Evaluating the role of subacromial impingement in rotator cuff tendinopathy: development and analysis of a novel rat model.

BACKGROUND: Subacromial impingement of the rotator cuff caused by variations in acromial anatomy or altered glenohumeral kinematics leads to inflammation and degeneration of the rotator cuff, ultimately contributing to the development of tendinopathy. However, the underlying cellular and molecular changes in the impinged tendon remain poorly understood. Because the rat is an accepted model for rotator cuff studies, we have developed a rat model to study rotator cuff tendinopathy. METHODS: Forty-four adult male Sprague-Dawley rats were allocated to one of 4 study groups: intact control group (group 1, n = 11); bilateral subacromial surgical clip placement to induce supraspinatus impingement for 2 weeks (group 2, n = 11), 4 weeks (group 3, n = 11), and 8 weeks (group 4, n = 11). Bilateral shoulder specimens were harvested for biomechanical testing, histology, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: Radiography confirmed that all microvascular clips remained in stable position in the subacromial space. Gross inspection of supraspinatus tendon specimens in the impingement groups revealed changes in tendon morphology at the enthesis and midsubstance. Biomechanical evaluation demonstrated decreased supraspinatus tendon failure force and tissue stiffness at all time points compared with control tendons. Semiquantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 8 weeks. qRT-PCR analysis of impinged tendon specimens demonstrated upregulation of gene expression for Col3 and Mmp14 in the impingement groups compared with control groups. In muscle samples, significant upregulation was seen in the expression of genes that are commonly associated with muscle atrophy (MuRF1 and Ube2b) and fatty infiltration (Fabp4, Pparg2, and Klf15). CONCLUSION: This new rat subacromial impingement model creates cellular and molecular changes consistent with the development of rotator cuff tendinopathy. The results of this study may serve as a baseline for future investigation.

Upregulated expression of transforming growth factor-ß receptor I/II in an endemic Osteoarthropathy in China.

BACKGROUND: Kashin-Beck disease (KBD) is a chronic, deforming, endemic osteochondropathy that begins in patients as young as 2-3 years of age. The pathogenesis of KBD remains unclear, although selenium (Se) deficiency and T-2 toxin food contamination are both linked to the disease. In the present study, we evaluated transforming growth factor-ß receptor (TGF-ßR I and II) levels in clinical samples of KBD and in pre-clinical disease models. METHODS: Human specimens were obtained from the hand phalanges of eight donors with KBD and eight control donors. Animal models of the disease were established using Sprague-Dawley rats, which were fed an Se-deficient diet for 4 weeks and later administered the T-2 toxin. Cartilage cellularity and morphology were examined by hematoxylin and eosin staining. Expression and localization of TGF-ßRI and II were evaluated using immunohistochemical staining and western blotting. RESULTS: In the KBD samples, chondral necrosis was detected based on cartilage cell disappearance and alkalinity loss in the matrix ground substance. In the necrotic areas, TGF-ßRI and II staining were strong. Positive percentages of TGF-ßRI and II staining were higher in the cartilage samples of KBD donors than in those of control donors. TGF-ßRI and II staining was also increased in cartilage samples from rats administered T-2 toxin or fed on Se-deficient plus T-2 toxin diets. CONCLUSION: TGF-ßRI and II may be involved in the pathophysiology of KBD. This study provides new insights into the pathways that contribute to KBD development.

Kartogenin Enhances Collagen Organization and Mechanical Strength of the Repaired Enthesis in a Murine Model of Rotator Cuff Repair.

