Increased versican content is associated with tendinosis pathology in the patellar tendon of athletes with jumper's knee

Abstract

Expansion of the extracellular matrix is a prominent but poorly characterized feature of tendinosis. The present study aimed to characterize the extent and distribution of the large aggregating proteoglycan versican in patients with patellar tendinosis. We obtained tendon from tendinopathy patients undergoing debridement of the patellar tendon and from controls undergoing intramedullary tibial nailing. Versican content was investigated by Western blotting and immunohistochemistry. Microvessel thickness and density were determined using computer-assisted image analysis. Markers for smooth muscle actin, endothelial cells (CD31) and proliferating cells (Ki67) were examined immunohistochemically. Western blot analysis and immunohistochemical staining revealed elevated versican content in the proximal patellar tendon of tendinosis patients (P=0.042). Versican content was enriched in regions of fibrocartilage metaplasia and fibroblast proliferation, as well as in the perivascular matrix of proliferating microvessels and within the media and intima of arterioles. Microvessel density was higher in tendinosis tissue compared with control tissue. Versican deposition is a prominent feature of patellar tendinosis. Because this molecule is not only a component of normal fibrocartilagenous matrices but also implicated in a variety of soft tissue pathologies, future studies should further detail both pathological and adaptive roles of versican in tendons.

Figure 1

Detection of versican in normal and pathological tendon. Western blots of purified tendon proteoglycan extracts (~2ug/lane) were probed with 2B1 (versican C-terminal domain) or LF-99 (N-terminal domain). Lanes represent normal (N) and pathological (P) human patellar tendon samples. Molecular weights are shown in kDa. Similar results were obtained in gels from 5 different patients.

Figure 2

Representative micrographs demonstrating zones of versican deposition in association with specific features of tendinosis pathology; MRI from a jumper’s knee patient is shown for orientation. Zone 1 contains banded distribution of versican (brown staining) in association with chondrocytic cells (Haematoxylin counterstain), bordered distally by microvascular hyperplasia (Zone 2). Zone 2 is interspersed and bordered by tenocyte hypercellularity and a generalized increase in versican content (Zone 3). In surrounding normal tendon versican staining is fainter and is localized to the endotendon. Scale bar on micrographs = 50 microns, on MRI = 1cm.

Figure 3

Association of versican with vascular proliferation A: Diffuse versican labeling (brown) around an abnormally dense capillary bed within the tendon proper, with more intense labeling directly adjacent to the vessels (arrow). B: CD31 labeling endothelial cells serial to A. C: Higher power view of the same region showing Ki67 labeling of endothelial cells in the microvascular walls. D: Intense versican labeling (asterisk) in a region of arteriolar proliferation. E: Serial to D, confirming the presence of α-SMA (brown) in the intima media. Scale bars = 50 microns

Figure 4

Versican distribution in arterioles from normal tendon and tendinosis tissue. A: Normal arteriole – versican is localized to the surrounding connective tissue, and to a single layer of endothelial cells (tunica intima). B: Abnormal arteriole – versican surrounds an irregularly thickened endothelial layer and extends into the smooth muscle layer (tunica media), and is also markedly increased in the outer layers. Scale bar = 50 microns.

Figure 5

Association of versican with microvascular hyperplasia and myofibroblasts. A: Versican in association with an abnormal microvascular bed (arrow), and remodeling tissue (asterisk). B: CD31 confirms that the upper structure is vascular, whereas the lower structure is not. C: Intensely labeling of α-SMA in the vascular structure indicating arterioles. α-SMA is also positive in scattered myofibroblasts in the adjacent remodeling area (rectangle). D: Higher power view of rectangular area in C. Scale bars = 50 microns.

Figure 6

Quantitation of vessel diameters in the proximal 0.35mm² of the patellar tendon. Morphometric analysis demonstrates increased presence of microvessels in patellar tendinosis.