Abstract
Matrix stiffness has been found to regulate cell morphology, while both cell morphology and matrix stiffness are verified as important factors directing BMSCs (bone marrow mesenchymal stem cells) differentiation. This study aimed to investigate whether matrix stiffness depended on cell morphology to regulate osteogenesis and adipogenesis of BMSCs on 2D substrates. First, we seeded BMSCs on tissue culture plates (TCPs) with different fibronectin (FN) concentrations and cytoskeleton inhibitor cytochalasin D, and FN was found to promote cell spreading and osteogenesis while inhibiting adipogenesis of BMSCs through F-actin reorganization. Based on these, we modulated BMSCs morphology on 0.5kPa and 32kPa CytoSoft® substrates through FN. High concentration of FN (300μg/ml) coated on 0.5kPa substrates promoted cell spreading to similar levels with 32kPa substrates coated with 100μg/ml of FN, and cells in both groups dominantly commit osteogenesis. On the other hand, low FN concentration (30μg/ml) on 32kPa substrates induced restricted cell morphology similar with 0.5kPa substrates coated with 100μg/ml of FN, and cells in both groups mainly commit adipogenesis. Immunofluorescence indicated nuclear translocation and higher intensity of YAP/TAZ when cells spread to larger areas, regardless of matrix stiffness. However, when cell spreading areas were fixed as similar levels, matrix stiffness didn't significantly affect YAP/TAZ intensity or location. Matrix stiffness failed to regulate BMSCs differentiation and YAP/TAZ activity without corresponding cell morphology. Cell spreading area could mediate effects of matrix stiffness on osteogenesis and adipogenesis of BMSCs.
Highlights
Tissue engineering requires precise control of stem cell fate decisions, which remains a challenge in the status quo
We found bone mesenchymal stem cells (BMSCs) on 0.5 kPa substrates coated with 300μg/ml of FN manifested spreading morphology with cells on 32 kPa substrates coated with 100 μg/ml of FN, and cells in both groups dominantly commit osteogenesis
BMSCs on 32 kPa substrates coated with 30μg/ml of FN manifested restricted morphology with cells in on 0.5 kPa substrates with 100μg/ml of FN, and in both groups cells mainly commit adipogenesis
Summary
Tissue engineering requires precise control of stem cell fate decisions, which remains a challenge in the status quo. Anchoring integrin to surrounding matrix, BMSCs exert traction force through cytoskeleton system to perceive their mechanical microenvironment, and matrix stiffness determines the resistance they receive [2]. A rigid substrate of 25-40 kPa produces high resistant force to BMSCs and induces FAs (focal adhesions) assembly and F-actin polymerization that promotes nuclear translocation of transcriptional regulators YAP/TAZ, thereby promoting osteogenesis of BMSCs, while soft substrates of 0.5-5 kPa inhibit the mechanotransduction process above and favor adipogenic differentiation [2,3,4]. Cytoskeleton dynamics induced by matrix stiffness simultaneously results in cell morphology change: cells spread to large areas with high cytoskeletal tension on rigid substrates while rounding up with reduced cytoskeletal tension on soft substrates [5]
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