PURPOSE: To investigate the use of kartogenin (KGN) in augmenting healing of the repaired enthesis after rotator cuff repair in a murine model. METHODS: Seventy-two C57BL/6 wild-type mice underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin sealant (control group; n = 36) or fibrin sealant containing 100 µmol/L of KGN (experimental group; n = 36) applied at the repair site. Postoperatively, mice were allowed free cage activity without immobilization. Mice were humanely killed at 2 and 4 weeks postoperatively. Repair site integrity was evaluated histologically through fibrocartilage formation and collagen fiber organization and biomechanically through load-to-failure testing of the supraspinatus tendon-bone construct. RESULTS: At 2 weeks, no differences were noted in percent area of fibrocartilage, collagen organization, or ultimate strength between groups. At 4 weeks, superior collagen fiber organization (based on collagen birefringence [17.3 ± 2.0 vs 7.0 ± 6.5 integrated density/µm2; P < .01]) and higher ultimate failure loads (3.5 ± 0.6 N vs 2.3 ± 1.1 N; P = .04) were seen in the KGN group. The percent area of fibrocartilage (13.2 ± 8.4% vs 4.4 ± 5.4%; P = .04) was higher in the control group compared with the KGN group. CONCLUSIONS: Rotator cuff repair augmentation with KGN improved the collagen fiber organization and biomechanical strength of the tendon-bone interface at 4 weeks in a murine model. CLINICAL RELEVANCE: These findings have implications for improving the structural integrity of the repaired enthesis and potentially reducing the retear rate after rotator cuff repair, which can ultimately lead to improvements in clinical outcomes.

Biomechanical, Histologic, and Molecular Evaluation of Tendon Healing in a New Murine Model of Rotator Cuff Repair.

PURPOSE: To develop a clinically relevant, robust murine model of rotator cuff tendon repair to examine cellular and molecular mechanisms of healing. METHODS: Sixty C57BL/6 male mice underwent rotator cuff transection and repair using microsurgical techniques. A modified Kessler suturing technique was used prior to tendon detachment. Sutures were passed through 2 intersecting bone tunnels that were made at the tendon attachment site. Mice were sacrificed at 2 and 4 weeks with subsequent biomechanical, histologic, micro-CT, and gene expression evaluations. RESULTS: Failure forces in the 2- and 4-week groups were 36% and 75% of the intact tendon, respectively. Histologic evaluation revealed complete reattachment of the tendon with no observable gap. Healing occurred by formation of fibrovascular tissue at the tendon-bone interface, similar to larger animal models. Molecular analysis revealed gene expression consistent with gradual healing of the reattached tendon over a period of 4 weeks. Comparisons were made using 1-way analysis of variance. CONCLUSIONS: This model is distinguished by use of microsurgical suturing techniques, which provides a robust, reproducible, and economic animal model to study various aspects of rotator cuff pathology. CLINICAL RELEVANCE: Improvement of clinical outcomes of rotator cuff pathology requires in-depth understanding of the underlying cellular and molecular mechanisms of healing. This study presents a robust murine model of supraspinatus repair to serve as a standard research tool for basic and translational investigations into signaling pathways, gene expression, and the effect of biologic augmentation approaches.

Involvement of Indian hedgehog signaling in mesenchymal stem cell-augmented rotator cuff tendon repair in an athymic rat model.

BACKGROUND: Bone marrow aspirate has been used in recent years to augment tendon-to-bone healing, including in rotator cuff repair. However, the healing mechanism in cell-based therapy has not been elucidated in detail. METHODS: Sixteen athymic nude rats were randomly allocated to 2 groups: experimental (human mesenchymal stem cells in fibrin glue carrier) and control (fibrin glue only). Animals were sacrificed at 2 and 4 weeks. Immunohistochemical staining was performed to evaluate Indian hedgehog (Ihh) signaling and SOX9 signaling in the healing enthesis. Macrophages were identified using CD68 and CD163 staining, and proliferating cells were identified using proliferating cell nuclear antigen staining. RESULTS: More organized and stronger staining for collagen II and a higher abundance of SOX9+ cells were observed at the enthesis in the experimental group at 2 weeks. There was significantly higher Gli1 and Patched1 expression in the experimental group at the enthesis at 2 weeks and higher numbers of Ihh+ cells in the enthesis of the experimental group vs control at both 2 weeks and 4 weeks postoperatively. There were more CD68+ cells localized to the tendon midsubstance at 2 weeks compared with 4 weeks, and there was a higher level of CD163 staining in the tendon midsubstance in the experimental group than in the control group at 4 weeks. CONCLUSION: Stem cell application had a positive effect on fibrocartilage formation at the healing rotator cuff repair site. Both SOX9 and Ihh signaling appear to play an important role in the healing process.

The Effect of Purified Human Bone Marrow-Derived Mesenchymal Stem Cells on Rotator Cuff Tendon Healing in an Athymic Rat.

