INTELLECTUAL MERIT: Mimicking elements of stem cell microenvironments to elicit regenerative control is one of the major challenges in biomaterials research. Of the major environmental cues, the presentation of membrane associated ligands involved in cell-cell interactions (e.g. cadherin, notch) and the sequestering of soluble factors (e.g. transforming growth factor-beta) at the cell surface have not yet been well established in 3D culture systems. As the 3D context is more appropriate for biomimicry of the stem cell niche, our long-term goal is to integrate microsphere-supported biomembrane systems into 3D matrices. This will be accomplished using a composite 3D hydrogel scaffold architecture that integrates laterally mobile ligands/bound factors positioned in microsphere-supported biomembrane systems known as proteolipobeads. For the first time, this will allow for the laterally mobile presentation of various ligands/membrane-bound factors to stem cells within a 3D format via novel, molecularly engineered interfaces with controlled surface densities. Thus, the specific focus of this proposal is to build a platform that incorporates laterally mobile N-Cadherin fragments and the TGF-beta3 receptor, TGF-beta-R3 (i.e., beta-glycan), in proteolipobeads suspended in a 3D matrix for presentation to human mesenchymal stem cells (hMSCs). The proposed mode of N-cadherin and TGF-beta-R3 display will be used to investigate the effects of N-cadherin-mediated cell-cell communication events and TGF-beta3 sequestering on MSC chondrogenesis in vitro and serve as the proof-of-concept for this transformative technology. The specific hypothesis behind the proposed research is that the introduction of proteolipobead assemblies into 3D scaffolds is a viable biomimetic means to present ligands/bound factors to stem cells and mimic cellular communication in the stem cell niche. The objectives are designed to examine if this mode of ligand presentation can be achieved and to assess the effects on stem cell viability and differentiation. Objective 1 is to embed N-cadherin/TGF-beta-R3 proteolipobeads in carboxymethylcellulose 3D constructs and probe N-Cadherin display and TGF-beta3 growth factor sequestration. Objective 2 is to investigate the interaction between N-cadherin/TGF-beta-R3 biomembrane-microspheres and hMSCs by monitoring MSC viability and chondrogenic differentiation.
BROADER IMPACTS: The benefits to society of the proposed activity are that contributions from the research could help cross major technological barriers in stem cell bioengineering, so that humanity can ultimately guide stem cell regeneration processes. Essentially a whole dimension in stem cell ligand/bound factor presentation has not yet been achieved, and the proposed scaffold design may lead to new molecular methods for control of stem cells in 3D culture. In addition, this work may lead to innovative therapies for cartilage repair, which is a significant clinical problem. To go beyond the impact of the research outcomes, we will implement a new mechanism for advancing discovery while promoting training and learning. This program will be specifically tailored to inner-city middle and high school students and aimed at stimulating a fascination with the molecular basis for color. This educational outreach integrates UV-VIS pigment identification field studies of sanctioned and renegade street graffiti art in the CCNY (Harlem) neighborhood. A concurrently running online dye and pigment database will be created, that will be further augmented with molecular structure and absorption-relevant molecular orbital visualizations using computational chemistry packages. The very same pigments, dyes, and fluorophores have dual uses in paints and as molecular reporter groups in the proposed studies. Thus, the participating labs are well positioned to provide the initial research setting along this learning pathway for local students who take an interest in the molecular basis of color. The results of this research in stem cell biomaterial engineering will be broadly disseminated via oral presentations at conferences, and in regular student research poster symposia at CCNY.
