In the present study, we explored the effects of lopinavir/ritonavir on gingival epithelium growth and differentiation as determined from the expression patterns
of key proliferation and differentiation markers. Gingival keratinocytes were isolated from human gingival tissue as previously described . Briefly, a mixed pool of gingival tissues was obtained from patients undergoing dental surgery. To maintain confidentiality, gingival samples were devoid of any identification such as name, race, age and religion. Approval to collect patient samples was obtained from the Penn State University College of Medicine Institutional Review Board (IRB# 25284). The connective tissue and dermis were removed from the epithelium and discarded. The epithelial tissue was washed three
selleck chemicals times with phosphate-buffered saline (PBS) containing 50 μg/mL gentamycin sulphate (Gibco BRL, Bethesda, MD, USA) and 1X nystatin (Sigma Chemical Co., St Louis, MO, USA). The epithelial Fulvestrant clinical trial tissue was then minced with scissors and trypsinized into a single-cell suspension in a spinner flask. The suspension was removed, 20 mL of E medium containing 5% fetal calf serum (FCS) was added and cells were pelleted by centrifugation. The supernatant was aspirated and the cell pellet was resuspended in 1 mL of 154 medium (Cascade Biologics Inc., Portland, OR, USA) supplemented with the Human Keratinocyte Growth Supplement Kit (Cascade Biologics, Inc.) and then added to a 10-cm tissue culture plate containing an additional 7 mL of 154 medium. This process was repeated three times. When cultures became ≈70% confluent they were split 1:3; when the plates of the
first passage were more than 70% confluent, the cells were used for growing raft cultures. Raft cultures were grown as previously described [20–22]. Briefly, human gingival epithelial keratinocytes were seeded onto collagen matrices containing J2 3T3 mouse fibroblast feeders. When the epithelial keratinocytes were attached to the dermal equivalent, the collagen matrices were lifted onto stainless-steel grids at the air–liquid interface. The raft cultures were fed by diffusion from below with E medium supplemented with lopinavir/ritonavir. We chose the peak concentration of Anidulafungin (LY303366) this drug in blood serum (Cmax) as our baseline concentration plus two lower and two higher concentrations for our treatments. Earlier studies showed that the drug level is almost the same in blood serum and in saliva [23–25]. We also assumed that the blood level of lopinavir/ritonavir would be the same as in the saliva. The Cmax of lopinavir/ritonavir is 9.8±3.7 μg/mL . In the first set of experiments, the rafts were treated from the first day with a range of lopinavir/ritonavir concentrations: 3, 6, 9.8, 13.5 and 16 μg/mL. Vehicle control rafts were fed with E medium containing an equivalent volume of 70% ethanol.