Osteogenic Effect of Pregabalin in Human Primary Mesenchymal Stem Cells, Osteoblasts, and Osteosarcoma Cells

Abstract

Seventy million patients worldwide are suffering from epilepsy. The long-term use of antiepileptic drugs causes the alteration of the bone tissue and its metabolism, thus increasing the risk of fractures. Clinical and pre-clinical studies have highlighted conflicting data on the influence of the relatively new antiepileptic drug pregabalin (Lyrica^®). The objective of the present study was therefore to investigate its cytotoxicity in primary human osteoblasts (hOB). HOB and human mesenchymal stem cells (hMSC) were isolated from patients. The human osteosarcoma cells MG63 were included as established cell line. Cells were incubated with pregabalin at concentrations ranging from 0 to 40 μg/mL. Time-dependent cell proliferation was measured by automatic cell counting, and metabolism was determined by XTT assay and osseous differentiation by alkaline phosphatase (ALP) activity. Histological examinations of calcium deposit were performed with ALP, Alizarin Red, and von Kossa staining. A concentration-dependent increase in the proliferation of hOB and hMSC was observed after treatment with pregabalin. All cells showed a significant increase in cell metabolism. The osteogenic differentiation, confirmed by the increase of calcium deposit, was promoted by the administration of pregabalin. This effect was already significant at the therapeutic plasma concentration of pregabalin (10 μg/mL). In contrast to the other antiepileptic drugs, pregabalin showed no osteocatabolic effects. Conflicting in-vivo data must therefore be attributed to systemic effects of pregabalin.

Keywords: antiepileptic drugs; bone defects; human bone cells; osteoblastogenesis; pregabalin.

Figure 1

HMSC (A), hOB (B) and MG63 (C) mean cell count per well after treatment with pregabalin at the concentrations 0, 2.5, 5, 10, 20, 40 µg/mL (C0, C1, C2, C3, C4, C5) in the interval of two days. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test on the 2nd–14th day after seeding. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01. Values are given as mean ± standard deviation.

Figure 2

Metabolism of hMSC (A), hOB (B) and MG63 (C) cells after treatment with pregabalin at the concentrations 0, 2.5, 10, 20, 40 µg/mL (C0, C1, C2, C3, C4, C5) was determined by XTT assay. Column height represents XTT metabolism of hMSC, hOB, and MG63 cells depending on the concentration of pregabalin treatment. XTT assay was performed 96 h after cell seeding. Values are given as mean ± standard deviation. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01. *** corresponds to a p-value of p < 0.001.

Figure 3

The fold change of ALP activity of pregabalin-treated hMSC (A), hOB (B) and MG63 (C) cells 0, 2.5, 5, 10, 20, 40 µg/mL (C0, C1, C2, C3, C4, C5) relative to untreated cells set at (C0). ALP activity assay was performed on the 10th day after cell seeding. Values are given as mean ± standard deviation. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01. *** corresponds to a p-value of p < 0.001.

Figure 4

ALP staining of hOB (A) and hMSC (B) cultured with pregabalin (C1–C5) and without (C0) for 21 days in chamber slides. Treatment with the highest pregabalin concentration enhanced ALP-staining. Densitometric quantification of ALP-staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01.

Figure 4

ALP staining of hOB (A) and hMSC (B) cultured with pregabalin (C1–C5) and without (C0) for 21 days in chamber slides. Treatment with the highest pregabalin concentration enhanced ALP-staining. Densitometric quantification of ALP-staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01.

Figure 5

Von-Kossa staining of untreated hOB (C0) (A) and hMSC (B) treated with pregabalin (C1–C5) for 21 days in chamber slides. Treatment with the highest pregabalin concentration enhanced von-Kossa staining. Densitometric quantification of von-Kossa-staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01.

Figure 5

Von-Kossa staining of untreated hOB (C0) (A) and hMSC (B) treated with pregabalin (C1–C5) for 21 days in chamber slides. Treatment with the highest pregabalin concentration enhanced von-Kossa staining. Densitometric quantification of von-Kossa-staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. * corresponds to a value of p < 0.05. ** corresponds to a value of p < 0.01.

Figure 6

Alizarin red staining of untreated hOB (A) and hMSC (B) treated with pregabalin (C1–C5) for 21 days in chamber slides. Treatment with the highest pregabalin concentration (C5: 40 µg/mL) enhanced Alizarin Red staining. Densitometric quantification of Alizarin red staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. ** corresponds to a value of p < 0.01. *** corresponds to a p-value of p < 0.001.

Figure 6

Alizarin red staining of untreated hOB (A) and hMSC (B) treated with pregabalin (C1–C5) for 21 days in chamber slides. Treatment with the highest pregabalin concentration (C5: 40 µg/mL) enhanced Alizarin Red staining. Densitometric quantification of Alizarin red staining in hOB and hMSC calculated by ImageJ software. Values are given as mean ± standard deviation from 10 sample images for each group. Significance in the difference between C5, C4, C3, C2, C1 vs. C0 was determined by Student’s t-test. ** corresponds to a value of p < 0.01. *** corresponds to a p-value of p < 0.001.