The effects of NF-κB suppression on the early healing response following intrasynovial tendon repair in a canine model.

The highly variable clinical outcomes noted after intrasynovial tendon repair have been associated with an early inflammatory response leading to the development of fibrovascular adhesions. Prior efforts to broadly suppress this inflammatory response have been largely unsuccessful. Recent studies have shown that selective inhibition of IkappaB kinase beta (IKK-ß), an upstream activator of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) signaling, mitigates the early inflammatory response and leads to improved tendon healing outcomes. In the current study, we test the hypothesis that oral treatment with the IKK-ß inhibitor ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinenitrile an inhibitor) will modulate the postoperative inflammatory response and improve intrasynovial flexor tendon healing. To test this hypothesis, the flexor digitorum profundus tendon of 21 canines was transected and repaired within the intrasynovial region and assessed after 3 and 14 days. Histomorphometry, gene expression analyses, immunohistochemistry, and quantitative polarized light imaging were used to examine ACHP-mediated changes. ACHP led to reduction in phosphorylated p-65, indicating that NF-κB activity was suppressed. ACHP enhanced expression of inflammation-related genes at 3 days and suppressed expression of these genes at 14 days. Histomorphometry revealed enhanced cellular proliferation and neovascularization in ACHP-treated tendons compared with time-matched controls. These findings demonstrate that ACHP effectively suppressed NF-κB signaling and modulated early inflammation, leading to increased cellular proliferation and neovascularization without stimulating the formation of fibrovascular adhesions. Together, these data suggest that ACHP treatment accelerated the inflammatory and proliferative phases of tendon healing following intrasynovial flexor tendon repair. Clinical Significance: Using a clinically relevant large-animal model, this study revealed that targeted inhibition of nuclear factor kappa-light chain enhancer of activated B cells signaling with ACHP provides a new therapeutic strategy for enhancing the repair of sutured intrasynovial tendons.

Early Joint Use Following Elbow Dislocation Limits Range-of-Motion Loss and Tissue Pathology in Posttraumatic Joint Contracture.

BACKGROUND: Simple elbow dislocation occurs at an incidence of 2.9 to 5.21 dislocations per 100,000 person-years, with as many as 62% of these patients experiencing long-term elbow joint contracture, stiffness, and/or pain. Poor outcomes and the need for secondary surgical intervention can often be prevented nonoperatively with early or immediate active mobilization and physical therapy. However, immobilization or limited mobilization may be necessary following trauma, and it is unknown how different periods of immobilization affect pathological changes in elbow joint tissue and how these changes relate to range of motion (ROM). The purpose of this study was to investigate the effects of varying the initiation of free mobilization on elbow ROM and histological features in an animal model of elbow posttraumatic joint contracture. METHODS: Traumatic elbow dislocation was surgically induced unilaterally in rats. Injured forelimbs were immobilized in bandages for 3, 7, 14, or 21 days; free mobilization was then allowed until 42 days after injury. Post-mortem joint ROM testing and histological analysis were performed. One-way analysis of variance was used to compare ROM data between control and injured groups, and Pearson correlations were performed between ROM parameters and histological outcomes. RESULTS: Longer immobilization periods resulted in greater ROM reductions. The anterior and posterior capsule showed increases in cellularity, fibroblasts, adhesions, fibrosis, and thickness, whereas the measured outcomes in cartilage were mostly unaffected. All measured histological characteristics of the capsule were negatively correlated with ROM, indicating that higher degrees of pathology corresponded with less ROM. CONCLUSIONS: Longer immobilization periods resulted in greater ROM reductions, which correlated with worse histological outcomes in the capsule in an animal model of posttraumatic elbow contracture. The subtle differences in the timing of ROM and capsule tissue changes revealed in the present study provide new insight into the distinct timelines of biomechanical changes as well as regional tissue pathology. CLINICAL RELEVANCE: This study showed that beginning active mobilization 3 days after injury minimized posttraumatic joint contracture, thereby supporting an immediate-motion clinical treatment strategy (when possible). Furthermore, uninjured but pathologically altered periarticular tissues near the injury location may contribute to more severe contracture during longer immobilization periods as the disease state progresses.

Segmental Colitis Associated With Diverticulosis Masquerading as Polyploid-Appearing Mucosa in the Rectosigmoid Area on Endoscopy and as Focal Thickening on Imaging.

