Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: Requirement of endogenous acetylcholine for outgrowth initiation

To better understand the mechanisms of skin re-epithelization, we developed a simple technique that assays the outgrowth of human keratinocytes. Second-passage foreskin keratinocytes were inoculated at high cell density into 3-mm wells cut from agarose gels in standard 6-well tissue culture dishes. The cells settled on the dish bottom and formed a confluent colony. The cells at the periphery of the colony flattened, spread their cytoplasm, and moved away over the dish surface under the agarose gel. The morphology of migrating keratinocytes was observed microscopically through the transparent agarose, and the migration distance was measured after the gels were removed and after cells were fixed and stained. To determine which cell activities were involved in the outgrowth, the effects of cholinergic compounds on keratinocyte outgrowth were compared with their effects on keratinocyte proliferation, cell-plastic attachment, and spreading measured in separate sets of experiments. Outgrowth was inhibited by the specific inhibitor of acetylcholine synthesis bromoacetylcholine (0.05 mM) and restored by 5 mM exogenous acetylcholine. The irreversible muscarinic antagonist propylbenzilylcholine mustard (0.05 mM) abolished the restorative effects of exogenous acetylcholine, and also inhibited outgrowth of intact keratinocytes. In keratinocyte cell cultures, bromoacetylcholine stopped cell division. Propylbenzilylcholine mustard increased cell number, but interfered with cell-plastic attachment and spreading. This suggests that cell-matrix attachment, spreading, and locomotion of human keratinocytes, but not mitosis, mediate the earliest stages of skin re-epithelization, and that endogenous acetylcholine regulates these keratinocyte functions. Specifically, keratinocyte acetylcholine is required to initiate outgrowth.