A biopatterning technology and a design methodology will be developed that will be directly translatable to developing off-the-shelf therapies for driving differentiation and regeneration of multi-tissue units such as muscle-tendon-bone (MTB). Inkjet-based biopatterning will enable the creation of persistent, spatially- defined patterns of multiple paracrine signaling factors (PSFs) at physiologically-relevant concentrations, at sub-millimeter resolution. PSFs include growth factors and their binding proteins, proteoglycans and other extracellular matrix molecules, and proteases, all of which interact to direct cell behavior in the pericellular environment. Printed PSFs will be organized in multiple adjacent regions of a delivery matrix, with each region targeting a different phenotype to be induced. An exogenous or endogenous stem cell population exposed to these patterned constructs will be simultaneously driven toward multiple differentiative fates in register to these patterns, either in vitro in culture or in vivo within the body. As a therapy, these patterned constructs could be stored on the shelf in a dry state and then, when needed, be rehydrated and implanted either directly without cells or used as a cellular delivery scaffold for stem cell engineering. As a paradigm application, biopatterning will be demonstrated by engineering an MTB unit in vitro and in vivo. Pattern designs for an MTB will first be determined with the aid of a systematic design methodology applied to in vitro studies to identify a minimum set of spatially-patterned PSF cues out of a very large number of design possibilities, and then, these selected designs will be validated in vivo in an ecoptic subcutaneous mouse model. As an additional in vivo validation and as a demonstration of the feasibility of this technology to be translated into a clinically-relevant therapy, PSF patterned constructs will be implanted, with and without exogenous primary mouse muscle-derived stem cells MDSCs, into a mouse Achilles tendon defect model, and histologically assessed for regeneration of this MTB unit.