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.

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.

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.

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.

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.

Widespread diversity in the transcriptomes of functionally divergent limb tendons.

KEY POINTS: Tendon is a hypocellular, matrix-rich tissue that has been excluded from comparative transcriptional atlases. These atlases have provided important knowledge about biological heterogeneity between tissues, and our study addresses this important gap. We performed measures on four of the most studied tendons, the Achilles, forepaw flexor, patellar and supraspinatus tendons of both mice and rats. These tendons are functionally distinct and are also among the most commonly injured, and therefore of important translational interest. Approximately one-third of the filtered transcriptome was differentially regulated between Achilles, forepaw flexor, patellar and supraspinatus tendons within either mice or rats. Nearly two-thirds of the transcripts that are expressed in anatomically similar tendons were different between mice and rats. The overall findings from this study identified that although tendons across the body share a common anatomical definition based on their physical location between skeletal muscle and bone, tendon is a surprisingly genetically heterogeneous tissue. ABSTRACT: Tendon is a functionally important connective tissue that transmits force between skeletal muscle and bone. Previous studies have evaluated the architectural designs and mechanical properties of different tendons throughout the body. However, less is known about the underlying transcriptional differences between tendons that may dictate their designs and properties. Therefore, our objective was to develop a comprehensive atlas of the transcriptome of limb tendons in adult mice and rats using systems biology techniques. We selected the Achilles, forepaw digit flexor, patellar, and supraspinatus tendons due to their divergent functions and high rates of injury and tendinopathies in patients. Using RNA sequencing data, we generated the Comparative Tendon Transcriptional Database (CTTDb) that identified substantial diversity in the transcriptomes of tendons both within and across species. Approximately 30% of filtered transcripts were differentially regulated between tendons of a given species, and nearly 60% of the filtered transcripts present in anatomically similar tendons were different between species. Many of the genes that differed between tendons and across species are important in tissue specification and limb morphogenesis, tendon cell biology and tenogenesis, growth factor signalling, and production and maintenance of the extracellular matrix. This study indicates that tendon is a surprisingly heterogenous tissue with substantial genetic variation based on anatomical location and species.

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.

Adaptive and innate immune cell responses in tendons and lymph nodes after tendon injury and repair.

Tendon injuries are a common clinical condition with limited treatment options. The cellular components of the innate immune system, such as neutrophils and macrophages, have been studied in tendon injuries. However, the adaptive immune system, comprising specialized lymphocytes, plays an important role in orchestrating the healing of numerous tissues, but less is known about these cells in tendon healing. To gain a greater understanding of the biological processes that regulate tendon healing, we determined how the cellular components of the adaptive and innate immune system respond to a tendon injury using two-month-old male mice. We observed that lymphatic vasculature is present in the epitenon and superficial regions of Achilles tendons, and that the lymphatics drain into the popliteal lymph node. We then created an acute Achilles tenotomy followed by repair, and collected tendons and popliteal lymph nodes 1, 2, and 4 wk after injury. Tendon injury resulted in a robust adaptive immune cell response that followed an initial innate immune cell response in tendons and lymph nodes. Monocytes, neutrophils, and macrophages initially accumulated at 1 wk after injury in tendons, while dendritic cells and CD4+ T cells peaked at 2 wk after injury. B cells and CD8+ T cells progressively increased over time. In parallel, immune cells of the popliteal lymph node demonstrated a similarly coordinated response to the injury. These results suggest that there is an adaptive immune response to tendon injury, and adaptive immune cells may play a role in regulating tendon healing.NEW & NOTEWORTHY While the innate immune system, consisting of macrophages and related hematopoietic cells, has been studied in tendon injury, less is known about the adaptive immune system. Using a mouse model of Achilles tendon tenotomy and repair, we observed an adaptive immune cell response, consisting of CD4+ and CD8+ T cells, and B cells, which occur through 4 wk after tendon injury. This response appeared to be coordinated by the draining popliteal lymph node.

The MRL/MpJ Mouse Strain Is Not Protected From Muscle Atrophy and Weakness After Rotator Cuff Tear.

