Effects of leukocyte-rich platelet-rich plasma and leukocyte-poor platelet-rich plasma on the healing of bone-tendon interface of rotator cuff in a mice model.

Platelet-rich plasma (PRP) is widely used clinically to treat tendon injuries, and often contains leukocytes. However, the debate regarding the concentration of leukocytes in PRP is still ongoing. This study aimed to evaluate the therapeutic effects of leukocyte-rich platelet-rich plasma (LR-PRP) and leukocyte-poor platelet-rich plasma (LP-PRP) on the healing of the bone-tendon interface (BTI) of the rotator cuff. A total of 102 C57BL/6 mice were used. Thirty mice were used to prepare the PRP, while 72 underwent acute supraspinatus tendon injury repair. The animals were then randomly assigned to three groups: LR-PRP, LP-PRP and control groups. The mice were euthanized at 4 and 8 weeks postoperatively, and histological, immunological and biomechanical analyses were performed. The histological results showed that the fusion effect at the bone-tendon interface at 4 and 8 weeks after surgery was greater in the PRP groups and significantly increased at 4 weeks; however, at 8 weeks, the area of the fibrocartilage layer in the LP-PRP group increased significantly. M2 macrophages were observed at the repaired insertion for all the groups at 4 weeks. At 8 weeks, M2 macrophages withdrew back to the tendon in the control group, but some M2 macrophages were retained at the repaired site in the LR-PRP and LP-PRP groups. Enzyme-linked immunoassay results showed that the concentrations of IL-1ß and TNF-α in the LR-PRP group were significantly higher than those in the other groups at 4 and 8 weeks, while the concentrations of IL-1ß and TNF-α in the LP-PRP group were significantly lower than those in the control group. The biomechanical properties of the BTI were significantly improved in the PRP group. Significantly higher failure load and ultimate strength were seen in the LR-PRP and LP-PRP groups than in the control group at 4 and 8 weeks postoperatively. Thus, LR-RPR can effectively enhance the early stage of bone-tendon interface healing after rotator cuff repair, and LP-PRP could enhance the later stages of healing after rotator cuff injury.

Characterization of the distributions of collagen and PGs content in the decellularized book-shaped enthesis scaffolds by SR-FTIR.

BACKGROUND: Bone-tendon interface (enthesis) plays a pivotal role in relaxing load transfer between otherwise structurally and functionally distinct tissue types. Currently, decellularized extracellular matrix (DEM) from enthesis provide a natural three-dimensional scaffold with tissue-specific orientations of extracellular matrix molecules for enthesis regeneration, however, the distributions of collagen and PGs content in the decellularized book-shaped enthesis scaffolds from rabbit rotator cuff by SR-FTIR have not been reported. METHODS: Native enthesis tissues (NET) harvested from rabbit rotator cuff were sectioned into cuboid (about 30 mm × 1.2 mm × 10 mm) for decalcification. The decellularized book-shaped enthesis scaffolds and intrinsic ultrastructure were evaluated by histological staining and scanning electron microscopy (SEM), respectively. The distributions of collagen and PGs content in the decellularized book-shaped enthesis scaffolds from rabbit rotator cuff were also measured innovatively by SR-FTIR. RESULTS: The decellularized book-shaped enthesis scaffolds from rabbit rotator cuff were successfully obtained. Histomorphology and SEM evaluated the effect of decellularization and the structure of extracellular matrix during decellularization. After mechanical testing, the failure load in the NET group showed significantly higher than that in the DEM group (P < 0.05). Meanwhile, the stiffness of the DEM group was significantly lower than the NET group. Furthermore, the distributions of collagen and PGs content in the decellularized book-shaped enthesis scaffolds were decreased obviously after decellularization by SR-FTIR quantitative analysis. CONCLUSION: SR-FTIR was applied innovatively to characterize the histological morphology of native enthesis tissues from rabbit rotator cuff. Moreover, this technology can be applied for quantitative mapping of the distribution of collagen and PGs content in the decellularized book-shaped enthesis scaffolds.

Subacromial bursal preservation can enhance rotator cuff tendon regeneration: a comparative rat supraspinatus tendon defect model study.

