IJUP08 - Universidade do Porto

Tetracyclines modulation of the osteogenic differentiation – in
vitro evaluation
I. Côrte-Real 1 , A. L. Marinho 1 , P. S. Gomes 1 , J. Rodrigues 1 , M. H. Fernandes 1
1 Laboratório de Farmacologia e Biocompatibilidade Celular, Faculty of Dental Medicine,
University of Porto, Portugal.
Tetracyclines are broad-spectrum antimicrobial agents that are characterized as modulators
of several biological effects in cellular mechanisms, unrelated to the inhibition of
ribosomal protein synthesis – their principal antibacterial mechanism of action [1]. High
chelation activity with cations may account for several of the non-antimicrobial properties,
at the same time that accounts for the high affinity to mineralized tissues, namely teeth and
bone [2]. Although the established affinity, little is known about the direct or indirect
modulation of bone metabolism by tetracyclines.
The aim of this work was to evaluate the proliferation and differentiation events of
osteoblastic-induced human bone marrow cells, in the presence of therapeutic
concentrations of doxycycline or minocycline (semi-synthetic tetracyclines).
Human osteoblastic cells were grown for 35 days, in the presence of doxycycline (1-25
µg/ml) or minocycline (1-50 µg/ml), on the surface of standard tissue culture plates. Cell
cultures were evaluated for proliferation and differentiation events.
Both pharmacological agents, at low concentrations (1�µg/ml), reported an increased cell
proliferation. After an initial lag phase, the increased cell growth was verified in
association with expression of high levels of alkaline phosphatase and a proportional
increased amount of mineralized extracellular matrix. Exposure to 5 µg/ml of doxycycline
induced similar results, while the same concentration of minocycline impaired osteoblastic
function. Higher levels of both tetracyclines greatly impaired cell proliferation and
function in a dose-dependent way.
Currently, characterization of tetracyclines’ influence during the in vitro osteogenic
development is being evaluated by flow cytometric analysis of the stromal precursor cell
marker STRO-1 and alkaline phosphatase expression, by osteoblastic-derived human bone
marrow cells. This double staining allows to differentiate between four STRO-1/alkaline
phosphatase subsets, which correspond to specific osteogenic differentiation stages.
Overall, the treatment of human osteoblastic cells with low concentrations of doxycycline
or minocycline induced cell proliferation without impairing functional activity, analyzed
by alkaline phosphatase activity and extent of matrix mineralization. Its therapeutic usage
might combine a targeted antimicrobial activity into the bone tissue with the induction of
osteoblastic proliferation, without interfering with cell-specific biological activity. Flow
cytometry analysis will contribute to evaluate tetracyclines influence in cell behavior, at
different stages of the osteogenic differentiation.
References:
[1] Nelson, M. (1998), Chemical and Biological Dynamics of Tetracyclines. Adv Dent Res, 12, 5-
11.
[2] White, J., Pearce, F. (1982), Characterization of chlortetracycline as a calcium ionophore.
Biochemistry, 21, 6309-6312.
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