The effect of recombinant human bone morphogenetic protein-4 on the osteoblastic differentiation of mouse calvarial cells affected by Porphyromonas gingivalis.
BACKGROUND
A number of studies have shown effective bone regeneration induced by bone morphogenetic proteins (BMPs), but it is not clear whether the presence of periodontopathic bacteria has any significant modulation effect on the bone regeneration ability of BMPs. The present study examined whether pretreatment of mouse calvarial cells with Porphyromonas gingivalis extracts can make a difference in their osteoblastic differentiation exerted by recombinant human bone morphogenetic protein-4 (rhBMP-4).


METHODS
Primary mouse calvarial osteoblastic (MCO) cells were cultured until they reached confluence. At confluence, cells were untreated or pretreated with 1 microgram/ml of sonicated P gingivalis extracts (SPEs) for 2 days. After washing, the cells were further incubated in the presence of rhBMP-4 (0 to 100 ng/ml) for 3 days. At the end of the treatment, the cells were harvested and lysed for measurement of the alkaline phosphatase (ALP) activity. Total RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) analysis for expression of ALP mRNA was conducted. The amount of prostaglandin E2 (PGE2) secreted into the culture supernatant was determined using an enzyme immunoassay.


RESULTS
The stimulatory effect of rhBMP-4 on ALP activity was observed in both untreated MCO cells and in cells pretreated with 1 microgram/ml of SPEs in a dose-dependent manner. The ALP activities were significantly reduced in the cells pretreated with SPEs at all concentrations of rhBMP-4 used in the study when compared to untreated cells. Similar results were obtained in the RT-PCR analysis for ALP mRNA. Cells pretreated with SPEs released significantly larger amounts of PGE2 than untreated cells, but the treatment with 100 ng/ml of rhBMP-4 had no significant effect on the amount of PGE2 released. These results suggest that the stimulatory effect of rhBMP-4 on osteoblastic differentiation might be significantly reduced by P gingivalis, possibly through the endogenous PGE2 pathway, but rhBMP-4 still has a stimulatory effect on osteoblastic differentiation of mouse calvarial cells affected by P gingivalis.


CONCLUSIONS
Our results suggest that supplemental BMPs would be beneficial for improved treatment of osseous defects, although their biologic effect might be significantly reduced by periodontopathic bacteria.
Kim
Chang-Sung
CS
Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, Korea.
Choi
Seong-Ho
SH
Choi
Bong-Kyu
BK
Chai
Jung-kiu
JK
Park
Joon-Bong
JB
Kim
Chong-Kwan
CK
Cho
Kyoo-Sung
KS
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000704:Analysis of Variance; D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D008813:Mice, Inbred ICR; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000382:microbiology*; D010012:Osteogenesis / Q000187:drug effects*; D016966:Porphyromonas gingivalis / Q000472:pathogenicity*; D012313:RNA / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013010:Sonication; D018709:Statistics, Nonparametric
C525809:BMP4 protein, human; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D012313:RNA; D000469:Alkaline Phosphatase; D015232:Dinoprostone