Localized delivery of miRNAs targets cyclooxygenases and reduces flexor tendon adhesions.

The formation of adhesions during healing of an injured tendon remains a difficult problem in clinical practice. Local anti-inflammation gene delivery provides high local gene concentration, reduces the inflammatory response of the injured tendon microenvironment, and decreases systemic side effects to enhance in vivo efficacy. In this study, we designed a novel local sustained gene delivery system by using cyclooxygenase (COX-1 and COX-2)-engineered miRNA plasmid/nanoparticles embedded in hyaluronic acid (HA) hydrogel to reduce flexor tendon adhesions. The local sustained gene delivery system significantly downregulates COX-1 and COX-2 expression in the tendon tissue and the surrounding subcutaneous tissue. More importantly, this plasmid/nanoparticle hydrogel system significantly reduced tissue adhesion formation. This approach offers an effective therapeutic strategy to reduce tendon adhesions by directly targeting the down-regulation of COX-1 and COX-2 expression within the microenvironment of the injured tendon. STATEMENT OF SIGNIFICANCE: A local sustained gene delivery system was developed to regulate the expression of targeted genes in the specific time and location for tendon adhesion treatment. The engineered miRNA plasmid/nanoparticles embedded in hyaluronic acid hydrogel were synthesized to downregulate the expression of cyclooxygenases in the tendon tissue during the early stage of tendon healing with inflammatory response. This plasmid/nanoparticle hydrogel system offers an effective therapeutic strategy to attenuate the formation of tendon adhesion through direct downregulation of COX-1 and COX-2 expression within the microenvironment of the injured tendon.

Postnatal tendon growth and remodeling require platelet-derived growth factor receptor signaling.

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in the fundamental biological activities of many cells that compose musculoskeletal tissues. However, little is known about the role of PDGFR signaling during tendon growth and remodeling in adult animals. Using the hindlimb synergist ablation model of tendon growth, our objectives were to determine the role of PDGFR signaling in the adaptation of tendons subjected to a mechanical growth stimulus, as well as to investigate the biological mechanisms behind this response. We demonstrate that both PDGFRs, PDGFRα and PDGFRß, are expressed in tendon fibroblasts and that the inhibition of PDGFR signaling suppresses the normal growth of tendon tissue in response to mechanical growth cues due to defects in fibroblast proliferation and migration. We also identify membrane type-1 matrix metalloproteinase (MT1-MMP) as an essential proteinase for the migration of tendon fibroblasts through their extracellular matrix. Furthermore, we report that MT1-MMP translation is regulated by phosphoinositide 3-kinase/Akt signaling, while ERK1/2 controls posttranslational trafficking of MT1-MMP to the plasma membrane of tendon fibroblasts. Taken together, these findings demonstrate that PDGFR signaling is necessary for postnatal tendon growth and remodeling and that MT1-MMP is a critical mediator of tendon fibroblast migration and a potential target for the treatment of tendon injuries and diseases.

Temporary inductions of matrix metalloprotease-3 (MMP-3) expression and cell apoptosis are associated with tendon degeneration or rupture after corticosteroid injection.

Corticosteroid injections are widely used to treat enthesopathy and tendinitis, but are also associated with possible side effects, such as tendon degeneration or rupture. However, the mechanism of tendon degeneration or rupture after corticosteroid injection remains controversial. The purpose of this study was to reveal the mechanism of tendon degeneration or rupture after injection of triamcinolone acetonide (TA) or prednisolone (PSL). Forty-two rats were divided into 3 groups: A normal saline injection group (control group), a TA injection group, and a PSL injection group; the normal saline or corticosteroid was injected around the Achilles tendon. One or 3 weeks after injection, the tendons were subjected to biomechanical testing and histological analysis. At 1 week, the biomechanical strength was significantly lower in the corticosteroid groups. Histological analysis, at 1-week post-injection, showed collagen attenuation, increased expression of MMP-3 and apoptotic cells in the corticosteroid groups. The histological changes and biomechanical weaknesses of the tendon were not seen at 3 weeks. These alterations appeared to be involved in tendon degeneration or rupture after corticosteroid injection.

Apoptosis occurs throughout the diseased rotator cuff.