Segmental colitis associated with diverticulosis (SCAD) is an inflammatory disease affecting segments of the large bowel with diverticular disease. SCAD presents several challenges in diagnoses and treatment because it often mimics a range of disorders including inflammatory bowel disease and malignancy. Here, we present the case of a 72-year-old man with lower abdominal pain and bloody stools whose initial abdominal workup showed nonspecific large bowel thickening and concerns for malignancy. Ultimately, the patient was diagnosed with mild SCAD and treated conservatively with a resolution of symptoms. He had no symptoms at the three-month and 1-year follow-ups. This case highlights the importance of including SCAD in the initial differential diagnosis to allow accurate identification and treatment.

Pleiotropic Effects of Simvastatin and Losartan in Preclinical Models of Post-Traumatic Elbow Contracture.

Elbow trauma can lead to post-traumatic joint contracture (PTJC), which is characterized by loss of motion associated with capsule/ligament fibrosis and cartilage damage. Unfortunately, current therapies are often unsuccessful or cause complications. This study aimed to determine the effects of prophylactically administered simvastatin (SV) and losartan (LS) in two preclinical models of elbow PTJC: an in vivo elbow-specific rat injury model and an in vitro collagen gel contraction assay. The in vivo elbow rat (n = 3-10/group) injury model evaluated the effects of orally administered SV and LS at two dosing strategies [i.e., low dose/high frequency/short duration (D1) vs. high dose/low frequency/long duration (D2)] on post-mortem elbow range of motion (via biomechanical testing) as well as capsule fibrosis and cartilage damage (via histopathology). The in vitro gel contraction assay coupled with live/dead staining (n = 3-19/group) evaluated the effects of SV and LS at various concentrations (i.e., 1, 10, 100 µM) and durations (i.e., continuous, short, or delayed) on the contractibility and viability of fibroblasts/myofibroblasts [i.e., NIH3T3 fibroblasts with endogenous transforming growth factor-beta 1 (TGFß1)]. In vivo, no drug strategy prevented elbow contracture biomechanically. Histologically, only SV-D2 modestly reduced capsule fibrosis but maintained elevated cellularity and tissue hypertrophy, and both SV strategies lessened cartilage damage. SV modest benefits were localized to the anterior region, not the posterior, of the joint. Neither LS strategy had meaningful benefits in capsule nor cartilage. In vitro, irrespective of the presence of TGFß1, SV (≥10 µM) prevented gel contraction partly by decreasing cell viability (100 µM). In contrast, LS did not prevent gel contraction or affect cell viability. This study demonstrates that SV, but not LS, might be suitable prophylactic drug therapy in two preclinical models of elbow PTJC. Results provide initial insight to guide future preclinical studies aimed at preventing or mitigating elbow PTJC.

Females and males exhibit similar functional, mechanical, and morphological outcomes in a rat model of posttraumatic elbow contracture.

Posttraumatic joint contracture (PTJC) is a debilitating condition characterized by loss of joint motion following injury. Previous work in a rat model of elbow PTJC investigated disease etiology, progression, and recovery in only male animals; this study explored sex-based differences. Rat elbows were subjected to a unilateral anterior capsulotomy and lateral collateral ligament transection followed by 42 days of immobilization and 42 days of free mobilization. Grip strength and gait were collected throughout the free mobilization period while joint mechanical testing, microcomputed tomography and histological analysis were performed postmortem. Overall, few differences were seen between sexes in functional, mechanical, and morphological outcomes with PTJC being similarly debilitating in male and female animals. Functional measures of grip strength and gait showed that, while some baseline differences existed between sexes, traumatic injury produced similar deficits that remained significantly different long-term when compared to control animals. Similarly, male and female animals both had significant reductions in joint range of motion due to injury. Ectopic calcification (EC), which had not been previously evaluated in this injury model, was present in all limbs on the lateral side. Injury caused increased EC volume but did not alter mineral density regardless of sex. Furthermore, histological analysis of the anterior capsule showed minor differences between sexes for inflammation and thickness but not for other histological parameters. A quantitative understanding of sex-based differences associated with this injury model will help inform future therapeutics aimed at reducing or preventing elbow PTJC.

Adipose stem cells exhibit mechanical memory and reduce fibrotic contracture in a rat elbow injury model.

