# Aligned Nanofiber Topography Directs the Tenogenic Differentiation of Mesenchymal Stem Cells
## 3 Results
### MSCs Exhibit Tenocyte-Like Morphology on Suspended Fibrous Scaffolds  
MSCs were guided by the three dimensional structure of the fibers and fiber orientation as assessed by phase-contrast microscopy (Figure 2). MSCs on parallel fibers elongated along the nanofibers in a tenocyte-like morphology within 24 h of seeding (Figure 2A) and maintained by day 7 (Figure 2B). Morphological changes were similar on day 14. Parallel orientation of fibers promoted an elongated tenocyte-like morphology and maintained parallel MSC orientation between adjacent cells. Similar spindle-like MSC morphology was observed on perpendicular fibrous scaffolds. Additional cell morphologies were observed on perpendicular suspended nanofibers as MSCs spread across multiple fibers at intersections of fibers, on day 1 (Figure 2C) and day 7 (Figure 2D). Figure 1: STEP scaffold characterization of fiber diameter. A schematic of an aligned parallel fibrous scaffold (**Ai**) and an aligned perpendicular fibrous scaffold (**Aii**); SEM images of aligned parallel fibrous scaffold (**Bi**) and aligned perpendicular fibrous scaffold (**Bii**); Scale bar 5 μm and 3 μm, respectively; Histogram of aligned parallel and aligned perpendicular fiber diameters, measured with ImageJ, \(n\) = 1400 (**C**). Cell migration and associated cell shape were better observed under time-lapse microscopy where we could clearly observe the cell-fiber interaction and changes in cell morphology (Figure 3). Time-lapse microscopy recording was started 24 h after seeding for parallel fibers (Video S1: Mesenchymal stem cell migration on aligned parallel nanofibers) and perpendicular fibers (Video S2: Mesenchymal stem cell migration on aligned perpendicular nanofibers). At this time, cell elongation was observed as a spindle-like morphology on parallel orientation of fibers and maintained MSC spacing between cells on adjacent fibers (Figure 3A-D). Perpendicular orientation of fibers allowed cells to migrate and spread along multiple fibers, temporarily change morphology, and switch directions at fiber intersections (Figure 3E-H). Fibers of perpendicular orientation promoted changes in cell migration direction and cell morphology through interaction with intersections and spreading along multiple fibers. The elongated tenocyte-like morphology was predominant before and after cells migrated through the intersections. MSC monolayers showed typical polygonal morphology on standard tissue culture treated plastic (data not shown). Viability was maintained throughout 14 days of culture on both parallel (Figure 4A) and perpendicular fibers (Figure 4B) showing no toxicity of cells on the polystyrene nanofibers. Figure 2: MSC morphology on aligned scaffolds. Representative phase-contrast microscopy images of equine bone marrow MSCs (mesenchymal stem cells) on aligned parallel oriented fibrous scaffolds (**A**,**B**) and aligned perpendicular fibrous scaffolds (**C**,**D**) on day 1 and day 7 of culture. Arrows, elongated cells; arrowheads, cell spreading. Insets show enlarged image of the cell. Scale bar 200 μm. MSCs cultured for 12 days on fibrous scaffolds elongated along aligned parallel fibers in both parallel and perpendicular oriented fibrous scaffolds as observed by fluorescence microscopy (Figure 5). Intracellular F-actin stained with rhodamine phalloidin and nuclei stained with DAPI were observed along the nanofibers and in the monolayers, showed cell attachment and interaction with fibers. Perpendicular fibrous scaffolds provided fiber intersections for cell interaction (Figure 5C,D), and parallel scaffolds maintained MSC spacing between fibers (Figure 5A,B) leading to parallel cell orientation and spacing between fibers. The above methods have shown that fiber alignment provides environmental cues, resulting in cell elongation/tenocyte-like morphology. Figure 4: Viability of MSCs on aligned scaffolds. MSCs stained with calcein AM on parallel scaffolds (**A**) and perpendicular scaffolds (**B**) after 14 days of culture.