Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model.

Abstract

The therapeutic success of periodontal regenerative therapy may be compromised by our limited understanding of the wound healing process. Wound healing requires the coordination of complex cellular and molecular interactions. Recently, using an in vitro wound model, our laboratory has shown that gingival fibroblasts (GF) fill an in vitro wound more rapidly than periodontal ligament cells (PDL). This suggests that there may be differences in the levels of proliferation for these 2 cell types during the wound healing process. Such specific cell type differences may be significant in clinical outcomes of regenerative therapy. Therefore, the aim of this research was to characterize and compare the levels of both proliferation and cellular wound fill between GF and PDL using our in vitro wound model. Primary cultures of human PDL and GF cells were established from explanted tissue, and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, or 9 days in media containing either 0.1% or 10% fetal bovine serum (FBS). At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU), fixed, and nuclei stained to measure DNA synthesis (as a measure for proliferation). Cells were counter stained with cytoplasmic stain to measure cell number. Quantitative analysis distant from (area of interest [AOI 1]), next to (AOI 2), and within the wound boundaries (AOI 3 and 4) was accomplished using computer-assisted histomorphometry. The levels of proliferation and cellular fill for each cell type were assessed relative to time and AOI. Overall, the PDL displayed greater (P <0.01) levels of proliferation than the GF. For both cell types, proliferation was found to be significantly (P<0.001) greater at day 2 compared to other time points. PDL displayed greater levels of proliferation than GF in all AOI, with this difference reaching significance (P<0.02) within the cell layer (AOI 1 and 2). When comparing levels of cellular fill in 10% FBS, GF displayed greater wound fill than the PDL. This difference was significant at day 6 (P <0.05) for both the marginal (AOI 3) and central (AOI 4) portions of the wound. These findings, demonstrating unique differences between PDL and GF with respect to proliferation and wound fill in an in vitro model, suggest that there may be cell-specific differences in cellular activity critical to periodontal wound healing. In addition, the results of this study show that the cellular proliferation response may not accurately reflect the overall wound healing response.