Fibrosis is driven by a misdirected cell response causing the overproduction of extracellular matrix and tissue dysfunction. Numerous pharmacological strategies have attempted to prevent fibrosis but have attained limited efficacy with some detrimental side effects. While stem cell treatments have provided more encouraging results, they have exhibited high variability and have not always improved tissue function. To enhance stem cell efficacy, we evaluated whether mechanical memory could direct cell response. We hypothesized that mechanically pre-conditioning on a soft matrix (soft priming) will delay adipose-derived stem cell (ASC) transition to a pro-fibrotic phenotype, expanding their regenerative potential, and improving healing in a complex tissue environment. Primary ASCs isolated from rat and human subcutaneous fat exhibited mechanical memory, demonstrated by a delayed cell response to stiffness following two weeks of soft priming including decreased cell area, actin coherency, and extracellular matrix production compared to cells on stiff substrates. Soft primed ASCs injected into our rat model of post-traumatic elbow contracture decreased histological evidence of anterior capsule fibrosis and increased elbow range-of-motion when evaluated by joint mechanics. These findings suggest that exploiting mechanical memory by strategically controlling the culture environment during cell expansion may improve the efficacy of stem cell-based therapies targeting fibrosis.

Decellularized bovine placentome for portacavally-interposed heterotopic liver transplantation in rats.

Scaffolds from healthy placentae offer advantages for tissue engineering with undamaged matrix, associated cytoprotective molecules, and embedded vessels for revascularization. As size disparities in human placenta and small recipients hamper preclinical studies, we studied alternative of bovine placentomes in smaller size ranges. Multiple cow placentomes were decellularized and anatomical integrity was analyzed. Tissue engineering used inbred donor rat livers. Placentomes were hepatized and immediately transplanted in rats with perfusion from portal vein and drainage into inferior vena cava. Cows yielded 99 ±â€¯16 placentomes each. Of these, approximately 25% had 3 to 9 cm diameter and 7 to 63 ml volume, which was suitable for transplantation. After decellularization, angiography and casts documented 100% of vessels and vascular networks were well-perfused without disruptions or leaks. The residual matrix also remained intact for transplantation of placentomes. Perfusion in transplanted placentomes was maintained over up to 30 days. Liver tissue reassembled with restoration of hepatic acinar and sinusoidal structure. Transplanted tissue was intact without apoptosis, or necrosis. Hepatic functions were maintained. Preservation of hepatic homeostasis was verified by cytofluorimetric analysis of hepatocyte ploidy. The prevalence in healthy and transplanted liver of diploid, tetraploid and higher ploidy classes was similar with 57%, 41% and 2% versus 51%, 46.5% and 2.6%, respectively, p = 0.77, ANOVA. CONCLUSIONS: Cow placentomes will allow therapeutic development with disease models in small animals. This will also advance drug or toxicology studies. Portasystemic interposition of engineered liver will be particularly suitable for treating hepatic insufficiencies (metabolic, secretory or detoxification needs), including for children or smaller adults.

Temporal Patterns of Motion in Flexion-extension and Pronation-supination in a Rat Model of Posttraumatic Elbow Contracture.

