ABSTRACT. Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.

Tendon and ligament (T/L) injuries present an important clinical challenge due to their intrinsically poor healing capacity. Natural healing typically leads to the formation of scar-like tissue possessing inferior mechanical properties. Therefore, tissue engineering has gained considerable attention as a promising alternative for T/L repair. In this study, we fabricated braided nanofibrous scaffolds (BNFSs) as a potential construct for T/L tissue engineering. Scaffolds were fabricated by braiding 3, 4, or 5 aligned bundles of electrospun poly(L-lactic acid) nanofibers, thus introducing an additional degree of flexibility to alter the mechanical properties of individual scaffolds. We observed that the Young's modulus, yield stress, and ultimate stress were all increased in the 3-bundle compared to the 4- and 5-bundle BNFSs. Interestingly, acellular BNFSs mimicked the normal tri-phasic mechanical behavior of native tendon and ligament (T/L) during loading. When cultured on the BNFSs, human mesenchymal stem cells (hMSCs) adhered, aligned parallel to the length of the nanofibers, and displayed a concomitant realignment of the actin cytoskeleton. In addition, the BNFSs supported hMSC proliferation and induced an upregulation in the expression of key pluripotency genes. When cultured on BNFSs in the presence of tenogenic growth factors and stimulated with cyclic tensile strain, hMSCs differentiated into the tenogenic lineage, evidenced most notably by the significant upregulation of Scleraxis gene expression. These results demonstrate that BNFSs provide a versatile scaffold capable of supporting both stem cell expansion and differentiation for T/L tissue engineering applications.

This review focuses on the role of nano-structure and nano-scale materials for tissue engineering applications. We detail a scaffold production method (electrospinning) for the production of nanofiber-based scaffolds that can approximate many critical features of the normal cellular microenvironment, and so foster and direct tissue formation. Further, we describe new and emerging methods to increase the applicability of these scaffolds for in vitro and in vivo application. This discussion includes a focus on methods to further functionalize scaffolds to promote cell infiltration, methods to tune scaffold mechanics to meet in vivo demands, and methods to control the release of pharmaceuticals and other biologic agents to modulate the wound environment and foster tissue regeneration. This review provides a perspective in the state-of-the-art of the production, application, and functionalization of these unique nanofibrous structures, and outlines future directions in this growing field.

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.