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.

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.

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.

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.

Achilles Tendons Display Region-Specific Transcriptomic Signatures Associated With Distinct Mechanical Properties.

BACKGROUND: Previous studies have examined the transcriptomes and mechanical properties of whole tendons in different regions of the body. However, less is known about these characteristics within a single tendon. PURPOSE: To develop a regional transcriptomic atlas and evaluate the region-specific mechanical properties of Achilles tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Achilles tendons from 2-month-old male Sprague Dawley rats were used. Tendons were isolated and divided into proximal, middle, and distal thirds for RNA sequencing (n = 5). For mechanical testing, the Achilles muscle-tendon-calcaneus unit was mounted in a custom-designed materials testing system with the unit clamped over the musculotendinous junction (MTJ) and the calcaneus secured at 90° of dorsiflexion (n = 9). Tendons were stretched to 20 N at a constant speed of 0.0167 mm/s. Cross-sectional area, strain, stress, and Young modulus were determined in each tendon region. RESULTS: An open-access, interactive transcriptional atlas was generated that revealed distinct gene expression signatures in each tendon region. The proximal and distal regions had the largest differences in gene expression, with 2596 genes significantly differentially regulated at least 1.5-fold (q < .01). The proximal tendon displayed increased expression of genes resembling a tendon phenotype and increased expression of nerve cell markers. The distal region displayed increases in genes involved in extracellular matrix synthesis and remodeling, immune cell regulation, and a phenotype similar to cartilage and bone. There was a 3.72-fold increase in Young modulus from the proximal to middle region (P < .01) and an additional 1.34-fold increase from the middle to distal region (P = .027). CONCLUSION: Within a single tendon, there are region-specific transcriptomic signatures and mechanical properties, and there is likely a gradient in the biological and functional phenotype from the proximal origin at the MTJ to the distal insertion at the enthesis. CLINICAL RELEVANCE: These findings improve our understanding of the underlying biological heterogeneity of tendon tissue and will help inform the future targeted use of regenerative medicine and tissue engineering strategies for patients with tendon disorders.

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.

Distinct Inflammatory Macrophage Populations Sequentially Infiltrate Bone-to-Tendon Interface Tissue After Anterior Cruciate Ligament (ACL) Reconstruction Surgery in Mice.

Macrophages are important for repair of injured tissues, but their role in healing after surgical repair of musculoskeletal tissues is not well understood. We used single-cell RNA sequencing (RNA-seq), flow cytometry, and transcriptomics to characterize functional phenotypes of macrophages in a mouse anterior cruciate ligament reconstruction (ACLR) model that involves bone injury followed by a healing phase of bone and fibrovascular interface tissue formation that results in bone-to-tendon attachment. We identified a novel "surgery-induced" highly inflammatory CD9+ IL1+ macrophage population that expresses neutrophil-related genes, peaks 1 day after surgery, and slowly resolves while transitioning to a more homeostatic phenotype. In contrast, CX3CR1+ CCR2+ macrophages accumulated more slowly and unexpectedly expressed an interferon signature, which can suppress bone formation. Deletion of Ccr2 resulted in an increased amount of bone in the surgical bone tunnel at the tendon interface, suggestive of improved healing. The "surgery-induced macrophages" identify a new cell type in the early phase of inflammation related to bone injury, which in other tissues is dominated by blood-derived neutrophils. The complex patterns of macrophage and inflammatory pathway activation after ACLR set the stage for developing therapeutic strategies to target specific cell populations and inflammatory pathways to improve surgical outcomes. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Evaluation of SS-31 as a Potential Strategy for Tendinopathy Treatment: An In Vitro Model.