BACKGROUND: The role of subacromial bursa in rotator cuff surgery is unknown. This study aimed to assess the subacromial bursa's role in the healing of supraspinatus tendon injury in a rat model. METHODS: Twenty-three male Sprague-Dawley rats (9 weeks old; weight, approximately 296 g) were used in this study. Three rats used as biomechanical study controls were killed at 12 weeks of age. A supraspinatus tendon defect was made bilaterally in 20 rats, whereas an additional subacromial bursa sectioning was performed on the left side. Six rats were killed for biomechanical testing and 4 were killed for histologic observation at 3 and 9 weeks, respectively. RESULTS: The regenerated tendon in the bursal preservation group showed significantly superior biomechanical properties in maximum load to failure at 3 and 9 weeks and stiffness at 9 weeks after surgery compared with the bursal removal group. The modified Bonar scale scores showed better regenerated supraspinatus tendons in the bursal preservation group. CONCLUSION: The present study found that the subacromial bursa plays an important role in rotator cuff regeneration in this rat supraspinatus injury model. Extensive bursectomy of the subacromial bursa may not be recommended in rotator cuff repair surgery, though future in vivo human studies are needed to confirm these observations.

SR-FTIR as a tool for quantitative mapping of the content and distribution of extracellular matrix in decellularized book-shape bioscaffolds.

BACKGROUND: To evaluate synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a tool for quantitative mapping of the content and distribution of the extracellular matrix in decellularized fibrocartilage bioscaffolds, and to provide a new platform for quantitatively characterizing bioscaffolds for tissue engineering. METHODS: Fibrocartilage was harvested and cut into book-shape bioscaffolds (N = 54), which were then decellularized. The structures and distribution of collagen fibrous and intrinsic ultrastructure in decellularized fibrocartilage bioscaffolds were evaluated by histological staining and scanning electron microscopy (SEM), respectively. The content of collagen and proteoglycan in the cellularized or decellularized bioscaffolds were also measured by SR-FTIR and biochemical assay. RESULTS: Book-shape fibrocartilage decellularized bioscaffolds were successfully obtained. Histological examination revealed that the structure of extracellular matrix endured during decellularization. Histology and DNA quantification analysis confirmed substantial removal of cells during decellularization. SEM demonstrated that intrinsic ultrastructure of the fibrocartilage bioscaffold was also well preserved. SR-FTIR quantitative analysis confirmed that decellularization had a significant effect on the content and distribution of collagen and proteoglycan in fibrocartilage bioscaffolds, these results are confirmed with the biochemical assay results. CONCLUSION: SR-FTIR imaging can capture the histological morphology of decellularized bioscaffolds. Moreover, it can be used for quantitative mapping of the content and distribution of collagen in the bioscaffolds.

The effects of primary cilia-mediated mechanical stimulation on nestin+-BMSCs during bone-tendon healing.

INTRODUCTION: Mechanical stimulation has been proven to promote bone-tendon interface (BTI) healing, but the mechanism remains unclear. OBJECTIVE: To investigate the effects of mechanical stimulation on the biological behavior of nestin+-bone mesenchymal stem cells (BMSCs) during the BTI healing, and to reveal the mechanisms of mechanical stimulation affecting BTI healing by primary cilia on the nestin+-BMSCs. METHODS: Transgenic tracing mice (nestin creERT2:: IFT88fl/fl/ROSA26 YFP) with primary cilia on nestin+-BMSCs conditioned knocked out were constructed, and the littermates (nestin creERT2:: ROSA26 YFP) with normal cilia on nestin+-BMSCs were the control. After establishing mouse supraspinatus insertion injury models, samples were collected at week-2 (n = 5 per group), 4 and 8 (n = 15 per group, respectively). In vivo, the repair efficiency was evaluated by histology, imaging, biomechanics, and the migration of nestin+-BMSCs, detected by immunofluorescence staining. In vitro, nestin+ BMSCs were sorted and stimulated by tensile force to study the mechanisms of primary cilium-mediated mechanosensitive basis. RESULTS: Mechanical stimulation (MS) accelerated the recruitment of nestin+-BMSCs and promoted osteogenic and chondrogenic capacity. Histological, imaging and biomechanical results showed that the BTI healing quality of the IFT88+/+, MS group was better than that of the other groups. After the conditionally knockout IFT88 in nestin+-BMSCs, the repair ability of the BTI was obviously deteriorated, even though mechanical stimulation did not increase significantly (IFT88-/-, MS group). In vitro results showed the tensile loading enhanced the proliferation, migration and osteogenic or chondrogenic gene expression of nestin+-BMSCs with normal cilia. On the other hand, osteogenesis and chondrogenic expression were significantly decreased after inhibiting actin- Hippo/YAP pathway components. CONCLUSION: The primary cilia mediated mechanical stimulation regulated osteogenic and chondrogenic differentiation potential of nestin+-BMSCs through the actin- Hippo/YAP pathway, and then promoted the BTI healing process.

Effect of Platelet-Rich Plasma at Different Initiation Times on Healing of the Bone-Tendon Interface of the Rotator Cuff in a Mouse Model.

