Chondrocyte death after mechanically overloading degenerated human intervertebral disk explants is associated with a structurally impaired pericellular matrix

Abstract

A type VI collagen-rich pericellular matrix (PCM) encloses both intervertebral disk (IVD) and articular cartilage chondrocytes. In the latter, the PCM protects the chondrocytes from mechanical overload, whereas tissue degeneration is associated with PCM destruction. As little is known about the IVD PCM, we investigated chondrocyte survival after mechanical overload as well as PCM structural integrity as a function of clinical tissue degeneration. The hypothesis was that IVD degeneration may affect PCM integrity and overload-related chondrocyte survival. Cylindrical human IVD explants from patients undergoing surgical procedures for lumbar disk degeneration, disk prolapse, or spinal trauma were generated and scored. Mechanical overload was applied by single uniaxial 50% compression followed by immediate release, and the explants were live-dead stained (n = 20 explants). Type VI collagen, the major PCM component, was fluorescent stained and the extent was determined, in which individual cells were enclosed by a recognizable PCM; this was termed PCM fraction. More than 50% of chondrocytes in all degenerative IVD explants displayed <25% PCM fraction and a lower PCM fraction correlated with higher cell numbers (p < 0.001), suggesting a PCM structural impairment in IVD degeneration that is associated with chondrocyte clustering. Mechanical overload-induced significantly increased cell death (p = 0.005), and the PCM fraction was significantly lower in overload-induced cell death than in live cells (p = 0.042), suggesting that a fully present PCM has a protective role in mechanical overload. Collectively, human IVD degeneration is associated with a structural impairment of the PCM, which may promote cell death under mechanical overload.

Biochemical changes of the pericellular matrix and spatial chondrocyte organization—Two highly interconnected hallmarks of osteoarthritis

“…Similar observations have been made for PCM‐collagen type VI 24 . It can thus be assumed that the PCM is destroyed during the course of OA, 24 which has also been suggested for the PCM in the intervertebral disk 25 and meniscus 26 . Collagen type VI and perlecan are thus well established and investigated PCM components in both healthy and OA affected tissue 24,27 ; several studies have emphasized their involvement and connection to the biomechanical properties of the PCM 28,29 .…”

“…Interestingly, in the absence of calcium, the microfibrils change their native organization, becoming loose and diffuse (own data not shown). When interpreting the constant levels of collagen type III and fibrillin‐1, it has to be borne in mind that the more pathological the spatial pattern, the higher the number of cells in the tissue 25 . This increase in cells can be highly attributed to cells with an impaired PCM, as shown by the optical readout for the categories 25% to 75% or less than 25% PCM signal circumference.…”

“…The analysis for structural integrity of the PCM was performed using a blinded optical approach to the histological side views, as previously described by Hofmann et al 25 Due to the arciform collagen fiber and cell orientation, these sections allow for better visualization of the developing patterns, especially in deeper cartilage layers where the orientation of the cells is perpendicular to the articular surface rather than parallel to it as in the superficial zone.…”

“…Two rectangular regions of interest of approximately 500 × 500 μm were selected at random from each mosaic image per cellular pattern. PCM integrity assessment was performed as previously described 25 . The percentage of the circumference of the cell that was surrounded by the staining signal for the PCM was thereby determined for the immunolabelled PCM components collagen type VI, perlecan, collagen type III, biglycan, and fibrillin‐1.…”

“…Distributions of variables were assessed by histograms. The weighted arithmetic mean of the averaged cellular PCM integrity in the histological analyses was calculated as previously described 25 . Data are graphically presented as boxplots and bar diagrams.…”