BACKGROUND: Studies in our laboratory have demonstrated mitochondrial dysfunction in human and animal models of supraspinatus tendinopathy. SS-31 (elamipretide) has been reported to improve mitochondrial function and to be effective in clinical trials for several diseases. The potential of SS-31 in treating tendinopathy has not been explored. HYPOTHESIS: SS-31 would improve mitochondrial function in human tenocytes sampled from patients with tendinopathy. STUDY DESIGN: Controlled laboratory study. METHODS: Healthy tenocytes were obtained from normal hamstring tendon biopsy specimens in 9 patients undergoing anterior cruciate ligament reconstruction, and tenocytes were collected from degenerative supraspinatus tendon biopsy specimens in 9 patients undergoing rotator cuff repair. Tenocytes were cultured, used at passage 1, and assigned to 4 groups: healthy tenocytes, healthy tenocytes with 1µM SS-31 treatment for 72 hours, degenerative tenocytes, and degenerative tenocytes with 1µM SS-31 treatment for 72 hours. The outcomes included measurements of mitochondrial potential, mitochondrial morphology by transmission electron microscopy imaging, reactive oxygen species and superoxidative dismutase activity, gene expression, and cell viability. RESULTS: An increase in the cell fraction with depolarized mitochondria was found in degenerative tenocytes (P = .014), followed by a decrease after SS-31 treatment (P = .018). Transmission electron microscopy images demonstrated morphological changes with a decreased number and size of mitochondria per cell in the degenerative tenocytes (P = .018) and with improvement after SS-31 treatment. There was no significant difference in the level of reactive oxygen species between healthy and degenerative tenocytes in culture, but superoxidative dismutase activity was significantly decreased in the degenerative group (P = .006), which then increased after SS-31 treatment (P = .012). These findings suggested that mitochondrial dysfunction may be reversed by SS-31 treatment. The gene expression of matrix metalloproteinase-1 (matrix remodeling, P = .029) and fatty acid-binding protein 4 (fatty infiltration, P = .046) was significantly upregulated in the degenerative tenocytes and reduced by SS-31 treatment (P = .048; P = .007). Gene expression for hypoxia-inducible factor1 α and the proapoptotic regulator Bcl-2-associated X protein was increased in the degenerative tenocytes. There was a significant decrease in cell viability in degenerative tenocytes as compared with the healthy tenocytes, with small improvement after treatment with SS-31. CONCLUSION: There are changes in mitochondrial structure and function in tenocytes derived from degenerative tendons, and SS-31, as a mitochondrial protectant, could improve mitochondrial function and promote the healing of tendinopathy. CLINICAL RELEVANCE: Mitochondrial dysfunction appears to play a role in the development of tendinopathy, and SS-31, as a mitochondrial protective agent, may be a therapeutic agent in the treatment of tendinopathy.

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.

Use of small animal PET-CT imaging for in vivo assessment of tendon-to-bone healing: A pilot study.

BACKGROUND: The availability of non-invasive means to evaluate and monitor tendon-bone healing processes in-vivo is limited. Micro Positron-Emission-Tomography (µPET) using 18F-Fluoride is a minimally invasive imaging modality, with which osteoblast activity and bone turnover can be assessed. The aim of this study was to investigate the use of serial in-vivo µPET/CT scans to evaluate bone turnover along the graft-tunnel interface in a rat ACL (anterior cruciate ligament) reconstruction model. METHODS: Unilateral autograft ACL reconstruction was performed in six rats. µPET/CT-scans using 18F-Fluoride were performed 7, 14, 21, and 28 days postoperatively. Standard uptake values (SUV) were calculated for three tunnel regions (intraarticular aperture (IAA), mid-tunnel, and extraarticular aperture (EAA)) of the proximal tibia. Animals were sacrificed at 28 days and evaluated with µCT and histological analysis. RESULTS: SUVs in both bone tunnels showed an increased 18F-Fluoride uptake at 7 days when compared to 14, 21, and 28 days. SUVs showed a gradient on the tibial side, with most bone turnover in the IAA and least in the EAA. At 7, 14, 21, and 28 days, there were significantly higher SUV values in the IAA compared to the EAA (p = .01, < .01, < .01, < .01). SUVs positively correlated with new bone volumetric density obtained with µCT (r = 0.449, p = .013). Volumetric density of newly formed bone detected on µCT correlated with osteoblast numbers observed along the tunnels in histological sections (r = 0.452, p < .016). CONCLUSIONS: Serial in-vivo µPET/CT-scanning has the potential to provide insight into bone turnover and therefore osteoblastic activity during the healing process. As a result, it allows us to directly measure the effect of interventional strategies in tendon-bone healing.

