Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands.

OBJECTIVE: To determine and correlate subchondral bone mineral density and overlying cartilage structure and tensile integrity in mature healthy equine stifle (low magnitude loading) and metacarpophalangeal (high magnitude loading) joints. ANIMALS: 8 healthy horses, 2 to 3 years of age. PROCEDURE: Osteochondral samples were acquired from the medial femoral condyle (FC) and medial trochlear ridge (TR) of the stifle joint and from the dorsal (MC3D) and palmar (MC3P) aspects of the distal medial third metacarpal condyles of the metacarpophalangeal joint. Articular cartilage surface fibrillation (evaluated via India ink staining) and tensile biomechanical properties were determined. The volumetric bone mineral density (vBMD) of the underlying subchondral plate was assessed via dual-energy x-ray absorptiometry. RESULTS: Cartilage staining (fibrillation), tensile moduli, tensile strength, and vBMD were greater in the MC3D and MC3P locations, compared with the FC and TR locations, whereas tensile strain at failure was less in MC3D and MC3P locations than FC and TR locations. Cartilage tensile moduli correlated positively with vBMD, whereas cartilage staining and tensile strain at failure correlated negatively with vBMD. CONCLUSIONS AND CLINICAL RELEVANCE: In areas of high joint loading, the subchondral bone had high vBMD and the articular cartilage surface layer had high tensile stiffness but signs of structural wear (fibrillation and low failure strain). The site-dependent variations and relationships in this study support the concept that articular cartilage and subchondral bone normally adapt to physiologic loading in a coordinated way.

In vitro physical stimulation of tissue-engineered and native cartilage.

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Dynamic shear stimulation of bovine cartilage biosynthesis of proteoglycan 4.

OBJECTIVE: The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules at the articular surface and in synovial fluid. The objective of this study was to determine the effects of dynamic shear stimulation on PRG4 biosynthesis by bovine cartilage explants. METHODS: Cartilage disks with intact articular surfaces were harvested from immature bovines. Some disks were subjected to 24 hours (day 1) of loading, consisting of a step load of 20% static compression either alone or with superimposed dynamic shear (3% amplitude and 0.1 Hz), while other disks were cultured free-swelling as controls. After the 24-hour loading period, disks were terminated or were further incubated for up to 72 hours (days 2-4) in free-swelling culture to assess chondrocyte responses to, and following, unloading. PRG4 products secreted into culture medium were quantified by enzyme-linked immunosorbent assay and characterized by Western blotting. Chondrocytes expressing PRG4 were localized by immunohistochemistry, and depth-associated variations in chondrocyte PRG4 expression were quantified by image analysis. RESULTS: Dynamic shear stimulation increased PRG4 secretion to 3-4 times that of unloaded controls and statically compressed samples. Sheared cartilage secreted more PRG4 of 345 kd relative to smaller molecular weight species, as compared with unloaded controls. Immunohistochemistry revealed that shear stimulation also increased the total number of cells expressing PRG4 by inducing expression by cells at a depth of 200-400 microm. CONCLUSION: The paradigm that certain mechanical stimuli up-regulate biosynthesis in cartilage appears operative not only for load-bearing matrix constituents, but also for PRG4 molecules that mediate lubrication.

Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal condyle.

OBJECTIVE: Determine (1) the site-associated response of articular cartilage of the equine distal metacarpal condyle to training at a young age as assessed by changes in indentation stiffness and alterations in cartilage structure and composition, and (2) relationships between indentation stiffness and indices of cartilage structure and composition. METHOD: Experimental animals (n=6) were trained on a track (increasing exercise to 1km/day by 5 months); controls (n=6) were pasture-reared. Animals were euthanized at 18 months and four osteochondral samples were harvested per metacarpal condyle from dorsal-medial, dorsal-lateral, palmar-medial, and palmar-lateral aspects. Cartilage was analyzed for India ink staining (quantified as reflectance score (RS)), short-term indentation stiffness (sphere-ended, 0.4mm diameter), thickness, and biochemical composition. RESULTS: Cartilage structural, biochemical and biomechanical properties varied markedly with site in the joint. Sites just medial and just lateral to the sagittal ridge showed signs of early degeneration, with relatively low RS, indentation stiffness, and collagen content, and relatively high water content. Effects of exercise and side (left vs right) were not detected for any measure. Overall, indentation stiffness correlated positively with RS and collagen content, and inversely with thickness and water content. CONCLUSION: Gentle exercise-imposed mechanical stimulation did not markedly affect articular cartilage function or structure. However, the marked site-associated variation suggests that biomechanical environment can initiate degenerative changes in immature cartilage during joint growth and maturation.