This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The central hypothesis of this proposal is that living skin equivalents (LSE) consisting of living cells may require an activation process in vitro to optimize their in vivo effects and accelerate healing in difficult clinical situations.
Specific Aims : 1) To determine the ideal conditions for in vitro activation of a living bilayered living skin equivalent (LSE)2) To use the optimally stimulated construct in difficult to heal human wounds3) To characterize novel genes that are expressed by injury to the LSE and for application to human woundsAt the completion of these studies, we will have produced and validated the best way to optimize the use of a living bilayered skin equivalent for use in human wounds. This knowledge could be applicable to other types of tissue engineering constructs. The overall goal of this pilot application is to explore a shift in paradigm with regard to the use of living skin equivalents (LSE). The underlying hypothesis is that it is possible to stimulate constructs and render them more effective.
Specific aim 1 will identify optimal ways to stimulate bioengineered skin constructs, using novel and established methods we have established in our laboratory. The focus of that specific aim will be an already available living skn equivalent (LSC). Data generated from specific aim 1 will be used in testing the use of stimulated LSC in a clinical trial of difficult to heal wounds, making use of our GMP facility to properly prepare the LSC. Assessment of efficacy and healing will be done by measurements of the healing rate from the edge of the wound, as well as size reduction and complete wound closure. As part of specific aim 3, we will also perform a thorough gene analysis of LSC during the process of epiboly. We regard the epiboly model as a potentially very useful tool for identifying genes of interest in the migrating keratinocyte population. Here too, the experimental design is dynamic. We can block the epiboly by various methods, including the antibodies to vitronectin and its integrin receptor, or even explore ways to render the construct unable to 'heal'. Radiation injury can be explored, for example. In published work, we have found ways to make the construct dormant and unable to reepithelialize. RNA isolated from the BSC will be used for affymetrix gene chip analysis, capable of detecting 33,000 genes. The COBRE has already purchaced a gene microarray scanner that will facilitate our studies. The hope is that we can determine specific genes that are either upregulated or downregulated in this dynamic keratinocyte migration (or lack thereof) model. These data have definite mechanistic significance. Ideally, one could use the information for developing a putative keratinocyte gene signature that could later be applied to and explored in human wounds. All of the proposed aims and experiments are within our capabilities, as we have developed a team that can move easily from bench work to animal experimentation, to human wounds. We believe this is a major strength of this pilot proposal. Another major strength is the underlying hypothesis. Many complex wounds either do not respond to treatment at all, or with great delay. Given the expenses associated with these new technologies, it is imperative that the wound bed be optimized. The PI described the problem of wound bed preparation in a previous report. This notion, which basically is meant to underline and stress the need to improve wound care before advanced therapies are used, has received considerable success. It is our hypothesis that we now need to explore a way to stimulate LSC to optimize their success in chronic wounds. We believe that in the future this could become an essential step in the use of tissue engineering treatment of complex wounds or other situations where the wound microenvironment is severely compromised. enroll 6 volunteers to have biopsies of their thighs as an additional control. The protocol for those biopsies will be the same as described for the patients.
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