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.

In Vivo Imaging of Fibroblast Activity Using a 68Ga-Labeled Fibroblast Activation Protein Alpha (FAP-α) Inhibitor: Study in a Mouse Rotator Cuff Repair Model.

BACKGROUND: Rotator cuff repair site failure is a well-established clinical concern. Tendon-to-bone healing is initiated by inflammatory mediators followed by matrix synthesis by fibroblasts. The kinetics of fibroblast accumulation and activity are currently poorly understood. METHODS: Ninety-six mice underwent supraspinatus tendon repair. Six were used for imaging using a novel 68Gallium (Ga)-labeled fibroblast activation protein alpha (FAP-α) inhibitor and positron emission tomography-computed tomography (PET/CT) at days 0 (before surgery), 3, 7, 14, and 28. Sixty-eight animals were divided into 4 groups to be evaluated at 3, 7, 14, or 28 days. Twenty-two native shoulders from mice without surgery were used as the control group (intact tendon). Six animals from each group were used for histological analysis; 6 from each group were used for evaluation of fibroblastic response-related gene expression; and 10 mice each from the intact, 14-day, and 28-day groups were used for biomechanical testing. RESULTS: There was minimal localization of 68Ga-labeled FAP-α inhibitor in the shoulders at day 0 (before surgery). There was significantly increased uptake in the shoulders with surgery compared with the contralateral sides without surgery at 3, 7, and 14 days. 68Ga-labeled FAP-α inhibitor uptake in the surgically treated shoulders increased gradually and peaked at 14 days followed by a decrease at 28 days. Gene expression for smooth muscle alpha (α)-2 (acta2), FAP-α, and fibronectin increased postsurgery followed by a drop at 28 days. Immunohistochemical analysis showed that FAP-α-positive cell density followed a similar temporal trend, peaking at 14 days. All trends matched closely with the PET/CT results. Biomechanical testing demonstrated a gradual increase in failure load during the healing process. CONCLUSIONS: 68Ga-labeled FAP-α inhibitor PET/CT allows facile, high-contrast in vivo 3-dimensional imaging of fibroblastic activity in a mouse rotator cuff repair model. CLINICAL RELEVANCE: Noninvasive imaging of activated fibroblasts using labeled radiotracers may be a valuable tool to follow the progression of healing at the bone-tendon interface.

The Effects of Tensioning of the Anterior Cruciate Ligament Graft on Healing after Soft Tissue Reconstruction.

The purpose of this study is to determine the effect of the magnitude of static mechanical tension on the anterior cruciate ligament (ACL) graft at the time of surgery on healing within the graft tunnels. Ninety male rats underwent unilateral ACL resection followed by reconstruction with a soft tissue tendon autograft. The ACL graft mechanical environment was modulated by different ACL graft pretension levels at the time of surgery (no pretension: 0N; moderate tension: 5N; over tension: 10N). External fixators were used to eliminate graft and joint motion during cage activity. Graft-tunnel healing was assessed at 3- and 6-week postoperatively, and articular joint surfaces were assessed at 9 weeks. Our results demonstrate that the ACL graft-tunnel healing was sensitive to different static graft pretension levels as demonstrated by different load-to-failure and stiffness properties among the different pretension levels. Pretensioning the graft to 5N (7-8% of the rat ACL ultimate load to failure) resulted in the best graft-tunnel healing as shown by higher graft-tunnel failure load and stiffness. Higher bone volume fraction was also seen in the 5N group relative to other pretension levels. Histological analysis of the graft-tunnel interface revealed differences in cellularity of the ACL graft between the 5N group and the other two groups. Furthermore, the highest graft pretension level (10N) resulted in loss of proteoglycan content among articular joint surfaces. In conclusion, we found that ACL graft-tunnel healing is sensitive to the magnitude of graft pretension at the time of surgery in a preclinical model of ACL reconstruction with joint immobilization. The combination of high-graft tension and immobilization is also deleterious for the articular surface. Further study is necessary to understand the interaction between the magnitude of graft tensioning and joint motion.