PURPOSE: To evaluate the ability of purified human bone marrow-derived mesenchymal stem cells (MSCs) to augment healing of an acute small- to medium-sized rotator cuff repair in a small-animal model, evaluating the structure and composition of the healing tendon-bone interface with histologic and biomechanical analyses. METHODS: Fifty-two athymic rats underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin glue (control group) or fibrin glue with 106 human MSCs (experimental group) applied at the repair site. Flow cytometry verified the stem cell phenotype of the cells as CD73+, CD90+, CD105+, CD14-, CD34-, and CD45-. Rats were killed at 2 and 4 weeks, with 10 from each group used for biomechanical testing and 3 for histologic analysis. RESULTS: Safranin O staining identified increased fibrocartilage formation at the repair site at 2 weeks in the human MSC group (18.6% ± 2.9% vs 9.1% ± 1.6%, P = .026). Picrosirius staining identified decreased energy (36.88 ± 4.99 J vs 54.97 ± 8.33 J, P = .04) and increased coherence in the human MSC group (26.96% ± 15.32% vs 14.53% ± 4.10%, P = .05), indicating improved collagen orientation. Biomechanical testing showed a significant increase in failure load (11.5 ± 2.4 N vs 8.5 ± 2.4 N, P = .002) and stiffness (7.1 ± 1.2 N/mm vs 5.7 ± 2.1 N/mm, P < .001) in the experimental group compared with the control group at 2 weeks. These effects dissipated by 4 weeks, with no significant differences in fibrocartilage formation (35% ± 5.0% vs 26.6% ± 0.6%, P = .172) or biomechanical load to failure (24.6 ± 7.1 N vs 21.5 ± 4.1 N, P = .361) or stiffness (13.5 ± 3.1 N/mm vs 16.1 ± 5.6 N/mm, P = .384). All failures occurred at the bone-tendon interface. CONCLUSIONS: Rotator cuff repair augmentation with purified human MSCs improved early histologic appearance and biomechanical strength of the repair at 2 weeks, although the effects dissipated by 4 weeks with no significant differences between groups. CLINICAL RELEVANCE: Human MSCs may improve early rotator cuff healing during the first 2 weeks after repair.

rhPDGF-BB promotes early healing in a rat rotator cuff repair model.

BACKGROUND: Tendon-bone healing after rotator cuff repair occurs by fibrovascular scar tissue formation, which is weaker than a normal tendon-bone insertion site. Growth factors play a role in tissue formation and have the potential to augment soft tissue healing in the perioperative period. QUESTIONS/PURPOSES: Our study aim was to determine if rhPDGF-BB delivery on a collagen scaffold can improve tendon-to-bone healing after supraspinatus tendon repair compared with no growth factor in rats as measured by (1) gross observations; (2) histologic analysis; and (3) biomechanical testing. METHODS: Ninety-five male Sprague-Dawley rats underwent acute repair of the supraspinatus tendon. Rats were randomized into one of five groups: control (ie, repair only), scaffold only, and three different platelet-derived growth factor (PDGF) doses on the collagen scaffold. Animals were euthanized 5 days after surgery to assess cellular proliferation and angiogenesis. The remaining animals were analyzed at 4 weeks to assess repair site integrity by gross visualization, fibrocartilage formation with safranin-O staining, and collagen fiber organization with picrosirius red staining, and to determine the biomechanical properties (ie, load-to-failure testing) of the supraspinatus tendon-bone construct. RESULTS: The repaired supraspinatus tendon was in continuity with the bone in all animals. At 5 days, rhPDGF-BB delivery on a scaffold demonstrated a dose-dependent response in cellular proliferation and angiogenesis compared with the control and scaffold groups. At 28 days, with the numbers available, rhPDGF-BB had no effect on increasing fibrocartilage formation or improving collagen fiber maturity at the tendon-bone insertion site compared with controls. The control group had higher tensile loads to failure and stiffness (35.5 ± 8.8 N and 20.3 ± 4.5 N/mm) than all the groups receiving the scaffold, including the PDGF groups (scaffold: 27 ± 6.4 N, p = 0.021 and 13 ± 5.7 N/mm, p = 0.01; 30 µg/mL PDGF: 26.5 ± 7.5 N, p = 0.014 and 13.3 ± 3.2 N/mm, p = 0.01; 100 µg/mL PDGF: 25.7 ± 6.1 N, p = 0.005 and 11.6 ± 3.3 N/mm, p = 0.01; 300 µg/mL PDGF: 27 ± 6.9 N, p = 0.014 and 12.7 ± 4.1 N/mm, p = 0.01). CONCLUSIONS: rhPDGF-BB delivery on a collagen scaffold enhanced cellular proliferation and angiogenesis during the early phase of healing, but this did not result in either a more structurally organized or stronger attachment site at later stages of healing. The collagen scaffold had a detrimental effect on healing strength at 28 days, and its relatively larger size compared with the rat tendon may have caused mechanical impingement and extrinsic compression of the healing tendon. Future studies should be performed in larger animal models where healing occurs more slowly. CLINICAL RELEVANCE: Augmenting the healing environment to improve the structural integrity and to reduce the retear rate after rotator cuff repair may be realized with continued understanding and optimization of growth factor delivery systems.

