Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix

TLDR

The study quantified whether calf articular chondrocytes can synthesize and assemble a mechanically functional cartilage‑like extracellular matrix in high‑density agarose gel culture. The authors cultured chondrocytes at ~10^7 cells/ml in agarose gels for 10 weeks, monitoring cell proliferation, proteoglycan synthesis and loss, GAG deposition, and measuring dynamic mechanical stiffness, oscillatory streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling over time. Biosynthetic activity peaked early and then matched native cartilage levels, with most proteoglycans retained in the gel; matrix rich in proteoglycans and collagen formed around cells, and mechanical and electrokinetic properties (equilibrium modulus, dynamic stiffness, streaming potential) increased more than fivefold while permeability dropped tenfold, reaching about 25 % of native cartilage by day 35, indicating a mechanically functional matrix had formed.

Abstract

Abstract 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 (∼ 10 7 cell/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 (∼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 ∼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 mechanically functional matrix.