The number of adhesions per cell in cells around the PCL substrate did decrease drastically between 24 and 48 h but remained higher than all other substrates. Open in a separate window Figure 2 Focal adhesion number, size, and morphology over time on each of the evaluated substrates. created. The effect of each substrate characteristic on osteoblastic differentiation was then examined. We found that, of the characteristics examined, only HAp density, and indeed a specific density (85 particles/cm2), significantly increased osteoblastic differentiation. Further, an increase in focal adhesion maturation and turnover was observed in cells cultured on this substrate. Moreover, -catenin translocation from the membrane bound cell fraction to the nucleus was more rapid in cells around the 85 particle/cm2 substrate compared to cells on tissue culture polystyrene. Together, these data suggest that particle density is usually one pivotal factor in determining a substrates overall osteogenic potential. Additionally, the observed increase in osteoblastic differentiation is usually a at least partly the result of -catenin translocation and transcriptional activity suggesting a -catenin mediated mechanism by which substrate surface characteristics are transduced. such as PCL and HAp have yielded conflicting results. One reason it is still challenging to create novel substrates with increased osteogenic potential is because it is still unknown how substrate surface characteristics are transduced and how this signal then regulates osteogenesis (Pavalko et al., 2003; Kashte et al., 2017). Numerous attempts have been made to determine the mechanism by which substrate surface characteristics are transduced into intracellular signals. This has given rise to the concept that surface characteristics induce cytoskeletal changes that then alter nuclear morphology and gene expression (Schaffler and Kennedy, 2012). Previous data also suggest -catenin, a protein found at cell adhesions Liensinine Perchlorate and a key contributor in the Wnt signaling pathway, transduces substrate surface characteristics, but this concept has yet to be validated (Alenghat and Ingber, 2002; Perez-Moreno et al., 2003). The canonical Wnt signaling pathway relies on -catenin translocation to the nucleus to regulate transcription factors, which in the case of osteoblastic cells, regulate pro-osteogenic genes (Cadigan and Waterman, 2012). Another role of -catenin is usually its contribution to the formation and stabilization of cell Liensinine Perchlorate adhesions such as focal adhesions and cadherins (Mbalaviele et al., 2006; Thompson et al., 2012). At E-cadherins, -catenin binds the cadherin directly at the N-terminus, where it is then stabilized by Liensinine Perchlorate -catenin. -catenin then either binds directly to actin or indirectly to vinculin that then binds actin (Jamora and Fuchs, 2002). More ENOX1 recent studies have also suggested that vinculin may directly bind -catenin after activation and that the -catenin/vinculin complex is usually capable of supporting mechanical tension (Peng et al., 2010; Bertocchi et al., 2019). Interestingly, vinculin is also a component of another adhesion complex, focal adhesions, although the role of -catenin, if any, at focal adhesions is still unknown (Kanchanawong et al., 2010). Focal adhesion complexes are composed of 50 proteins and are known to contribute to mechanosensing within the cell (Zamir and Geiger, 2001). The transmembrane portion is usually comprised of integrins that bind to the extracellular matrix. Previous studies suggest that integrins respond differentially to various surface characteristics, with different forms of integrins adhering preferentially to pro-osteogenic substrates compared to sub-optimal substrates (Hyzy et al., 2017). Focal adhesions, well-known mechanosensors, also increase in size in response to increased actin fiber tension. This phenomena, known as the growth model of force-induced focal adhesion, is usually driven by actomyosin-mediated tension (Besser and Safran, 2006; Geiger et al., 2009; Kim and Wirtz, 2013; Kuo, 2014). Previous studies examining human mesenchymal stem cell differentiation have observed a correlation between nanopost density, focal adhesion formation and maturation, and the differentiation state of the cell (Di Cio and Gautrot, 2016). These studies found the median densities often elicit the greatest increase in focal adhesion maturation and differentiation. In addition, evidence presented by Dubrovskyi et al. suggests that -catenin may localize to focal adhesions as well, binding paxillin during Rac activation (Dubrovskyi et al., 2013). However, the contribution -catenin may have facilitating focal adhesion formation and binding of actin stress fibers has not been fully explored. The relationship between focal adhesion maturation, osteoblastic differentiation, and -catenin localization has led to the examination of whether focal adhesions transduce substrate surface characteristics to mediate osteogenesis and, if so, the mechanism by which this occurs (Perez-Moreno et.