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

Abstract

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 stiffness <10 MPa. Like chondrocytes, 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.

A Physiology-Inspired Multifactorial Toolbox in Soft-to-Hard Musculoskeletal Interface Tissue Engineering

“…Round OBs were found in stiffer substrates (N10 MPa), Chon presented a similar morphology with long extensions and few points of contact, and MSCs presented longer cytoskeletal arrangements in less stiff surfaces (b10 MPa). Reproduced, with permission, from [38]. The same effect was demonstrated assessing MSC response to tunable polyacrylamide hydrogels coated with fibronectin with increasing stiffness.…”

“…Surface stiffness was shown to modulate stem cell adhesion, proliferation, and differentiation ( Figure 3A). Indeed, MSCs tend to become more spread on substrates with higher stiffness (ranging from 50 to 90 kPa) and differentiate toward chondrogenic [37,38] and osteogenic lineages [38,39] ( Figure 3A, a-b), whereas softer surfaces (∼30-50 kPa) result in a tenogenic-like phenotype [39,40] ( Figure 3A, c). These results support the creation of material stiffness gradients to guide stem cell differentiation along tendon-to-bone and bone-cartilage bioengineered constructs.…”

Regulation and Directing Stem Cell Fate by Tissue Engineering Functional Microenvironments: Scaffold Physical and Chemical Cues

“…Stem cells differentiate into muscle cells on soft substrates and osteoblasts on harder substrates [86,87]. Another study supported this finding, and stem cell on soft materials when stiffness is less than 0.05 kPa could promote neural differentiation effectively, while hard stiffness materials (>40 kPa) promoted osteogenic differentiation effectively [88,89], which could be related to the Wnt signal pathway [90]. However, there is no agreement on the optimal stiffness for stem cells to differentiate into neurons, muscle cells, cartilage cells, and osteoblasts [86,91].…”

Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering

“…An alternative but attractive approach would be to functionalize a routinely used hydrogel bioink such as alginate with cECM. Alginate is a naturally derived polysaccharide and a versatile biomaterial owing to its excellent biocompatibility, gelation properties, tuneable stiffness,, injectability, printability and ability to support the differentiation of encapsulated cells . Although widely used for cartilage tissue engineering applications, alginate is often modified, e.g., by immobilization of cell adhesion ligands such as arginine‐glycine‐asparagine (RGD) sequences for enhanced cell attachment or combined with other naturally derived biomaterials such as chitosan, hyaluronic acid, or collagen for improved chondrogenesis.…”