Chronic rotator cuff tears are a common source of shoulder pain and disability. Patients with rotator cuff tears often have substantial weakness, fibrosis, and fat accumulation, which limit successful surgical repair and postoperative rehabilitation. The Murphy Roths Large (MRL) strain of mice have demonstrated superior healing and protection against pathological changes in several disease and injury conditions. We tested the hypothesis that, compared with the commonly used C57Bl/6 (B6) strain, MRL mice would have less muscle fiber atrophy and fat accumulation, and be protected against the loss in force production that occurs after cuff tear. Adult male B6 and MRL mice were subjected to a rotator cuff tear, and changes in muscle fiber contractility and histology were measured. RNA sequencing and shotgun metabolomics and lipidomics were also performed. The muscles were harvested one month after tear. B6 and MRL mice had a 40% reduction in relative muscle force production after rotator cuff tear. RNA sequencing identified an increase in fibrosis-associated genes and a reduction in mitochondrial metabolism genes. The markers of glycolytic metabolism increased in B6 mice, while MRL mice appeared to increase amino acid metabolism after tear. There was an accumulation of lipid after injury, although there was a divergent response between B6 and MRL mice in the types of lipid species that accrued. There were strain-specific differences between the transcriptome, metabolome, and lipidome of B6 and MRL mice, but these differences did not protect MRL mice from weakness and pathological changes after rotator cuff tear. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:811-822, 2020.

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.

Long-term Evaluation of Meniscal Tissue Formation in 3-dimensional-Printed Scaffolds With Sequential Release of Connective Tissue Growth Factor and TGF-ß3 in an Ovine Model.

BACKGROUND: Artificial meniscal scaffolds are being developed to prevent development of osteoarthritis after meniscectomy. Previously, it was reported that 3-dimensional (3D) anatomic scaffolds loaded with connective tissue growth factor (CTGF) and transforming growth factor ß3 (TGF-ß3) achieved meniscal regeneration in an ovine model. This was a relatively short-term study (3 months postoperative), and outcome analyses did not include magnetic resonance imaging (MRI). PURPOSE: To evaluate long-term outcome of meniscal replacement with growth factor-laden poly-ε-caprolactone (PCL) scaffolds. STUDY DESIGN: Controlled laboratory study. METHODS: Anatomically shaped ovine meniscal scaffolds were fabricated from PCL with a 3D printer based on MRI data. Skeletally mature sheep (N = 34) were randomly allocated to 3 groups: scaffold without growth factor (0-µg group), scaffold with CTGF microspheres (µS) (5 µg) + TGF-ß3 µS (5 µg) (5-µg group), and scaffold with CTGF µS (10 µg) + TGF-ß3 µS (10 µg) (10-µg group). Unilateral medial meniscal replacement was performed. Animals were euthanized at 6 or 12 months. Regenerated meniscus, articular cartilage status, and synovial reaction were evaluated quantitatively with gross inspection, histology, and MRI. Kruskal-Wallis and Dunn tests were used to compare the 3 groups. RESULTS: Remnants of the PCL scaffold were evident in the 6-month specimens and were decreased but still present at 12 months in most animals. There were no significant differences among groups in gross inspection, histology, or MRI for either meniscal regeneration or articular cartilage protection. All experimental groups exhibited articular cartilage degeneration as compared with control (nonoperated). In terms of synovitis, there were no clear differences among groups, suggesting that growth factors did not increase inflammation and fibrosis. MRI revealed that meniscal extrusion was observed in most animals (82.7%). CONCLUSION: Previously, the combination of CTGF and TGF-ß3 was shown to stimulate mesenchymal stem cells into a fibrochondrocyte lineage. CTGF and TGF-ß3 did not aggravate synovitis, suggesting no adverse response to the combination of 3D-printed PCL scaffold combined with CTGF and TGF-ß3. Further work will be required to improve scaffold fixation to avoid meniscal extrusion. CLINICAL RELEVANCE: A significant advantage of this technique is the ability to print custom-fit scaffolds from MRI-generated templates. In addition, average-size menisci could be printed and available for off-the-shelf applications. Based on the 1-year duration of the study, the approach appears to be promising for meniscal regeneration in humans.