Recent progress in graphene-material-based optical sensors.

Graphene material has been widely used for optical sensors owing to its excellent properties, including high-energy transfer efficiency, large surface area, and great biocompatibility. Different analytes such as nucleic acids, proteins, and small molecules can be detected by graphene-material-based optical sensors. This review provides a comprehensive discussion of graphene-material-based optical sensors focusing on detection mechanisms and biosensor designs. Challenges and future perspectives for graphene-material-based optical sensors are also presented.

Establishment of a Posttraumatic Osteoarthritis Model in Mice Induced by Noninvasive Anterior Cruciate Ligament Tear.

BACKGROUND: Animal models that use open surgical transection of the anterior cruciate ligament (ACL) do not accurately simulate the clinical condition regarding the pivot-shift mechanism and the associated inflammatory response that occurs before reconstruction. PURPOSE/HYPOTHESIS: The purpose was to characterize a reproducible manual, nonsurgical method to mimic an isolated ACL tear in a clinically relevant model and to evaluate the development of progressive posttraumatic osteoarthritis due to ACL injury. It was hypothesized that the ACL could be reproducibly torn with minimal damage to other ligaments and that there would be progressive development of degenerative joint disease after ACL injury. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 37 mice (strain C57BL/6) were used to compare the manual procedure with sham surgery (sham group; n = 10) and with the established surgical ACL transection (ACLT) procedure (surgical group; n = 27). In the sham group, a closed manual procedure was performed on the right knee and sham surgery on the left knee. In the surgical group, the closed manual procedure was performed on the right knee and surgical ACLT on the left knee. Dissection using India ink, histological assessment with safranin O and hematoxylin-eosin staining, radiological evaluation through radiographs and microfocus computed tomography scans, and gait analyses were performed to assess cartilage/ligament status. Osteoarthritis Research Society International (OARSI) and synovitis scores, anterior tibial translation, range of motion, bone microstructure, osteophyte volume, and pain were assessed at 2, 4, and 8 weeks postoperatively. RESULTS: The manual procedure successfully resulted in an ACL rupture and associated meniscal injury. The posterior cruciate, lateral collateral, and medial collateral ligaments were intact in all dissected knees. Two weeks after ACL tear, the surgical group showed a significantly higher synovitis score, whereas 8 weeks after ACL tear, the manual group showed a significantly higher volume of osteophytes. No significant differences were found between the groups in terms of OARSI score, anterior tibial translation, range of motion, bone microstructure computed tomography values, and stride distance/irregularity. CONCLUSION: This procedure can be used to create an ACL tear model without causing grossly evident injuries to other ligaments and avoiding the risk of cartilage damage from surgical instruments. CLINICAL RELEVANCE: This procedure offers a more clinically relevant ACL tear model and facilitates simple, inexpensive, and reproducible development of posttraumatic osteoarthritis.

Bone-ACL-bone allograft for anterior cruciate ligament reconstruction: Short-term evaluation in a rabbit model with microcomputed tomography.

