transforming-growth-factor-beta and Rotator-Cuff-Injuries

The rotator cuff undergoes natural degeneration with age, leading to age-related rotator cuff tear; however, the precise mechanism remains unclear. Transforming growth factor-beta (TGF-β) concentrations rise with age and TGF-β contributes to the pathophysiology of skeletal muscle. TGF-β has also been shown to suppress expression of the myokine, apelin, in skin fibroblasts. We hypothesized that TGF-β expression in the rotator cuff changes with age and regulates apelin expression, thereby contributing to rotator cuff degeneration.. We used quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) to measure the expression of apelin and tendon-related genes (Tnmd, Col1a1, and Col3a1) in the rotator cuff of young (12 weeks), adult (24 weeks), and old (48 weeks) rats. Using Q-RT-PCR and enzyme-linked immunosorbent assay, we also measured Tgfb mRNA and TGF-β protein levels, respectively. Furthermore, we used Q-RT-PCR to measure apelin mRNA levels in rotator cuff-derived cells after treatment with 0 (control) and 10 ng/mL recombinant TGF-β.. Apelin mRNA levels were significantly lower in old compared to young and adult rats. Similarly, tendon-related genes, Tnmd, Col1a1, and Col3a1, were significantly lower in adult and old rats than young rats. In contrast, Tgfb mRNA and TGF-β protein were significantly higher in old compared to young rats. Stimulation with exogenous TGF-β significantly decreased Apelin mRNA expression compared to control.. TGF-β regulates apelin expression in the rotator cuff and may play a key role in the degenerative pathology of the rotator cuff with age.

Topics: Animals; Apelin; Rats; RNA, Messenger; Rotator Cuff; Rotator Cuff Injuries; Transforming Growth Factor beta; Transforming Growth Factors

Bone morphogenetic protein 2 (BMP-2) plays an important role in the tendon-to-bone repair process. However, there is no previous literature on acceleration of the tendon-to-bone repair process by BMP-2 delivered by β-tricalcium phosphate (β-TCP). The aim of this study was to investigate the accelerated effect of recombinant human BMP-2 (rhBMP-2) delivered by β-TCP on the tendon-to-bone repair process.. The infraspinatus tendon of elderly female Japanese white rabbits was detached from its insertion site on the humerus. A bone tunnel (4.2 mm) was created at the original insertion site of the tendon, which was repaired using the McLaughlin procedure after filling in β-TCP (porosity 75%) without BMP-2 (control group) or with 10 µg rhBMP-2 (BMP group). The rabbits were sacrificed at the second, fourth, and eighth weeks after surgery for histologic analysis and biomechanical testing. We also evaluated the maturity of the tendon-to-bone junction using the tendon-to-bone maturity score.. Histologic analysis revealed no significant difference between the groups at 2 and 8 weeks but a more abundant organized fibrocartilage at the tendon-to-bone junction in the BMP group at 4 weeks. The tendon-to-bone maturity score improved sequentially. The interface of the BMP group at 4 weeks had significantly improved biomechanical properties than that of the control group.. The tendon-to-bone repair process was facilitated by the use of rhBMP-2 delivered by β-TCP at 4 weeks.

Topics: Animals; Biocompatible Materials; Bone Morphogenetic Protein 2; Calcium Phosphates; Disease Models, Animal; Drug Delivery Systems; Female; Humerus; Plastic Surgery Procedures; Rabbits; Recombinant Proteins; Rotator Cuff; Rotator Cuff Injuries; Shoulder Joint; Tendons; Transforming Growth Factor beta