BACKGROUND: Even though apoptosis is known to be closely associated with rotator cuff tears, the differences in apoptosis according to the location within the torn supraspinatus tendon are still unknown. PURPOSE: To elucidate where apoptosis begins within the supraspinatus tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon tissues were collected from 14 patients undergoing arthroscopic rotator cuff repair surgery and 7 patients undergoing surgery for proximal humeral fracture who served as controls. In the patients with rotator cuff tears, the samples were harvested at 3 sites: the most lateral torn margin, 1 cm medial from the torn margin, and at the posterior torn corner. Caspase 3/7, 8, and 9 and cytochrome c activities were measured to determine the intracellular apoptosis pathway. Apoptotic cells were determined by in situ TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining, and immunohistochemistry was performed. RESULTS: The apoptotic activities of tendons from the experimental subjects were significantly higher than those of the controls. There were, however, no significant differences between the 3 sample sites. Immunohistochemistry also revealed strong expression of increased caspase 3/7, 8, and 9 and cytochrome c but no significant difference between them. CONCLUSION: This study shows that the intracellular apoptotic pathway is not only through the cell membrane receptor but also via intracellular mitochondria cascade. CLINICAL RELEVANCE: Because apoptosis occurs regardless of the location within the rotator cuff, debridement of the torn margin to obtain a healthy tendon may not be needed. Further study should focus on not only the technique of tying the torn tendon back to the bone but also biological augmentation to reverse or prevent further apoptosis within rotator cuff tendon.

MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix.

The extracellular matrix (ECM) of skeletal muscle and tendon is composed of different types of collagen molecules that play important roles in the transmission of forces throughout the body, and in the repair and regeneration of injured tissues. Fibroblasts are the primary cells in muscle and tendon that maintain, repair, and modify the ECM in response to mechanical loading, injury, and inactivity. Matrix metalloproteinases (MMPs) are enzymes that digest collagen and other structural molecules, which are synthesized and excreted by fibroblasts. MMPs are required for baseline ECM homeostasis, but disruption of MMP regulation due to injury or disease can alter the normal ECM architecture and prevent proper force transmission. Chronic injuries and diseases of muscles and tendons can be severely debilitating, and current therapeutic modalities to enhance healing are quite limited. This review will discuss the mechanobiology of MMPs, and the potential use of MMP inhibitors to improve the treatment of injured and diseased skeletal muscle and tendon tissue.

Bone marrow-derived matrix metalloproteinase-9 is associated with fibrous adhesion formation after murine flexor tendon injury.

The pathogenesis of adhesions following primary tendon repair is poorly understood, but is thought to involve dysregulation of matrix metalloproteinases (Mmps). We have previously demonstrated that Mmp9 gene expression is increased during the inflammatory phase following murine flexor digitorum (FDL) tendon repair in association with increased adhesions. To further investigate the role of Mmp9, the cellular, molecular, and biomechanical features of healing were examined in WT and Mmp9(-/-) mice using the FDL tendon repair model. Adhesions persisted in WT, but were reduced in Mmp9(-/-) mice by 21 days without any decrease in strength. Deletion of Mmp9 resulted in accelerated expression of neo-tendon associated genes, Gdf5 and Smad8, and delayed expression of collagen I and collagen III. Furthermore, WT bone marrow cells (GFP(+)) migrated specifically to the tendon repair site. Transplanting myeloablated Mmp9(-/-) mice with WT marrow cells resulted in greater adhesions than observed in Mmp9(-/-) mice and similar to those seen in WT mice. These studies show that Mmp9 is primarily derived from bone marrow cells that migrate to the repair site, and mediates adhesion formation in injured tendons. Mmp9 is a potential target to limit adhesion formation in tendon healing.

Metalloproteases and rotator cuff disease.

The molecular changes occurring in rotator cuff tears are still unknown, but much attention has been paid to better understand the role of matrix metalloproteinases (MMP) in the development of tendinopathy. These are potent enzymes that, once activated, can completely degrade all components of the connective tissue, modify the extracellular matrix (ECM), and mediatethe development of painful tendinopathy and tendon rupture. To control the local activity of activated proteinases, the same cells produce tissue inhibitors of metalloproteinases (TIMP) that bind to the enzymes and prevent degradation. The balance between the activities of MMPs and TIMPs regulates tendon remodeling, whereas an imbalance produces a collagen dis-regulation and disturbances intendons. ADAMs (a disintegrin and metalloproteinase) are cell membrane-linked enzymes with proteolytic and cell signaling functions. ADAMTSs (ADAM with thrombospondin motifs) are secreted into the circulation, and constitute a heterogenous family of proteases with both anabolic and catabolic functions. Biologic modulation of endogenous MMP activity to basal levels may reduce pathologic tissue degradation and favorably influence healing after rotator cuff repair. Further studies are needed to better define the mechanism of action, and whether these new strategies are safe and effective in larger models.

