Image analysis of remesothelialization following chemical wounding of cultured human peritoneal mesothelial cells: the role of hyaluronan synthesis.

BACKGROUND

To understand what happens during the wound healing process of the mesothelium, we have developed an in vitro wounding model of cultured human peritoneal mesothelial cells (HPMCs) utilizing an image acquisition and analysis system. Using this system, cell mobility and hyaluronan synthesis were quantified and their interrelationship discussed.

METHODS

1N NaOH was used to create circular wounds in cultured HPMC monolayers, which were then exposed for 30 minutes to the peritoneal dialysis solutions or fetal calf serum (FCS)-free M199 culture medium, followed by incubation with 0.3% FCS/M199 culture medium for up to 96 hours. Digitalized microscopic date was captured every 30 minutes to quantify the wound healing process. In separate experiments, the HPMC monolayers were stained with biotin-conjugated hyaluronan-binding protein (B-HABP) at a regular time interval.

RESULTS

Centripetal migration of the HPMCs into the wound area was the predominant process involved in wound repair with proliferation playing a secondary role. Two noticeable observations were made from the digital video movies: (1) cell mobility varied and was dependent upon the morphology and location of the cell relative to the wound edge, and (2) cell migration continued even after wound closure. Staining for B-HABP was confined to the remesothelialized area when wound closure was complete at 24 hours. At 48 hours after wound closure, the stained area was even more visible, although somewhat diffuse; thereafter, staining was reduced to almost background levels.

CONCLUSION

The cell culture model of wound healing used in our study has enabled us to demonstrate quantitative image data of the cellular processes that occur during wound healing. We have been able to continuously observe cell migration, proliferation, and transformation. Synthesis and subsequent decomposition of hyaluronan appears to be related to the mobility of the wounded and intact HPMCs in this model system.