The standard grafts used for anterior cruciate ligament (ACL) reconstruction are tendon, either patellar tendon, hamstring, or quadriceps. However, the microstructure and composition of tendon differs from ligament. Ideally, the ACL would be replaced with the same tissue. To evaluate the incorporation of a bone-ACL-bone (B-ACL-B) graft for ACL reconstruction, we performed a controlled laboratory study in a rabbit model with microcomputed tomography (µCT). Forty-six New Zealand white rabbits were used, with 17 donor rabbits to harvest bilateral B-ACL-B allografts and 29 rabbits undergoing unilateral ACL reconstruction with B-ACL-B allograft. Knee specimens were collected for biomechanical testing (n = 14) at 4 and 8 weeks and for µCT analysis (n = 15) at 2, 4, and 8 weeks after surgery. Gross inspection and µCT examination confirmed bone blocks in the appropriate anatomic position. Biomechanical tests revealed no difference in mean load-to-failure force for B-ACL-B allografts between 4 and 8 weeks. Progressive healing occurred between the bone block and the tunnel as demonstrated by a gradual increase on average bone-volume fraction and total mineral density (TMD) in both femoral and tibial tunnels. Remodeling of the bone block was evidenced by a significant decrease in TMD of both tibial and femoral bone blocks. This is a report of a novel rabbit B-ACL-B allograft reconstruction model demonstrating early signs of graft remodeling and incorporation. Clinical Relevance: This study demonstrates ACL reconstruction using an anatomically matched ACL allograft, rather than a tendon graft, may be possible based on early findings in this lapine model.

One-Stage Arthroscopic Multiple Ligament Reconstruction for Schenck IV Knee Dislocation.

PURPOSE: Schenck IV knee dislocation patients have dissatisfactory knee function and return-to-sport rate with the existing treatment methods. The purpose of this study was to illustrate a one-stage arthroscopic multiple ligament reconstruction method for treating Schenck IV knee dislocations. METHODS: A retrospective case series study was performed. All patients with a history of Schenck IV knee dislocation who underwent one-stage arthroscopic multi-ligament reconstruction from 2010 to 2018 were followed for 24 months. The outcomes, including general patient data, Lysholm scores, International Knee Documentation Committee (IKDC) scores, visual analog scale (VAS) pain scores, knee active range of motion, and complications, were reviewed. The data was analyzed with paired-samples t-test. RESULTS: A total of 12 patients, comprising nine males and three females, were followed up and reviewed. The mean age at the time of the surgical procedure was 40.3 ± 9.0 (22-57) years. The mean body mass index (BMI) was 24.6 ± 4.9 (15.2-32.5) kg/m2 . The mean IKDC score and Lysholm score before surgery were 30.4 ± 6.1 (21-42) and 28.2 ± 6.2 (22-39), respectively. The average operation time was 121.8 minutes. The mean IKDC score and Lysholm score at the 24-month follow-up were 80.6 ± 6.5 (68-92) and 82.0 ± 7.5 (72-95), respectively. There were significant differences in the IKDC and Lysholm scores between the preoperative and 24-month postoperative time points (p < 0.01). The mean knee range of motion was 124.6° ± 6.6° (115°-135°) at the 24-month follow-up. No major complications occurred. CONCLUSIONS: The results of this retrospective study suggest that the new arthroscopic one-stage multi-ligament reconstruction technique is an effective way to treat Schenck IV knee dislocation with satisfactory postoperative knee function.

Short-Term Evaluation of Bone-ACL-Bone Complex Allograft in ACL Reconstruction in a Rabbit Model.

The study is to evaluate incorporation of a bone-anterior cruciate ligament-bone (B-ACL-B) allograft in anterior cruciate ligament (ACL) reconstruction in a rabbit model. A total of 61 New Zealand white rabbits were used, with 23 donor rabbits for harvesting B-ACL-B allografts and 38 recipient rabbits undergoing unilateral ACL reconstruction with B-ACL-B allograft. Animals were euthanized for biomechanical testing, micro-computed tomography examination, histological analysis, multi-photon microscopy and transmission electron microscopy testing at 2, 4 and 8 weeks after surgery. Gross inspection and radiographs confirmed the intact ACL allograft in the proper anatomic position. Progressive healing occurred between the bone block and the bone tunnel as demonstrated by a gradual increase in average bone volume fraction and total mineral density at 4 and 8 weeks. Histological analysis showed new bone formation at the bone block-tunnel interface, with maintenance of the native ACL enthesis. Ultrastructural analysis demonstrated the maintenance of overall collagen matrix alignment, while there was repopulation with smaller diameter collagen fibrils. There was no significant difference between 4 and 8 weeks in mean failure force (p = 0.39) or stiffness (p = 0.15) for the B-ACL-B allografts. This study demonstrates the restoration of the normal anatomy of the ACL and progressive graft incorporation and remodeling using a B-ACL-B allograft for ACL reconstruction in the rabbit knee.

