Michael Lavagnino

Activation of stress‐activated protein kinases (SAPK) in tendon cells following cyclic strain: the effects of strain frequency, strain magnitude, and cytosolic calcium

Cyclic strain has been shown to benefit tendon health. However, repetitive loading has also been implicated in the etiology of tendon overuse injuries. Recent studies demonstrated that in several cell lines cyclic strain was associated with an activation of stress‐activated protein kinases (SAPKs). These SAPKs, in turn, were shown to be important upstream regulators of a variety of cell processes including apoptosis. To examine the effect of repetitive loading on SAPK activation in tendon cells in vitro, canine patellar tendon cells were cyclically strained, and the cellular stress response evaluated by measuring c‐Jun N‐terminal kinase (JNK) activation. The effects of strain frequency and strain magnitude as well as the role of calcium signaling in this mechanotransduction mechanism were also examined. Cyclic strain resulted in an immediate activation of JNK, which peaked at 30 min and returned to resting levels by 2 h. This activation was regulated by a magnitude‐dependent but not frequency‐dependent response and appeared to be mediated through a calcium‐dependent mechanotransduction pathway. While transient JNK activation is associated with normal cell processes, persistent JNK activation has been linked to the initiation of the apoptotic cascade. A similar mechanism could be responsible for initiating the pathological events (localized cell death) seen in tendon overuse injury.

The mechanobiological aetiopathogenesis of tendinopathy: is it the over-stimulation or the under-stimulation of tendon cells?

While there is a significant amount of information available on the clinical presentation(s) and pathological changes associated with tendinopathy, the precise aetiopathogenesis of this condition remains a topic of debate. Classically, the aetiology of tendinopathy has been linked to the performance of repetitive activities (so‐called overuse injuries). This has led many investigators to suggest that it is the mechanobiologic over‐stimulation of tendon cells that is the initial stimulus for the degradative processes which have been shown to accompany tendinopathy. Although several studies have been able to demonstrate that the in vitro over‐stimulation of tendon cells in monolayer can result in a pattern(s) of gene expression seen in clinical cases of tendinopathy, the strain magnitudes and durations used in these in vitro studies, as well as the model systems, may not be clinically relevant. Using a rat tail tendon model, we have studied the in vitro mechanobiologic response of tendon cells in situ to various tensile loading regimes. These studies have led to the hypothesis that the aetiopathogenic stimulus for the degenerative cascade which precedes the overt pathologic development of tendinopathy is the catabolic response of tendon cells to mechanobiologic under‐stimulation as a result of microscopic damage to the collagen fibres of the tendon. In this review, we examine the rationale for this hypothesis and provide evidence in support of this theory.

Effect of Amplitude and Frequency of Cyclic Tensile Strain on the Inhibition of MMP-1 mRNA Expression in Tendon Cells: An In Vitro Study

To determine the effect of cyclic strain amplitude and frequency on MMP-1 (interstitial collagenase) expression in tendon cells, rat tail tendons (RTT) were immobilized or cyclically displaced to various amplitudes (1, 3, or 6% strain at 0.017 Hz) or frequencies (1% strain at 0.017, 0.17, or 1.0 Hz) for 24 hr. Stress-deprivation for 24 hr resulted in a marked upregulation in MMP-1 expression. Cyclic tensile loading at 0.017 Hz was found to significantly inhibit, but not completely eliminate, MMP-1 expression at 1% strain. MMP-1 expression was completely eliminated at 3 and 6% strain. Increasing the frequency of application of the 1% strain to 0.17 or 1.0 Hz completely eliminated MMP-1 expression. Disruption of the actin cytoskeleton with cytochalasin D abolished all inhibitory effects of cyclic strain on MMP-1 expression. The results of our study demonstrate that MMP-1 expression in tendon cells can be modulated by varying amplitudes and frequencies of cyclic tensile strain, presumably through a cytoskeletally based mechanotransduction pathway.

Ex vivo static tensile loading inhibits MMP-1 expression in rat tail tendon cells through a cytoskeletally based mechanotransduction mechanism

To determine the effect of various degrees of ex vivo static tensile loading on the expression of collagenase (MMP‐1) in tendon cells, rat tail tendons were statically loaded in tension at 0.16, 0.77, 1.38 or 2.6 MPa for 24 h. Northern blot analysis was used to assay for mRNA expression of MMP‐1 in freshly harvested, 24 h load deprived, and 24 h statically loaded tendons. Western blot analysis was used to assay for pro‐MMP‐1 and MMP‐1 protein expression in fresh and 24 h load deprived tendons. Freshly harvested rat tail tendons demonstrated no evidence of MMP‐1 mRNA expression and no evidence of the pro‐MMP‐1 or MMP‐1 protein. Ex vivo load deprivation for 24 h resulted in a marked increase in the mRNA expression of MMP‐1 which coincided with a marked increase of both pro‐MMP‐1 and MMP‐1 protein expression. When tendons were subjected to ex vivo static tensile loading during the 24 h culture period, a significant inhibition of this upregulation of MMP‐1 mRNA expression was found with increasing ioad (p < 0.05). A strong (r2 = 0.78) and significant (p < 0.001) inverse correlation existed between the level of static tensile load and the expression of MMP‐1. Disruption of the actin cytoskeleton with cytochalasin D abolished the inhibitory effect of ex vivo static tensile loading on MMP‐1 expression. The results of this study suggest that up‐regulation of MMP‐1 expression in tendon cells ex vivo can be inhibited by static tensile loading, presumably through a cytoskeletally based mechanotransduction pathway.