Background: Platelet-rich plasma (PRP) has demonstrated beneficial effects on healing of the bone-tendon interface (BTI). Purpose: To determine the optimal initiation time for PRP application after rotator cuff repair in an animal model. Study Design: Controlled laboratory study. Methods: A total of 136 C57BL/6 mice were included; 40 mice were used to prepare PRP, while 96 mice underwent acute supraspinatus tendon (SST) repair. The animals were randomly divided into 4 groups: a control group and 3 groups in which PRP was injected into the injury interface immediately after surgery, on the 7th postoperative day (PRP-7d), and on the 14th postoperative day. At 4 and 8 weeks postoperatively, the animals were sacrificed, blood was collected by eyeball removal, and samples of the SST-humerus complex were collected. Histological, imaging, immunological, and biomechanical data were compared among the groups using 1-way analysis of variance with the Bonferroni post hoc test. Results: Histological analysis revealed that the fibrocartilage layer at the BTI was larger in the PRP-7d group compared to the other groups at both 4 and 8 weeks postoperatively. Moreover, the PRP-7d group exhibited improved proteoglycan content and distribution compared to the other groups. Enzyme-linked immunosorbent assay results demonstrated that at 4 weeks postoperatively, higher concentrations of transforming growth factor-ß1 and platelet-derived growth factor-BB (PDGF-BB) were seen in the PRP-7d group versus the PRP-14d and control gruops (P < .05), and at 8 weeks postoperatively, the concentration of PDGF-BB was higher in the PRP-7d group versus the control group (P < .05). Biomechanical testing at 4 weeks postoperatively revealed that the failure load and ultimate strength of the SST-humerus complex were superior in the PRP-7d group compared to the other groups (P < .05), at 8 weeks, PRP-7d group was superior to the control group (P < .05). Additionally, at 8 weeks postoperatively, the PRP-7d group exhibited a greater trabecular number and trabecular thickness at the BTI compared to the PRP-14d and control gruops (P < .05). Conclusion: PRP promoted healing of the BTI after a rotator cuff injury at an early stage. Clinical Relevance: A PRP injection on the 7th postoperative day demonstrated superior therapeutic effects compared with injections at other time points.

Effects of adipose-derived cell supplementation on tendon-bone healing in a rat model of chronic rotator cuff tear with suprascapular nerve injury.

OBJECTIVE: To investigate the effect of adipose-derived cells (ADCs) on tendon-bone healing in a rat model of chronic rotator cuff tear (RCT) with suprascapular nerve (SN) injury. METHODS: Adult rats underwent right shoulder surgery whereby the supraspinatus was detached, and SN injury was induced. ADCs were cultured from the animals' abdominal fat. At 6 weeks post-surgery, the animals underwent surgical tendon repair; the ADC (+ve) group (n = 18) received an ADC injection, and the ADC (-ve) group (n = 18) received a saline injection. Shoulders were harvested at 10, 14, and 18 weeks and underwent histological, fluorescent, and biomechanical analyses. RESULTS: In the ADC (+ve) group, a firm enthesis, including dense mature fibrocartilage and well-aligned cells, were observed in the bone-tendon junction and fatty infiltration was less than in the ADC (-ve) group. Mean maximum stress and linear stiffness was greater in the ADC (+ve) compared with the ADC (-ve) group at 18 weeks. CONCLUSION: ADC supplementation showed a positive effect on tendon-bone healing in a rat model of chronic RCT with accompanying SN injury. Therefore, ADC injection may possibly accelerate recovery in massive RCT injuries.

The effect of local sympatholysis on bone-tendon interface healing in a murine rotator cuff repair model.

