Cell size and shape affect cellular processes such as cell survival, growth and differentiation, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Articular cartilage lesions, which can progress to osteoarthritis, are a particular challenge for regenerative medicine strategies, as cartilage function stems from its complex depth-dependent microstructural organization, mechanical properties, and biochemical composition. Fibrous scaffolds offer a template for cartilage extracellular matrix production; however, the success of homogeneous scaffolds is limited by their inability to mimic the cartilage's zone-specific organization and properties. We fabricated trilaminar scaffolds by sequential electrospinning and varying fiber size and orientation in a continuous construct, to create scaffolds that mimicked the structural organization and mechanical properties of cartilage's collagen fibrillar network. Trilaminar composite scaffolds were then compared to homogeneous aligned or randomly oriented fiber scaffolds to assess in vitro cartilage formation. Bovine chondrocytes proliferated and produced a type II collagen and a sulfated glycosaminoglycan-rich extracellular matrix on all scaffolds. Furthermore, all scaffolds promoted significant upregulation of aggrecan and type II collagen gene expression while downregulating that of type I collagen. Compressive testing at physiological strain levels further demonstrated that the mechanical properties of trilaminar composite scaffolds approached those of native cartilage. Our results demonstrate that trilaminar composite scaffolds mimic key organizational characteristics of native cartilage, support in vitro cartilage formation, and have superior mechanical properties to homogenous scaffolds. We propose that these scaffolds offer promise in regenerative medicine strategies to repair articular cartilage lesions.

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