Clinical Orthopaedics and Related Research ®

A Publication of The Association of Bone and Joint Surgeons ®

Symposium: Molecular and Clinical Developments in Tendinopathy 15 articles

Articles

The Basic Science of Tendinopathy

Yinghua Xu MBBS, George A. C. Murrell MD, DPhil Tendinopathy is a common clinical problem with athletes and in many occupational settings. Tendinopathy can occur in any tendon, often near its insertion or enthesis where there is an area of stress concentration, and is directly related to the volume of repetitive load to which the tendon is exposed. Recent studies indicate tendinopathy is more likely to occur in situations that increase the “dose” of load to the tendon enthesis – including increased activity, weight, advancing age, and genetic factors. The cells in tendinopathic tendon are rounder, more numerous, and show evidence of oxidative damage and more apoptosis. These cells also produce a matrix that is thicker and weaker with more water, more immature and cartilage-like matrix proteins, and less organization. There is now evidence of a population of regenerating stem cells within tendon. These studies suggest prevention of tendinopathy should be directed at reducing the volume of repetitive loads to below that which induces oxidative-induced apoptosis and cartilage-like genes. The management strategies might involve agents or cells that induce tendon stem cell proliferation, repair and restoration of matrix integrity.

Coordinate Regulation of IL-1β and MMP-13 in Rat Tendons Following Subrupture Fatigue Damage

Hui B. Sun PhD, Yonghui Li PhD, David T. Fung PhD, Robert J. Majeska PhD, Mitchell B. Schaffler PhD, Evan L. Flatow MD Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1β. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1β. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1β, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1β. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1β expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1β in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1β-dependent and IL-1β-independent pathways.

Loss of Homeostatic Tension Induces Apoptosis in Tendon Cells: An In Vitro Study

Monika Egerbacher DVM, PhD, Steven P. Arnoczky DVM, Oscar Caballero MS, Michael Lavagnino PhD, Keri L. Gardner MS Apoptosis (programmed cell death) has been identified as a histopathologic feature of tendinopathy. While the precise mechanism(s) that triggers the apoptotic cascade in tendon cells has not been identified, it has been theorized that loss of cellular homeostatic tension following microscopic damage to individual tendon fibrils could be the stimulus for initiating the pathologic events associated with tendinopathy. To determine if loss of homeostatic tension following stress deprivation could induce apoptosis in tendon cells, rat tail tendons were stress-deprived or cyclically loaded (3% strain at 0.17 Hz) for 24 hours under tissue culture conditions. Caspase-3 (an upstream mediator of apoptosis) mRNA expression was evaluated using quantitative polymerase chain reaction and caspase-3 protein synthesis was identified using immunohistochemistry. Apoptotic cells were identified histologically using an antibody for single-stranded DNA. Stress deprivation for 24 hours resulted in an increase in caspase-3 mRNA expression when compared to fresh controls or cyclically loaded tendons. Stress deprivation also increased the percentage of apoptotic cells (10.59% ± 2.80) compared to controls (1.87% ± 1.07) or cyclically loaded tendons (3.73% ± 0.87). These data suggest loss of homeostatic tension following stress deprivation induces apoptosis in rat tail tendon cells.

VEGF Expression in Patellar Tendinopathy: A Preliminary Study

Alexander Scott MSc, BSc(PT), Øystein Lian MD, PhD, Roald Bahr MD, PhD, David A. Hart PhD, Vincent Duronio PhD Vascular function and angiogenesis are regulated by vascular endothelial growth factor-A (VEGF). The purpose of this preliminary study was to address the following questions: Is VEGF expression in the patellar tendon more prevalent in patients with patellar tendinopathy than in individuals with normal, pain-free patellar tendons? Which cell populations express VEGF in normal and tendinopathic tendon? Is there a difference in symptom duration between VEGF+ and VEGF− tendons? We collected patellar tendon tissue from 22 patients undergoing open débridement of the patellar tendon and from 10 patients undergoing intramedullary nailing of the tibia. VEGF expression was assessed immunohistochemically. Relevant inflammatory and repair cell types were immunolabeled. VEGF expression was absent from control tendons, but was present in a subset of patients with histopathological evidence of angiofibroblastic tendinosis. VEGF was expressed in the intimal layer of tendon vessels, but was absent in other cell types. Patients demonstrating VEGF expression in the patellar tendon had a shorter symptom duration (12 ± 7.8 months) than patients with no detectable VEGF (32.8 ± 23.5 months). VEGF may contribute to the vascular hyperplasia that is a cardinal feature of symptomatic tendinosis, particularly in cases with more recent onset.