Evaluation of sex differences in rodent anterior cruciate ligament injury.

The relative contributions of sex differences in anatomy, biomechanics, and hormones to the increased risk of anterior cruciate ligament (ACL) injury in female athletes remains unknown. The purpose of this study is to investigate sex differences in anatomy and biomechanics of the native and reconstructed ACL using our established murine model. A total of 140 12-week-old wild-type C57Bl/6 (70 male vs. 70 female) mice were used for this study. ACL reconstruction was performed on 120 mice who were split into four groups: Group 1 (30 males sacrificed at 14 days), Group 2 (30 females sacrificed at 14 days), Group 3 (30 males sacrificed at 28 days), and Group 4 (30 females sacrificed at 28 days). Tendon graft-to-bone healing was assessed by biomechanical, histological, and micro-CT analysis. Twenty mice were used for baseline testing. Females showed significantly higher anterior (p < 0.05) and total displacement (p < 0.05). Males demonstrated a significantly higher load-to-failure force of native ACLs compared to females (p < 0.05). There was no significant difference in load-to-failure force in the ACL autograft. There were no significant sex differences in histological analysis of graft integration or tibial slope. The increased knee laxity and reduced load-to-failure of the native ACL observed in the female mice are consistent with some of the proposed risk factors driving the increased risk of ACL injury in females. Understanding the relative contributions of factors driving sex differences in material properties of the ACL will provide insight into the sex differences in ACL injury and future prevention strategies.

Xianglian pill modulates gut microbial production of succinate and induces regulatory T cells to alleviate ulcerative colitis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Xianglian pill (XLP), a traditional Chinese formula, is widely used as treatment for ulcerative colitis (UC) in China. However, the mechanism of its therapeutic effect is still unclear. AIM OF THE STUDY: Our previous studies showed a low oral bioavailability and a predominant distribution of major XLP ingredients in the gut. In the present study, we aimed to explore the mechanism of action of XLP on UC with respect to the regulation of gut microecology. MATERIALS AND METHODS: UC model rats established using 5% dextran sulfate sodium were treated with XLP. After the treatment period, bodyweight, colon length, histopathology, and inflammatory changes were evaluated. Further, changes in gut microbiota structure were detected via 16S rRNA sequencing, and microbial metabolites in feces were analyzed via a metabolomic assay. Antibiotic intervention and fecal microbiota transplantation were also employed to explore the involvement of gut microbiota, while the level of regulatory T cells (Tregs) in mesenteric lymph nodes was determined via flow cytometry. Transcriptome sequencing was also performed to determine colonic gene changes. RESULTS: XLP alleviated colonic injury, inflammation, and gut microbial dysbiosis in UC model rats and also changed microbial metabolite levels. Particularly, it significantly decreased succinate level in the tyrosine pathway. We also observed that fecal microbiota derived from XLP-treated rats conferred resilience to UC model rats. However, this therapeutic effect of XLP on UC was inhibited by succinate. Moreover, XLP increased the level of anti-inflammatory cellular Tregs via gut microbiota. However, this beneficial effect was counteracted by succinate supplementation. Further, XLP induced the differentiation of Treg possibly by the regulation of the PHD2/HIF-1α pathway via decreasing microbial succinate production. CONCLUSIONS: Our findings indicated that XLP exerts its therapeutic effects on UC mainly via the gut microbiota-succinate-Treg differentiation axis.

GLI1 Deficiency Impairs the Tendon-Bone Healing after Anterior Cruciate Ligament Reconstruction: In Vivo Study Using Gli1-Transgenic Mice.

