BIOMIMETIC TISSUES--ORAL MUCOSACONJUNCTIVA, AND SKIN

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Grafting skin into the oral cavity is commonly used for vestibular extension procedures, repair of trauma induced defects, and to reconstruct large oral mucosal defects resulting from excision of malignant tumors. However, skin grafted into the oral cavity maintains its original structure and function, and often patients experience hair growth and sweating from the grafted skins. Ideally, oral mucosal tissues would be used for such grafts, but this is limited by a lack of sufficient donor tissue. Like the tissues in the oral cavity, the conjunctiva is a mucosal tissue that lines the eye and the eyelids. It is comprised of a non-keratinizing stratified squamous epithelium, similar to the buccal mucosa. Two conditions produce diffuse conjunctival scarring, which result in painful visual loss: ocular cicatricial pemphigoid and alkali burns of the ocular surface. There is currently no satisfactory treatment for either condition. It would highly desirable if both the oral mucosa and the conjunctiva could be replaced with bioengineered substitutes. The problem arises when trying to build replacements for tissues that are continuously renewing, such as the epithelia of the oral mucosa and conjunctiva. All continuously renewing epithelial tissues contain stem cells, which are the undifferentiated cells capable of self-renewal and responsible for the maintenance of the differentiating cell population, which makes up the epithelial tissue architecture. In stratified squamous epithelia, stem cells reside in the proliferative basal cell compartment, and it has been generally accepted that they divide less frequently than the rest of the proliferative cell population and that they repopulate the epithelia after damage. Thus, it is critical when bioengineering epithelial tissues that we include the appropriate epithelial stem cells in the initial phase of making the tissues. Unfortunately, to date, isolating epithelial stem cells has been problematic. Previously, using tritiated thymidine, we were able to label a subpopulation of slowly- cycling basal cells in mouse skin and oral mucosa that we called label- retaining cells (LRCs), which showed many of the characteristics of stem cells. This tagging method has been used for the last decade to identify stem cells in several other epithelia, including the limbs of the eye. Recently, we have devised a method that enriches the epidermal stem cell population from 2 percent to over 50 percent, and further that these selected cells show the growth and repopulation potential expected for epidermal stem cells. In this study, we propose to use our novel enrichment method to select stem cells from oral mucosal and limbal epithelia, to determine that these cells show the clonogenic properties of stem cells in vitro, and they can be used to bioengineer tissues for use as long term replacements, and to determine whether stem cells respond to intrinsic or extrinsic factors when maintaining specific tissue junctions.