# Aligned Nanofiber Topography Directs the Tenogenic Differentiation of Mesenchymal Stem Cells
## 4 Discussion  
Aligned parallel and perpendicular suspended fiber networks were used to investigate cell response to microenvironmental cues of orientation and alignment; specifically tenogenic differentiation of equine bone marrow-derived MSCs on 3D nanofibers. We found that MSCs adhered to and elongated on fibrous scaffolds of both parallel and perpendicular oriented fibers, maintained viability, and migrated along both individual and multiple fibers regardless of fiber orientation. Previous studies using aligned fibrous scaffolds have shown that MSCs elongate along aligned fibrous scaffolds to promote tenocyte-like morphologies and tendon differentiation, while random electrospun fibrous scaffolds promote polygonal cell morphology and random cell orientation [16; 31; 32]. Instead of randomly oriented fibers, we used aligned perpendicular fibers resulting in primarily two different morphologies. These findings show that alignment cues affected cell morphology and cell alignment, providing preliminary evidence of tenogenesis. Further evidence of tenogenesis was demonstrated by an increasing trend of SCX and COL I gene expression, in response to fiber orientation from both parallel and perpendicular fibers, over time. Topographical cues from the fibers control cytoskeletal rearrangement and result in changes in cell morphology that contribute towards differentiation [33; 34; 35]. Using aligned rope-like silk scaffolds, [PERSON] et al. have previously demonstrated the association between cell morphology and tendon differentiation with increased expression of COL I with increase in SCX [36]. However, it was unexpected to have similar changes in gene expression of MSCs from the parallel and perpendicular oriented fibers. This can be explained by the similarities in the cell-fiber interactions. The cells on both the parallel and perpendicular fibers interacted with the individual fibers due to fiber density. Previous studies have investigated tendon gene expression from cells cultured on fibrous scaffolds where tendon gene expression was not significantly altered, although this was a comparison between aligned  
Figure 7: MSC production of the key matrix components. Collagen content quantification in media from cells in monolayer, aligned parallel scaffolds and aligned perpendicular scaffolds measured on days 1, 7, and 14 (**A**). \(p\) = 0.0038 for increased collagen production between days 1, 7, and 14, regardless of fiber orientation; GAG content quantification in media from cells in monolayer, aligned parallel scaffolds and aligned perpendicular scaffolds measured on days 1, 7, and 14 (**B**). \(p\) = 0.0038 for increased GAG production between all three days and fiber orientation. \(p\)-value \(\leq\) 0.05 were considered significant. Groups not connected by the same letter are significantly different. Error bars represent SEM. and random scaffolds [37]. In another study, [PERSON] et al. compared aligned (parallel) and grid (perpendicular) architectural cues and observed an increase of gene expression of SCX and COL I from day 6 to day 12, with no significant differences in tendon gene expression between designs [22], similar to results observed in our scaffolds. Our findings suggest that alignment, rather than orientation, may be the predominant cue influencing tenogenic differentiation. We investigated whether morphology and gene expression also correlated to the production of ECM components. We found equine MSCs produced increased amounts of collagen and GAGs over time in response to fibrous scaffolds, in accordance with previous studies. Although our study involved differences in fiber orientation compared to others, we found that the production of ECM components increased similarly. [PERSON] et al. showed an increase of collagen production from fibroblast like cells on aligned scaffolds compared to unaligned nanofiber scaffolds [38]. Therefore, increased collagen production was expected with both scaffold designs being highly aligned.