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

Background: 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 i nteractions. Recently, using an in vitro wound model, our laboratory has sh own that gingival fibroblasts (GF) fill an in vitro wound more rapidly than periodontal ligament cells (PDL). This suggests that there may be differen ces 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 resea rch was to characterize and compare the levels of both proliferation and ce llular wound fill between GF and PDL using our in vitro wound model. Methods: Primary cultures of human PDL and GF cells were established from e xplanted tissue, and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds we re 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 da ys 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 (A OI 2), and within the wound boundaries (AOI 3 and 4) was accomplished using computer-assisted histomorphometry. Results: The levels of proliferation and cellular fill for each cell type w ere assessed relative to time and AOI. Overall, the PDL displayed greater ( P <0.01) levels of proliferation than the GF For both cell types, prolifera tion was found to be significantly (P<0.001) greater at day 2 compared to o ther time points. PDL displayed greater levels of proliferation than GF in all AOI, with this difference reaching significance (P<0.02) within the cel l layer (AOI 1 and 2). When comparing levels of cellular fill in 10% FBS, G F displayed greater wound fill than the PDL. This difference was significan t at day 6 (P <0.05) for both the marginal (AOI 3) and central (AOI 4) port ions of the wound. Conclusions: These findings, demonstrating unique differences between PDL a nd GF with respect to proliferation and wound fill in an in vitro model, su ggest that there may be cell-specific differences in cellular activity crit ical to periodontal wound healing. In addition, the results of this study s how that the cellular proliferation response may not accurately reflect the overall wound healing response.