It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m(2) and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell's immediate mechanistic environment arising from alignment and geometry of fibers.

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Various biomaterial technologies are promising for tissue engineering, including electrospinning, but commercial scale-up of throughput is difficult. The goal of the study was to evaluate meltblown fabrics as candidate scaffolds for rotator cuff tendon tissue engineering. Meltblown poly(lactic acid) fabrics were produced with several polymer crystallinities and airflow velocities [500(low), 900(medium) or 1400(high) mair/h/m fabric]. Fiber diameter, alignment, and baseline bidirectional tensile mechanical properties were evaluated. Attachment and spreading of human adipose-derived stem cells (hASCs) were evaluated over 3 days immediately following seeding. After initial screening, the fabric with the greatest Young's modulus and yield stress was selected for 28-day in vitro culture and for evaluation of tendon-like extracellular matrix production and development of mechanical properties. As expected, airflow velocity of the polymer during meltblowing demonstrated an inverse relationship with fiber diameter. All fabrics exhibited fiber alignment parallel to the direction of collector rotation. All fabrics demonstrated mechanical anisotropy at baseline. Cells attached, proliferated, and spread on all fabrics over the initial three-day culture period. Consistent with the observed loss of integrity of the unseeded biomaterial, hASC-seeded scaffolds demonstrated a significant decrease in Young's modulus over 28 days of culture. However, dsDNA, sulfated glycosaminoglycan, and collagen content increased significantly over 28 days. Histology and polarized light microscopy demonstrated collagen deposition and alignment throughout the thickness of the scaffolds. While fiber diameters approximated an order of magnitude greater than those previously reported for electrospun scaffolds intended for tendon tissue engineering, they were still within the range of collagen fiber diameters found in healthy tendon. The extent of matrix production and alignment was similar to that previously observed for multilayered electrospun scaffolds. While the Young's modulus of scaffolds after 28 days of culture was lower than native rotator cuff tendon, it approximated that reported previously following culture of electrospun scaffolds and was on the same order of magnitude as of current Food and Drug Administration-approved patches for rotator cuff augmentation. Together, these data suggest that with minor polymer and parameter modifications, meltblown scaffolds could provide an economical, high-throughput production alternative method to electrospinning for use in rotator cuff tendon tissue engineering.