Project Summary/Abstract: During the process of fabricating an engineered tissue construct, it is critical to be able to assess the initial cell seeding and subsequent cell functions in real time. This TR&D will focus on the development of biosensor tech- nology that will leverage the widespread use of scaffolds in tissue engineering to produce sensor-enabled scaf- folds in different materials and configurations to serve our CPs/SPs. Our key innovation is to incorporate sensing functions into the scaffolds that can be used to monitor multiple specific biological properties dynamically and longitudinally, thus enabling the potential to control and modify cell seeding, nutrient exchange, metabolic waste removal and differentiation cues in real-time. We will accomplish this in two Specific Aims: Specific Aim 1: Generation of sensing scaffolds to monitor and automate cellular seeding. During the automated fabrication process of a TE construct, it is critical to be able to assess cell attachment in real-time in order to validate the seeding process or take remedial action if called for. Because different scaffold biomaterials and syntheses provide different microenvironmental cues for cells, assessing the attachment and distribution of cells within the scaffold during TEMP assembly can help optimize the process. We will design beacon-type aptamers that bind and react to cell whole cells or alternately to cell surface molecules and are therefore usable as short- term sensors in this application. The aptamers will have a molecular beacon-type of reporter chemistry that will be activated by conformational changes upon binding their target cell and will provide a fluorescent feedback signal proportional to the number of cells seeded to the scaffold. Our aptamer sensor that can be attached to or integrated into scaffolds/biomaterials/bioinks. Variations of the technology will include optic light guide aug- mented scaffolds to assess the interior of thick structures. Specific Aim 2: Sensor platforms to measure O2, glucose and lactate. O2, glucose and lactate are 3 key markers of metabolic activity. Monitoring of these is essential during differentiation and/or maturation of tissues to deter- mine both their functionality and biochemical quality, and to, again, provide signals for remedial action if needed. We will develop O2, glucose, and lactate optodes using novel chemistries, and integrate them with scaffolds in a variety of application-specific ways to measure levels of O2, glucose, and lactate in the tissue interior, at the tissue surface and eventually in the bulk medium. We will also develop a system to measure oxygen uptake rate at the surface of the engineered construct. The sensors will use biocompatible materials and will be integrated into the scaffolds or reversibly attached to them using novel micro-anchors. The sensors will be tested for stability during scaffold storage, and for biocompatibility and stability during tissue differentiation an...