Use of Human Placenta-Derived Cells in a Preclinical Model of Tendon Injury.

BACKGROUND: Emerging data suggest that human cells derived from extraembryonic tissues may have favorable musculoskeletal repair properties. The purpose of this study was to determine whether the injection of human placenta-derived mesenchymal-like stromal cells, termed placental expanded cells (PLX-PAD), would improve tendon healing in a preclinical model of tendinopathy. METHODS: Sixty male Sprague-Dawley rats underwent bilateral patellar tendon injection with either saline solution (control) or PLX-PAD cells (2 × 10 cells/100 µL) 6 days after collagenase injection to induce tendon degeneration. Animals were killed at specific time points for biomechanical, histological, and gene expression analyses of the healing patellar tendons. RESULTS: Biomechanical testing 2 weeks after the collagenase injury demonstrated better biomechanical properties in the tendons treated with PLX-PAD cells. The load to failure of the PLX-PAD-treated tendons was higher than that of the saline-solution-treated controls at 2 weeks (77.01 ± 10.51 versus 58.87 ± 11.97 N, p = 0.01). There was no significant difference between the 2 groups at 4 weeks. There were no differences in stiffness at either time point. Semiquantitative histological analysis demonstrated no significant differences in collagen organization or cellularity between the PLX-PAD and saline-solution-treated tendons. Gene expression analysis demonstrated higher levels of interleukin-1ß (IL-1ß) and IL-6 early in the healing process in the PLX-PAD-treated tendons. CONCLUSIONS: Human placenta-derived cell therapy induced an early inflammatory response and a transient beneficial effect on tendon failure load in a model of collagenase-induced tendon degeneration. CLINICAL RELEVANCE: Human extraembryonic tissues, such as the placenta, are an emerging source of cells for musculoskeletal repair and may hold promise as a point-of-care cell therapy for tendon injuries.

Postoperative Tendon Loading With Treadmill Running Delays Tendon-to-Bone Healing: Immunohistochemical Evaluation in a Murine Rotator Cuff Repair Model.

Mechanical stress has an important effect on tendon-to-bone healing. The purpose of the present study was to compare tendon-to-bone healing in animals exposed to either tendon unloading (botulinum toxin injection) or excessive loading (treadmill running) in a murine rotator cuff repair model. Forty-eight C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair. Mice in the unloaded group were injected with botulinum toxin to the supraspinatus muscle. The contralateral shoulder of the unloaded group was used as a control. Mice were euthanized at 1, 2, and 4 weeks after surgery and evaluated with hematoxylin-eosin and immunohistochemical (IHC) staining for Ihh, Gli1, Wnt3a, and ß-catenin. The positive staining area on IHC and the Modified Tendon Maturing Score were measured. The score of the unloaded group was significantly higher (better healing) than that of the treadmill group at 4 weeks. Ihh and the glioma-associated oncogene homolog 1 (Gli1) positive area in the unloaded group were significantly higher than those of the control group at 1 week. The peak time-points of the Ihh and Gli1 positive area was 1 week for the unloaded group and 2 weeks for the treadmill group. The Wnt3a positive area in the unloaded group was significantly higher than that of the control group at 2 weeks. The ß-catenin positive area in the unloaded group was significantly higher than that of the treadmill group and the control group at 1 week. Our data indicated that the unloaded group has superior tendon maturation compared to the treadmill running group. Excessive tendon loading may delay the tendon healing process by affecting the activity of Ihh and Wnt/ß-Catenin pathways. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1628-1637, 2019.

Development of Tube Radial Distribution Chromatography Based on Phase-Separation Multiphase Flow Created via Pressure Loss.

A tube radial distribution chromatography (TRDC) method based on phase-separated multiphase flow created through phase transformation via temperature change has been developed. These systems typically required a temperature-controlling device containing a water bath and a stirrer. Herein, we proposed a novel TRDC system without a cooling device, where the phase transformation was achieved via pressure loss in a capillary tube of 50 µm inner diameter and 550 cm length. Model analytes were successfully separated with the developed TRDC system, which provided a simplified platform and helped to clarify the operating principle of TRDC based on phase transformation in a capillary tube.