Assessment of Mitochondrial Dysfunction in a Murine Model of Supraspinatus Tendinopathy.

BACKGROUND: The purpose of this study was to assess mitochondrial dysfunction in a murine model of supraspinatus tendinopathy. METHODS: Eighty-four mice (168 limbs) were included in the study. Supraspinatus tendinopathy was induced by inserting a microsurgical clip in the subacromial space of 63 mice bilaterally (126 limbs). Forty-two of these limbs were harvested at 4 weeks postoperatively, 42 underwent clip removal at 4 weeks after the initial procedure and were harvested at 2 weeks, and 42 underwent clip removal at 4 weeks and were harvested at 4 weeks. Forty-two limbs in the remaining 21 mice did not undergo surgical intervention and were utilized as the control group. Outcomes included biomechanical, histological, gene expression, superoxide dismutase (SOD) activity, and transmission electron microscopy (TEM) analyses. RESULTS: Radiographs confirmed stable clip position in the subacromial space at 4 weeks. Biomechanical testing demonstrated a 60% decrease in failure force of the supraspinatus tendons at 4 weeks compared with the control group. The failure force gradually increased at 2 and 4 weeks after clip removal. Histological analysis demonstrated inflammation surrounding the tendon with higher modified Bonar scores at 4 weeks after clip placement followed by gradual improvement following clip removal. The expression of mitochondrial-related genes was decreased at 4 weeks after clip placement and then significantly increased after clip removal. SOD activity decreased significantly at 4 weeks after clip placement but increased following clip removal. TEM images demonstrated alterations in morphology and the number of mitochondria and cristae at 4 weeks after clip placement with improvement after clip removal. CONCLUSIONS: Mitochondrial dysfunction appears to be associated with the development of tendinopathy. CLINICAL RELEVANCE: Mitochondrial protection may offer a potential strategy for delaying the development of tendinopathy and promoting tendon healing.

Chronic subacromial impingement leads to supraspinatus muscle functional and morphological changes: Evaluation in a murine model.

Muscle atrophy and fatty infiltration have been directly correlated with higher rates of incomplete or failed healing following surgical repair of the rotator cuff. The purpose of this study was to evaluate clinically relevant functional and morphological changes in the supraspinatus muscle at various time points in this model of rotator cuff tendinopathy. Subacromial impingement was induced in 47, male C57BL/6 mice (total 94 limbs) by implantation of a metal clip in the subacromial space. Specimens were evaluated at 4, 6, and 12 weeks postoperatively. Gait analysis was used to measure various kinematic parameters. Supraspinatus muscle wet weight, histology, and quantitative reverse-transcription polymerase chain reaction analysis of genes related to muscle atrophy and adipogenesis were performed to characterize the structural, cellular, and molecular changes. Muscle atrophy and fatty infiltration was evident beginning at 6 weeks, with progression out to 12 weeks. Gait analysis identified significant functional changes in many aspects of gait and abnormal stance tracing as early as 4 weeks, verifying alterations in upper extremity function. We have demonstrated that clinically relevant changes to the supraspinatus muscle are seen starting 6 weeks after induction of subacromial impingement. Furthermore, the gait analysis provides key functional outcome measurements that may be useful for future evaluation of new therapeutic strategies.

Expression of alarmins in a murine rotator cuff tendinopathy model.

