Department of Periodontology, Operative and Preventive Dentistry, University of Bonn, Bonn, Germany.
Department of Prosthodontics, Preclinical Education and Dental Materials Sciences, University of Bonn, Bonn, Germany.
Mol Oral Microbiol. 2018 Apr;33(2):133-142. doi: 10.1111/omi.12203. Epub 2017 Dec 26.
The present in vitro study examines molecular processes that are relevant during bone homeostasis after Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infection with a focus on the differentiation level of osteoblasts. Regenerative processes are often hindered by the recurrence of bacterial infections, which can ultimately provoke a severe destruction of bone tissue. To obtain more detailed insights into such a complex scenario, we have used undifferentiated MG63 osteoblast-like cells as an experimental paradigm to examine the impact of two oral pathogens, A. actinomycetemcomitans and P. gingivalis, on proliferation, cytotoxicity and osteogenic differentiation. Cell culture experiments were performed to analyze cellular behavior. The level of genes interfering with bone tissue integrity (matrix metalloproteinases and their tissue inhibitors) and osteogenic markers (alkaline phosphatase, Runx2, human β-defensin-2) was compared in undifferentiated versus differentiated MG63 cells using real-time polymerase chain reaction. Functional activity of matrix metalloproteinases was quantified by zymography. Western blot analysis was used to verify the phosphorylation state of mitogen-activated protein kinases p38 and extracellular-signal-regulated kinases 1/2. When co-cultured with undifferentiated MG63 cells, oral pathogens provoked distinct cellular effects. Only A. actinomycetemcomitans reduced cell proliferation, increased cell death, and induced osteogenic differentiation. A comparison of matrix metalloproteinase network stability in the presence of oral pathogens revealed a partial sensitivity towards P. gingivalis but not A. actinomycetemcomitans. So, beside the proof of concept that MG63 cells co-cultured with oral pathogens can serve as an in vitro model for mimicking destructive and regenerative events after bacterial infections, our data indicate that double infections might counterbalance otherwise positive effects.
本体外研究着眼于成骨细胞分化水平，检查了伴放线放线杆菌和牙龈卟啉单胞菌感染后骨稳态相关的分子过程。再生过程常因细菌感染的复发而受阻，这最终可能导致骨组织的严重破坏。为了更详细地了解这种复杂的情况，我们使用未分化的 MG63 成骨样细胞作为实验范例，研究了两种口腔病原体，伴放线放线杆菌和牙龈卟啉单胞菌对增殖、细胞毒性和成骨分化的影响。进行细胞培养实验以分析细胞行为。使用实时聚合酶链反应比较未分化与分化的 MG63 细胞中干扰骨组织完整性的基因（基质金属蛋白酶及其组织抑制剂）和成骨标志物（碱性磷酸酶、Runx2、人β-防御素-2）的水平。通过明胶酶谱法定量测定基质金属蛋白酶的功能活性。使用 Western blot 分析验证丝裂原活化蛋白激酶 p38 和细胞外信号调节激酶 1/2 的磷酸化状态。当与未分化的 MG63 细胞共培养时，口腔病原体引起了不同的细胞效应。只有伴放线放线杆菌降低细胞增殖、增加细胞死亡并诱导成骨分化。在存在口腔病原体的情况下比较基质金属蛋白酶网络稳定性时，发现牙龈卟啉单胞菌具有部分敏感性，但伴放线放线杆菌没有。因此，除了证明与口腔病原体共培养的 MG63 细胞可作为体外模型来模拟细菌感染后的破坏性和再生性事件之外，我们的数据还表明，双重感染可能会抵消其他积极影响。