BACKGROUND: The elbow is highly susceptible to contracture, which affects up to 50% of patients who experience elbow trauma. Previously, we developed a rat model to study elbow contracture that exhibited features similar to the human condition, including persistently decreased ROM and increased capsule thickness/adhesions. However, elbow ROM was not quantitatively evaluated over time throughout contracture development and subsequent mobilization of the joint. QUESTIONS/PURPOSES: The purposes of this study were (1) to quantify the time-dependent mechanics of contracture, including comparison of contracture after immobilization and free mobilization; and (2) to determine what changes occur in capsule and joint surface morphology that may support the altered joint mechanics. METHODS: A total of 96 male Long-Evans rats were randomized into control and injury (unilateral soft tissue injury/immobilization) groups. Flexion-extension and pronation-supination joint mechanics (n = 8/group) were evaluated after 3, 7, 21, or 42 days of immobilization (IM) or after 42 days of IM with either 21 or 42 days of free mobilization (63 or 84 FM, respectively). After measuring joint mechanics, a subset of these limbs (n = 3/group) was prepared for histologic analysis and blinded sections were scored to evaluate capsule and joint surface morphology. Joint mechanics and capsule histology at 42 IM and 84 FM were reported previously but are included to demonstrate the full timeline of elbow contracture. RESULTS: In flexion-extension, injured limb ROM was decreased compared with control (103° ± 11°) by 21 IM (70° ± 13°) (p = 0.001). Despite an increase in injured limb ROM from 42 IM (55° ± 14°) to 63 FM (83° ± 10°) (p < 0.001), injured limb ROM was still decreased compared with control (103° ± 11°) (p = 0.002). Interestingly, ROM recovery plateaued because there was no difference between injured limbs at 63 (83° ± 10°) and 84 FM (73° ± 19°) (p > 0.999). In pronation-supination, increased injured limb ROM occurred until 7 IM (202° ± 32°) compared with control (155° ± 22°) (p = 0.001), representative of joint instability. However, injured limb ROM decreased from 21 (182° ± 25°) to 42 IM (123° ± 47°) (p = 0.001), but was not different compared with control (155° ± 22°) (p = 0.108). Histologic evaluation showed morphologic changes in the anterior capsule (increased adhesions, myofibroblasts, thickness) and nonopposing joint surfaces (surface irregularities with tissue overgrowth, reduced matrix), but these changes did not increase with time. CONCLUSIONS: Overall, flexion-extension and pronation-supination exhibited distinct time-dependent patterns during contracture development and joint mobilization. Histologic evaluation showed tissue changes, but did not fully explain the patterns in contracture mechanics. Future work will use this rat model to evaluate the periarticular soft tissues of the elbow to isolate tissue-specific contributions to contracture to ultimately develop strategies for tissue-targeted treatments. CLINICAL RELEVANCE: A rat model of posttraumatic elbow contracture quantitatively described contracture development/progression and reiterates the need for rehabilitation strategies that consider both flexion-extension and pronation-supination elbow motion.

Sclerostin Antibody Treatment Enhances Rotator Cuff Tendon-to-Bone Healing in an Animal Model.

BACKGROUND: Rotator cuff tears are a common source of pain and disability, and poor healing after repair leads to high retear rates. Bone loss in the humeral head before and after repair has been associated with poor healing. The purpose of the current study was to mitigate bone loss near the repaired cuff and improve healing outcomes. METHODS: Sclerostin antibody (Scl-Ab) treatment, previously shown to increase bone formation and strength in the setting of osteoporosis, was used in the current study to address bone loss and enhance rotator cuff healing in an animal model. Scl-Ab was administered subcutaneously at the time of rotator cuff repair and every 2 weeks until the animals were sacrificed. The effect of Scl-Ab treatment was evaluated after 2, 4, and 8 weeks of healing, using bone morphometric analysis, biomechanical evaluation, histological analysis, and gene expression outcomes. RESULTS: Injury and repair led to a reduction in bone mineral density after 2 and 4 weeks of healing in the control and Scl-Ab treatment groups. After 8 weeks of healing, animals receiving Scl-Ab treatment had 30% greater bone mineral density than the controls. A decrease in biomechanical properties was observed in both groups after 4 weeks of healing compared with healthy tendon-to-bone attachments. After 8 weeks of healing, Scl-Ab-treated animals had improved strength (38%) and stiffness (43%) compared with control animals. Histological assessment showed that Scl-Ab promoted better integration of tendon and bone by 8 weeks of healing. Scl-Ab had significant effects on gene expression in bone, indicative of enhanced bone formation, and no effect on the expression of genes in tendon. CONCLUSIONS: This study provides evidence that Scl-Ab treatment improves tendon-to-bone healing at the rotator cuff by increasing attachment-site bone mineral density, leading to improved biomechanical properties. CLINICAL RELEVANCE: Scl-Ab treatment may improve outcomes after rotator cuff repair.

Persistent motion loss after free joint mobilization in a rat model of post-traumatic elbow contracture.