The aim of this study was to investigate the presence of alarmins in a novel murine rotator cuff tendinopathy model. Alarmins have been described as essential early activators of an immune response to tissue damage. Subacromial impingement was induced in both shoulders of 37 male C57Bl/6 mice by placement of a small metal clip in the subacromial space. Animals were allocated to different time points up to 6 weeks. The morphology and cellularity of the supraspinatus tendon were evaluated by hematoxylin-eosin staining, alcian blue, and picrosirius red. The expression and localization of alarmins interleukin-33 (IL-33), c (HMGB1), hypoxia-inducible factor-1 subunit α (HIF1α), and S100A9 were evaluated by immunohistochemical staining and quantitative polymerase chain reaction. The percentage of positively stained cells with HMGB1 and IL-33 was significantly increased in the impingement group at 1w, 4w, and 6w. HIF1α staining was higher in the impingement group at 1w and 6w compared with the control group. HMGB1 gene expression was higher in the 5d impingement group and 6w impingement group. The gene expression of HIF1α was upregulated at all-time points in the impingement group (5d, 2w, 4w, and 6w). The expression of the S100A9 gene was also upregulated in the 5d impingement group. This is the first study to demonstrate the involvement of alarmins in the early phase of tendinopathy using a reproducible animal model. Alarmins may play an important role in the early phases of the development of tendinopathy They may represent potential therapeutic targets for treatment of tendinopathy.

Effect of Lubricin Mimetics on the Inhibition of Osteoarthritis in a Rat Anterior Cruciate Ligament Transection Model.

BACKGROUND: Lubricin, a mucinous glycoprotein, plays a chondroprotective role as a constituent of synovial fluid. Structural analogs have been synthesized to mimic the structure and function of native lubricin in an effort to recapitulate this effect with the goal of delaying progression of osteoarthritis (OA). PURPOSE: To investigate the efficacy of intra-articular injections of lubricin mimetics in slowing or preventing the progression of posttraumatic OA by using a rat anterior cruciate ligament transection model. STUDY DESIGN: Controlled laboratory design. METHODS: Four lubricin mimetics were investigated, differing from one another in their binding orientations and steric interactions. Eighty skeletally mature Sprague-Dawley rats underwent bilateral anterior cruciate ligament transections and were randomly allocated to receive intra-articular injections (50 µL/injection) of 1 of the 4 mimetics in the right knee and equal volumes of saline injection in the contralateral knee (control). All rats were euthanized 8 weeks postoperatively and assessed via biomechanical analysis, which evaluated comparative friction coefficients across the 4 groups, and histological evaluation of articular cartilage, osteophytes, and synovitis. The Osteoarthritis Research Society International (OARSI) histopathological assessment system was used to evaluate the degree of articular cartilage degeneration and osteophytes, while synovitis was assessed through a semiquantitative scoring system. Binding efficacy of the 4 mimetics was assessed in vitro and in vivo through the immunohistochemical localization of polyethylene glycol. Articular cartilage degeneration and synovitis scoring data analyses were performed with generalized estimating equation modeling. RESULTS: Injection of the group 3 mimetic (random 24 + 400 + 30) directly correlated with improved OARSI scores for femoral articular cartilage degeneration when compared with saline-injected contralateral control knees (P = .0410). No lubricin mimetic group demonstrated statistically significant differences in OARSI scores for tibial articular cartilage degeneration. Injection of the group 4 mimetic (AB 24 + 400 + 30) led to a statistically significant difference in osteophyte OARSI score (P = .0019). None of the 4 lubricin mimetics injections incited an additive synovial inflammatory response. Immunohistochemical staining substantiated the binding capacity of all 4 mimetics, while in vivo experimentation revealed that the group 1 and 3 mimetics were still retained within the joint 4 weeks after injection. There were no differences in friction coefficients between any pair of groups and no significant trends based on lubricin mimetic structure. CONCLUSION: We demonstrated that the tribosupplementation of a traumatically injured knee with a specific lubricin structural analog may attenuate the natural progression of OA. CLINICAL RELEVANCE: The current lack of efficacious clinical options to counter the onset and subsequent development of OA suggests that further investigation into the synthesis and behavior of lubricin analogs could yield novel translational applications.

Matrix Metalloproteinase Inhibition With Doxycycline Affects the Progression of Posttraumatic Osteoarthritis After Anterior Cruciate Ligament Rupture: Evaluation in a New Nonsurgical Murine ACL Rupture Model.