Role of metalloproteinases in rotator cuff tear.

The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPS) in the pathophysiology of rotator cuff tears has not been established yet. Recent advances empathize about the role of MMPs and TIMPS in extracellular matrix (ECM) remodeling and degradation in rotator cuff tears pathogenesis and healing after surgical repair. An increase in MMPs synthesis and the resulting MMPs mediated alterations in the ECM of tendons have been implicated in the etiopathogenesis of tendinopathy, and there is an increase in the expression of MMPs and a decrease in TIMP messenger ribonucleic acid expression in tenocytes from degenerative or ruptured tendons. Importantly, MMPs are amenable to inhibition by cheap, safe, and widely available drugs such as the tetracycline antibiotics and bisphosphonates. A better understanding of relationship and activity of these molecules could provide better strategies to optimize outcomes of rotator cuff therapy.

Expression of matrix metalloproteinases 1, 3, and 9 in differing extents of tendon retraction in the torn rotator cuff.

PURPOSE: Differing extents of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiologic context of tendon degeneration and the extent of tendon retraction are unclear. Tendon integrity depends on the extracellular matrix, which is regulated by matrix metalloproteinases (MMP). It is unknown which enzymes play a role in tendon degeneration. The hypotheses are that (1) the expression of MMPs 1, 3, and 9 is altered in the torn rotator cuff when compared with healthy tendon samples; and (2) that there is a relationship between MMP expression and the extent of tendon retraction in the torn cuff. METHODS: Rotator cuff tendon samples of 33 patients with full-thickness rotator cuff tears (Bateman grade III) were harvested during reconstructive surgery. Samples were dehydrated and paraffin-embedded. Immunohistologic determination of MMP 1, 3, and 9 expression was performed by staining sample slices with MMP antibody. The extent of tendon retraction was determined intraoperatively according to Patte's classification and patients were assigned to 4 groups (control group, and by tendon retraction grade Patte I-III). The control group consisted of six healthy tendon samples. RESULTS: Expression of MMPs 1 and 9 was significantly higher in torn cuff samples than in healthy tendons whereas MMP 3 expression was significantly decreased (P < 0.05). MMP 9 expression significantly increased with rising extent of tendon retraction in the torn cuff (P < 0.05). No significant association was found between expression of MMPs 1 and 3 and the rising extent of tendon retraction by Patte's classification. CONCLUSION: Elevated expression of MMPs 1 and 9 as well as decreased MMP 3 expression can be detected in torn rotator cuff tendon tissue. There is a significant association between the extent of tendon retraction and MMP 9 expression. The results of this study give evidence that early surgical treatment of small and partial-thickness rotator cuff tears is required.

Stem cells genetically modified with the developmental gene MT1-MMP improve regeneration of the supraspinatus tendon-to-bone insertion site.

BACKGROUND: Rotator cuffs heal through a scar tissue interface after repair, which makes them prone to failure. Membrane type 1 matrix metalloproteinase (MT1-MMP) is upregulated during embryogenesis in areas that develop into tendon-bone insertion sites. HYPOTHESIS: Bone marrow-derived stem cells in the presence of the developmental signal from MT1-MMP will drive the healing process toward regeneration and away from scar formation. STUDY DESIGN: Controlled laboratory study. METHODS: Sixty Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon. Thirty animals received mesenchymal stem cells (MSCs) in a fibrin glue carrier, and 30 received adenoviral MT1-MMP (Ad-MT1-MMP)-transduced MSCs. Animals were sacrificed at 2 weeks and 4 weeks and evaluated for the presence of fibrocartilage and collagen fiber organization at the insertion. Biomechanical testing was performed to determine the structural and material properties of the repaired tissue. Statistical analysis was performed with a Wilcoxon rank-sum test with significance set at P = .05. RESULTS: There were no differences between the Ad-MT1-MMP and MSC groups in any outcome variable at 2 weeks. At 4 weeks, the Ad-MT1-MMP group had more fibrocartilage (P = .05), higher ultimate load to failure (P = .01), higher ultimate stress to failure (P = .005), and higher stiffness values (P = .02) as compared with the MSC group. CONCLUSION: Mesenchymal stem cells genetically modified to overexpress the developmental gene MT1-MMP can augment rotator cuff healing at 4 weeks by the presence of more fibrocartilage at the insertion and improved biomechanical strength. CLINICAL RELEVANCE: Biologic augmentation of repaired rotator cuffs with MT1-MMP-transduced MSCs may reduce the incidence of retears. However, further studies are needed to determine if this remains safe and effective in larger models.