Background: Although neuroregulation plays an important role in tissue healing, the key neuroregulatory pathways and related neurotransmitters involved in bone-tendon interface (BTI) healing are still unknown. It is reported that sympathetic nerves can regulate cartilage and bone metabolism, which are the basic aspects of BTI repair after injury, through the release of norepinephrine (NE). Thus, the purpose of this study was to explore the effect of local sympatholysis (LS) on BTI healing in a murine rotator cuff repair model. Methods: Specifically, C57BL/6 mice underwent unilateral supraspinatus tendon (SST) detachment and repair was established on a total of 174 mature C57BL/6 mice (12 weeks old): 54 mice were used to examine the sympathetic fibers and its neurotransmitter NE for the representation of sympathetic innervation of BTI, while the rest of them were randomly allocated into (LS) group and control group to verify the effect of sympathetic denervation during BTI healing. The LS group were intervened with fibrin sealant containing 10 â€‹ng/ml guanethidine, while the control group received fibrin sealant only. Mice were euthanized at postoperative 2, 4 and 8 weeks for immunofluorescent, qRT-PCR, ELISA, Micro-computed tomography (CT), histology and biomechanical evaluations. Results: Immunofluorescence, qRT-PCR and ELISA evaluations indicated that there were the expression of tyrosine hydroxylase (TH), NE and ß2-adrenergic receptor (ß2-AR) at the BTI site. All the above showed a trend of increasing at the early postoperative stage and they started to decrease with the healing time after a significant peak. Meanwhile, local sympathetic denervation of BTI was achieved after the use of guanethidine as shown in the NE ELISA outcomes in two groups. QRT-PCR analysis revealed that the healing interface in the LS group expressed more transcription factors, such as Runx2, Bmp2, Sox9, and Aggrecan, than the control group. Further, radiographic data showed that the LS group significantly possessed higher bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and lower trabecular spacing (Tb.Sp) than the control group. Also, histological test results showed that there was more fibrocartilage regenerated at the healing interface in the LS group compared with the control group. Mechanical testing results demonstrated that the failure load, ultimate strength and stiffness in the LS group were significantly higher at postoperative week 4 (P â€‹< â€‹0.05), but not at postoperative week 8 (P â€‹> â€‹0.05), compared to the control group. Conclusion: The regulation of sympathetic innervation was involved in the healing process of injured BTI, and local sympathetic denervation by using guanethidine was beneficial for BTI healing outcomes.The translational potential of this article: This is the first study to evaluate the expression and specific role of sympathetic innervation during BTI healing. The findings of this study also imply that the antagonists of ß2-AR could serve as a potential therapeutic strategy for BTI healing. Also, we firstly successfully constructed a local sympathetic denervation mouse model by using guanethidine loaded fibrin sealant, which provided a new effective methodology for future neuroskeletal biology study.

Hot spots and frontiers in bone-tendon interface research: a bibliometric analysis and visualization from 2000 to 2023.

Objective: In this research, we investigated the current status, hotspots, frontiers, and trends of research in the field of bone-tendon interface (BTI) from 2000 to 2023, based on bibliometrics and visualization and analysis in CiteSpace, VOSviewer, and a bibliometric package in R software. Methods: We collected and organized the papers in the Web of Science core collection (WoSCC) for the past 23 years (2000-2023), and extracted and analyzed the papers related to BTI. The extracted papers were bibliometrically analyzed using CiteSpace for overall publication trends, authors, countries/regions, journals, keywords, research hotspots, and frontiers. Results: A total of 1,995 papers met the inclusion criteria. The number of papers published and the number of citations in the field of BTI have continued to grow steadily over the past 23 years. In terms of research contribution, the United States leads in terms of the number and quality of publications, number of citations, and collaborations with other countries, while the United Kingdom and the Netherlands lead in terms of the average number of citations. The University of Leeds publishes the largest number of papers, and among the institutions hosting the 100 most cited papers Hospital for Special Surgery takes the top spot. MCGONAGLE D has published the highest number of papers (73) in the last 10 years. The top three clusters include #0 "psoriatic arthritis", #1 "rotator cuff repair", and #2 "tissue engineering". The structure and function of the BTI and its key mechanisms in the healing process are the key to research, while new therapies such as mechanical stimulation, platelet-rich plasma, mesenchymal stem cells, and biological scaffolds are hot topics and trends in research. Conclusion: Over the past 23 years, global research on the BTI has expanded in both breadth and depth. The focus of research has shifted from studies concentrating on the structure of the BTI and the disease itself to new therapies such as biomaterial-based alternative treatments, mechanical stimulation, platelet-rich plasma, etc.

Effect of suprascapular nerve injury on muscle and regenerated enthesis in a rat rotator cuff tear model.

BACKGROUND: Massive rotator cuff tears (RCTs) are complicated by muscle atrophy, fibrosis, and intramuscular fatty degeneration, which are associated with postoperative tendon-to-bone healing failure and poor clinical outcomes. We evaluated muscle and enthesis changes in large tears with or without suprascapular nerve (SN) injury in a rat model. METHODS: Sixty-two adult Sprague-Dawley rats were divided into SN injury (+) and SN injury (-) groups (n=31 each), comprising tendon (supraspinatus [SSP]/infraspinatus [ISP]) and nerve resection and tendon resection only cases, respectively. Muscle weight measurement, histological evaluation, and biomechanical testing were performed 4, 8, and 12 weeks postoperatively. Ultrastructural analysis with block face imaging was performed 8 weeks postoperatively. RESULTS: SSP/ISP muscles in the SN injury (+) group appeared atrophic, with increased fatty tissue and decreased muscle weight, compared to those in the control and SN injury (-) groups. Immunoreactivity was only positive in the SN injury (+) group. Myofibril arrangement irregularity and mitochondrial swelling severity, along with number of fatty cells, were higher in the SN injury (+) group than in the SN injury (-) group. The bone-tendon junction enthesis was firm in the SN injury (-) group; this was atrophic and thinner in the SN injury (+) group, with decreased cell density and immature fibrocartilage. Mechanically, the tendon-bone insertion was significantly weaker in the SN injury (+) group than in the control and SN injury (+) groups. CONCLUSIONS: In clinical settings, SN injury may cause severe fatty changes and inhibition of postoperative tendon healing in large RCTs. Level of evidence: Basic research, controlled laboratory study.

