Hyaluronic acid as a macromolecular crowding agent for production of cell-derived matrices.

Abstract

Cell-derived matrices (CDM) provide an exogenous source of human extracellular matrix (ECM), with applications as cell delivery vehicles, substrate coatings for cell attachment and differentiation, and as biomaterial scaffolds. However, commercial application of CDMs has been hindered due to the prolonged culture time required for sufficient ECM accumulation. One approach to increasing matrix deposition in vitro is macromolecular crowding (MMC), which is a biophysical phenomenon that limits the diffusion of ECM precursor proteins, resulting in increased ECM accumulation at the cell layer. Hyaluronic acid (HA), a natural MMC highly expressed in vivo during fetal development, has been shown to play a role in ECM production, but has not been investigated as a macromolecule for increasing cell-mediated ECM deposition in vitro. In the current study, we hypothesized that HA can act as a MMC, and increase cell-mediated ECM production. Human dermal fibroblasts were cultured for 3, 7, or 14 days with 0%, 0.05%, or 0.5% high molecular weight HA. Ficoll 70/400 was used as a positive control. SDS-PAGE, Sircol and hydroxyproline assays indicated that 0.05% HA-treated cultures had significantly higher mean collagen deposition at 14 days, whereas Ficoll 70/400-treated cultures had significantly lower collagen production compared to the HA and untreated controls. However, fluorescent immunostaining of ECM proteins and quantification of mean gray values did not indicate statistically significant differences in ECM production in HA or Ficoll 70/400-treated cultures compared to untreated controls. Raman imaging (a marker-free spectral imaging method) indicated that HA increased ECM deposition in human dermal fibroblasts. These results are consistent with decreases in CDM stiffness observed in Ficoll 70/400-treated cultures by atomic force microscopy. Overall, these results indicate that there are macromolecule- and cell type- dependent effects on matrix assembly, turnover, and stiffness in cell-derived matrices.