Epithelial tissues, both cutaneous and mucosal, provide underlying tissues with protection from the environment. It is particularly important in the oral cavity, where masticatory functions increase damage, that the epithelial lining is intact and injuries are quickly repaired, in order to prevent micro-organisms and toxic material from entering the underlying Barasertib in vivo tissues. Epithelial cells undergo a complicated, well-defined programme of differentiation that allows the expression of structural proteins designed to preserve the integrity and

function of these tissues [15]. Damage cannot be completely avoided in an environment such as the oral cavity, and epithelial turnover rates in the oral cavity are second only to those of the small intestine [16]. Typically, this allows a rapid wound healing response of compromised tissue. It is possible that changes in the turnover rate and wound healing abilities of the oral epithelium in response to HAART may affect the occurrence of oral disease. The epithelium is predominantly comprised of cytokeratins. The expression of hypoxia-inducible factor cancer cytokeratins depends on the type of tissue, its proliferation and differentiation state and pathological

conditions [17, 18]. In short, examining the cytokeratin profile of a tissue provides a snapshot of the proliferation and differentiation state of that tissue. The effect of ZDV on the oral epithelium is currently unknown. In the present study, the organotypic (raft) tissue culture model system derived from primary gingival cells was used to examine, for the first time, the effect of ZDV on gingival epithelium growth, and the expression patterns of differentiation and proliferation markers. Primary gingival keratinocytes were isolated from a mixed pool of tissues obtained from patients undergoing dental surgery in accordance with Penn State University College of Medicine Institutional Review Board (IRB #25284) procedures. The tissue was washed three times in phosphate-buffered saline (PBS) containing 50 μg/mL

gentamycin sulfate (Gibco BRL, Bethesda, MD) and 1× nystatin (Sigma Chemical Co., St Louis, MO) The connective tissue and dermis were removed, leaving the epithelium. DNA ligase The epithelial tissue was then minced with a scalpel and trypsinized in a sterile glass universal container with a stir bar containing 25 mL of 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco BRL). The sample was stirred on a magnetic stirrer at 37°C and incubated for 45 min. The supernatant was removed and neutralized with 25 mL of E-medium plus 5% fetal bovine serum (FBS) [19], and cells were pelleted by centrifugation. The supernatant was removed and the cell pellet was re-suspended in 10 mL of 154 medium (Cascade Biologics, Inc., Portland, OR) and then added to a 100-cm2 tissue culture plate. The procedure was repeated an additional two times.