Eccentric Contraction Enhances Healing of the Bone-Tendon Interface After Rotator Cuff Repair in Mice.

BACKGROUND: Various muscle contraction modalities have differing effects on the musculoskeletal system. To understand the magnitude of these effects, the authors investigated the effects of eccentric and concentric contractions on the bone-tendon interface after rotator cuff repair in mice. HYPOTHESIS: Eccentric contraction promotes healing of the bone-tendon interface after rotator cuff repair in mice better than other muscle contraction patterns. STUDY DESIGN: Controlled laboratory study. METHODS: The authors performed acute supraspinatus tendon repair of the right shoulder in 104 C57BL/6 mice. Animals were randomized into 4 groups postoperatively: control group (Con group), horizontal running group (Horz group), +15° uphill running group (Up group), and -15° downhill running group (Down group), with 26 animals in each group. At 4 and 8 weeks postoperatively, the authors removed the eyeball, collected blood samples, and extracted the supraspinatus tendon-humerus complex for histological, immunological, bone morphological, and biomechanical tests. RESULTS: At 4 and 8 weeks postoperatively, the Down group exhibited a better collagen cell arrangement and fibrocartilage layer than the other 3 groups. At 4 weeks postoperatively, anti-inflammatory macrophages (M2 macrophages) were observed at the repair site in all groups except for the Con group. At 8 weeks postoperatively, M2 macrophages were withdrawn from the tendon site in all groups. The transforming growth factor ß1 concentration in the Down group was greater than that in the other 3 groups at 4 weeks postoperatively, and it was higher than that in the Con group at 8 weeks postoperatively. The bone volume fraction, number of trabeculae, and thickness of trabeculae at the repair site in the Down group, as well as the ultimate strength and failure load in the biomechanical tests, were greater than those in the other 3 groups at 8 weeks postoperatively. CONCLUSION: Eccentric contraction promotes healing of the bone-tendon interface after rotator cuff repair in mice better than other muscle contraction patterns. CLINICAL RELEVANCE: After clinical rotator cuff repair, patients can be rehabilitated by eccentric training to speed up the functional recovery of the shoulder joint.

Rotator cuff healing is regulated by the lymphatic vasculature.

Background: Despite great advances in surgical techniques for rotator cuff tear (RCT) over the past decades, the postoperative failure rate of RCT is still high due to the poor healing competence of bone-tendon interface (BTI). The lymphatic vasculature plays a regulatory role in inflammatory disease and affects tissue healing. However, whether lymphangiogenesis and the role of lymphatic vasculature in the physiopathological process of rotator cuff （RC）injury remains unknown. Methods: In this study, we constructed a mouse RC injury model and the BTI samples were collected for measurement. Firstly, immunofluorescence was used to investigate the temporal and spatial distribution of lymphangiogenesis in BTI area at different post-injury time points. Subsequently, the mice of experimental group were gavaged with the lymphatic inhibitors (SAR131675) on the first postoperative day to inhibit lymphangiogenesis, while the control group was treated with the vehicle. At postoperative week 2 and 4, the samples were collected for immunofluorescence staining to evaluate lymphatic angiogenesis inhibition. At postoperative week 4 and 8, The supraspinatus (SS) tendon-humeral complexes were collected for bone morphometric, histological and biomechanical tests to assess the healing outcome of the BTI. Results: Immunofluorescence results showed that the lymphatic proliferation in the BTI injury area and increased in consistence with the healing time, and the lymphatic hyperplasia area significantly diminished at postoperative week 4. The lymphatic hyperplasia area in the SAR group was significantly lower than that in the control group both at 2 and 4 weeks postoperatively. Moreover, the administration of SAR131675 significantly impeded RC healing, as evidenced by lower histological scores, lower bone morphometric parameters, and worse biomechanical properties in comparison with that in control group at postoperative weeks 4 and 8. Conclusion: Lymphangiogenesis plays a positive role in RC healing, and targeting the lymphatic drainage at healing site may be a new therapeutic approach to promote RC injury repair. The translational potential of this article: This is the first study to assess the specific role of lymphatic vessels in RC healing, and improving lymphatic drainage may be a potential new therapeutic approach to facilitate repair of BTI. Further, our study provides a reference for possible future treatment of BTI by intervening the lymphatic system.