Hedgehog (Hh) signaling plays a fundamental role in the enthesis formation process and GLI-Kruppel family member GLI1 (Gli1) is a key downstream mediator. However, the role of Gli1 in tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) is unknown. To evaluate the tendon-bone healing after ACLR in Gli1LacZ/LacZ (GLI1-NULL) mice, and compare Gli1LacZ/WT (GLI1-HET) and Gli1WT/WT wild type (WT) mice, a total of 45 mice, 15 mice each of GLI1-NULL, GLI1-HET and WT were used in this study. All mice underwent microsurgical ACLR at 12 weeks of age. Mice were euthanized at 4 weeks after surgery and were used for biomechanical testing, histological evaluation, and micro-CT analysis. The GLI1-NULL group had significantly lower biomechanical failure force, poorer histological healing, and lower BV/TV when compared with the WT and GLI1-HET groups. These significant differences were only observed at the femoral tunnel. Immunohistology staining showed positive expression of Indian hedgehog (IHH) and Patched 1(PTCH1) in all three groups, which indicated the activation of the Hh signal pathway. The GLI1 was negative in the GLI1-NULL group, validating the absence of GLI1 protein in these mice. These results proved that activation of the Hh signaling pathway occurs during ACL graft healing, and the function of Gli1 was necessary for tendon-bone healing. Healing in the femoral tunnel is more obviously impaired by Gli1 deficiency. Our findings provide further insight into the molecular mechanism of tendon-bone healing and suggest that Gli1 might represent a novel therapeutic target to improve tendon-bone healing after ACLR.

A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction.

We designed and validated a novel device for applying flexion-extension cycles to a rat knee in an in vivo model of anterior cruciate ligament reconstruction (ACL-R). Our device is intended to simulate rehabilitation motion and exercise post ACL-R to optimize physical rehabilitation treatments for the improved healing of tendon graft ligament reconstructions. The device was validated for repeatability of the knee kinematic motion by measuring the force versus angular rotation response from repeated trials using cadaver rats. The average maximum force required for rotating an ACL reconstructed rat knee through 100 degrees of flexion-extension was 0.4 N with 95% variability for all trials within ±0.1 N.

Role of coracoacromial ligament and related structures in glenohumeral stability: a cadaveric study.

This study sought to determine the role of the coracoacromial ligament and related arch structures in glenohumeral joint stabilization. Eight fresh-frozen cadaver specimens were tested at multiple angles of glenohumeral abduction and rotation for translations (in the direction of and perpendicular to a 50-N force) in intact, vented shoulders and after three interventions: coracoacromial veil release, coracoacromial ligament release, and anterior acromioplasty. After releasing the veil, an inferior force significantly increased inferior translation at lower angles of abduction with no additional increase after coracoacromial ligament section or acromioplasty. After ligament release or acromioplasty, a superior force increased superior translation at all angles. Few increases in anterior or posterior translations were observed. The coracoacromial veil interacts with the structures of the coracoacromial arch and glenohumeral capsule to limit inferior humeral translation. Likewise, the coracoacromial ligament and the acromian serve to limit superior translation. Attempts to preserve these structures may help improve surgical outcomes.

SS-31 as a Mitochondrial Protectant in the Treatment of Tendinopathy: Evaluation in a Murine Supraspinatus Tendinopathy Model.

BACKGROUND: Prior studies have demonstrated mitochondrial dysfunction in tendinopathy. The objective of this investigation was to explore the potential of SS-31 (elamipretide), a mitochondrial protectant, to improve mitochondrial function and promote tendon healing in a murine supraspinatus tendinopathy model. METHODS: One hundred and twenty-six mice (252 limbs) were divided into 6 groups (42 limbs/group) that received (I) 4 weeks of impingement; (II) 8 weeks of impingement; (III) 8 weeks of impingement including 4 weeks of SS-31 treatment (5 mg/kg/d) starting after 4 weeks of impingement; (IV) 4 weeks of impingement ending with clip removal, followed by harvesting 4 weeks later; and (V) 4 weeks of impingement ending with clip removal, followed by 4 weeks of SS-31 treatment and harvesting; and a control group. Specimens were prepared for biomechanical testing, histological analysis, transmission electron microscopy, measurement of superoxidative dismutase (SOD) activity, and measurement of gene expression. RESULTS: Failure force decreased after impingement, compared with the intact tendon, and the decrease was partially reversed after clip removal, SS-31 treatment, and the 2 treatments combined. A similar pattern was observed for stiffness. Histological analysis demonstrated higher modified Bonar scores in the impingement groups; however, the changes in tendon morphology were partially reversed following all treatments, especially the combined treatment. Decreased mitochondrial number and altered organization and density of cristae were observed in the impingement groups. Mitochondrial structure and number became more normal, with improvement in morphology of the cristae, after clip removal and/or SS-31 treatment. SOD activity decreased after impingement, compared with the control group, then increased significantly again after treatment, especially in the combined treatment group. Mitochondria-related gene expression decreased in the impingement groups and increased again after treatment. CONCLUSIONS: The mitochondrial protectant SS-31 improved mitochondrial function, promoting tendon healing, especially when combined with removal of subacromial impingement. CLINICAL RELEVANCE: Improving mitochondrial function with agents such as SS-31 may represent an effective treatment to promote healing in the setting of supraspinatus tendinopathy.

