Kim Chang-Sung, Choi Seong-Ho, Choi Bong-Kyu, Chai Jung-kiu, Park Joon-Bong, Kim Chong-Kwan, Cho Kyoo-Sung
Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, Korea.
J Periodontol. 2002 Oct;73(10):1126-32. doi: 10.1902/jop.2002.73.10.1126.
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).
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.
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.
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.
多项研究表明骨形态发生蛋白（BMPs）可诱导有效的骨再生，但尚不清楚牙周病原菌的存在是否对BMPs的骨再生能力有显著的调节作用。本研究检测了用牙龈卟啉单胞菌提取物预处理小鼠颅骨细胞是否会对重组人骨形态发生蛋白-4（rhBMP-4）诱导的成骨细胞分化产生影响。
原代小鼠颅骨成骨细胞（MCO）培养至汇合。汇合时，细胞不做处理或用1微克/毫升的超声破碎牙龈卟啉单胞菌提取物（SPEs）预处理2天。洗涤后，细胞在rhBMP-4（0至100纳克/毫升）存在的情况下进一步孵育3天。处理结束时，收获细胞并裂解以测量碱性磷酸酶（ALP）活性。提取总RNA，并进行逆转录-聚合酶链反应（RT-PCR）分析以检测ALP mRNA的表达。使用酶免疫测定法测定分泌到培养上清液中的前列腺素E2（PGE2）的量。
在未处理的MCO细胞和用1微克/毫升SPEs预处理的细胞中均观察到rhBMP-4对ALP活性的刺激作用，且呈剂量依赖性。与未处理的细胞相比，在本研究中使用的所有rhBMP-4浓度下，用SPEs预处理的细胞中的ALP活性均显著降低。在ALP mRNA的RT-PCR分析中也获得了类似的结果。用SPEs预处理的细胞释放的PGE2量明显多于未处理的细胞，但用100纳克/毫升的rhBMP-4处理对释放的PGE2量没有显著影响。这些结果表明，牙龈卟啉单胞菌可能通过内源性PGE2途径显著降低rhBMP-4对成骨细胞分化的刺激作用，但rhBMP-4对受牙龈卟啉单胞菌影响的小鼠颅骨细胞的成骨细胞分化仍有刺激作用。
我们的结果表明，补充BMPs对改善骨缺损的治疗有益，尽管其生物学效应可能会被牙周病原菌显著降低。
