The effects of platelet-derived growth factor-BB on periodontal cells in an in vitro wound model.
BACKGROUND
Platelet-derived growth factor (PDGF-BB) has been shown to enhance periodontal regeneration. Principles of guided tissue regeneration dictate that one of the goals of therapy is to modulate the wound healing processes to favor repopulation of the wound with cells derived from the periodontal ligament rather than from the gingival tissues. Using an in vitro wound model, gingival fibroblasts (GF) have been shown to fill a wound space significantly faster than periodontal ligament cells (PDL). There are no data reported directly comparing the response of these 2 cell types to PDGF-BB within such a wound model. Therefore, the aims of this research were: 1) to characterize both the proliferative and wound fill (WF) effects of PDGF-BB within an in vitro model and 2) to compare specific growth factor effects between GF and PDL.


METHODS
Primary cultures of both human PDL and GF were derived from explanted tissues 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, and 9 days in media containing 0.1% fetal bovine serum (FBS) and 1 of 5 concentrations of PDGF-BB. At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU) fixed, and nuclei were stained to measure BrdU incorporation (as a measure for proliferation). Cells were counter-stained with cytoplasmic stain to measure cell number. Quantitative analyses within the wound boundaries, marginally (area of interest [AOI] 1) and centrally (AOI 2), were accomplished using computer-assisted histomorphometry.


RESULTS
PDL exhibited a significantly greater proliferative response to PDGF-BB in both AOI when compared to GF (P <0.0001). The PDL exhibited increased levels of proliferation at concentrations of PDGF-BB greater than or equal to 10 ng/ml. By contrast, GF displayed no increase in proliferation in response to stimulation with PDGF-BB at any of the concentrations tested when compared to negative controls. The wound fill (WF) responses to PDGF-BB were similar between PDL and GF, with both cell types responding in an all or none fashion when measured at day 2, and in a concentration-dependent manner at later time points. The only significant difference in WF between PDL and GF occurred in AOI 2 in negative control medium (0 ng/ml of PDGF-BB), with GFs having greater (P <0.01) levels of WF over the 9 days.


CONCLUSIONS
The findings from this study demonstrate differing effects of PDGF-BB on the proliferation of PDL and GF in this in vitro model. These results suggest that there may be cell-specific differences critical to periodontal wound healing that may be exploited in the development of new therapies.
Mumford
J H
JH
Department of Periodontics, Wilford Hall Medical Center, Lackland AFB, San Antonio, TX 78229, USA.
Carnes
D L
DL
Cochran
D L
DL
Oates
T W
TW
D000077214:Becaplermin; D001769:Blood; D001973:Bromodeoxyuridine; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D004396:Coloring Agents; D003470:Culture Media; D003593:Cytoplasm / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D010513:Periodontal Ligament / Q000166:cytology / Q000187:drug effects*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D012038:Regeneration / Q000187:drug effects; D013223:Statistics as Topic; D013997:Time Factors; D014945:Wound Healing / Q000187:drug effects
D004396:Coloring Agents; D003470:Culture Media; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin; D004247:DNA; D001973:Bromodeoxyuridine