BACKGROUND: Post-traumatic joint contracture (PTJC) in the elbow is a challenging clinical problem due to the anatomical and biomechanical complexity of the elbow joint. METHODS: We previously established an animal model to study elbow PTJC, wherein surgically induced soft tissue damage, followed by 6 weeks of unilateral immobilization in Long-Evans rats, led to stiffened and contracted joints that exhibited features similar to the human condition. In this study, after 6 weeks of immobilization, we remobilized the animal (ie, external bandage removed and free cage activity) for an additional 6 weeks, after which the limbs were evaluated mechanically and histologically. The objective of this study was to evaluate whether this decreased joint motion would persist after 6 weeks of free mobilization (FM). RESULTS: After FM, flexion-extension demonstrated decreased total range of motion (ROM) and neutral zone length, and increased ROM midpoint for injured limbs compared with control and contralateral limbs. Specifically, after FM total ROM demonstrated a significant decrease of approximately 22% and 26% compared with control and contralateral limbs for injury I (anterior capsulotomy) and injury II (anterior capsulotomy with lateral collateral ligament transection), respectively. Histologic evaluation showed increased adhesion, fibrosis, and thickness of the capsule tissue in the injured limbs after FM compared with control and contralateral limbs, which is consistent with patterns previously reported in human tissue. CONCLUSION: Even with FM, injured limbs in this model demonstrate persistent joint motion loss and histologic results similar to the human condition. Future work will use this animal model to investigate the mechanisms responsible for PTJC and responses to therapeutic intervention.

The effect of mesenchymal stromal cell sheets on the inflammatory stage of flexor tendon healing.

BACKGROUND: The clinical outcomes following intrasynovial flexor tendon repair are highly variable. Excessive inflammation is a principal factor underlying the formation of adhesions at the repair surface and affecting matrix regeneration at the repair center that limit tendon excursion and impair tendon healing. A previous in-vitro study revealed that adipose-derived mesenchymal stromal cells (ASCs) modulate tendon fibroblast response to macrophage-induced inflammation. The goal of the current study was therefore to explore the effectiveness of autologous ASCs on the inflammatory stage of intrasynovial tendon healing in vivo using a clinically relevant animal model. METHODS: Zone II flexor tendon transections and suture repairs were performed in a canine model. Autologous ASC sheets were delivered to the surface of repaired tendons. Seven days after repair, the effects of ASCs on tendon healing, with a focus on the inflammatory response, were evaluated using gene expression assays, immunostaining, and histological assessments. RESULTS: ASCs delivered via the cell sheet infiltrated the host tendon, including the repair surface and the space between the tendon ends, as viewed histologically by tracking GFP-expressing ASCs. Gene expression results demonstrated that ASCs promoted a regenerative/anti-inflammatory M2 macrophage phenotype and regulated tendon matrix remodeling. Specifically, there were significant increases in M2-stimulator (IL-4), marker (CD163 and MRC1), and effector (VEGF) gene expression in ASC-sheet treated tendons compared with nontreated tendons. When examining changes in extracellular matrix expression, tendon injury caused a significant increase in scar-associated COL3A1 expression and reductions in COL2A1 and ACAN expression. The ASC treatment effectively counteracted these changes, returning the expression levels of these genes closer to normal. Immunostaining further confirmed that ASC treatment increased CD163+ M2 cells in the repaired tendons and suppressed cell apoptosis at the repair site. CONCLUSIONS: This study provides a novel approach for delivering ASCs with outcomes indicating potential for substantial modulation of the inflammatory environment and enhancement of tendon healing after flexor tendon repair.

Development and use of an animal model to study post-traumatic stiffness and contracture of the elbow.

Post-traumatic joint stiffness (PTJS) of the elbow is a debilitating condition that poses unique treatment challenges. While previous research has implicated capsular tissue in PTJS, much regarding the development and progression of this condition remains unknown. The objective of this study was to develop an animal model of post-traumatic elbow contracture and evaluate its potential for studying the etiology of PTJS. The Long-Evans rat was identified as the most appropriate species/breed for development due to anatomical and functional similarities to the human elbow joint. Two surgical protocols of varying severity were utilized to replicate soft tissue damage seen in elbow subluxation/dislocation injuries, including anterior capsulotomy and lateral collateral ligament transection, followed by 6 weeks of unilateral joint immobilization. Following sacrifice, flexion-extension mechanical joint testing demonstrated decreased range-of-motion and increased stiffness for injured-immobilized limbs compared to control and sham animals, where functional impact correlated with severity of injury. Histological evaluation showed increased cellularity, adhesion, and thickness of capsule tissue in injured limbs, consistent with clinical evidence. To our knowledge, this is the first animal model capable of examining challenges unique to the anatomically and biomechanically complex elbow joint. Future studies will use this animal model to investigate mechanisms responsible for PTJS.

