Multi-layered periodontal pocket epithelium reconstituted in vitro: histology and cytokeratin profiles.

Abstract

In order to study inter-individual differences in bacterial adhesion/invasion of periodontal tissues, an in vitro model for culturing multi-layered pocket epithelium without feeder layers or stromal equivalents (including the evaluation of their cytokeratin profiles) was developed. Pocket epithelium was collected and grown until confluent in Falcon flasks using keratinocyte-serum free medium (KSFM), without a feeder layer. In the second passage, oral keratinocytes were re-grown in a 2 compartment system using either a clear polyester (transwell-clear [TCL]) or a collagen (transwell-col [TCO]) membrane as culture surface. After the first week, the calcium concentration was raised to 1.2 mM and in half the wells, the KSFM was supplemented with 10% fetal calf serum (FCS). Histology and immunohistochemistry were performed after 1, 2, and 3 weeks of additional growth. In general, all conditions resulted in a structured epithelium consisting of 3 to 5 layers, but important differences were observed between the membrane types and between the media. CK4 was rarely and only lightly expressed while CK18 and 19 (characteristic of junctional epithelium) were very strongly expressed in the older (2 and 3 weeks) cultures. CK13 and 14 (characteristic of any stratifiable epithelial cell) also tended to increase over time; CK13 seemed to be stronger in KSFM with FCS while the contrary was true for CK14. The multi-layer created by the combination TCL/KSFM + 10% FCS resembled a junctional epithelium most, while that grown on TCO without FCS mimicked the sulcular epithelium. It seems possible to create a histiotypic culture resembling either periodontal pocket or junctional epithelium without the use of stromal equivalents or feeder layers which make this approach more cumbersome. This multi-layered culture offers a model to investigate the permeability of pocket epithelium and the adhesion and penetration of bacteria under well-defined environmental conditions.