Controlling Osteogenic Stem Cell Differentiation via Soft Bioinspired Hydrogels

Abstract

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is guided by various physical and biochemical factors. Among these factors, modulus (i.e., rigidiy) of the ECM has gained significant attention as a physical osteoinductive signal that can contribute to endochondral ossification of a cartilaginous skeletal template. However, MSCs also participate in intramembranous bone formation, which occurs de novo from within or on a more compliant tissue environment. To further understand the role of the matrix interactions in this process, we evaluated osteogenic differentiation of hMSCs cultured on low moduli (102, 390 or 970 Pa) poly(N-isopropylacrylamide) (p(NIPAAm)) based semi-interpenetrating networks (sIPN) modified with the integrin engaging peptide bsp-RGD(15) (0, 105 or 210 µM). Cell adhesion, proliferation, and osteogenic differentiation of hMSCs, as measured by alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone sialoprotein-2 (iBSP), and osteocalcien (OCN) protein expression, was highest on substrates with the highest modulus and peptide concentrations. However, within this range of substrate stiffness, many osteogenic cellular functions were enhanced by increasing either the modulus or the peptide density. These findings suggest that within a compliant and low modulus substrate, a high affinity adhesive ligand serves as a substitute for a rigid matrix to foster osteogenic differentiation.

Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior

“…These results were consistent with those previously reported [36]. Hydrogel shear modulus was directly correlated to both PEG-NB monomer concentration and PEG-DT crosslinker density (with 50%–100% density corresponding to 0.5–1 mole ratio of thiol-to-norbornene) in the precursor solution, and these results were confirmed by determining equilibrium mass swelling ratio (Figure 3e and Figure 4b).…”

“…cell area and morphology) and perturbations (e.g. proliferation and cytoskeletal organization) have been demonstrated to be accurate qualitative measures of hMSC mechanosensing in response to substrate stiffness and ligand density, we examined these properties here using automated data collection techniques that augmented the enhanced-throughput capability of our array-based screening system [11, 12, 15, 24]. Hydrogel arrays were formed using gold-coated glass slides patterned with alkanethiolate self-assembled monolayers (SAMs), and differential wettability was used to define the geometry and confine the contents of each spot in the array (Figure 1).…”

“…Using these arrays, we screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term cell culture. hMSC adhesion was chosen as a model biological outcome to demonstrate the utility of hydrogel arrays, as recent studies have extensively detailed the influence of substrate properties on hMSC behavior [10–12] [5, 13–17]. …”

“…Over the past two decades, many researchers have demonstrated a positive correlation between the substrate stiffness and the intracellular cytoskeletal tension, indicating that cells sense and respond to changes in matrix stiffness by modulating cytoskeletal assembly and contractility (a concept referred to as “mechanosensing”) [11, 12]. We and others have also demonstrated control of hMSC behavior by regulating mechanosensing: hMSCs cultured on surfaces that promoted high cytoskeletal tension (via high substrate stiffness, cell adhesion molecule affinity, or cell adhesion molecule density) resulted in well-spread cells that possessed highly-organized f-actin stress fibers and large and well-defined focal adhesions, while substrates that promoted low cytoskeletal tension resulted in rounded cells with diffused actin and small, unstable focal adhesions.…”

Numerical modeling of cell differentiation and proliferation in force-induced substrates via encapsulated magnetic nanoparticles

“…Since hydrogels become stiffer as they are strained, cell response strongly depends on the compression and tension loads applied in such substrates. This stiffening can be regulated by applying an internal force in the cell micro-environment to modulate and control its differentiation and proliferation [29,52,68]. A Neo-Hookean hyperelastic material model is here employed to model nonlinear behavior of hydrogel substrate materials undergoing deformations [17].…”