Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffnesschondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process

Essential roles of Pdia3/PLAA receptor complex and CaMKII IN 1α,25(OH)₂D₃ and Wnt5a calcium-dependent signaling pathways in osteoblasts and chondrocytes

The vitamin D metabolite 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] plays an important role in the regulation of musculoskeletal growth and differentiation. 1α,25(OH)2D3 mediates its effects on cells, including chondrocytes and osteoblasts, through the classical nuclear 1α,25(OH)2D3 receptor. Additionally, recent evidence indicates that several cellular responses to 1α,25(OH)2D3 are mediated via a rapid, calcium-dependent membrane-mediated pathway. These actions of 1α,25(OH)2D3 can be blocked by antibodies to protein-disulfide isomerase family A, member 3 (Pdia3), indicating that it is part of the receptor complex; however, the pathway which is activated by this receptor is not fully understood. The overall goal of this thesis was to examine the roles of phospholipase A2 activating protein (PLAA) and calcium calmodulin-dependent kinase II (CaMKII) in 1α,25(OH)2D3 rapid membrane-mediated signaling. We further investigated the interaction between two pathways regulating growth plate cartilage and endochondral bone formation, 1α,25(OH)2D3 and Wnt5a, at the receptor complex level. Results indicated that PLAA was required for mediating 1α,25(OH)2D3 signal from Pdia3. Furthermore, CaM and CaMKII were identified as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral bone formation. This study demonstrated that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components in osteoblasts and chondrocytes suggesting that these pathways may interact.Ph.D

Integrin expression is stiffness- and cell-type- dependent.

<p>Comparison of integrin mRNA levels in MSCs, OBs, and chondrocytes cultured on surfaces of varying stiffness. *<i>P</i> < 0.05 vs. 18 MA, #<i>P</i> <0.05 vs. 29 MA, $<i>P</i> <0.05 vs. 40 MA.</p

Integrin-dependent osteoblast differentiation.

<p>Levels of chondrogenic (A) and osteoblastic (B) mRNA. (C-F) Cell number and osteogenic protein levels in wild type human MSCs (WT) and silenced integrin β1 MSCs (shITGB1) cultured on surfaces of varying stiffness. *<i>P</i> < 0.05 vs. 18 MA, #<i>P</i> <0.05 vs. wild type.</p

Osteoblastic differentiation on MA-MMA networks.

<p>(A-B) mRNA levels for osteoblast-specific marker RUNX2. (C-J) MSC and HOB response to substrate stiffness seen in cell number and osteogenic protein levels. *<i>P</i> < 0.05 vs. 18 MA, #<i>P</i> <0.05 vs. 29 MA, $<i>P</i> <0.05 vs. 40 MA.</p

Toughness vs. elastic modulus for load-bearing biological tissues (green) and MA-MMA networks (black).

<p>Modified from data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170312#pone.0170312.ref023" target="_blank">23</a>].</p

Cytoskeleton arrangement was altered by substrate stiffness.

<p>Representative staining of F-actin by phalloidin (green) and nuclei by DAPI (blue) in human MSCs (A-D), HOBs (E-H), and chondrocytes (I-L) cultured on surfaces of varying stiffness. (Scale bars: 100 μm for A,B,D; 50 μm for all others.)</p