The alignment and fusion assembly of adipose-derived stem cells on mechanically patterned matrices

Abstract

Cell patterning is typically accomplished by selectively depositing proteins for cell adhesion only on patterned regions; however in tissues, cells are also influenced by mechanical stimuli, which can also result in patterned arrangements of cells. We developed a mechanically-patterned hydrogel to observe and compare it to extracellular matrix (ECM) ligand patterns to determine how to best regulate and improve cell type-specific behaviors. Ligand-based patterning on hydrogels was not robust over prolonged culture, but cells on mechanically-patterned hydrogels differentially sorted based on stiffness preference: myocytes and adipose-derived stem cells (ASCs) underwent stiffness-mediated migration, i.e. durotaxis, and remained on myogenic hydrogel regions. Myocytes developed aligned striations and fused on myogenic stripes of the mechanically-patterned hydrogel. ASCs aligned and underwent myogenesis, but their fusion rate increased, as did the number of cells fusing into a myotube as a result of their alignment. Conversely, neuronal cells did not exhibit durotaxis and could be seen on soft regions of the hydrogel for prolonged culture time. These results suggest that mechanically-patterned hydrogels could provide a platform to create tissue engineered, innervated micro-muscles of neural and muscle phenotypes juxtaposed next to each other in order better recreate a muscle niche.

Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels

“…Because the average y velocity of cells on the 2.9-kPa/mm hydrogel was not significantly different from zero, we can conclude that the gradient did not induce biased migration. These data improve upon the durotactic threshold suggested in previous studies (17,19) by narrowing the range in which the threshold potentially occurs to above 2.9 kPa/mm and below 8.2 kPa/mm. To rule out the possibility that cell attachment or cell proliferation is biased by the stiffness gradients, the centroid of all measured cells was calculated at low (20,000 cells per hydrogel) and high (50,000 cells per hydrogel) density cell seeding on the 2.9-kPa/mm hydrogels (depicted and quantified in Fig.…”

“…S1B). Although other systems have used polymerized PA above prepolymerized PA (17), those systems had very small diffusion distances from the surface of the gel to the underlying prepolymerized gel (∼25 μm), allowing (i) an equilibrium concentration to form before polymerization occurs and (ii) the mechanical properties of the underlying stiffness layer to influence those of the upper layer.…”

“…When mimicking the stiffness of neural (∼1 kPa), muscle (∼12 kPa), and bone (∼30 kPa) tissues (15,16), substrate stiffness can induce differentiation toward those specific tissue types. Furthermore, stem cells will migrate toward regions of higher stiffness in a process known as "durotaxis" (17)(18)(19), whereas neurons have shown preference for softer regions (20,21). Additionally, some cancer cell lines are inversely sensitive to substrate stiffness and thus show markedly different migration phenotypes (22)(23)(24).…”

Fibroblasts probe substrate rigidity with filopodia extensions before occupying an area

“…However, many of these systems require lengthy time-lapse recording over a wide area to capture the brief moment when a cell encounters a rigidity border. Whereas some recent studies have developed substrates with micropatterned rigidity (15)(16)(17)34), the present composite substrate has several distinct advantages. First, it traps single cells at a simulated rigidity border, which allows prolonged observation of cell behavior.…”

Dynamic phototuning of 3D hydrogel stiffness

“…Dynamic changes to both the structure and composition of the ECM result in alterations to the biophysical properties of the matrix, which are known to have significant influence on cellular behavior, including migration (1), alignment (2), proliferation (3), morphology (4), and differentiation of progenitors (5,6). Temporal changes in matrix stiffness occur in tissue development (7), liver fibrosis (8), tumor progression (9, 10), and myocardial infarction (11).…”