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Abstract
Chondrocytes cultured in agarose have been shown to maintain their phenotypic expression during long term culture as manifested in the synthesis of a matrix containing cartilage-specific collagens and large aggregating proteoglycans. This thesis adopted the chondrocyte/agarose system to investigate (1) the development during long term culture of functional mechanical and electromechanical properties of the matrix (2) the molecular basis of these properties and (3) the effect of mechanical compression on biosynthesis in this tissue. Under free-swelling conditions, chondrocytes seeded at high density (10 $-$ 20 $\times$ 10$\sp6$ cells per ml of gel) increased matrix glycosaminoglycan (GAG) content many-fold. Measured physical properties changed in a manner consistent with increasing matrix content; the equilibrium modulus and streaming potential rose to many times ($>$5$\times$) values measured just after casting; the hydraulic permeability decreased to a fraction ($\sim$1/10) of that of the cell-laden porous agarose at the beginning of the culture. By 1 month in culture, the magnitude of the dynamic stiffness, and streaming potential and permeability approached that of articular cartilage.
A molecular model was developed to predict the contribution of electrostatic repulsion forces between matrix GAG molecules to the swelling pressure of proteoglycan (PG) solutions and to the equilibrium modulus of intact articular cartilage. The results indicated that such electrostatic interactions can completely account for literature values of the swelling pressure of PG solutions, and comprise $\sim$1/2 the measured equilibrium modulus of cartilage. The molecular structure incorporated into the model was seen to be essential for a quantitative prediction of these mechanical properties of cartilage and its PG constituents since a macroscopic model based on Donnan equilibrium failed to adequately account for these measured properties.
Static and dynamic compression was applied to chondrocyte/agarose (CA) disks for short times (10-16 hours) in culture in the presence of radiolabel precursors used as markers for biosynthesis. At late times in culture, when matrix was present, static compression of CA disks (by up to 50%) reduced the synthetic rate of PG and protein, while dynamic compression (of $\sim$3%) increased biosynthetic rates. These responses were similar to those previously found with cartilage explants. At early times before significant matrix deposition, there was little or no effect of compression on biosynthesis. This suggested a dependence on the presence of matrix for these effects of mechanical stimuli.
Another series of experiments was initiated to investigate the role of minor matrix constituents in the matrix assembly. Preliminary results support a suggested hypothesis that type IX collagen is a bridging molecule between type II collagen fibrils and proteoglycan. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)