Exosomes from CD133+ human urine-derived stem cells combined adhesive hydrogel facilitate rotator cuff healing by mediating bone marrow mesenchymal stem cells.

Background: The inadequate regeneration of natural tissue (mainly fibrocartilage) between tendon and bone during rotator cuff (RC) repair results in an unsatisfactory quality of RC healing. Cell-free therapy based on stem cell exosomes is a safer and more promising approach for tissue regeneration. Here, we investigated the effect of exosomes from human urine-derived stem cells (USCs) and their subpopulations (CD133+USCs) on RC healing. Methods: USCs were isolated from urine and sorted by flow cytometry to obtain CD133+ urine-derived stem cells (CD133+ USCs). Urine-derived stem cell exosomes (USC-Exos) and CD133+ urine-derived stem cell exosomes (CD133+ USC-Exos) were subsequently isolated from the cell supernatant and identified by transmission electron microscopy (TEM), particle size analysis, and Western blot. We performed in vitro functional assays to evaluate the effects of USC-Exos and CD133+ USC-Exos on human bone marrow mesenchymal stem cells (BMSCs) proliferation, migration, osteogenic differentiation, and chondrogenic differentiation. In vivo experiments were performed by local injection of exosome-hydrogel complexes for the treatment of RC injury. The effects of CD133+ USC-Exos and USC-Exos on RC healing were assessed from imaging, histological, and biomechanical tests. Results: CD133+ USCs were positive for CD29, CD44, CD73, CD90, CD133, but negative for CD34 and CD45. Differentiation ability test results showed that both USCs and CD133+ USCs had the potential for osteogenic, chondrogenic, and adipogenic differentiation, but CD133+ USCs had stronger chondrogenic differentiation ability. CD133+ USC-Exos and USC-Exos could be efficiently taken up by BMSCs and promote their migration, osteogenic and chondrogenic differentiation. However, CD133+ USC-Exos could promote the chondrogenic differentiation of BMSCs more than USC-Exos. Compared with USC-Exos, CD133+ USC-Exos could promote the healing of bone-tendon interface (BTI) more effectively, which might be related to its ability to promote the differentiation of BMSCs into chondroblasts. Although the two exosomes exhibited the same effect in promoting subchondral bone repair in BTI, the CD133+ USC-Exos group had higher histological scores and stronger biomechanical properties. Conclusion: CD133+ USC-Exos hydrogel complex may become a promising therapeutic approach for RC healing based on stem cell exosomes. The translational potential of this article: This is the first study to assess the specific role of CD133+ USC-Exos in RC healing which may be related to the activation of BMSCs by CD133+ USC-Exos towards chondrogenic differentiation. Further, our study provides a reference for possible future treatment of BTI by applying CD133+ USC-Exos hydrogel complex.

Effect of Mechanical Stimulation Combined With Platelet-Rich Plasma on Healing of the Rotator Cuff in a Murine Model.

BACKGROUND: Mechanical stimulation and platelet-rich plasma (PRP) have been shown to be beneficial for healing of the bone-tendon interface (BTI), but few studies have explored the efficacy of a combination of these applications. We investigated the effect of mechanical stimulation combined with PRP on rotator cuff repair in mice. HYPOTHESIS: Mechanical stimulation combined with PRP can enhance BTI healing in a murine model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 160 C57BL/6 mice were used. Overall, 40 mice were used to prepare PRP, while 120 mice underwent acute supraspinatus tendon (SST) repair. The animals were randomly assigned to 4 groups: control group, mechanical stimulation group, PRP group, and mechanical stimulation combined with PRP group (combination group). At 4 and 8 weeks postoperatively, animals were sacrificed, the eyeballs were removed to collect blood, and the SST-humeral complexes were collected. Histological, biomechanical, immunological, and bone morphometric tests were performed. RESULTS: Histologically, at 4 and 8 weeks after surgery, the area of the fibrocartilage layer at the BTI in the combination group was larger than in the other groups. The content and distribution of proteoglycans in this layer in the combination group were significantly greater than in the other groups. At 8 weeks postoperatively, trabecular number, and trabecular bone thickness of the subchondral bone area of interest at the BTI of the combination group were greater than those of the other groups, bone volume fraction of the combination group was greater than the control group. On biomechanical testing at 4 and 8 weeks after surgery, the failure load and ultimate strength of the SST-humeral complex in the combination group were higher than in the other groups. Enzyme-linked immunosorbent assay results showed that, at 4 weeks postoperatively, the serum concentrations of transforming growth factor beta 1 and platelet-derived growth factor (PDGF) in the combination group were significantly higher than in the other groups; at 8 weeks, the PDGF-AB concentration in the combination group was higher than in the control and mechanical stimulation groups. CONCLUSION: Mechanical stimulation combined with PRP can effectively promote the early stage of healing after a rotator cuff injury. CLINICAL RELEVANCE: These findings imply that mechanical stimulation combined with PRP can serve as a potential therapeutic strategy for rotator cuff healing.