Diseased tendons are characterised by fibrotic scar tissue, which adversely affects tendon structure and function and increases the likelihood of re-injury. The mechanisms and expression profiles of fibrosis in diseased tendon is understudied compared to pulmonary and renal tissues, where transforming growth factor (TGF)β and its associated superfamily are known to be key drivers of fibrosis and modulate extracellular matrix homeostasis. We hypothesised that differential expression of TGFβ superfamily members would exist between samples of human rotator cuff tendons with established disease compared to healthy control tendons.. Healthy and diseased rotator cuff tendons were collected from patients presenting to an orthopaedic referral centre. Diseased tendinopathic (intact) and healthy rotator cuff tendons were collected via ultrasound-guided biopsy and torn tendons were collected during routine surgical debridement. Immunohistochemistry and quantitative real-time polymerase chain reaction were used to investigate the protein and gene expression profiles of TGFβ superfamily members in these healthy and diseased tendons.. TGFβ superfamily members were dysregulated in diseased compared to healthy tendons. Specifically, TGFβ-1, TGFβ receptor (R)1 and TGFβ R2 proteins were reduced (p < 0.01) in diseased compared to healthy tendons. At the mRNA level, TGFβ R1 was significantly reduced in samples of diseased tendons, whereas TGFβ R2 was increased (p < 0.01). BMP-2, BMP-7 and CTGF mRNA remained unchanged with tendon disease.. We propose that downregulation of TGFβ pathways in established tendon disease may be a protective response to limit disease-associated fibrosis. The disruption of the TGFβ axis with disease suggests associated downstream pathways may be important for maintaining healthy tendon homeostasis. The findings from our study suggest that patients with established tendon disease would be unlikely to benefit from therapeutic TGFβ blockade, which has been investigated as a treatment strategy in several animal models. Future studies should investigate the expression profile of fibrotic mediators in earlier stages of tendon disease to improve understanding of the targetable mechanisms underpinning tendon fibrosis.

Topics: Adolescent; Adult; Aged; Cohort Studies; Female; Gene Expression Regulation; Humans; Male; Middle Aged; Rotator Cuff; Rotator Cuff Injuries; Tendons; Transforming Growth Factor beta; Young Adult

Rotator cuff tears represent a large burden of muscle-tendon injuries in our aging population. While small tears can be repaired surgically with good outcomes, critical size tears are marked by muscle atrophy, fibrosis, and fatty infiltration, which can lead to failed repair, frequent re-injury, and chronic disability. Previous animal studies have indicated that Transforming Growth Factor-β (TGF-β) signaling may play an important role in the development of these muscle pathologies after injury. Here, we demonstrated that inhibition of TGF-β1 signaling with the small molecule inhibitor SB431542 in a mouse model of massive rotator cuff tear results in decreased fibrosis, fatty infiltration, and muscle weight loss. These observed phenotypic changes were accompanied by decreased fibrotic, adipogenic, and atrophy-related gene expression in the injured muscle of mice treated with SB431542. We further demonstrated that treatment with SB431542 reduces the number of fibro/adipogenic progenitor (FAP) cells-an important cellular origin of rotator cuff muscle fibrosis and fatty infiltration, in injured muscle by promoting apoptosis of FAPs. Together, these data indicate that the TGF-β pathway is a critical regulator of the degenerative muscle changes seen after massive rotator cuff tears. TGF-β promotes rotator cuff muscle fibrosis and fatty infiltration by preventing FAP apoptosis. TGF-β regulated FAP apoptosis may serve as an important target pathway in the future development of novel therapeutics to improve muscle outcomes following rotator cuff tear.

Topics: Adipose Tissue; Animals; Apoptosis; Benzamides; Dioxoles; Disease Models, Animal; Female; Fibrosis; Gene Expression Regulation; Mice; Muscular Atrophy; Rotator Cuff; Rotator Cuff Injuries; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Muscle atrophy, fatty infiltration, and fibrosis of the muscle have been described as important factors governing outcome after rotator cuff injury and repair. Muscle fibrosis is also thought to have a role in determining muscle compliance at the time of surgery. The transforming growth factor-β (TGF-β) pathways are highly conserved pathways that exert a potent level of control over muscle gene expression and are critical regulators of fibrosis in multiple organ systems. It has been shown that TGF-β can regulate important pathways of muscle atrophy, including the Akt/mammalian target of rapamycin pathway. The purpose of this study was to evaluate the expression of TGF-β and its downstream effectors of fibrosis after a massive rotator cuff tear (RCT) in a previously established rat model.. To simulate a massive RCT, infraspinatus and supraspinatus tenotomy and suprascapular nerve transection were performed on Sprague-Dawley rats with use of a validated model. Two and 6 weeks after surgery, supraspinatus muscles were harvested to study alterations in TGF-β signaling by Western blotting, quantitative polymerase chain reaction, and histologic analysis.. There was a significant increase in fibrosis in the rotator cuff muscle after RCT in our animal model. There was a concomitant increase in TGF-β gene and protein expression at both 2 and 6 weeks after RCT. Evaluation of the TGF-β signaling pathway revealed an increase in SMAD2 activation but not in SMAD3. There was an increase in profibrotic markers collagen I, collagen III, and α-smooth muscle actin.. TGF-β signaling is significantly upregulated in rat supraspinatus muscles after RCTs.

