EFFECT OF INJURIOUS COMPRESSION OF THE SUPERFICIAL ZONE AND DEEPER ZONES OF BOVINE ARTICULAR CARTILAGE EXPLANTS *(**)Rolauffs B, *DiMicco M, *Kisiday J, *Frank E, **Cole AA, Grodzinsky, A *M.I.T., Cambridge, MA, 77 Massachusetts Ave. NE 47-377, Cambridge, MA 02139
[email protected] **Rush Medical College, Chicago, Illinois, U.S.A. INTRODUCTION Mechanical injury to cartilage explants in vitro causes tissue swelling consistent with damage to the collagen network [1], increased turnover of matrix proteoglycans and proteins [2], and altered cell viability [3]. In vivo, mechanical injury to articular cartilage leads to an increased risk of osteoarthritis [4], a degenerative joint disease, in which the articular surface and in particular the superficial zone is gradually lost [5]. The superficial zone differs from deeper cartilage zones in cellular morphology, orientation 6 and density [7], collagen network organization [6], proteoglycan content [8], lubricating gene product expression [9] and response to catabolic stimuli [10]. The objective of this study was to examine the effects of injurious compression on the mechanical properties of the superficial zone of articular cartilage. METHODS Explant & Culture: Cartilage discs 3mm in diameter (thickness as in Fig.1) were manually harvested from the superficial (S) Superficial Zone (n=59) and deeper zones (D) (n=55) of Thickness of Explants (µm) Deeper Zones 1600 both medial (MC) and lateral (LC) 1200 condyles and the patello-femoral 800 groove (G) from 3 distal femura of 1-2 400 weeks old calves. Discs were cultured 0 in low-glucose DMEM, 10% FBS and Medial Lateral Groove Ascorbate. Condyle Condyle Fig.1 Explant thickness (average SE).
Mechanical Testing & Injury: After 5 days of culture, the biomechanical properties of 36 discs of the superficial zone and 36 discs of the deeper zones were assessed individually in uniaxial unconfined compression using an incubator-housed tissue loading instrument [11]. Discs were subjected to three sequential compression steps to final strains of 20%, 23%, and 26%, and the resulting equilibrium loads at these strains were used to compute the unconfined compression modulus. At 26% offset strain (offset load 50g), discs were then subjected to 3% dynamic strain amplitude at 1.0, 0.8, 0.5, 0.3, 0.2, 0.1 HZ, to compute the dynamic stiffness. A 50% injurious compression was then applied to 18 discs at 1mm/s strain rate, and then immediately released. After 20 minutes of unconfined re-swelling, the unconfined equilibrium modulus and dynamic stiffness of the 18 injured and 18 non-injured control discs in each zone were remeasured. Radiolabel Incorporation: All injured and control discs were then radiolabeled for 18 hrs using 35SO4-2 and 3Hproline as measures of GAG and protein synthesis, respectively, washed, digested with protease K and analyzed by scintillation counting. Data are presented as normalized to tissue thickness (volume). Biochemical analysis: Sulfated GAG content was determined by dimethyl-methylene blue dye-binding assay. RESULTS Mechanical Testing: The calculated equilibrium modulus (Fig.2) differed significantly Equilibrium Modulus (MPa)Modulus before Modulus Equilibrium Injury (n=36) Modulus Injury Equilibrium Modulus after Injury after (n=18) (p
