Response of MG63 osteoblasts on bacterial challenge is dependent on the state of differentiation.

Abstract

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.