Abstract
Event Abstract Back to Event Designing dense connective tissues: identifying microenvironmental factors that direct human MSC differentiation in 3D Jenna Usprech1 and Craig A. Simmons1, 2 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Department of Mechanical and Industrial Engineering, Canada Introduction: Microenvironmental factors such as biomaterial physical and biochemical properties, external mechanical stimuli, and soluble chemical cues integrate in vivo to direct the differentiation of mesenchymal stromal cells (MSCs). Identifying what factor combinations promote the differentiation of MSCs into myofibroblasts (the tissue producing cells of developing valves, ligaments, skin, tendons and periodontal ligaments) is a significant challenge when designing for dense, type I collagen-rich connective tissues. To date, select combinations of microenvironmental factors have been studied in 2D, but systematic consideration of the integration of factors in 3D culture has not been explored fully. Materials and Methods: We developed a 3D screening platform to systematically study MSC responses to microenvironmental stimuli. Permutations of the cell adhesion sequences RGD, DGEA and YIGSR at different concentrations (0-2 mM) were incorporated into 5-11 wt% (5-22 kPa compressive moduli) polyethylene glycol norbornene (PEG-NB) hydrogel arrays. Since PEG-NB is inert and easily crosslinked with UV light, adhesion ligands can be tuned independently from biomaterial properties, enabling us to identify the contribution of individual factors in a combinatorial fashion. Human bone marrow-derived MSCs were encapsulated and cultured in the 3D arrays for 7 days with 0 or 5 ng/mL TGF-β1. The extent of α-smooth muscle actin (αSMA) expression and collagen type I deposition were modeled using least squares estimation and regression on 3D confocal data (Figure 1A) that was analysed using Imaris (Figure 1B). Each experiment was replicated four times and statistical analyses were performed in JMP on the pooled data. Results and Discussion: Regression analyses of the screening data revealed that in the presence of TGF-β1, PEG-NB hydrogel wt% was the most significant factor influencing myofibroblast differentiation (p < 0.0001), with greater αSMA staining intensity manifesting at lower gel wt%. These data are in contrast to MSC myofibroblastic differentiation in 2D, which is promoted by stiffer substrates. With TGF-β1, RGD concentration was correlated with αSMA staining intensity in a positive, biphasic manner and demonstrated a significant, non-linear dependence with PEG-NB wt% (p = 0.02) (Figure 2A). Interestingly, αSMA staining intensity could only be predictably modeled by regression in cultures that were supplemented with TGF-β1. These observations suggest that while MSCs can express αSMA in the absence of exogenous TGF-β1, αSMA expression is only sensitive to material properties in the presence of TGF-β1. Collagen deposition was predominantly negatively correlated with PEG-NB hydrogel wt% (p<0.0001) with or without TGF-β1, with slight non-linear dependence between RGD and DGEA (p = 0.01) (Figure 2B) or linear dependence with RGD (p = 0.04) in the absence or presence of TGF-β1, respectively. Conclusion: The newly developed 3D screening platform enables the systematic identification of optimal conditions for connective tissue engineering when considering multiple interacting microenvironmental factors. The data presented here demonstrate that conditions which promote a myofibroblast phenotype in 2D do not translate completely to the more complex and integrative 3D environment. Keywords: Cell Differentiation, screening, 3D scaffold, instructive microenvironment Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials in constructing tissue substitutes Citation: Usprech J and Simmons CA (2016). Designing dense connective tissues: identifying microenvironmental factors that direct human MSC differentiation in 3D. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02714 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jenna Usprech Craig A Simmons Google Jenna Usprech Craig A Simmons Google Scholar Jenna Usprech Craig A Simmons PubMed Jenna Usprech Craig A Simmons Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
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