In situ cell nucleus deformation in tendons under tensile load; a morphological analysis using confocal laser microscopy

Cell and cell nucleus deformations have been implicated in the mechanotransduction of mechanical loads acting on tissues. While in situ cell nucleus deformation in response to increasing tissue strains has been examined in articular cartilage this phenomenon has not been investigated in tendons. To examine in situ cell nuclei deformation in tendons undergoing tensile strain rat tail tendons were harvested from adult Sprague–Dawley rats and stained with acridine orange to highlight the cell nuclei. The tendons were mounted on a custom‐designed, low‐load, tensile testing device affixed to the mechanical stage of a confocal laser microscope. Cells within the tendons were isolated for analysis. Images of individual cells were captured at 0% strain as well as sequentially at 2%, 4% and 6% grip‐to‐grip tendon strain. Digital images of the cell nuclei were then measured in the x (length) and y (height) axis and deformation expressed as a percentage of cell nuclei strain. In addition, centroid‐to‐centroid distances of adjacent cell nuclei within each image were measured and used to calculate local tissue strain. There was a weak (r2 = 0.34) but significant (p < 0.01) correlation between local tissue strain and cell nucleus strain in the x axis. The results of this study support the hypothesis that in situ cell nucleus deformation does occur during tensile loading of tendons. This deformation may play a significant role in the mechanical signal transduction pathway of this tissue.

Matrix metalloproteinase inhibitors prevent a decrease in the mechanical properties of stress-deprived tendons: an in vitro experimental study.

Background An increase in matrix metalloproteinases (MMPs) and the resulting degradation of the extracellular matrix have been implicated in the pathogenesis of tendinopathy. Studies have documented the beneficial effects of MMP inhibitors used to treat pathologic conditions in which MMP activity has had a negative effect on connective tissues. Hypothesis Matrix metalloproteinase inhibitors will prevent the decrease in material properties associated with tendon stress deprivation by inhibiting MMP activity. Study Design Controlled laboratory study. Methods Rat tail tendons were subjected to 7 days of in vitro stress deprivation with and without the addition of 1 of 2 broad-spectrum MMP inhibitors (doxycycline and ilomastat). The material properties (ultimate tensile stress, strain, and tensile modulus) of the tendons were compared with each other and with fresh control tendons. In addition, tendons from each group were evaluated for MMP-13 messenger RNA expression, MMP-13 protein synthesis, MMP-13 activity, and pericellular matrix morphology. Results Both MMP inhibitors resulted in a statistically significant reduction in MMP activity in 7 day stress-deprived tendons when compared with nontreated, stress-deprived tendons. Similarly, tendons treated with either ilomastat or doxycycline had significantly improved material properties. MMP-13 messenger RNA expression and protein synthesis were not significantly affected by either MMP inhibitor. Both MMP inhibitors were able to maintain the integrity of the pericellular matrix when compared with nontreated, stress-deprived tendons. Conclusion Matrix metalloproteinase inhibitors prevented the activation of MMP-13 and significantly inhibited pericellular matrix degeneration and the loss of material properties associated with stress deprivation. Clinical Relevance Matrix metalloproteinase inhibitors may play a supportive role in the treatment of tendinopathy by limiting the MMP-mediated degradation of the extracellular matrix.

Tensile Fixation Strengths of Absorbable Meniscal Repair Devices as a Function of Hydrolysis Time An in Vitro Experimental Study

To determine the effect of hydrolysis time on the fixation strength of absorbable meniscal repair devices, adult bovine menisci were repaired with five devices and a suture. The ultimate tensile strength of the repair was then tested in six specimens immediately or after 6, 12, or 24 weeks of incubation at 37°C in a saline solution containing antibiotics, antimycotics, and protease inhibitors. Immediately after implantation the Bionx Meniscus Arrow had a significantly higher failure strength (57.7 13.8 N) than the Linvatec BioStinger (35.1 6.7 N), the Innovasive Clearfix screw (34.9 13 N), the Surgical Dynamics S D sorb staple (9.4 4.6 N), and the Mitek Meniscal Repair System (polydioxanone) (27.2 6.0 N). However, there was no significant difference between the Bionx Meniscus Arrow and a 2—0 polydioxanone vertical suture (51.6 2.7 N). The polydioxanone-based implants demonstrated a significant decrease in failure strength at 12 and 24 weeks. Similarly, the Surgical Dynamics S D sorb staple lost all fixation strength by 24 weeks. The remaining devices showed no significant loss of failure strength over the 24-week period, suggesting that 24 weeks of hydrolysis does not adversely affect the ultimate holding power of poly L-lactide-based meniscal fixation devices.

