Comparative Evaluation of the Cytotoxic and Angiogenic Effects of Minocycline and Clindamycin: An In Vitro Study

Abstract

Introduction: This study aimed to compare the cytocompatibility and angiogenic potential of 2 antibiotics (clindamycin [CLIN] and minocycline [MINO]) at distinct concentrations on dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs).

Methods: DPSCs and HUVECs were exposed to cell culture media modified with CLIN or MINO at concentrations ranging from 30 μg/mL-1000 μg/mL. Cell toxicity and proliferation were investigated using the lactate dehydrogenase and tetrazolium reduction assays, respectively. A capillarylike tube formation in vitro assay was conducted to determine the angiogenic potential associated with each antibiotic. Additionally, selected morphometric angiogenesis parameters were determined using dedicated software (WimTube; Onimagin Technologies SCA, Córdoba, Spain). All statistical analyses were performed using 1-way analysis of variance and the Tukey post hoc test (α= .05).

Results: The collected data showed that compared with the control (cell culture media, alpha-minimum essential medium Eagle) increasing the antibiotic concentration significantly decreased cell viability and proliferation of both DPSCs and HUVECs. In terms of angiogenic potential, when tested at 30 μg/mL and 50 μg/mL, CLIN significantly amplified tube formation when compared with MINO with angiogenesis parameters (ie, tube length and tube number) similar to the effect promoted by exogenous vascular endothelial growth factor (50 ng/mL).

Conclusions: CLIN was less cytotoxic when compared with MINO at higher concentrations. Of note, CLIN did not hinder the proangiogenic activity induced by vascular endothelial growth factor to the same extent as MINO, suggesting that the replacement of MINO by CLIN might translate into positive implications in the overall regenerative outcome.

Keywords: Angiogenesis; clindamycin; disinfection; minocycline; pulp; regeneration; stem cells.

Figure 1.

Cell proliferation for both cell types after incubation with different antibiotics’ concentrations. A) HUVEC and B) DPSC. MINO and CLIN at 250 μg/ml showed a decrease in cell proliferation for HUVEC; whereas, DPSC showed decreased proliferation at 500 μg/ml. The same letters indicate a nonsignificant difference compared with the results on the same day.

Figure 2.

Cell toxicity measured by LDH release for both cell types after incubation with different antibiotics’ concentrations. A) HUVEC and B) DPSC. MINO and CLIN showed approximately a 50% toxicity at 100 μg/mL for HUVEC; whereas, for DPSC, it was 500 μg/mL. The same letters indicate a non-significant difference compared with the results on different day.

Figure 3.

Brightfield and fluorescence images of capillary-like tube formation. Prior to the start of the assay, the endothelial cells were treated with calcein AM to visualize the cells using fluorescence microscopy. Increasing concentrations of antibiotics resulted in decreases in the capillary-like network structure. However, for MINO, even at lower concentrations, cells showed apoptosis, and tubes detach from the matrix and break apart (bar = 200 μm).

Figure 4.

(A) Morphological parameters of angiogenesis estimated by processing the binary skeleton using WimTubeTM angiogenesis analysis. (B-F) The quantitative evaluation of morphological features of a capillary-like network structure after treating HUVECs with different concentrations of antibiotics were calculated in 4 different images using WimTubeTM angiogenesis image analysis. (B) Total tube length relative to the control (C) Total number of tubes (D) Total branching point (E) Total number of loops (F) Relationship of cell proliferation and angiogenesis.