Despite important advances within the past decade, current tissue engineered skin equivalents represent little more than a fragile, short-term dressing for patients that need viable skin replacements. The major weakness in current skin equivalents is that the constituent cells are cultured and applied under conditions that are very different from that of natural skin. It is believed that this is principally due to our limited understanding of the roles that 3-D scaffold topography and mechanical stimuli have on the intercellular organization, connectivity, and communication of engineered tissues. In this project an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy, and optical coherence elastography is utilized to uniquely visualize the structural and functional relationships of cells within 3-D engineered skin constructs, and measure the evolving biomechanical properties. Second, this project investigates and longitudinally images in 3-D the growth of engineered skin constructs on varying microtopographic substrates. This will provide fundamental insight into the mechanical influences at the dermal-epidermal junction on the keratinocytes and fibroblasts. Third, the effects of mechanical stimuli on these constructs will be investigated, defining how varying stimuli affect the 3-D cell dynamics and tissue organization over time. such stimulation effects will provide a more physiologically-relevant culture condition. Finally, this project will longitudinally image the cell and tissue responses in vivo following the grafting of the skin constructs to host pre-clinical models, contributing significantly to our understanding of how engineered tissue grafts interface with biological hosts.
The field of tissue engineering has made significant advances over the last two decades, and has developed potential solutions in many applications to improve our health and quality of life. However, many of the microscopic cellular and microstructural changes that occur during the growth and use of engineered tissues remain unknown, or poorly understood, because we lack advanced imaging and visualization tools and techniques to track the dynamic cell and tissue changes in real-time, at high resolution, in three-dimensions, and over extended time periods. This project developed an advanced microscopy system that integrated several different types of optical imaging techniques and computational algorithms to comprehensively visualize microscopic structure and function within engineered skin tissues. A new computational algorithm co-registered image information so that images acquired over many months could all be related to one another. Imaging was also performed in living tissue after the engineered skin tissues were applied to help improve wound healing and skin regeneration. The intellectual merit of this project included new optical imaging and visualization techniques, coupled with new ways to grow engineered skin, and track the changes in living tissue over time. The broader impact of this research included new ways to fabricate better, more robust, engineered skin, as well as ways to improve our success for using engineered skin grafts to heal wounds or replace diseased tissues.
