Macromolecular crowding as a means to assess the effectiveness of chondrogenic media

Abstract

Chondrocyte-based tissue engineering requires in vitro cell expansion, which is associated with phenotypic losses, decrease in Collagen Type II synthesis and increase in Collagen Type I synthesis. Another major obstacle in clinical translation of chondrocyte-based therapies is the lack of extracellular matrix (ECM) in the engineered cartilage substitutes. Various research and commercially available media claim that they can maintain chondrogenic phenotype, whereas macromolecular crowding (MMC) has been shown to increase tissue-specific ECM deposition and maintain cell phenotype in vitro. Herein, we hypothesised that the combination of chondrogenic media with MMC will enable chondrogenic phenotype maintenance during in vitro expansion and increase cartilage-specific ECM deposition, enabling that way the development of a tissue-engineered cartilage substitute. Immunocytochemistry analysis of Passage 3 human chondrocytes in normal media in monolayer revealed that MMC significantly increased Collagen Type I deposition, whereas no statistical difference was observed in Collagen Type II deposition. When Passage 3 human chondrocytes were cultured in normal media and alginate beads, immunocytochemistry analysis revealed that MMC increased, albeit not significantly, both Collagen Type I and Collagen Type II deposition. Subsequently, human chondrocytes were expanded up to Passage 6 in either fetal bovine serum or human serum and redifferentiated using commercially available chondrogenic media in either monolayer or alginate beads. Immunocytochemistry analysis revealed that MMC, independently of the serum used, significantly increased Collagen Type I deposition in humanredifferentiated monolayer and alginate bead chondrocyte cultures, whereas almost no Collagen Type II was detected. These data clearly illustrate that an optimal chondrogenic medium is still elusive.

Physicochemical cues are not potent regulators of human dermal fibroblast trans-differentiation

“…When cells were cultured in the presence of MMC, although lower cell number and metabolic activity values were observed, cell viability was above 95%, as with every other condition. It is worth noting that carrageenan has been used extensively as MMC agent in stem [60] , [61] , [62] , [63] , [64] and permanently differentiated [90] , [91] , [92] , [93] , [94] , [95] cell cultures and in preclinical setting [96] without any negative effects.…”

Physical and mechanical cues affecting biomaterial-mediated plasmid DNA delivery: insights into non-viral delivery systems

“…(Table 4 ) The most evident effect is related to biomaterial stability, water content, swelling ratio and thereby DNA diffusion and release [ 111 ]. Furthermore, be they seeded on a substrate [ 192 , 193 ] or embedded in 3D structures [ 9 , 188 ], cells respond to variable stiffness by rearranging their cytoskeleton. Increased substrate stiffness caused reinforcement and acquisition of orientational order of actin stress fibers [ 190 , 193 ], which in turn influence cell polarity and cell shape [ 192 ].…”

“…inert component) and collagen can be used. Alginate self-polymerises after the addition of calcium carbonate [ 9 ]: by varying the concentration of the latter, stiffness can be controlled [ 202 ].…”

“…Not only do the biomaterials increase localisation at the desired site, offer a support for cells to grow and a mechanical support [2,8], but they also directly modulate transfection efficiency. As a matter of fact, cell embedding [9], cell adhesion [10,11] and migration [12,13] influence cytoskeletal arrangement and focal adhesions, ultimately affecting DNA internalisation and nuclear transport. Biomaterial-mediated gene delivery systems generally result in a higher transfection [14][15][16] and even reduced cytotoxicity [15], than traditional ones.…”