Topics: Animals; Disease Models, Animal; Female; Fibrosis; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rotator Cuff; Rotator Cuff Injuries; Tendon Injuries; Transforming Growth Factor beta; Up-Regulation

The purpose of this study was to examine the role of two of the three transforming growth factor beta (TGF-β) isoforms at the healing tendon-to-bone insertion. The supraspinatus tendons of 64 rats were transected at their bony insertions and repaired to the humeral head. One shoulder of each rat received an osmotic pump for sustained delivery of the following factors at the repair site: (1) TGF-β1 and neutralizing antibodies to TGF-β2 and 3 (TGF-β1 group), (2) TGF-β3 and neutralizing antibodies to TGF-β1 and 2 (TGF-β3 group), (3) neutralizing antibodies to TGF-β1, 2, and 3 (anti-TGF-β group), and (4) saline (saline group). The contralateral shoulders received saline to serve as paired controls. The repairs were evaluated at multiple time points postmortem using histology-based assays and biomechanical testing. Treated shoulders in the TGF-β1 group showed increased type III collagen production compared to the paired control shoulders, indicative of a scar-mediated response. There was a trend toward reduced mechanical properties in the TGF-β1 group, but these changes did not reach statistical significance. The anti-TGF-β group showed no difference in tissue volume, but significantly inferior mechanical properties, compared to the paired control shoulders. The TGF-β3 group did not show any differences compared to the paired control shoulders. Although TGF-β isoforms play important roles in tendon-to-bone development and healing, application of exogenous TGF-β isoforms and neutralizing antibodies to the subacromial space using osmotic pumps did not improve supraspinatus tendon-to-bone healing.

Topics: Animals; Biomechanical Phenomena; Collagen Type III; Histological Techniques; Immunohistochemistry; Male; Protein Isoforms; Rats; Rats, Sprague-Dawley; Rotator Cuff; Rotator Cuff Injuries; Statistics, Nonparametric; Tendon Injuries; Transforming Growth Factor beta; Wound Healing

Rotator cuff repair is known to have a high failure rate. Little is known about the natural healing process of the rotator cuff repair site, hence little can be done to improve the tendon's ability to heal. The purpose of this study was to investigate the collagen formation at the early repair site and to localize TGFbeta-1 and 3 during early healing and compare their levels to cell proliferation and histological changes. Bilateral supraspinatus tendons were transected and repaired in 60 rats. Specimens were harvested and evaluated at 0, 1, 3, 7, 10, 28, and 56 days. Histological sections were evaluated for cell morphology. Immunohistochemistry and in situ hybridization was performed to localize protein and mRNA for collagen types I and III and TGFbeta-1 and 3. Proliferating cell nuclear antigen (PCNA) assay was performed to measure cell proliferation, and cells were counted to determine cell density. Biomechanical properties were evaluated. Repair tissue demonstrated an initial inflammatory response with multinucleated cells present at 1 and 3 days, and lymphocytes and plasma cells presents at 7 and 10 days. Capillary proliferation began at 3 days and peaked at 10 days. Ultimate force increased significantly over the time period studied. Collagen I protein and mRNA significantly increased at 10 days, and reached a plateau by 28 and 56 days. Collagen III showed a similar trend, with an early increase, and remained high until 56 days. TGFbeta-1 was localized to the forming scar tissue and showed a distinct peak at 10 days. TGFbeta-3 was not seen at the healing insertion site. Cell proliferation and density followed the same trend as TGFbeta-1. A wound healing response does occur at the healing rotator cuff insertion site, however, the characteristics of the tendon after healing differ significantly from the uninjured tendon insertion site at the longest time-point studied. A distinctive collagen remodeling process occurred with an initial increase in the formation of collagen types I and III followed by a decrease toward baseline levels seen at time 0. Growth factor TGFbeta-1 was localized to repair tissue and coincided with a peak in cell proliferation and cellularity. Repair sites remained unorganized histologically and biomechanically inferior in comparison to previously described uninjured insertion sites.