The Role of Indian Hedgehog Signaling in Tendon Response to Subacromial Impingement: Evaluation Using a Mouse Model.

BACKGROUND: The underlying cellular and molecular mechanisms involved in the development of tendinopathy due to subacromial supraspinatus tendon (SST) impingement and the response to subsequent removal of impingement remain unknown. PURPOSE: To investigate the involvement of Indian hedgehog (IHH) signaling in the development of SST tendinopathy and the subsequent healing process after the relief of subacromial impingement in a novel mouse shoulder impingement model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 male wild-type C57BL/6 mice were used in this study. Supraspinatus tendinopathy was induced by inserting a microsurgical clip into the subacromial space bilaterally. Eleven mice were sacrificed at 4 weeks after surgery to establish impingement baseline; 24 mice underwent clip removal at 4 weeks after surgery and then were euthanized at 2 or 4 weeks after clip removal. Thirteen mice without surgical intervention were utilized as the control group. All SSTs were evaluated with biomechanical testing; quantitative histomorphometry after staining with hematoxylin and eosin, Alcian blue, and picrosirius red; and immunohistochemical staining (factor VIII, IHH, Patched1 [PTCH1], and glioma-associated oncogene homolog 1 [GLI1]). RESULTS: The mean failure force and stiffness in the 4-week impingement group decreased significantly compared with the control group (P < .001) and gradually increased at 2 and 4 weeks after clip removal. Histological analysis demonstrated increased cellularity and disorganized collagen fibers in the SST, with higher modified Bonar scores at 4 weeks, followed by gradual improvement after clip removal. The IHH-positive area and PTCH1- and GLI1-positive cell percentages significantly increased after 4 weeks of clip impingement (20.64% vs 2.06%, P < .001; 53.9% vs 28.03%, P = .016; and 30% vs 12.19%, P = .036, respectively) and continuously increased after clip removal. CONCLUSION: The authors' findings suggest that the hedgehog signaling pathway and its downstream signaling mediator and target GLI1 may play a role in the development and healing process of rotator cuff tendinopathy due to extrinsic rotator cuff impingement. CLINICAL RELEVANCE: This study suggests the potential for the hedgehog pathway, together with its downstream targets, as candidates for further study as potential therapeutic targets in the treatment of supraspinatus tendinopathy.

Transforming growth factor-ß receptors mediates matrix degradation and abnormal hypertrophy in T-2 toxin-induced hypertrophic chondrocytes.

This study investigated whether transforming growth factor-ß receptor I (TGF-ßRI) and TGF-ßRII mediate matrix degradation and abnormal hypertrophy in T-2 toxin-induced hypertrophic chondrocytes. Hypertrophic chondrocytes were exposed to TGF-ßRI and TGF-ßRII binding inhibitor (GW788388) for 24 h prior to exposure to different concentrations of T-2 toxin (0, 10, 25, and 50 ng/mL for 48 h). Hypertrophic chondrocytes were assessed based on the expression of matrix-degrading and terminal differentiation-related genes and cell viability. Matrix metalloproteinases (MMPs, MMP-13, MMP-1, and MMP-9) were reduced in the GW788388+T-2 toxin group compared to the T-2 toxin group. The expression of terminal differentiation-related genes (MMP-2, MMP-10, and collagen X) was increased in hypertrophic chondrocytes in the inhibited groups compared to that in the T-2 toxin group. The survival rate of chondrocytes decreased significantly in a dose-dependent manner. GW788388 did not significantly block the reduced cell viability in hypertrophic chondrocytes exposed to T-2 toxin. The upregulated expression of TGF-ßRI and TGF-ßRII mediates the abnormal chondrocyte hypertrophy and extracellular matrix degeneration observed in T-2 toxin-induced hypertrophic chondrocytes.