BACKGROUND: Doxycycline has broad-spectrum activity as a matrix metalloproteinase (MMP) inhibitor and thus could reduce the progression of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) rupture. HYPOTHESIS: Doxycycline would inhibit progression of PTOA in a murine ACL rupture model. STUDY DESIGN: Controlled laboratory study. METHODS: For the in vitro study, cadaveric C57BL/6 male mice knees (N = 108) were used for the development of a nonsurgical ACL rupture model. For the in vivo study, 24 C57BL/6 male mice then underwent ACL rupture with our manual procedure and were divided into 4 groups: untreated control; doxycycline, 10 mg/kg/d; doxycycline, 50 mg/kg/d; and doxycycline, 100 mg/kg/d. Doxycycline was administered in drinking water beginning immediately after ACL rupture. Radiographic imaging and paw prints were evaluated at 3, 7, 14, and 28 days. The foot length and toe spread were analyzed as measures of function. Histology and MMP-13 immunohistochemistry were done at 4 weeks. RESULTS: Radiographs demonstrated anterior tibial subluxation and meniscal extrusion after ACL rupture, confirming knee joint instability without fractures. Statistically significant differences in gait were found between the intact and experimental groups. Histologic examination demonstrated cartilage damage, meniscal tears, and mild osteoarthritis after ACL rupture, similar to what occurs in human patients. Hypertrophy of the posterior horn of the medial and lateral meniscus was found, and tears of the posterior horn of the menisci were common. All doxycycline groups had a lower score than the untreated control group, indicating less cartilage damage. The posterior tibia of the untreated group had the most cartilage damage as compared with the 3 doxycycline groups, with a significant difference between the untreated and 50-mg/kg/d doxycycline groups, suggesting that the latter dose may protect against proteoglycan loss and decrease the progression of osteoarthritis. The nondoxycycline group had the highest synovial inflammation score among all groups, indicating that doxycycline has an inhibitory effect on synovitis. There was significantly lower MMP-13 expression on the tibia in the doxycycline-treated groups, with a positive correlation between doxycycline concentration and MMP-13 inhibition. CONCLUSION: Modulation of MMP-13 activity by doxycycline treatment may offer a novel biological pathway to decrease the progression of PTOA after ACL rupture. CLINICAL RELEVANCE: Doxycycline is an approved, readily available drug with infrequent side effects of photosensitivity and gastrointestinal symptoms. Future clinical trials could evaluate doxycycline to reduce or prevent progressive cartilage damage after ACL rupture.

Identification of Inflammatory Mediators in Tendinopathy Using a Murine Subacromial Impingement Model.

Subacromial impingement is associated with a spectrum of disorders-including rotator cuff disease-but their relationship is complex. We have established a novel murine model of subacromial impingement to study supraspinatus tendinopathy. The purpose of this study was to evaluate changes in gene expression in this murine shoulder impingement model to further elucidate the mechanisms underlying the development of tendinopathy. Twenty-eight C57BL/6 mice were used in this study. All mice underwent bilateral surgery with insertion of a small metal clip in the subacromial space or a sham procedure. The supraspinatus tendons underwent histological analyses, biomechanical testing, and RNA extraction for multiplex gene expression analysis (NanoString, Seattle, WA). Histology demonstrated increased cellularity and disorganized collagen fibers of the supraspinatus tendon in the clip impingement group. Mean load to failure (5.20 vs. 1.50 N, p < 0.001) and mean stiffness (4.95 vs. 1.47 N/mm, p < 0.001) were lower in the impingement group than the sham group. NanoString analyses revealed 111 differentially expressed genes (DEGs) between the impingement and sham groups. DEGs of interest included Mmp3 (expression ratio [ER]: 2.68, p = 0.002), Tgfb1 (ER: 1.76, p = 0.01), Col3a1 (ER: 1.66, p = 0.03), and Tgfbr2 (ER: 1.53, p = 0.01). Statement of clinical significance: We identified 111 DEGs that may contribute to the development of tendinopathy in this model. Further studies of these specific genes will allow identification of their roles in the initiation and regulation of tendon damage, and their potential to serve as novel therapeutic targets in the treatment of rotator cuff disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2575-2582, 2019.