Characterize the microstructure change after tendon enthesis injury using synchrotron radiation µCT.

The microstructure of the bone-tendon interface (BTI) deserves in-depth investigation. In this study, we first aimed to extend the application of synchrotron radiation µCT to characterize the gradient structure of supraspinatus tendon (SST) enthesis, from both tissue morphology to cell distribution. Second, to acquire detailed morphological information of SST enthesis when after injury. Our results showed that in normal enthesis, the phenotype of chondrocyte in BTI was dependent on its distance to subchondral bone. After injury, the fibrocartilage cells were disrupted, as evidenced by reduced lacunae size. Our observation may partly explain the loss of BTI mechanical properties after injury, and we believe the application of synchrotron radiation microcomputed tomography will have promising potential for characterizing the morphology changes in enthesis and for evaluating the therapeutic effects of interventions in preclinical studies.

Effect of Exercise Intensity on the Healing of the Bone-Tendon Interface: A Mouse Rotator Cuff Injury Model Study.

BACKGROUND: Injuries at the bone-tendon interface (BTI) are common findings in clinical practice. Rehabilitation procedures after BTI surgery are important but are controversial. PURPOSE: To investigate the effects of different exercise intensities on BTI healing by means of an established mouse rotator cuff injury model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 150 specific pathogen free male C57BL/6 mice, with supraspinatus insertion injury, were randomly assigned to 1 of 5 groups according to postoperative rehabilitation of different exercise intensities: (1) control group, (2) low-intensity exercise group, (3) moderate-intensity exercise group, (4) high-intensity exercise group, and (5) increasing-intensity exercise group (IG). The specimens were harvested 4 or 8 weeks postoperatively for microarchitectural, histological, molecular biological, and mechanical evaluations. RESULTS: Histological test results showed that the degrees of tissue fusion and polysaccharide protein distribution at the healing interface at 4 and 8 weeks after surgery were significantly better in the IG than in the other 4 groups. Synchrotron radiation micro-computed tomography showed that the quantity of subchondral bone at the enthesis (bone volume/total volume fraction, trabecular thickness, trabecular number) was higher and trabecular separation was lower in the IG than in the other 4 groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the healing interface in the IG expressed more transcription factors, such as sox 9, runx 2, and scleraxis, than the interfaces in the other groups. Although no significant difference was seen in the cross-sectional area between the groups at postoperative weeks 4 and 8 (P > .05), the tensile load, ultimate strength, and stiffness of the specimens in the IG were significantly better than those in the other 4 groups (P < .05). CONCLUSION: The rehabilitation program with increasing-intensity exercise was beneficial for BTI healing. CLINICAL RELEVANCE: The results of this study provide evidence supporting the use of a simple and progressive exercise rehabilitation program after rotator cuff surgery.

Treadmill running initiation times and bone-tendon interface repair in a murine rotator cuff repair model.

Postoperative exercise has been demonstrated to be beneficial for bone-tendon interface (BTI) healing, yet the debate regarding the optimal time to initiate exercise after tendon enthesis repair is ongoing. This study aimed to evaluate the initiation times for exercise after enthesis repair. A total of 192 C57BL/6 mice underwent acute supraspinatus tendon injury repair. The animals were then randomly assigned to four groups: free cage activity after repair (control group); treadmill running started on postoperative day 2 (2-day delayed group); treadmill running started on postoperative day 7 (7-day delayed group), and treadmill running started on postoperative day 14 (14-day delayed group). Mice were euthanized at 4 and 8 weeks postoperatively, and histological, biomechanical, and bone morphometric tests were performed. Higher failure loads and bone volume fractions were found for the 7-day delayed group and the 14-day delayed group at 4 weeks postoperatively. The 7-day delayed group had better biomechanical properties and higher bone volume fractions than the 2-day delayed group at 4 weeks postoperatively. Histologically, the 7-day delayed group exhibited lower modified tendon-to-bone maturity scores than the control group and the 2-day delayed group at 4 and 8 weeks postoperatively. Quantitative reverse-transcription polymerase chain reaction results showed that the 7-day delayed group had higher expressions of chondrogenic- and osteogenic-related genes. Statement of clinical significance: Postoperative treadmill running initiated on postoperative day 7 had a more prominent effect on BTI healing than other treatment regimens in this study and could accelerate BTI healing and rotator cuff repair.

Lugol's solution but not formaldehyde affects bone microstructure and bone mineral density parameters at the insertion site of the rotator cuff in rats.