Prolonged immobilization compromises up-regulation of repair genes after tendon rupture in a rat model.

It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase-polymerase chain reaction was performed at 8 and 17 days post-rupture to assess different growth factors [brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia-inducible factor-1alpha (HIF-1alpha)] in the healing region. At 8 days post-injury, tendon mRNA levels were comparable in both groups. However, by day 17, the mRNA levels for BDNF, bFGF, COX 1 and HIF-1alpha in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There were no significant differences in the expression of NGF, IGF-1 or COX 2 between the different groups, indicating that injury-associated expression of these molecules is not overtly influenced by loading. This study supports the notion that prolonged immobilization post-rupture hampers the healing process by compromising the up-regulation of repair gene expression in the healing tendon. It might be speculated that a shorter period of immobilization, i.e. 1 week, would not impair the healing process significantly. The findings support the current development of earlier and more active rehabilitation programs after tendon injuries.

Pathophysiology of cyclooxygenase inhibition in animal models.

Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins (PGs), which play a significant role in health and disease in the gastrointestinal tract (GI) and in the renal, skeletal, and ocular systems. COX-1 is constitutively expressed and found in most normal tissues, whereas COX-2 can be expressed at low levels in normal tissues and is highly induced by pro-inflammatory mediators. Inhibitors of COX activity include: (1) conventional nonselective, nonsteroidal anti-inflammatory drugs (ns-NSAIDs) and (2) COX-2 selective nonsteroidal anti-inflammatory drugs (COX-2 s-NSAIDs). Inhibition of COX-1 often elicits GI toxicity in animals and humans. Therefore, COX-2 s-NSAIDs were developed to provide a selective COX-2 agent, while minimizing the attendant COX-1-mediated GI toxicities. Rats and dogs overpredict COX inhibition for renal effects such as renal handling of electrolytes in humans. COX inhibitors are shown to have both beneficial and detrimental effects, such as on healing of ligament or tendon tears, on the skeletal system in animal models. Certain ophthalmic conditions such as glaucoma and keratitis are associated with increased COX-2 expression, suggesting a potential role in their pathophysiology.

Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons.

The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures.

Gene expression and matrix turnover in overused and damaged tendons.

Chronic, painful conditions affecting tendons, frequently known as tendinopathy, are very common types of sporting injury. The tendon extracellular matrix is substantially altered in tendinopathy, and these changes are thought to precede and underlie the clinical condition. The tendon cell response to repeated minor injuries or "overuse" is thought to be a major factor in the development of tendinopathy. Changes in matrix turnover may also be effected by the cellular response to physical load, altering the balance of matrix turnover and changing the structure and composition of the tendon. Matrix turnover is relatively high in tendons exposed to high mechanical demands, such as the supraspinatus and Achilles, and this is thought to represent either a repair or tissue maintenance function. Metalloproteinases are a large family of enzymes capable of degrading all of the tendon matrix components, and these are thought to play a major role in the degradation of matrix during development, adaptation and repair. It is proposed that some metalloproteinase enzymes are required for the health of the tendon, and others may be damaging, leading to degeneration of the tissue. Further research is required to investigate how these enzyme activities are regulated in tendon and altered in tendinopathy. A profile of all the metalloproteinases expressed and active in healthy and degenerate tendon is required and may lead to the development of new drug therapies for these common and debilitating sports injuries.

Expression and enzymatic activity of MMP-2 during healing process of the acute supraspinatus tendon tear in rabbits.