In Vivo Evaluation of Adipose-Derived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair.

Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor BMP2 led to impaired healing, suggesting that this growth factor should not be used in the tendon-to-bone repair setting.

Chronic Degeneration Leads to Poor Healing of Repaired Massive Rotator Cuff Tears in Rats.

BACKGROUND: Chronic rotator cuff tears present a clinical challenge, often with poor outcomes after surgical repair. Degenerative changes to the muscle, tendon, and bone are thought to hinder healing after surgical repair; additionally, the ability to overcome degenerative changes after surgical repair remains unclear. PURPOSE/HYPOTHESIS: The purpose of this study was to evaluate healing outcomes of muscle, tendon, and bone after tendon repair in a model of chronic rotator cuff disease and to compare these outcomes to those of acute rotator cuff injuries and repair. The hypothesis was that degenerative rotator cuff changes associated with chronic multitendon tears and muscle unloading would lead to poor structural and mechanical outcomes after repair compared with acute injuries and repair. STUDY DESIGN: Controlled laboratory study. METHODS: Chronic rotator cuff injuries, induced via detachment of the supraspinatus (SS) and infraspinatus (IS) tendons and injection of botulinum toxin A into the SS and IS muscle bellies, were created in the shoulders of rats. After 8 weeks of injury, tendons were surgically reattached to the humeral head, and an acute, dual-tendon injury and repair was performed on the contralateral side. After 8 weeks of healing, muscles were examined histologically, and tendon-to-bone samples were examined microscopically, histologically, and biomechanically and via micro-computed tomography. RESULTS: All repairs were intact at the time of dissection, with no evidence of gapping or ruptures. Tendon-to-bone healing after repair in our chronic injury model led to reduced bone quality and morphological disorganization at the repair site compared with acute injuries and repair. SS and IS muscles were atrophic at 8 weeks after repair of chronic injuries, indicating incomplete recovery after repair, whereas SS and IS muscles exhibited less atrophy and degeneration in the acute injury group at 8 weeks after repair. After chronic injuries and repair, humeral heads had decreased total mineral density and an altered trabecular structure, and the repair had decreased strength, stiffness, and toughness, compared with the acute injury and repair group. CONCLUSION: Chronic degenerative changes in rotator cuff muscles, tendons, and bone led to inferior healing characteristics after repair compared with acute injuries and repair. The changes were not reversible after repair in the time course studied, consistent with clinical impressions. CLINICAL RELEVANCE: High retear rates after rotator cuff repair are associated with tear size and chronicity. Understanding the mechanisms behind this association may allow for targeted tissue therapy for tissue degeneration that occurs in the setting of chronic tears.

The developing shoulder has a limited capacity to recover after a short duration of neonatal paralysis.

Mechanical stimuli are required for the proper development of the musculoskeletal system. Removal of muscle forces during fetal or early post-natal timepoints impairs the formation of bone, tendon, and their attachment (the enthesis). The goal of the current study was to examine the capacity of the shoulder to recover after a short duration of neonatal rotator cuff paralysis, a condition mimicking the clinical condition neonatal brachial plexus palsy. We asked if reapplication of muscle load to a transiently paralyzed muscle would allow for full recovery of tissue properties. CD-1 mice were injected with botulinum toxin A to paralyze the supraspinatus muscle from birth through 2 weeks and subsequently allowed to recover. The biomechanics of the enthesis was determined using tensile testing and the morphology of the shoulder joint was determined using microcomputed tomography and histology. A recovery period of at least 10 weeks was required to achieve control properties, demonstrating a limited capacity of the shoulder to recover after only two weeks of muscle paralysis. Although care must be taken when extrapolating results from an animal model to the human condition, the results of the current study imply that treatment of neonatal brachial plexus palsy should be aggressive, as even short periods of paralysis could lead to long-term deficiencies in enthesis biomechanics and shoulder morphology.

The early inflammatory response after flexor tendon healing: a gene expression and histological analysis.

