Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels

Abstract

Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate + matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-β3 supplementation. TGF-β3’s influence on pyridinoline content and mechanical properties was also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al., (2003). Transient TGF-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and highest binding affinity; collagen synthesis was elevated in TGF-β3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

Matrix Production in Large Engineered Cartilage Constructs Is Enhanced by Nutrient Channels and Excess Media Supply

“…This notion is supported by the variability of media supply rates found in the literature (Table 1). 7,11,13,[15][16][17][18][19][20][21] The aim of our study was to formulate and validate systemspecific computational models for estimating the nutrient supplies needed to engineer large tissues, with or without nutrient channels. These models may account for systemspecific ECM synthesis and nutrient consumption.…”

“…In our recent experimental study of cellular glucose consumption and matrix synthesis in small constructs (B4 · 2.34 mm), a glucose concentration of 12.5 mM was identified as the nutrient threshold below which the ECM synthesis ceased. 21,22 This threshold represented half the initial glucose concentration of 25 mM in standard cell culture media, suggesting that such standard conditions may easily lead to nutrient depletion in tissue engineering studies when the media/cell ratio is inadequate. Indeed, preliminary simulations conducted for this study suggested that our standard culture conditions fail to supply larger (B10 · 2.34 mm) constructs with adequate glucose levels for ECM elaboration.…”

“…23,24 These models employ a multiphasic mixture framework that accounts for chemical reactions, simulating glucose transport from the bath into the construct, glucose diffusion within the construct, and glucose consumption by chondrocytes. 21 Chondrocytes were not modeled explicitly, but their consumption of glucose was described by a sink rate uniformly distributed across the construct geometry to represent a similarly homogeneous cell-seeding density. In the standard 2-2-3-day media change schedule, the 3-day period simulated here represented the longest duration without nutrient replenishment.…”

“…In the standard 2-2-3-day media change schedule, the 3-day period simulated here represented the longest duration without nutrient replenishment. Based on the system-specific experimental parameters (glucose threshold and glucose consumption rate), 21,22 simulations were performed to identify the culture conditions that prevented the glucose concentration throughout the construct from dropping below the glucose threshold for ECM synthesis.…”

“…21 FE model geometries and meshes (CUBIT; Sandia National Laboratory) matched the geometries of several candidate channel and bath configurations ( Fig. 1): constructs (B10 · 2.34 mm) suspended within a media bath volume of 5, 10, or 15 mL (MV: 5, 10, and 15) and configured with 0, 3, 7, or 12 channels (CH: 0, 3, 7, 12), generating a total of 12 culture conditions.…”