Addition of Nitric Oxide Through Nitric Oxide-paracetamol Enhances Healing Rat Achilles Tendon

George A. C. Murrell MD, DPhil, Gongyao Tang MD, Richard C. Appleyard PhD, Piero Soldato MD, Min-Xia Wang MD Nitric oxide is an important messenger molecule in many physiological processes. The addition of NO via NO-flurbiprofen enhances the material properties of healing tendon, however, flurbiprofen has a detrimental effect on healing. We asked if NO delivered by a cyclooxygenase 3 inhibitor (paracetamol/acetaminophen) would enhance healing in a rat Achilles tendon healing model. Rats were injected subcutaneously daily with NO-paracetamol, paracetamol or vehicle from two days before surgery to the day of tissue harvesting. Paracetamol had no effect on tendon healing compared with vehicle alone. NO-paracetamol did not change the failure load, but did decrease the water content, enhance the collagen content, reduce the cross-sectional area and improve the ultimate stress of healing tendon compared with paracetamol and vehicle. The collagen organization of the healing tendon in the NO-paracetamol group, as determined by polarized light microscopy, was enhanced. Our data suggests NO-paracetamol increases the total collagen content and enhances organization while decreasing the cross-sectional area of healing rat Achilles tendon and is consistent with human clinical trials where NO has improved the symptoms and signs of tendinopathy.

Gene Expression in Rat Supraspinatus Tendon Recovers From Overuse With Rest

Scott A. Jelinsky PhD, Spencer P. Lake BS, Joanne M. Archambault PhD, Louis J. Soslowsky PhD Rest is a common treatment for overuse injuries, but its effectiveness on gene expression has not been systematically evaluated under controlled experimental conditions. We asked whether genes regulated in the supraspinatus tendon as a result of overuse would return to normal levels after 2 or 4 weeks of rest. We used a rat model of tendon overuse that generates reproducible changes in the histology, geometry, gene expression, and mechanical properties consistent with an overuse injury. Animals were subjected to the overuse protocol for 2 or 4 weeks followed by either 2 or 4 weeks of rest. Microarray analysis was used to measure global changes in gene expression after the overuse plus rest protocol. Genes upregulated as a result of the overuse returned to near normal levels after rest in most animals. The biochemical composition of the tendon was similar to normal after the imposed rest period, except for slightly lower collagen content. These results suggest as little as 2 weeks of rest is often sufficient to recover from the molecular and biochemical effects of 2 and 4 weeks of overuse in this rat model.

Heat Shock Protein and Apoptosis in Supraspinatus Tendinopathy

Neal L. Millar MBChB, MRCS (Glasg), Ai Q. Wei MBBS, Timothy J. Molloy PhD, Fiona Bonar MBChB, MRCPI, FRCPath(Lond), George A. C. Murrell MD, DPhil Heat shock proteins (HSPs) are often upregulated following oxidative and other forms of stress. Based on reports of excessive apoptosis in torn supraspinatus tendon and mechanically loaded tendon cells, we hypothesized heat shock proteins may be present in rodent and human models of tendinopathy due to their central role in caspase dependent apoptotic cell signaling. We used a running rat supraspinatus tendinopathy overuse model with custom microarrays to investigate the process at a genetic level. Additionally torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. We identified substantial upregulation of heat shock proteins and apoptotic genes in the rodent model. We further confirmed increased levels of heat shock protein and apoptotic regulatory genes in human supraspinatus and subscapularis tendon. This finding suggests heat shock proteins play a role in the cascade of stress-activated programmed cell death and degeneration in tendinopathy and may provide a novel target in preventing tendinopathies.

Loss of Homeostatic Strain Alters Mechanostat “Set Point” of Tendon Cells In Vitro

Steven P. Arnoczky DVM, Michael Lavagnino PhD, Monika Egerbacher DVM, PhD, Oscar Caballero MS, Keri Gardner MS, Marisa A. Shender Tendon cells respond to mechanical loads. The character (anabolic or catabolic) and sensitivity of this response is determined by the mechanostat set point of the cell, which is governed by the cytoskeleton and its interaction with the extracellular matrix. To determine if loss of cytoskeletal tension following stress deprivation decreases the mechanoresponsiveness of tendon cells, we cultured rat tail tendons under stress-deprived conditions for 48 hours and then cyclically loaded them for 24 hours at 1%, 3%, or 6% strain at 0.17 Hz. Stress deprivation upregulated MMP-13 mRNA expression and caused progressive loss of cell-matrix contact compared to fresh controls. The application of 1% strain to fresh tendons for 24 hours inhibited MMP-13 mRNA expression compared to stress-deprived tendons over the same period. However, when tendons were stress-deprived for 48 hours and then subjected to the same loading regime, the inhibition of MMP-13 mRNA expression was decreased. In stress-deprived tendons, it was necessary to increase the strain magnitude to 3% to achieve the same level of MMP-13 mRNA inhibition seen in fresh tendons exercised at 1% strain. The data suggest loss of cytoskeletal tension alters the mechanostat set point and decreases the mechanoresponsiveness of tendon cells.