We investigated the spontaneous healing process of a surgically created supraspinatus tendon tear in rabbits with specific reference to the expression of matrix metalloproteinase-2 (MMP-2) and its time-course change in enzymatic activity along with the expression of tissue inhibitors of metalloproteinases (TIMPs). A transverse, full thickness tear of the supraspinatus tendon was created and examined. Immunohistochemical analysis revealed that MMP-2 positive cells were mainly localized at both cutting ends of the tendon, and reparative tissue encroached into the gap from the bursal side. The expression of TIMP-1 was induced in the cells at not only the tendon edges but also the reparative tissue during the healing process. TIMP-2 was constitutively expressed in both the tendon and the reparative tissue. Gelatin zymography using tissue culture media demonstrated latent and active forms of MMP-2 and characteristic time-linked changes of the enzymatic activity. Western blotting confirmed the bands as the latent form of MMP-2. These results suggest that MMP-2 is expressed and activated during the healing process of acute supraspinatus tendon tear and can play an important role in the remodeling process.

Antioxidant enzyme peroxiredoxin 5 is upregulated in degenerative human tendon.

Peroxiredoxin 5 (PRDX5) is a novel thioredoxin peroxidase recently identified in a variety of human cells and tissues, which is considered to play an important role in oxidative stress protection mechanisms. However, little is known about its expression in tendon degeneration, a common and disabling condition that primarily affects older people, in which oxidative stress may be implicated. The present study demonstrated that normal human tendon expresses PRDX5 and its expression is significantly increased in degenerative tendon. In addition, we have localized PRDX5 to fibroblasts in normal tendon and to both fibroblasts and endothelial cells in degenerate tendon. The differential expression of PRDX5 in normal and degenerate tendon shows that a thioredoxin peroxidase with antioxidant properties is upregulated under pathophysiological conditions and suggests that oxidative stress may be involved in the pathogenesis of tendon degeneration. PRDX5 may play a protective role against oxidative stress during this pathophysiological process.

In vivo gene transfer and overexpression of focal adhesion kinase (pp125 FAK) mediated by recombinant adenovirus-induced tendon adhesion formation and epitenon cell change.

Adhesion formation is a frequent complication of tendon injury repair: however, little is known about its mechanisms. The intracellular focal adhesion kinase (FAK)-related signaling pathway may be one of the mechanisms involved in the induction of tendon adhesions. The replication deficient adenovirus containing the FAK gene (pp125 FAK) was constructed and named Adv-Fak. By in vitro transductions with the recombinant virus, overexpression of the FAK protein was documented in transduced cultured primary tendon cells. By in vivo direct injection of Adv-FAK into the space between the tendon and tendon sheath of White Leghorn chickens, FAK gene transfer with overexpression of the FAK protein was detected by immunohistological staining. The morphology of these stained cells changed from the normal flat shape to cuboid. The group with overexpressed adenovirus-mediated FAK had significant adhesion formation, as seen by increased work of flexion (118.197 +/- 29.616), compared with the group with overexpressed adenovirus-mediated beta-galactosidase (67.507 +/- 36.066) (p < 0.0393) and the group with adenovirus-mediated FAK antisense gene transfer (60.357 +/- 48.562) (p < 0.0211). Histological examination of the samples from tendons with Adv-FAK showed fibers between the tendon and tendon sheath; there were no fibers in the cavities of samples of injured tendons infected with Adv-beta gal. Moreover, at the application site of the former tendons, a thick fiber layer without epitenon cells was built up on the outer surface, whereas a thin fiber layer with clear epitenon cells was observed in the tendons to which Adv-beta gal was applied. Our results show that overexpression of FAK can induce tendon adhesion formation in vivo. This indicates that FAK and the FAK-related signaling pathway may be involved in the process of tendon adhesion formation. Understanding the details of this process may help to prevent tendon adhesion and improve healing.

Histochemical correlates of hamstring injuries.

This study reports the histochemical fiber type composition of the human hamstring muscles. Muscle specimens from necropsy specimens were obtained from seven locations in the hamstring, four locations in the quadriceps, and one location in the adductor magnus. The hamstring muscles are shown to have a relatively high proportion of Type II fibers. Type II fibers are more involved with exercise of higher intensity and force production and it is postulated that the hamstrings are capable of high intrinsic force production. The hamstrings are two-joint muscles and are, therefore, subject to increased stretch and force production extrinsically by motion at the hip and knee. It is proposed that high levels of tension in the hamstrings produced by intrinsic force production and extrinsic stretch may make them prone to injury in periods of intense muscular activity. This proposal is also relevant to other frequent athletic muscle injuries.