BACKGROUND: This study aimed to investigate whether rodent shoulder specimens fixed in formaldehyde for histological and histomorphometric investigations and specimens stained using Lugol's solution for soft tissue visualization by micro-computed tomography (microCT) are still eligible to be used for bone architecture analysis by microCT. METHODS: In this controlled laboratory study, 11 male Sprague-Dawley rats were used. After sacrifice and exarticulation both shoulders of healthy rats were assigned into three groups: (A) control group (n = 2); (B) formaldehyde group (n = 4); (C) Lugol group (n = 5). Half of the specimens of groups B and C were placed in a 4% buffered formaldehyde or Lugol's solution for 24 h, whereas the contralateral sides and all specimens of group A were stored without any additives. MicroCT of both sides performed in all specimens focused on bone mineral density (BMD) and bone microstructure parameters. RESULTS: BMD measurements revealed higher values in specimens after placement in Lugol's solution (p < 0.05). Bone microstructure analyses showed increased BV/TV and Tb.Th values in group C (p < 0.05). Specimens of group C resulted in clearly decreased Tb.Sp values (p < 0.05) in comparison to the control group. Formaldehyde fixation showed minimally altered BMD and bone microstructure measurements without reaching any significance. CONCLUSIONS: MicroCT scans of bone structures are recommended to be conducted natively and immediately after euthanizing rats. MicroCT scans of formaldehyde-fixed specimens must be performed with caution due to a possible slight shift of absolute values of BMD and bone microstructure. Bone analysis of specimens stained by Lugol's solution cannot be recommended.

Acceleration of Bone-Tendon Interface Healing by Low-Intensity Pulsed Ultrasound Is Mediated by Macrophages.

OBJECTIVE: Low-intensity pulsed ultrasound (LIPUS) has been proven to facilitate bone-tendon interface (BTI) healing and regulate some inflammatory cytokines. However, the role of macrophages, a key type of inflammatory cell, during treatment remains unknown. This study aimed to investigate the role of macrophages in the treatment of BTI injury with LIPUS in a rotator cuff tear animal model. METHODS: In this experimental and comparative study, a total of 160 C57BL/6 mature male mice that underwent supraspinatus tendon detachment and repair were randomly assigned to 4 groups: daily ultrasonic treatment and liposomal clodronate (LIPUS+LC), daily ultrasonic treatment and liposomes (LIPUS), daily mock sonication and liposomal clodronate (LC), and daily mock sonication and liposomes (control [CTL]). LIPUS treatment was initiated immediately postoperatively and continued daily until the end of the experimental period. RESULTS: The failure load and stiffness of the supraspinatus tendon-humerus junction were significantly higher in the LIPUS group than in the other groups at postoperative weeks 2 and 4, whereas those in the LIPUS+LC and LC groups were lower than those in the CTL group at postoperative week 4. The LIPUS, LIPUS+LC, and LC groups exhibited significantly more fibrocartilage than the CTL group at 2 weeks. Only the LIPUS group had more fibrocartilage than the CTL group at 4 weeks. Micro-computed tomography results indicated that LIPUS treatment could improve the bone quality of the attachment site after both 2 and 4 weeks. When macrophages were depleted by LC, the bone quality-promoting effect of LIPUS treatment was significantly reduced. CONCLUSION: The enhancement of BTI healing by LIPUS might be mediated by macrophages. IMPACT: In our study, LIPUS treatment appeared to accelerate BTI healing, which was associated with macrophages based on our murine rotator cuff repair model. The expressions of macrophage under LIPUS treatment may offer a potential mechanism to explain BTI healing and the effects of LIPUS on BTI healing.

Joining soft tissues to bone: Insights from modeling and simulations.

Entheses are complex multi-tissue regions of the musculoskeletal system serving the challenging task of connecting highly dissimilar materials such as the compliant tendon to the much stiffer bone, over a very small region. The first aim of this review is to highlight mathematical and computational models that have been developed to investigate the many attachment strategies present at entheses at different length scales. Entheses are also relevant in the medical context due to the high prevalence of orthopedic injuries requiring the reattachment of tendons or ligaments to bone, which are associated with a rather poor long-term clinical outcome. The second aim of the review is to report on the computational works analyzing the whole tendon to bone complex as well as targeting orthopedic relevant issues. Modeling approaches have provided important insights on anchoring mechanisms and surgical repair strategies, that would not have been revealed with experiments alone. We intend to demonstrate the necessity of including, in future models, an enriched description of enthesis biomechanical behavior in order to unravel additional mechanical cues underlying the development, the functioning and the maintaining of such a complex biological interface as well as to enhance the development of novel biomimetic adhesive, attachment procedures or tissue engineered implants.