Despite advances in surgical techniques over the past three decades, tendon repairs remain prone to poor clinical outcomes. Previous attempts to improve tendon healing have focused on the later stages of healing (i.e., proliferation and matrix synthesis). The early inflammatory phase of tendon healing, however, is not fully understood and its modulation during healing has not yet been studied. Therefore, the purpose of this work was to characterize the early inflammatory phase of flexor tendon healing with the goal of identifying inflammation-related targets for future treatments. Canine flexor tendons were transected and repaired using techniques identical to those used clinically. The inflammatory response was monitored for 9 days. Temporal changes in immune cell populations and gene expression of inflammation-, matrix degradation-, and extracellular matrix-related factors were examined. Gene expression patterns paralleled changes in repair-site cell populations. Of the observed changes, the most dramatic effect was a greater than 4,000-fold up-regulation in the expression of the pro-inflammatory factor IL-1ß. While an inflammatory response is likely necessary for healing to occur, high levels of pro-inflammatory cytokines may result in collateral tissue damage and impaired tendon healing. These findings suggest that future tendon treatment approaches consider modulation of the inflammatory phase of healing.

The effects of chronic unloading and gap formation on tendon-to-bone healing in a rat model of massive rotator cuff tears.

The objective of this study was to understand the effect of pre-repair rotator cuff chronicity on post-repair healing outcomes using a chronic and acute multi-tendon rat rotator cuff injury model. Full-thickness dual tendon injuries (supra- and infraspinatus) were created unilaterally in adult male Sprague Dawley rats, and left chronically detached for 8 or 16 weeks. After chronic detachment, tears were repaired and acute dual tendon injuries were created and immediately repaired on contralateral shoulders. Tissue level outcomes for bone, tendon, and muscle were assessed 4 or 8 weeks after repair using histology, microcomputed tomography, biomechanical testing, and biochemical assays. Substantial gap formation was seen in 35% of acute repairs and 44% of chronic repairs. Gap formation negatively correlated with mechanical and structural outcomes for both healing time points regardless of injury duration. Bone and histomorphometry, as well as biomechanics, were similar between acute and chronic injury and repair regardless of chronicity and duration of healing. This study was the first to implement a multi-tendon rotator cuff injury with surgical repair following both chronic and acute injuries. Massive tear in a rodent model resulted in gap formation regardless of injury duration which had detrimental effects on repair outcomes.

The effect of tear size and nerve injury on rotator cuff muscle fatty degeneration in a rodent animal model.

BACKGROUND: Irreversible muscle changes after rotator cuff tears is a well-known negative prognostic factor after shoulder surgery. Currently, little is known about the pathomechanism of fatty degeneration of the rotator cuff muscles after chronic cuff tears. The purposes of this study were to (1) develop a rodent animal model of chronic rotator cuff tears that can reproduce fatty degeneration of the cuff muscles seen clinically, (2) describe the effects of tear size and concomitant nerve injury on muscle degeneration, and (3) evaluate the changes in gene expression of relevant myogenic and adipogenic factors after rotator cuff tears using the animal model. MATERIALS AND METHODS: Rotator cuff tears were created in rodents with and without transection of the suprascapular nerve. The supraspinatus and infraspinatus muscles were examined at 2, 8, and 16 weeks after injury for histologic evidence of fatty degeneration and expression of myogenic and adipogenic genes. RESULTS: Histologic analysis revealed adipocytes, intramuscular fat globules, and intramyocellular fat droplets in the tenotomized and neurotomized supraspinatus and infraspinatus muscles. Changes increased with time and were most severe in the muscles with combined tenotomy and neurotomy. Adipogenic and myogenic transcription factors and markers were upregulated in muscles treated with tenotomy or tenotomy combined with neurotomy compared with normal muscles. CONCLUSIONS: The rodent animal model described in this study produces fatty degeneration of the rotator cuff muscles similar to human muscles after chronic cuff tears. The severity of changes was associated with tear size and concomitant nerve injury.

PSF suppresses tau exon 10 inclusion by interacting with a stem-loop structure downstream of exon 10.

Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA-protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5' splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.

Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model.

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon-to-bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF-ß3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF-ß3 may enhance tendon-to-bone healing. We hypothesized that: TGF-ß3 could be released in a controlled manner using a heparin/fibrin-based delivery system (HBDS); and delivery of TGF-ß3 at the healing tendon-to-bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF-ß3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon-to-bone insertions of rats. Animals were sacrificed at 14-56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF-ß3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF-ß3 to the healing tendon-to-bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF-ß3 delivered at a sustained rate using a HBDS enhanced tendon-to-bone healing in a rat model.