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Endogenous collagen cross-links influence cartilage biomechanical properties and resistance to degradation. Formalin fixation modifies collagen residues and forms new cross-links in a dose-dependent manner. We tested the hypothesis that magnetization transfer (MT) effects and T(2) depend on collagen cross-linking in cartilage. These parameters were measured in bovine nasal cartilage (BNC) prior to fixation, after 9 weeks of immersion in formalin solutions ranging in concentration from 0% to 10%, and after NaBH(3)CN reduction and washing. T(2) decreased by 59.4% +/- 1.1% upon fixation in 10% formalin, and was 32.2% +/- 5.2% shorter than initial values after washing. The apparent MT rate increased 25.9% +/- 3.7% and 52.8% +/- 7.1% over baseline under these conditions. Biochemical assays showed no significant differences in water, proteoglycan, natural cross-link, or collagen content between the 0% and 10% formalin-treated samples, while amino acid analysis demonstrated losses in (hydroxy)lysine and tyrosine, and new peaks consistent with methylene cross-links in fixed samples only. We conclude that formalin fixation of cartilage results in significant decreases in T(2) and increases in MT parameters that persist after removal of unreacted formaldehyde. The collagen cross-links thus created are associated with large changes in MT and T(2), indicating that interpretation of T(2) and MT values in terms of cartilage macromolecular content must be made with caution.

It has been hypothesized that the electric fields associated with the dynamic loading of cartilage may affect its growth, remodeling, and biosynthesis. While the application of exogenous fields has been shown to modulate cartilage biosynthesis, it is not known what range of field magnitudes and frequencies can alter biosynthesis and how they relate to the magnitudes and frequencies of endogenous fields. Such information is necessary to understand and identify mechanisms by which fields may act on cartilage metabolism. In this study, incorporation of 35S-methionine was used as a marker for electric field-induced changes in chondrocyte protein synthesis in disks of cartilage from the femoropatellar groove of 1 to 2-week-old calves. The cartilage was stimulated sinusoidally at 1, 10, 100, 10(3), and 10(4) Hz with current densities of 10-30 mA/cm2. Incorporation was assessed in control disks maintained in the absence of applied current at 37, 41, and 43 degrees C. The possibility that applied currents would induce synthesis of the same stress proteins that are caused by heating or other mechanisms was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examination of gel fluorographs. Total radiolabel incorporation in cartilage that had been stimulated relative to incorporation in the controls increased with current density magnitudes greater than 10 mA/cm2. The increase was greatest at 100 Hz and 1 kHz, and it depended on the position on the joint surface from which the cartilage samples were taken. Together, these results suggest that endogenous electric fields could affect cartilage biosynthesis. Stress proteins were not induced at any current density when the electrodes were electrically connected but chemically isolated from the media by agarose bridges. Stress proteins were observed for disks incubated at temperatures greater than 39 degrees C (no field) and when the stimulating platinum electrodes were in direct contact with the media bathing the cartilage disks. Therefore, the increase in incorporation of 35S-methionine due to applied fields with the use of chemically isolated electrodes did not appear to be associated with stress response.

The ability of chondrocytes from calf articular cartilage to synthesize and assemble a mechanically functional cartilage-like extracellular matrix was quantified in high cell density (approximately 10(7) cells/ml) agarose gel culture. The time evolution of chondrocyte proliferation, proteoglycan synthesis and loss to the media, and total deposition of glycosaminoglycan (GAG)-containing matrix within agarose gels was characterized during 10 weeks in culture. To assess whether the matrix deposited within the agarose gel was mechanically and electromechanically functional, we measured in parallel cultures the time evolution of dynamic mechanical stiffness and oscillatory streaming potential in uniaxial confined compression, and determined the intrinsic equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient of the developing cultures. Biosynthetic rates were initially high, but by 1 month had fallen to a level similar to that found in the parent calf articular cartilage from which the cells were extracted. The majority of the newly synthesized proteoglycans remained in the gel. Histological sections showed matrix rich in proteoglycans and collagen fibrils developing around individual cells. The equilibrium modulus, dynamic stiffness, and oscillatory streaming potential rose to many times (>5x) their initial values at the start of the culture; the hydraulic permeability decreased to a fraction (approximately 1/10) that of the cell-laden porous agarose at the beginning of the culture. By day 35 of culture, DNA concentration (cell density), GAG concentration, stiffness, and streaming potential were all approximately 25% that of calf articular cartilage. The frequency dependence of the dynamic stiffness and potential was similar to that of calf articular cartilage. Together, these results suggested the formation of a mechanically functional matrix.