Movin and Bonar Scores Assess the Same Characteristics of Tendon Histology

Nicola Maffulli MD, MS, PhD, FRCS (Orth), Umile Giuseppe Longo MD, Francesco Franceschi MD, Carla Rabitti MD, Vincenzo Denaro MD The Movin scoring system and its validated modifications and the Bonar scoring system are used to classify the histopathological findings of tendinopathy. We compared the reliability of these two different histopathological evaluation scores of tendon tissue. Tendon samples were harvested from 88 individuals (49 men, 39 women; mean age, 58.2 years) who underwent arthroscopic repair of a rotator cuff tear, and from five male patients who died of cardiovascular events (mean age, 69.6 years). A piece of supraspinatus tendon that was not directly involved in the tear was harvested en bloc within the intact middle portion of the tendon. Using hematoxylin and eosin staining and Alcian blue, slides were assessed using Bonar and Movin scores. The intraclass correlation was 0.921 (confidence interval 95% 0.790–0.963). Movin’s and Bonar’s scores have a high correlation and assess similar characteristics and variables of tendon abnormalities.

Treatment of Tendinopathy: What Works, What Does Not, and What is on the Horizon

Brett M. Andres MD, George A. C. Murrell MD, Dphil Tendinopathy is a broad term encompassing painful conditions occurring in and around tendons in response to overuse. Recent basic science research suggests little or no inflammation is present in these conditions. Thus, traditional treatment modalities aimed at controlling inflammation such as corticosteroid injections and nonsteroidal antiinflammatory medications (NSAIDS) may not be the most effective options. We performed a systematic review of the literature to determine the best treatment options for tendinopathy. We evaluated the effectiveness of NSAIDS, corticosteroid injections, exercise-based physical therapy, physical therapy modalities, shock wave therapy, sclerotherapy, nitric oxide patches, surgery, growth factors, and stem cell treatment. NSAIDS and corticosteroids appear to provide pain relief in the short term, but their effectiveness in the long term has not been demonstrated. We identified inconsistent results with shock wave therapy and physical therapy modalities such as ultrasound, iontophoresis and low-level laser therapy. Current data support the use of eccentric strengthening protocols, sclerotherapy, and nitric oxide patches, but larger, multicenter trials are needed to confirm the early results with these treatments. Preliminary work with growth factors and stem cells is promising, but further study is required in these fields. Surgery remains the last option due to the morbidity and inconsistent outcomes. The ideal treatment for tendinopathy remains unclear.,[object Object]

Collagens, Proteoglycans, MMP-2, MMP-9 and TIMPs in Human Achilles Tendon Rupture

Evgenia Karousou PhD, Mario Ronga MD, Davide Vigetti PhD, Alberto Passi MD, PhD, Nicola Maffulli MD, MS, PhD, FRCS(Orth) Tendon integrity depends on the extracellular matrix (ECM) metabolism which is regulated by proteolytic enzymes. However, it is unclear which enzymes play a role in tendon rupture. We studied the ECM of 19 ruptured human Achilles tendons, comparing the composition of specimens harvested close to the rupture with specimens harvested from an apparently healthy area in the same tendon. We compared gene expression of collagen Type I, decorin, and versican including enzymes involved in their metabolism as matrix metalloproteases (MMP-2 and -9) and tissue inhibitory of metalloproteinase (TIMP-1 and -2) using real-time PCR, zymography and FACE analysis. We found greater gene expression of proteoglycan core protein decorin and versican, collagen Type I, MMPs and TIMPs in the tendon rupture. Zymography analysis, reflecting expression of enzymatic activity, confirmed the gene expression data at protein level. Carbohydrate content was greater in the macroscopically healthy area than in the ruptured area. In the ruptured area, we found increased core protein synthesis but without the normal glycosaminoglycan production. The tissue in the area of rupture undergoes marked rearrangement at molecular levels and supports the role of MMPs in the pathology.

Successful Management of Tendinopathy With Injections of the MMP-inhibitor Aprotinin

John Orchard MD, PhD, FACSP, FACSM, FFSEM (UK), Andrew Massey MB BCh BAO BSc(Hons), Richard Brown MBBS Bsc(Med) (Hons) FRACGP FACSP, Adéline Cardon-Dunbar, Jamie Hofmann MD Aprotinin is a broad spectrum proteinase inhibitor (including matrix metalloproteinase [MMP] inhibitor) used for treating patellar and Achilles tendinopathies. One previous randomized control trial demonstrated aprotinin injections superior to both corticosteroid and saline injections in patellar tendinopathy (Level II), whereas results reported for aprotinin treatment in Achilles tendinopathy have been mixed. We performed a case review and followup questionnaire for 430 consecutive patients with tendinopathy treated by 997 aprotinin injections (30,000 KIU). A response rate of 72% was achieved with a minimum followup of 3 months (average, 12.2 months; range, 3–54 months). Seventy-six percent of patients had improved, 22% of patients reported no change, and 2% were worse. Sixty-four percent of patients thought aprotinin injections were helpful, while 36% believed they had neither a positive nor negative effect. Mid-Achilles tendinopathy patients (84% improvement) were more successfully treated than patellar tendinopathy patients (69% improvement). Despite stronger published evidence of benefit in patellar tendinopathy, clinical outcomes appeared better with aprotinin use in Achilles tendinopathies.,[object Object]