Individual and collective mechanosensing of extracellular matrix thickness in skeletal stem cell differentiation

Abstract

The elastic modulus of growth substrates and/or extracellular matrix (ECM) affects many cell types' spreading, proliferation and differentiation, including bone marrow stromal cells (BMSCs). However, the substrate modulus and geometry determine the stiffness a cell senses, including material thickness and cell crowding. In this study, we hypothesised that these factors would impact the traction force applied by BMSCs to the ECM and their differentiation. Soft and stiff polyacrylamide (PAAm) hydrogel matrices of different thicknesses were fabricated by varying the monomer ratio and PAAm mixture volume. Hydrogel stiffness and thickness were measured by nanoindentation and confocal microscopy. Cells were plated on collagen type I coated hydrogels to evaluate the effect of hydrogel mechanical properties on cell spreading (cell area), focal adhesions formation (immunocytochemistry), actin fibre formation (actin staining), proliferation (PicoGreen), and osteogenic (ALPL activity) and adipogenic (Oil red O) differentiation. The effect of hydrogel stiffness and thickness and cell crowding on cellular forces during osteogenic and adipogenic differentiation was evaluated by quantifying hydrogel deformations by Stro-1+ BMSCs at different seeding densities on soft, thin, and thick PAAm hydrogels after being incubated in basal, osteogenic and adipogenic media. 1 kPa (soft) hydrogels were thicker than 40 kPa (stiff) and exhibited the presence of wrinkles on the hydrogel surface, and the increase in the PAAm volume increased hydrogel thickness. The addition of fluorescent microbeads decreased the thickness of thick, soft, and stiff PAAm hydrogels and the elastic modulus of stiff, thin hydrogels. The elastic modulus of soft and stiff hydrogels increases on thinner hydrogels; ~19Pa (5µL) vs ~5Pa (50μL) for soft and ~200Pa (5μL) vs ~60Pa (50µL) for stiff hydrogels. BMSC on soft, thick hydrogels spread to a lesser extent than those on thin hydrogels with a mean cell area of 2600 ± 79 and 5600 ± 990 µm2, respectively and modified their morphology depending on the hydrogel mechanical properties. The hydrogel elasticity and thickness also influenced the formation of focal adhesions and actin fibres. ALPL activity, expression of osteogenic genes (ALPL and RUNX2) and Oil red O absorption showed that BMSCs differentiated into osteoblasts and adipocytes regardless of the hydrogel’s elasticity and thickness. Stro-1+ BMSCs highly deformed soft, thick, and soft, thin PAAm hydrogels but not stiff hydrogels. Hydrogel deformations decreased over time with the increase in cell crowding from ~14 pixels on day 0 to ~3 on week 7 on soft, thick hydrogels. These results were confirmed by evaluating the soft hydrogel deformations at different seeding densities for 24 hours in basal and osteogenic media; ~16 pixels (osteogenic) -21 pixels (basal) with cells at 1,000 cells/cm2 and ~6 pixels (basal) -8 pixels (osteogenic) pixels at 20,000 cells/cm2. Additionally, results showed that at low seeding density BMSCs perceived differences in soft hydrogel thickness and caused more significant deformations. (basal: ~10 pixels; osteogenic: ~11 pixels) compared to cells at higher seeding density (basal: ~4 pixels; osteogenic: ~3 pixels) on soft, thick hydrogels on day 14. Further evaluations highlighted that osteogenic or adipogenic supplements modified hydrogel displacements, especially on soft, thick matrices; ~18 pixels in basal media and ~14 pixels in osteogenic media and ~12 pixels in basal media and ~6 pixels in adipogenic media on day 14. Stro-1+ BMSCs mechanosense changes in substrate elasticity and thickness by modifying their morphology and slightly increasing their spreading. Despite cells detecting changes in hydrogel mechanical properties, no differences in osteogenic and adipogenic differentiation were encountered when modifying hydrogel elasticity and thickness. The quantification of hydrogel displacements suggests that cell mechanosensing is influenced by hydrogel elasticity and thickness, the increase in cell crowding and the differentiation state of stem cells. Indeed, cells perceive soft hydrogels as stiffer when the thickness is reduced and cell crowding increases, promoting osteogenic differentiation of Stro-1+ BMSCs. This may suggest that by modifying the substrate thickness and controlling cell crowding, it would be possible to promote differentiation for tissue repair.

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Identifiers

ORCID for Maria Luisa Hernandez Miranda: orcid.org/0000-0002-7366-898X

ORCID for Nicholas Evans: orcid.org/0000-0002-3255-4388

ORCID for Bram Sengers: orcid.org/0000-0001-5859-6984

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