The Effects ofROCKInhibition on Mesenchymal Stem Cell Chondrogenesis Are Culture Model Dependent

Abstract

Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (mRB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in mRB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSCbased cartilage regeneration in different culture models.

Mechanotransduction and Stiffness-Sensing: Mechanisms and Opportunities to Control Multiple Molecular Aspects of Cell Phenotype as a Design Cornerstone of Cell-Instructive Biomaterials for Articular Cartilage Repair

“…Increased chondrogenesis was always accompanied by enhanced N-cadherin expression, suggesting N-cadherin as a robust marker to, for example, select culture conditions that promote chondrogenesis. Interestingly, in another study [ 199 ], ROCK inhibition had minimal effects in the 2D or 3D models and varying the polymer used did not change the chondrogenic response to ROCK inhibition within each culture model. However, ROCK inhibition decreased chondrogenesis in a newly developed gelatin-based microribbon (μRB) model, which is a highly macroporous scaffold, in which encapsulated cells attach to the surface of individual μRBs and exhibit rapid cell spreading upon encapsulation [ 200 ].…”

“…However, this comparison is difficult, as the substrates used in both studies had comparable stiffnesses but different biomaterial types. Regardless, we identified one study that assessed hMSC chondrogenesis on the polymers’ gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol in 2D vs. 3D [ 199 ]. This study demonstrated that the expression levels of the chondrogenic differentiation markers collagen type II, ACAN, and SOX9 were comparable in 2D vs. 3D but much higher in 3D, and this behavior was observed for all four polymers.…”

“…Thus, it would be expectable that ROCK inhibition would decrease chondrogenesis in this model, as highly spreading cells are known to contain relatively high amounts of activated ROCK and more pronounced stress fiber formation [ 136 ]. In turn, the limited effects of ROCK inhibition on chondrogenesis in the 2D or 3D models in previous research [ 199 ] can perhaps be explained by the limited cell spreading known to occur in hydrogels [ 201 ].…”

Wdpcp Regulates Proliferation and Differentiation in the Developing Limb via Hedgehog Signaling

“…4F) and Cdh2 (N-Cadherin) (Fig. 4G) and are two markers of early chondrogenesis [37], and Cdh2 upregulation is a robust marker of early chondrogenic differentiation [38]. We noted signi cantly decreased expression of Cdh2 in Wdpcp-cKO versus control (Fig.…”

Enhancing the potential of aged human articular chondrocytes for high‐quality cartilage regeneration

“…Interestingly, the level of SOX9 was reduced with the increased formation of cell aggregates. It has been reported that the cells in pellets expressed significantly lower levels of N‐cadherin than monolayer culture 47 and N‐cadherin has been shown to be a critical regulator of chondrogenesis 48‐50 . In particular, our previous study showed that immunoblocking of N‐cadherin suppressed SOX9 expression 51 .…”