The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: An in vitro experimental study

Purpose. To determine the effect of stress deprivation and cyclic loading on TIMP-1/MMP-13 mRNA expression ratio in rat tail tendon (RTT) cells. Method. Adult RTTs were stress-deprived for 0, 24, 48, or 72 hours in the presence or absence of a MMP inhibitor (ilomastat), or subjected to 1%, 3%, or 6% strain for 24 h under tissue culture conditions. TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA expression were measured using quantitative PCR and TIMP/MMP ratios were calculated for each group. Results. The ratio of TIMP-1 to MMP-13 in control RTTs was 3.73:1 ± 0.73. Stress deprivation for 24 h significantly decreased the TIMP-1/MMP-13 ratio (0.25:1 ± 0.04) and MMP-13 expression continued to increase significantly with time of stress deprivation. Inhibition of MMP-13 mRNA expression with ilomastat in stress-deprived samples did not alter TIMP-1 expression when compared to normal controls. Cyclic loading significantly increased TIMP-1/MMP-13 expression at all strain levels examined. Conclusions. RTTs normally have a positive TIMP-1/MMP-13 expression ratio. While cyclic loading increased the TIMP-1/MMP-13 ratio, loss of cellular homeostatic tension inversed this ratio through a significant increase in MMP-13 mRNA expression rather than a decrease in TIMP expression. A negative TIMP/MMP ratio has been implicated in the pathogenesis of tendinopathy. Increasing the TIMP/MMP ratios in these patients through exercise may be beneficial in the management of tendinopathy.

Effect of in vitro stress-deprivation and cyclic loading on the length of tendon cell cilia in situ

To determine the effect of loading conditions on the length of primary cilia in tendon cells in situ, freshly harvested rat tail tendons were stress‐deprived (SD) for up to 72 h, cyclically loaded at 3% strain at 0.17 Hz for 24 h, or SD for 24 h followed by cyclic loading (CL) for 24 h. Tendon sections were stained for tubulin, and cilia measured microscopically. In fresh control tendons, cilia length ranged from 0.6 to 2.0 µm with a mean length of 1.1 µm. Following SD, cilia demonstrated an increase (p < 0.001) in overall length at 24 h when compared to controls. Cilia length did not increase with time of SD (p = 0.329). Cilia in cyclically loaded tendons were shorter (p < 0.001) compared to all SD time periods, but were not different from 0 time controls (p = 0.472). CL for 24 h decreased cilia length in 24 h SD tendons (p < 0.001) to levels similar to those of fresh controls (p = 0.274). The results of this study demonstrate that SD resulted in an immediate and significant increase in the length of primary cilia of tendon cells, which can be reversed by cyclic tensile loading. This suggests that, as in other tissues, cilia length in tendon cells is affected by mechanical signaling from the extracellular matrix.

Patellar Tendon Strain is Increased at the Site of the Jumper's Knee Lesion during Knee Flexion and Tendon Loading: Results and Cadaveric Testing of a Computational Model

Background Patellar tendinopathy (jumpers knee) is characterized by localized tenderness of the patellar tendon at its origin on the inferior pole of the patella and a characteristic increase in signal intensity on magnetic resonance imaging at this location. However, it is unclear why the lesion typically occurs in this area of the patellar tendon as surface strain gauge studies of the patellar tendon through the range of motion have produced conflicting results. Hypothesis The predicted patellar tendon strains that occur as a result of the tendon loads and patella-patellar tendon angles (PPTAs) experienced during a jump landing will be significantly increased in the area of the patellar tendon associated with patellar tendinopathy. Study Design Descriptive laboratory study. Methods A 2-dimensional, computational, finite element model of the patella-patellar tendon complex was developed using anatomic measurements taken from lateral radiographs of a normal knee. The patella was modeled with plane strain rigid elements, and the patellar tendon was modeled with 8-node plane strain elements with neo-Hookean material properties. A tie constraint was used to join the patellar tendon and patella. Patella-patellar tendon angles corresponding to knee flexion angles between 0° and 60° and patellar tendon strains ranging from 5% to 15% were used as input variables into the computational model. To determine if the location of increased strain predicted by the computational model could produce isolated tendon fascicle damage in that same area, 5 human cadaveric patella-patellar tendon-tibia specimens were loaded under conditions predicted by the model to significantly increase localized tendon strain. Pre- and posttesting ultrasound images of the patella–patellar tendon specimens were obtained to document the location of any injured fascicles. Results Localized tendon strain at the classic location of the jumpers knee lesion was found to increase in association with an increase in the magnitude of applied patellar tendon strain and a decrease in the PPTA. The principal stresses and strains predicted by the model for this localized area were tensile and not compressive in nature. Applying the tendon strain conditions and PPTA predicted by the model to significantly increase localized strain resulted in disruption of tendon fascicles in 3 of the 5 cadaveric specimens at the classic location of the patellar tendinopathy lesion. Conclusion The localized increase in patellar tendon strain that occurs in response to the application of tendon loads and decreased PPTA could induce microdamage at the classic location of the jumpers knee lesion. Clinical Relevance The association of decreasing PPTA with increasing localized tendon strain would implicate the role of knee-joint angle as well as tendon force in the etiopathogenesis of jumpers knee.