Regenerative inadequacy, or injury followed by inadequate regeneration of tissues, is seen after extensive battlefield injuries. After severe injury resulting in major tissue/bone loss complex tissues do not fully regenerate. The recent rise in casualties from the war in Iraq and Afghanistan has mandated changes in the medical strategies regarding the treatment of battlefield injuries. Blast injury related wounds and amputations have filled our medical hospitals with wounds that have either progressed to the point of amputation or the production of severe/debilitating scars. Our proposal offers a translational approach to understanding how wounds heal and how to develop strategies for enhancing wound healing. This involves the use of layer-by-layer assembly of factors onto inverse colloidal crystal (LBL-ICC) nanoparticles or other biomaterials containing chemotaxis, growth and differentiation factors which, after implantation at the wound site, supports the development of numerous artificial blastemas or regions of proliferation, differentiation and maturation of cells allowing for the regeneration of complex tissues such as the epidermis and dennis of skin. Since it is the combination of the intrinsic characteristics of stem cells and their microenvironment that shapes their properties and defines their potential particles engineered to act as biological sensors will be placed at specific sites within the wound and will be used to evaluate influx of microbial invaders, changes in PH, oxygen levels and potentially cytokines and growth factor levels. Human bone marrow derived mesenchymal stem cells, murine embryonic stem cells and human skin derived stem cell lines developed in our laboratory will be used in an in vitro 3D culture model to (I) evaluate the use of LBL-ICC particles to produce cytokine and chemokine gradients that influence the influx of cells and development of tissue in an in vitro 3-dimensional bioreactor and (2) Determine the appropriate mixture of growth factors, cytokines and reagents to be delivered by LBL-ICC beads which promote blastema formation and complex tissue growth in vitro. The novelty of our proposal lies in the creation of a controlled cellular milieu where the interaction of progenitor cells with mixtures of biodegradable polymers will be used to regenerate healthy, functional replacement tissue in the case of severe injury or loss of limb. The work outlined in this proposal will be used to develop procedures that will be applied to a murine wound model in future studies.