Objective   To investigate the effect of the supernatant of Actinobacillus actinomycetemcomitans (A.a) on the differentiation and gap junctional intercellular communication in osteoblasts in vitro.    Methods   Osteoblastic cells were maintained in BHI growth medium mixed with A.a culture supernatants. Neutral red was used to test cellular activity. Quantitative real-time PCR assay was used to detect the differentiation markers

including alkaline phosphatase (ALP)

osteocalcin (OCN) and bone sialoprotein (BSP)

and the immunohistochemistry for Ⅰ collagen. The change of gap junctional intercellular communication in osteoblastic cells was studied by scrape-loading and dye transfer technique (SLDT)

and the connexin43 expression was detected by Western blotting.    Results   Until concentration to 20% there was no changing on the viability of osteoblastic cells compared to BHI sample as control. SLDT showed the diffusion distance of fluorescent dye in cells cultured with A.a culture supernatants was shorter than those of control. Real-time PCR showed the mRNA expressions of ALP

BSP and OCN were decreased compared with those in control. Immunohistochemistry showed that Ⅰ collagen expression in the osteoblasts was decreased in A.a culture supernatants. The protein expression of Cx43 in sample treated with A.a culture supernatants was remarkable reduced than in sample treated with BHI.    Conclusion   A.a inhibits the differentiation and gap junctional intercellular communication in osteoblasts. 