related to osteogenic differentiation of BMSCs in vitro. In vivo, the SLPI group had higher histological scores at the tendon-bone interface by histological evaluation. Micro-CT showed more new bone formation and bone ingrowth around the grafted tendon in the SLPI group. Evaluation of the healing strength of the tendon-bone connection showed that the SLPI group had a higher maximum failure force and stiffness. CONCLUSION: SLPI can effectively promote early tendon-to-bone healing after ACL reconstruction via enhancing the migration and osteogenic differentiation of BMSCs. Cite this article: Bone Joint Res 2022;11(7):503-512.

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.

Caffeine intake does not appear to impair tendon-to-bone healing strength in a rat rotator cuff repair model.

Background: Tendon-to-bone (TtB) healing is essential for successful rotator cuff repair (RCR). This study aimed to investigate if caffeine intake impaired TtB healing in a rat RCR model. Methods: Seventy-two rats were randomized into a caffeinated group or a noncaffeinated group. Specimens received one week of oral caffeine solution or normal saline before RCR. All rats then underwent bilateral RCR. Caffeination or saline gavages continued until rats were sacrificed at 2, 4, and 8 weeks postoperatively. Load-to-failure (primary outcomes measure), maximum stress, and stiffness of the TtB interface were measured for one shoulder of each specimen. Six random shoulders from each group underwent histological assessment of TtB healing. Results: Load-to-failure and maximum stress of RCR did not appear to differ between groups at any time point. No difference in RCR stiffness was found between groups at 2 and 4 weeks; however, stiffness in the caffeinated group did appear to lower at 8 weeks (P = .04). Conclusion: Perioperative caffeine intake did not appear to affect load-to-failure strength of RCR in an animal model. Although our secondary outcome measures of maximum stress and stiffness also did not appear to be influenced by perioperative caffeine intake, there did appear to be a trend toward decreased RCR stiffness at 8 weeks postoperatively in specimens that received caffeine.