Duration of postoperative immobilization affects MMP activity at the healing graft-bone interface: Evaluation in a mouse ACL reconstruction model.

Excessive MMP activity may impair tendon-to-bone healing. However, little is known about the effect of joint motion on MMP activity after ACL reconstruction. The aim of this study was to determine the effect of different durations of knee immobilization on MMP activity in a mouse ACL reconstruction model using a fluorescent MMP probe which detects MMP 2, 3, 9, and 13 and near-infra red in vivo imaging. Sixty C57BL male mice underwent ACL reconstruction. Post-operatively, the animals were treated with free cage activity (Group 1), or with the use of an external fixator to restrict knee motion and weight bearing for 5 days (Group 2), 14 days (Group 3), and 28 days (Group 4). At days 3, 7, 16, 23, and 30, five mice underwent IVIS imaging. At days 3, 7, 16, and 30, histological analysis was also performed. Probe signal intensity in the whole limb peaked at day 7, followed by a decrease at day 16, and maintenance up to day 30. There was no significant difference among groups at any time point based on IVIS, but histologic localization of MMP probe signal showed significantly less activity in Group 2 and Group 3 compared to Group 4 in the bone tunnel at day 30. We demonstrated that short-term immobilization led to less MMP activity around the bone tunnel compared with prolonged immobilization. A short period of immobilization after ACL reconstruction might enhance graft-bone interface healing by mitigating excess MMP expression. These findings have implications for post-operative rehabilitation protocols following ACL reconstruction. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:325-334, 2019.

Micro patterning of hydroxyapatite by soft lithography on hydrogels for selective osteoconduction.

Mechanically robust hydrogels are promising biomaterials as artificial supportive tissue. These applications require selective and robust bonding of the hydrogels to living tissue. Recently, we achieved strong in vivo bone bonding of a tough double network (DN) hydrogel, a potential material for use as artificial cartilage and tendon, by hybridizing osteoconductive hydroxyapatite (HAp) in the gel surface layer. In this work, we report micro patterning of HAp at the surface of the DN hydrogel for selective osteoconduction. Utilizing the dissolution of HAp in an acidic environment, the soft lithography technique using an acid gel stamp was adopted to form a high-resolution HAp pattern on the gel. The HAp-patterned gel showed well-regulated migration and adhesion of cells in vitro. Moreover, the HAp-patterned gel showed selective and robust bonding to the rabbit bone tissue in vivo. This HAp soft lithography technique allows for simple and quick preparation of tailor-made osteoconductive hydrogels and can be applied to other hydrogels for selective bone bonding. STATEMENT OF SIGNIFICANCE: Hydrogels, preserving large amount of water, have been studied for next-generation artificial soft tissues. However, fixation of hydrogels to living tissue was unsolved issue for clinical application. Recently, we achieved robust bonding of a tough double network gel to bone in vivo by coating of osteoconductive hydroxyapatite in the gel surface layer. For further progress for practical use, we report the micro patterning of HAp at the surface of the DN hydrogel by using soft lithography technique, to perform selective bonding to only objective area without unnecessary coalescence. The HAp lithography technique is simple, quick and non-toxic method to prepare tailor-made osteoconductive hydrogels and has universality of species of hydrogels.

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.