Topics: Acute Disease; Animals; Biomechanical Phenomena; Cell Count; Cell Proliferation; Collagen Type I; Collagen Type III; Disease Models, Animal; Immunoenzyme Techniques; In Situ Hybridization; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rotator Cuff; Rotator Cuff Injuries; Tendon Injuries; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Degeneration of the rotator cuff is often associated with inflammation of the subacromial bursa and focal mineralization of the supraspinatus tendon. Portions of the supraspinatus tendon distant from the insertion site could transform into fibrous cartilage, causing rotator-cuff tears owing to mechanical instability. Indirect evidence is presented to link this pathology to ectopic production and secretion of bioactive bone morphogenetic proteins (BMPs) from sites within the subacromial bursa. Surgically removed specimens of subacromial bursa tissue from patients with chronic tears of the rotator cuff were analyzed by immunohistochemistry and reverse transcription-PCR. Bioactive BMP was detected in bursa extracts by a bioassay based on induction of alkaline phosphatase in the osteogenic/myogenic cell line C2C12. Topical and differential expression of BMP-2/4 and BMP-7 mRNA and protein was found in bursa tissue. The bioassay of C2C12 cells revealed amounts of active BMP high enough to induce osteogenic cell types, and blocking BMP with specific antibodies or soluble BMP receptors Alk-3 and Alk-6 abolished the inductive properties of the extract. Sufficient information was gathered to explain how ectopic expression of BMP might induce tissue transformation into ectopic bone/cartilage and, therefore, promote structural degeneration of the rotator cuff. Early surgical removal of the subacromial bursa might present an option to interrupt disease progression.

Topics: Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bursa, Synovial; Cells, Cultured; Chronic Disease; Cytokines; Extracellular Matrix Proteins; Histocytochemistry; Humans; Immunohistochemistry; Lacerations; Middle Aged; RNA, Messenger; Rotator Cuff Injuries; Transforming Growth Factor beta

The rotator cuff frequently sustains athletic and occupational injury, often resulting in chronic pain and disability. However, despite the high incidence of such shoulder problems, the pathophysiology of rotator cuff injury and healing has not yet been fully elucidated. The notable finding of this study was the presence of a contractile actin isoform, alpha-smooth muscle actin (SMA), in nonvascular cells in all of the seven torn human rotator cuff specimens evaluated immunohistochemically. Up to 95% of cells in any one region, and over 95% of elongated cells found in association with crimped collagen, contained SMA. Most of the cells staining positive for SMA in these sections had morphological features of the fibroblast, though a small number were chondrocyte-like. Treatment of cells growing out from human rotator cuff explants with TGF-beta1 significantly increased the amount of SMA evaluated by Western blot analysis. PDGF-BB and IFN-gamma had no effect on the cell content of SMA. This is the first documentation of the presence of SMA-positive cells in the human rotator cuff tendon. SMA has been found in a number of other healing connective tissues including skin, ligament, meniscus, cartilage, and other types of tendon. Of importance are previous findings that SMA-positive cells can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. The results of the present study thus suggest that SMA-containing cells could contribute to the retraction of the torn ends of a ruptured rotator cuff and play an important role in healing.

Topics: Actins; Adult; Aged; Blotting, Western; Cells, Cultured; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Middle Aged; Muscle, Smooth; Rotator Cuff; Rotator Cuff Injuries; Tendons; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wounds and Injuries

Inflammation in the subacromial bursa causes pain in patients suffering from rotator cuff tear, with this long-lasting inflammation leading to fibrosis and thickening of the subacromial bursa. Both inflammatory cytokines and mechanical stress, and impingement in the subacromial space, might induce and worsen this inflammation. However, little is known of the mechanism of this inflammation. In this study, we used immunohistological staining to demonstrate the expression of Interleukin-1 beta (IL-1 beta), Tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF) in subacromial bursa derived from the patients suffering from rotator cuff tear. On the other hand the expression of these inflammatory cytokines and growth factors were little detected only to a small degree in patients with anterior shoulder instability who did not have severe shoulder pain and impingement in the subacromial space. Our findings suggest that those inflammatory cytokines and growth factors may play an important role in inflammation of the subacromial bursa. Controlling the expression of these cytokines and growth factors might be important for treating patients suffering from shoulder pain due to rotator cuff tear.

Topics: Acromion; Adult; Aged; Bursa, Synovial; Bursitis; Cytokines; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Interleukin-1; Male; Middle Aged; Pain; Rotator Cuff; Rotator Cuff Injuries; Shoulder Impingement Syndrome; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha