Mesenchymal stem cells produce functional cartilage matrix in three-dimensional culture in regions of optimal nutrient supply

Abstract

Mesenchymal stem cells (MSCs) are a promising cell source for the treatment of musculoskeletal disease. However, MSC chondrogenesis in 3D culture generates constructs whose macroscopic (bulk) mechanical properties are inferior to constructs formed with chondrocytes. To investigate where and why these deficits in functionality arise, we assessed the local (microscopic) properties of cell-laden hydrogel constructs. Both chondrocyte-and MSC-laden constructs showed pronounced depth dependency, with ~3.5 and ~11.5 fold decreases in modulus from the surface to central regions, respectively. Importantly, in the surface region, properties were similar, suggesting that MSCs can produce matrix of mechanical equivalence to chondrocytes, but only in conditions of maximal nutrient support. Dynamic culture on an orbital shaker (which enhances diffusion) attenuated depth-dependent disparities in mechanics and improved the bulk properties compared to free swelling conditions (225 to 438 kPa for chondrocytes, 122 to 362 kPa for MSCs). However, properties in MSC-based constructs remained significantly lower due to persistent mechanical deficits in central regions. MSC viability in these central regions decreased markedly, with these changes apparent as early as day 21, while chondrocyte viability remained high. These findings suggest that, under optimal nutrient conditions, MSCs can undergo chondrogenesis and form functional tissue on par with that of the native tissue cell type. However, the lack of viability and matrix production in central regions suggests that chondrogenic MSCs do not yet fully recapitulate the advanced phenotype of the chondrocyte, a cell that is optimized to survive (and thrive) in a mechanically challenging and nutrient-poor environment.

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

“…Indeed, our work and that of others show that even small hydrogel constructs develop significant mechanical heterogeneities over relatively small thicknesses. [41][42][43] Thus, the in vitro culture conditions for molded constructs will need to be optimized to produce improved cell viability, better ECM deposition, and higher mechanical stiffness at the center. This may be accomplished by dynamic loading, 20 culture in hydrodynamic environments that encourage nutrient exchange, 24,44 or even the ordered introduction of channels within the construct to facilitate more consistent nutrient exchange to the central regions of these large constructs.…”

In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

“…The in vivo biochemical microenvironment of the NP, which is characterized by poor nutrition, low oxygen, and low pH, represents a challenge for MSC-based therapies, as there is evidence that these cells are particularly sensitive to Color images available online at www.liebertpub.com/tea microenvironmental stress. [41][42][43] Future work will establish how these environmental factors mediate MSC viability and biosynthetic potential, and explore ways to condition MSCs to maximize their regenerative potential upon delivery to the in vivo space. An additional challenge faced when delivering MSC-based therapies to the degenerate disc is the presence of chronic inflammation 44 ; there is evidence that inflammation may limit the regenerative potential of MSCs.…”

Functional consequences of glucose and oxygen deprivation on engineered mesenchymal stem cell-based cartilage constructs

“…Passage 2 cells (the two donors combined for 1 biological replicate) were trypsinized, resuspended in chemically defined media [12] at a density of 40×10 6 cells/mL, and mixed with 4% w/v molten Type VII agarose (49°C; Sigma-Aldrich, St. Louis, MO, in PBS) at a 1:1 ratio. The agarose/cell solution (2% agarose, 20×10 6 cells/mL) was cast between two parallel glass plates separated by either a 2.25 mm spacer (‘thick’; the same thickness as in work previously published by our group [12, 14, 15]) or 0.75 mm spacer (‘thin’; to reduce diffusion distances), and constructs (4 mm diameter) were formed using a biopsy punch. For each hydrogel assay to follow, technical replicates are denoted with number of constructs or samples ‘n.’…”

“…For thin constructs, images of both axial surfaces (construct top and bottom) were acquired at 2X and 10X magnification on Days 7, 14, 21, and 28. Percent viability (thin constructs) was calculated by counting the number of dead cells (ethidium homodimer-1, red) and live cells (calcein, green) in the 10X images [15]. Since viability differed greatly between the two surfaces, the sides of minimum and maximum viability were grouped for each condition.…”

“…However, when cultured in the same conditions, chondrocytes outperform MSCs, with higher viability, increased matrix production, and increased compressive moduli [12–14]. A recent study from our group, using a 3D agarose hydrogel model and local analysis of mechanical properties (compressive equilibrium modulus), showed that the properties of MSC-based constructs are higher at the construct periphery compared to the same region of constructs based on chondrocytes that were cultured identically [15]. The marked disparity in overall (bulk) construct properties arose from deficiencies in the central regions, where local mechanical properties in MSC-based constructs were significantly lower than those of chondrocyte-based constructs.…”