The Effects of Mechanical Loading and ADAMTS5 Activity in a Tendinopathy Model
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Tendinopathy is a chronic tendon disorder that accounts for ~50% of all sports-related “over-use” injuries and ~12% of work-related injuries (Bureau of Labor Statistics). Consistent findings in tendinopathic tissue are collagen fiber disorganization/rupture and mucoid degeneration (aggrecan rich deposits, or ARDs). Despite their prevalence, presently it is unknown whether ARDs are pathogenic. Aggrecan presence occurs at low levels in the tensile regions of tendon and undergoes rapid aggrecanase-mediated turnover in normal tendons. The lack of ADAMTS5 (aggrecanase-2) has been shown to lead to ARDs in collagen rich tissues (skin, cartilage). The objective of this dissertation was to test the hypothesis that accumulation of aggrecan is detrimental to normal and injured tendon function. The first aim was to examine the effect of ADAMTS5 on the properties of ﬂexor digitorum longus (FDL) and Achilles tendons in skeletally mature mice. FDL tendons of adamts5-/- (ts5-/-) mice showed a 33% larger cross-sectional area, increased collagen ﬁbril area fraction, and decreased material properties relative to those of age matched wild type mice. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in ts5-/- and wild type mice; however, the ts5-/- tendons exhibited a higher tensile modulus and a weakened enthesis. In ts5-/- mice, aggrecan accumulated in the pericellular matrix of tendon ﬁbroblasts within the tendon body as well as the insertion site. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of adamts5 in tendon is to remove pericellular and interﬁbrillar aggrecan to maintain the molecular architecture responsible for normal tissue function. The second aim was to develop an in vivo tendinopathy model characterized by ARDs and weakened biomechanical properties. TGFβ was used to create this model in wild type cage active mice by injecting the growth factor into the Achilles tendon body. At two weeks the cross-sectional area was increased, while the tensile stiffness and maximum stress were reduced relative to the respective properties of naïve (uninjured) wild type mice. The stiffness and maximum stress remained compromised compared to the naïve at 4 weeks. Histologic analysis demonstrated increased cellularity and aggrecan staining throughout the midportion of tendon near the injection site both two weeks and four weeks. Gene expression of aggrecan and MMP3 increased by greater than 100 fold relative to naïve at two weeks and remained elevated (> 75 fold increase) at 4 weeks, whereas gene expression for the collagen binding integrins (α1 and α2) was reduced throughout the 4-week period. The third aim was to examine the role of mechanobiology in the development of tendinopathy using the murine model developed in Aim 2. Treadmill exercise following induction of the tendinopathy caused an increase of cross-sectional area and decrease of stiffness and maximum stress at 2 weeks but at 4 weeks, the stiffness and maximum stress returned to naïve levels. The increased presence of aggrecan deposits that was seen at 2 weeks was no longer present at 4 weeks with the treadmill exercising. Aggrecan and MMP3 gene expression at 4 weeks was also significantly reduced compared to the tendinopathy model. Gene expression for the collagen binding integrins α1 and α2 had also returned to levels seen in the naïve mice. The fourth aim examined the adamts5-/- mice in the tendinopathy and mechanical stimulation models. Tendon cross-sectional area increased, while stiffness and maximum stress decreased at 2 weeks relative to the naïve ts5-/- mice. At 4 weeks, the maximum stress was still compromised. The histology showed increased cellularity and increased aggrecan staining at both 2 and 4 weeks. Aggrecan had reduced expression at 2 weeks compared to the wild type with mechanical stimulation but had a 4 fold increase in expression at 4 weeks. Type 1 and Type 3 collagens had significantly reduced expression at 4 weeks compared to the wild type with treatment. The integrin expression was considerably increased at both 2 weeks and 4 weeks. These results demonstrate the importance of both the ADAMTS5 enzyme and mechanical stimulation for restoration of tensile properties in this animal model of tendinopathy. In all cases where there was histologic evidence of aggrecan accumulation in the tendon matrix, the tissue mechanical properties, maximum stress and stiffness, were reduced. Hence, collectively, these studies confirm our hypothesis that aggrecan rich deposits are detrimental to tendon mechanical function.