Perceptual and acoustic voice characteristics of children with anhidrotic ectodermal dysplasia

This investigation examined perceptual and acoustic voice characteristics in the speech of 13 pre-pubescent children (6 males, 7 females) who were diagnosed with anhidrotic ectodermal dysplasia (ED) (mean age = 6 years, 2 months) and in the speech of 13 normal peers (6 males, 7 females) identified as the control group (mean age = 5 years, 4 months). For each subject, acoustic measurements of mean fundamental frequency (FO), mean jitter ratio, harmonics-to-noise ratio (HNR), maximum phonation duration (MPD), and bandwidth values for the first and second formants (BW F1 & BW F2) were extracted from spontaneous speech samples and/or the midportion of 3 vowels in isolation (/a, i, u/) and embedded in the carrier phrase “Say ha&barbelow;d again, Say hi&barbelow;d again, Say hu&barbelow;d again”. Speech samples were then presented to 5 experienced listeners for the perceptual task where they rated two parameters: (1) voice quality on a 4-point categorical scale (“normal, hoarse, breathy” and “rough”), and (2) severity on a 7-point equal-appearing interval scale. Significant differences between the two groups were found for mean jitter ratio, HNR, MPD, and perceptual ratings. Results of the perceptual task indicated that listeners perceived subjects diagnosed with ED as having voices characterized as “breathy” (36%), “hoarse” (32%), “rough” (24%) and “normal” (8%). Correlation analysis between voice category groups revealed a significant correlation between acoustic variables (mean jitter ratio and HNR) and the voice category groups “breathy” and “hoarse”. Within voice category groups, only the “breathy” group yielded a significant inverse correlation between average jitter ratio values and HNR.

The Role of Matrix Properties in Directing Valvular Interstitial Cell Phenotype

This thesis presents the development of hydrogel platforms to study the fibroblast-to-myofibroblast transition in valvular interstitial cells (VICs). These systems were used to characterize the effects of extracellular matrix cues on VICs, as well as the synergies between mechanical and biochemical signals. First, the impact of culture platform on VIC phenotype was assessed by culturing VICs in peptide-functionalized poly(ethylene glycol) hydrogels (2D and 3D) and comparing them to those cultured on tissue culture polystyrene (TCPS). Expression of the myofibroblast marker α-smooth muscle actin (αSMA), as well as by a global analysis of the transcriptional profiles1 demonstrated that TCPS caused significant perturbations in gene expression from the native VIC phenotype. The dimensionality of the hydrogel (2D vs 3D) was particularly influential in the regulation of genes related to cell structure and motility, developmental processes, proliferation and differentiation, and transport; these findings motivated the use of 3D cultures for the following experiments. The effect of matrix modulus, particularly matrix stiffening, on encapsulated VICs was investigated2. To vary the matrix modulus without dramatic changes in VIC morphology, a method was developed for in situ stiffening of cell-laden hydrogels using sequential gelation steps. In contrast with prior findings in 2D, increased stiffness resulted in lower levels of myofibroblast activation, and suggested that stiffness alone was not sufficient to cause pathological activation of VICs to the myofibroblast phenotype in 3D. To facilitate the investigation of additional stimuli in a physiologically-relevant context, a high-throughput technique to encapsulate VICs within 3D hydrogels was developed and used to study VIC response to dynamic changes in matricellular signals3. A thiol-ene photoclick reaction provided temporal control over the presentation of peptide ligands to study their effects on VIC morphology and myofibroblast properties. Collectively, these studies demonstrate the ability to study and direct VIC phenotype through the temporal presentation of mechanical and biochemical cues in 3D polymer matrices. References 1. Mabry, K. M., Payne, S. Z. \u26 Anseth, K. S. Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype. Submitted. 2. Mabry, K. M., Lawrence, R. L. \u26 Anseth, K. S. Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment. Biomaterials 49, 47–56 (2015). 3. Mabry, K. M., Schroeder, M. E., Payne, S. Z. \u26 Anseth, K. S. Three-dimensional high-throughput cell encapsulation platform t

Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels

Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011) [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled “Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype” (Mabry et al., 2015) [2]

Three-Dimensional High-Throughput Cell Encapsulation Platform to Study Changes in Cell-Matrix Interactions

In their native extracellular microenvironment, cells respond to a complex array of biochemical and mechanical cues that can vary in both time and space. High-throughput methods that allow characterization of cell-laden matrices are valuable tools to screen through many combinations of variables, ultimately helping to evolve and test hypotheses related to cell–ECM signaling. Here, we developed a platform for high-throughput encapsulation of cells in peptide-functionalized poly­(ethylene glycol) hydrogels. Hydrogels were synthesized using a thiol–ene, photoclick reaction, which allowed the cell matrix environment to be modified in real time. Matrix signals were dynamically altered by in situ tethering of RGDS (0–1.5 mM), a fibronectin-derived adhesive peptide that induced more elongation than RLD or IKVAV, and/or by increasing the matrix modulus (1 to 6 kPa). This method was demonstrated with aortic valvular interstitial cells (VICs), a population of cells responsible for the pathological fibrosis and matrix remodeling that leads to aortic stenosis. VIC response to cell–matrix interactions was characterized by quantifying cell morphology and the fraction of cells exhibiting α-smooth muscle actin (αSMA) stress fibers, a hallmark of the myofibroblast phenotype. VICs elongated in response to RGDS addition, with a dramatic change in morphology within 24 h. Myofibroblast activation was also dependent on RGDS addition, with VICs exhibiting high activation (16–24%) in 1 kPa gels with RGDS. Response to RGDS was path-dependent, with the amount of time exposed to the adhesive ligand important in determining VIC morphology and activation. Although VIC aspect ratios were dependent on the amount of time spent in a stiff vs soft gel, low levels of VIC activation (≤4%) were observed in any gels cultured in higher modulus (6 kPa vs 1 kPa) microenvironments

Photoresponsive Elastic Properties of Azobenzene-Containing Poly(ethylene-glycol)-Based Hydrogels

The elastic modulus of the extracellular matrix is a dynamic property that changes during various biological processes, such as disease progression or wound healing. Most cell culture platforms, however, have traditionally exhibited static properties, making it necessary to replate cells to study the effects of different elastic moduli on cell phenotype. Recently, much progress has been made in the development of substrates with mechanisms for either increasing or decreasing stiffness in situ, but there are fewer examples of substrates that can both stiffen and soften, which may be important for simulating the effects of repeated ECM injury and resolution. In the work presented here, poly­(ethylene glycol)-based hydrogels reversibly stiffen and soften with multiple light stimuli via photoisomerization of an azobenzene-containing cross-linker. Upon irradiation with cytocompatible doses of 365 nm light (10 mW/cm², 5 min), isomerization to the azobenzene <i>cis</i> configuration leads to a softening of the hydrogel up to 100–200 Pa (shear storage modulus, <i>G</i>′). This change in gel properties is maintained over a time scale of several hours due to the long half-life of the <i>cis</i> isomer. The initial modulus of the gel can be recovered upon irradiation with similar doses of visible light. With applications in mechanobiology in mind, cytocompatibility with a mechanoresponsive primary cell type is demonstrated. Porcine aortic valvular interstitial cells were encapsulated in the developed hydrogels and shown to exhibit high levels of survival, as well as a spread morphology. The developed hydrogels enable a route to the noninvasive control of substrate modulus independent of changes in the chemical composition or network connectivity, allowing for investigations of the effect of dynamic matrix stiffness on adhered cell behavior