Growth factors regulate expression of mineral associated genes in cementoblasts

Abstract

Background: Knowledge of the responsiveness of cells within the periodontal region to specific bioactive agents is important for improving regenerative therapies. The aim of this study was to determine the effect of specific growth factors, insulin-like growth factor-I (IGF-I), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) on cementoblasts in vitro and ex vivo.

Methods: Osteocalcin (OC) promoter driven SV40 transgenic mice were used to obtain immortalized cementoblasts. Growth factor effects on DNA synthesis were assayed by [3H]-thymidine incorporation. Northern analysis was used to determine the effects of growth factors on gene expression profile. Effects of growth factors on cementoblast induced biomineralization were determined in vitro (von Kossa stain) and ex vivo (re-implantation of cells in immunodeficient (SCID) mice).

Results: All growth factors stimulated DNA synthesis compared to control. Twenty-four hour exposure of cells to PDGF-BB or TGF-beta resulted in a decrease in bone sialoprotein (BSP) and osteocalcin (OCN) mRNAs while PDGF-BB also increased osteopontin (OPN) mRNA. Cells exposed to IGF-I for 24 hours exhibited decreased transcripts for OCN and OPN with an upregulation of BSP mRNA noted at 72 hours. In vitro mineralization was inhibited by continuous application of PDGF-BB or TGF-beta, while cells exposed to these factors prior to implantation into SCID mice still promoted biomineralization.

Conclusions: These data indicate IGF-I, PDGF-BB, and TGF-beta influence mitogenesis, phenotypic gene expression profile, and biomineralization potential of cementoblasts suggesting that such factors alone or in combination with other agents may provide trigger factors required for regenerating periodontal tissues.

Figures

Figure 1

Effect of growth factors on DNA synthesis measured by [3H]-thymidine incorporation. Each bar represents the mean ± SD. Results from a representative experiment of 3 repetitions are shown. * P <0.001: 10% FBS and PDGF-BB>TGF-β and IGF-I and 0% FBS; † P <0.001:TGF-β & IGF-I > 0% FBS.

Figure 2

Gene expression analysis. Total RNA was isolated and subjected to Northern analysis. Genes probed: OPN, osteopontin; BSP, bone sialoprotein; OCN, osteocalcin; and GAPDH, “housekeeping” gene to normalize loading of mRNA. Factors: IGF-I, insulin-like growth factor-I; PDGF-BB, platelet derived growth factor-BB;TGF-β, transforming growth factor-β; 2% w/AA: cells incubated with DMEM containing 2% FBS, ascorbic acid and β-glycerophosphate (positive control). A. 24-hour exposure to growth factors; B. 72-hour exposure to growth factors; C. normalization of OPN, BSP, and OCN mRNA to GAPDH mRNA at 24 and 72 hours post-treatment. Results from a representative experiment of 3 repetitions are shown.

Figure 3

Eight-day von Kossa mineralization assay. Cells were exposed to DMEM containing 2% FBS plus 50 μg/ml ascorbic acid and PDGF-BB platelet derived growth factor-BB, 30 ng/ml, or TGF-β transforming growth factor-β, 4 ng/ml, either in a continuous or pulse fashion. As a positive control (2% w/AA;(+)CN), cells were exposed to DMEM containing 2% FBS plus 50 μg/ml ascorbic acid (AA). Note positive black staining indicative of mineral nodule formation. As a negative control, AA was omitted (2%(-)CN). Note absence of stain. A. Continuous treatment: Note that cells exposed to either PDGF or TGF-β were not capable of promoting mineral nodule formation. B. Pulse application: Note decreased mineral nodule formation. Results from a representative experiment of 3 repetitions are shown, where, in each experiment triplicate wells were used for each growth factor.

Figure 4

Biomineralization by cementoblasts ex vivo. Histological sections obtained from implantation of cells and HA/TCP in immunodeficient SCID mice (original magnification, ×10). A. material harvested after 6 weeks post-implantation. No mineral formation observed. The scaffold and granular structure of the material remained. B. OC-CM 30 cells grown in DMEM containing 10% FBS and implanted within HA/TCP, harvested 1 week post-implantation. No evidence of mineralization was observed. C. OC-CM 30 cells grown in DMEM containing 10% FBS and seeded in HA/TCP, harvested 3 weeks post-implantation. No evidence of mineralization was observed. D. OC-CM 30 cells grown in DMEM containing 10% FBS and implanted within HA/TCP, harvested 6 weeks post-implantation (positive control). Extensive biomineralization was observed with a woven-type pattern to the mineralized tissue E and F. OC-CM 30 cells treated with PDGF-BB for 24 or 72 hours, implanted within HA/TCP, harvested 6 weeks post-implantation. Biomineralization was observed throughout the graft and particles, with “woven type” mineralization. Note abundance of blood vessels throughout the mineralized matrix, especially in the 72-hour specimen (F). G and H. OC-CM 30 cells treated with TGF-β for 24 or 72 hours and implanted within HA/TCP, harvested 6 weeks post-implantation. Biomineralization was observed throughout the graft and particles, with a pattern similar to OC-CM 30 group in 10% FBS.