Differential Maturation and Structure–Function Relationships in Mesenchymal Stem Cell- and Chondrocyte-Seeded Hydrogels

Abstract

Degenerative disease and damage to articular cartilage represents a growing concern in the aging population. New strategies for engineering cartilage have employed mesenchymal stem cells (MSCs) as a cell source. However, recent work has suggested that chondrocytes (CHs) produce extracellular matrix (ECM) with superior mechanical properties than MSCs do. Because MSC-biomaterial interactions are important for both initial cell viability and subsequent chondrogenesis, we compared the growth of MSC- and CH-based constructs in three distinct hydrogels-agarose (AG), photocrosslinkable hyaluronic acid (HA), and self-assembling peptide (Puramatrix, Pu). Bovine CHs and MSCs were isolated from the same group of donors and seeded in AG, Pu, and HA at 20 million cells/mL. Constructs were cultured for 8 weeks with biweekly analysis of construct physical properties, viability, ECM content, and mechanical properties. Correlation analysis was performed to determine quantitative relationships between formed matrix and mechanical properties for each cell type in each hydrogel. Results demonstrate that functional chondrogenesis, as evidenced by increasing mechanical properties, occurred in each MSC-seeded hydrogel. Interestingly, while CH-seeded constructs were strongly dependent on the 3D environment in which they were encapsulated, similar growth profiles were observed in each MSC-laden hydrogel. In every case, MSC-laden constructs possessed mechanical properties significantly lower than those of CH-seeded AG constructs. This finding suggests that methods for inducing MSC chondrogenesis have yet to be optimized to produce cells whose functional matrix-forming potential matches that of native CHs.

Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent

“…In contrast, we found juvenile bovine MSCs are inferior in terms of functional ECM production to donormatched chondrocytes in various hydrogels [17,23,25,33] but have not considered MSC age in our hyaluronic acid (HA) hydrogel system. Thus, although the literature demonstrates aging affects MSC and chondrocyte function, the relative effects of aging of bovine chondrocytes compared with MSCs is unknown.…”

“…Cell viability was analyzed (LIVE/DEAD staining kit; Invitrogen, Carlsbad, CA) [17], and mechanical testing (n = 4) was performed followed by biochemical assays (n = 4) for DNA, GAG, and collagen content and histologic analysis of proteoglycan and collagens (Fig. 1C).…”

“…HA gel solution was prepared at 1% (mass/volume) in phosphatebuffered saline (PBS) with 0.05% w/v of the photoinitiator I2959 (2-methyl-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone; Ciba-Geigy, Tarrytown, NY) and MSCs were suspended at 20 million cells/mL before being ultravioletpolymerized between glass plates separated by a 2.25-mm spacer [17]. Cylindrical constructs were removed from gel slabs using a sterile 4-mm-diameter biopsy punch (Miltex, York, PA).…”

“…Cartilage, pellet, and hydrogel samples were papaindigested and assayed for DNA, GAG, and collagen content using the Picogreen (Molecular Probes, Eugene, OR), 1,9-dimethylmethylene blue [19], and orthohydroxyproline assays [38,45], respectively, as described in Erickson et al [17] and Huang et al [25].…”

“…Pellets were cryosectioned to 12 lm. Sections were stained for proteoglycans with Alcian blue (pH 1.0) and for collagen by picrosirius red [17].…”