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

Subacromial impingement of the rotator cuff is understood as a contributing factor in the development of rotator cuff tendinopathy. However, changes that occur in the impinged tendon are poorly understood and warrant further study. To enable further study of rotator cuff tendinopathy, we performed a controlled laboratory study to determine feasibility and baseline characteristics of a new murine model for subacromial impingement. This model involves surgically inserting a microvascular clip into the subacromial space in adult C57Bl/6 mice. Along with a sham surgery arm, 90 study animals were distributed among time point groups for sacrifice up to 6 weeks. All animals underwent bilateral surgery (total N = 180). Biomechanical, histologic, and molecular analyses were performed to identify and quantify the progression of changes in the supraspinatus tendon. Decreases in failure force and stiffness were found in impinged tendon specimens compared to sham and no-surgery controls at all study time points. Semi-quantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 6 weeks. Quantitative real-time polymerase chain reaction analysis of impinged tendon specimens demonstrated persistently increased expression of genes related to matrix remodeling, inflammation, and tendon development. Overall, this novel murine subacromial impingement model creates changes consistent with acute tendonitis, which may mimic the early stages of rotator cuff tendinopathy. A robust, simple, and reproducible animal model of rotator cuff tendinopathy is a valuable research tool to allow further studies of cellular and molecular mechanisms and evaluation of therapeutic interventions in rotator cuff tendinopathy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2780-2788, 2018.

Tissue-specific endothelial cells: a promising approach for augmentation of soft tissue repair in orthopedics.

Biologics are playing an increasingly significant role in the practice of modern medicine and surgery in general and orthopedics in particular. Cell-based approaches are among the most important and widely used modalities in orthopedic biologics, with mesenchymal stem cells and other multi/pluripotent cells undergoing evaluation in numerous preclinical and clinical studies. On the other hand, fully differentiated endothelial cells (ECs) have been found to perform critical roles in homeostasis of visceral tissues through production of an adaptive panel of so-called "angiocrine factors." This newly discovered function of ECs renders them excellent candidates for novel approaches in cell-based biologics. Here, we present a review of the role of ECs and angiocrine factors in some visceral tissues, followed by an overview of current cell-based approaches and a discussion of the potential applications of ECs in soft tissue repair.

The Effect of Intraoperative Graft Coverage With Preserved Remnant Tissue on the Results of the Pivot-Shift Test After Anatomic Double-Bundle Anterior Cruciate Ligament Reconstruction: Quantitative Evaluations With an Electromagnetic Sensor System.

BACKGROUND: Remnant tissue preservation may be important in improving graft healing after anterior cruciate ligament (ACL) reconstruction, but it has yet to be established whether remnant tissue preservation improves the control of pivot-shift laxity. HYPOTHESIS: The amount of ACL graft coverage with preserved remnant tissue improves the control of pivot-shift laxity, as qualitatively determined with an electromagnetic device. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: The 3-dimensional kinematics were evaluated intraoperatively using an electromagnetic sensor system in 38 patients at the time of anatomic double-bundle ACL reconstruction with remnant tissue preservation and again at a minimum of 12 months postoperatively. The magnitude of the peak coupled anterior tibial translation (pCAT) and the maximal acceleration of posterior translation (APT) during the pivot-shift test were evaluated. The degree of graft coverage by remnant tissue was determined arthroscopically at the end of surgery, which was evaluated quantitatively using a scoring system (0-9 points). The relationship between the values during the pivot-shift test and preoperative and intraoperative factors were assessed. RESULTS: The mean (±SD) side-to-side difference of the pCAT (ΔpCAT) was significantly ( P < .0001) improved from 14.0 ± 5.0 mm to 2.6 ± 1.1 mm. Also, the mean side-to-side difference of the APT (ΔAPT) was significantly ( P < .0001) improved from 525.6 ± 99.7 mm/s2 to 32.9 ± 23.6 mm/s2. The mean initial graft coverage score was 5.3 ± 2.6. The correlation analysis demonstrated that the degree of initial graft coverage was significantly correlated with the ΔpCAT ( r = -0.517, P = .0007) and ΔAPT ( r = -0.532, P = .0005). The status of the reconstructed graft at second-look arthroscopic surgery showed no significant correlations with the degree of initial graft coverage or the results of the pivot-shift test. CONCLUSION: The present study demonstrated that the preservation of ACL remnant tissue in anatomic double-bundle ACL reconstruction appears to improve the control of pivot-shift laxity at a minimum of 12 months postoperatively, as measured by an electromagnetic device. This improvement was significantly affected by the degree of intraoperative graft coverage with preserved remnant tissue.