# Aligned Nanofiber Topography Directs the Tenogenic Differentiation of Mesenchymal Stem Cells
## 1 Introduction  
Tendon injury is a common clinical problem among veterinary and human patients with significant similarities in physiology and tendon pathologies. The inherent regenerative capacity of tendon is poor due to low cellularity and low vascularity of the tissue. These characteristics contribute to prolonged and incomplete healing with a high risk of re-injury [1]. Mesenchymal stem cells (MSCs) are used in tissue engineering and regenerative medicine applications for musculoskeletal tissue repair, particularly for their regenerative capabilities. MSCs can differentiate into tenocytes to promote tendon healing [2; 3; 4]. Engineered in vitro nanofiber scaffolds mimic the native topography of the extracellular matrix (ECM) microenvironmental cues that influence cell response and function including adhesion, migration, proliferation, and differentiation [5; 6]. Scaffold directed MSC differentiation is determined through a combination of morphology, panels of tendon-related markers, collagen production and alignment, and levels of glycosaminoglycan (GAG) content [7; 8]. Scleraxis (SCX), a transcription factor, is a key tendon-related marker which functions in tendon development and differentiation and promotes the expression and production of type I collagen (COL I) [9; 10; 11]. Type I collagen is the mainfibrillar collagen functioning as a structural protein in healthy tendon composition [12]. GAGs aid in collagen fiber growth and function; abnormal levels signify disease or injury [13]. A challenge of designing scaffolds is determining the specific topographical cues of fiber diameter, orientation, and alignment that are most suited for tendon studies. Techniques used to manufacture fibrous scaffolds vary in their ability to control these parameters and produce various combinations. Fiber alignment influences MSC-fiber interaction and guides cell behavior. [PERSON] et al. fabricated a nanofiber/microfiber composite scaffold that enhanced cell adhesion, cell spreading, and scaffold infiltration [14]. In another study, multilayered aligned electrospun scaffolds provided an anisotropic environment that promoted expression of tenomodulin, a marker of tendon maturation, and alignment of collagen throughout the scaffold in comparison to nonaligned scaffolds [15]. [PERSON] et al. showed that compared to random electrospun fibers, aligned nanofibers provided microenvironmental cues for differentiation into the teno-lineage [16]. MSCs seeded on aligned nanofibers showed evidence that topography alone promoted changes in cell morphology and orientation. Cell differentiation was only enhanced by addition of TGF-\(\beta\)3 [6] and printing of growth factors on aligned fibers [17]. These studies demonstrate that differentiation is a complex process sensitive to environmental cues, thus necessitating the need to optimize fibrous and growth factor parameters to mimic native environment to achieve specific lineages. Electrospinning is a common manufacturing technique used to engineer fibrous scaffolds for tissue engineering. Improvements to this technique, including the use of modified collectors and fiber manipulation, have increased the applicability of this technique [18; 19; 20; 21]. Besides electrospinning, other approaches including micropatterning, electro-chemically aligned have been used to study differentiation. For example, [PERSON] et al. studied the effects of architectural cues on aligned tissue formation using micropatterned aligned scaffolds of varying lengths and orientation and concluded that the combination of cues i.e. fiber length, boundaries, and orientation regulate tissue formation in a complex manner [22]. [PERSON] et al. investigated the effects of topography on tenogenesis with electro-chemically aligned threads, and showed alignment induced increased expression of sclarcasis and tenomodulin [23].