Beneficial Effects of Vitamins K and D3 on Redox Balance of Human Osteoblasts Cultured with Hydroxyapatite-Based Biomaterials

Abstract

Hydroxyapatite-based biomaterials are commonly used in surgery to repair bone damage. However, the introduction of biomaterials into the body can cause metabolic alterations, including redox imbalance. Because vitamins D3 and K (K1, MK-4, MK-7) have pronounced osteoinductive, anti-inflammatory, and antioxidant properties, it is suggested that they may reduce the adverse effects of biomaterials. The aim of this study was to investigate the effects of vitamins D3 and K, used alone and in combination, on the redox metabolism of human osteoblasts (hFOB 1.19 cell line) cultured in the presence of hydroxyapatite-based biomaterials (Maxgraft, Cerabone, Apatos, and Gen-Os). Culturing of the osteoblasts in the presence of hydroxyapatite-based biomaterials resulted in oxidative stress manifested by increased production of reactive oxygen species and decrease of glutathione level and glutathione peroxidase activity. Such redox imbalance leads to lipid peroxidation manifested by an increase of 4-hydroxynonenal level, which is known to influence the growth of bone cells. Vitamins D3 and K were shown to help maintain redox balance and prevent lipid peroxidation in osteoblasts cultured with hydroxyapatite-based biomaterials. The strongest effect was observed for the combination of vitamin D3 and MK-7. Moreover, vitamins promoted growth of the osteoblasts, manifested by increased DNA biosynthesis. Therefore, it is suggested that the use of vitamins D3 and K may protect redox balance and support the growth of osteoblasts affected by hydroxyapatite-based biomaterials.

Keywords: 4-hydroxynonenal; hydroxyapatite-based biomaterials; lipid peroxidation; osteoblast growth; oxidative stress; redox balance; vitamins.

Figure 1

Reactive oxygen species (ROS) level in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 8, 12, 16, and 20 days. The results are expressed as the μM/min/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus group hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)); p < 0.05.

Figure 2

Glutathione (GSH) level in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 8, 12, 16, and 20 days. The results are expressed as the nmol/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus group hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)); p < 0.05.

Figure 3

GSH-Px activity in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 8, 12, 16, and 20 days. The results are expressed as the mU/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus group hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)); p < 0.05.

Figure 4

4-HNE level in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 8, 12, 16, and 20 days. The results are expressed as the nmol/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control. p < 0.05; (a) statistically significant differences versus group hydroxyapatites ((Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)), p < 0.05.

Figure 5

DNA levels in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 4, 8, 12, 16, and 20 days. The results are expressed as the ng/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)), p < 0.05;

Figure 6

ALP activity in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 4, 8, 12, 16, and 20 days. The results are expressed as the nmol/min/mg of protein and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)), p < 0.05.

Figure 7

OC levels in osteoblasts after incubation with hydroxyapatites and treated with vitamins D3, K1, MK4, and MK7 after 8, 12, 16, and 20 days. The results are expressed as the ng/mL of medium and are shown as the mean ± SD (n = 5). The values for the control cells and the treated cells were significantly different according to unpaired Student’s t-test. * Statistically significant differences versus control, p < 0.05; (a) statistically significant differences versus group hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)), p < 0.05. (b) statistically significant differences versus group vitamin D₃ with hydroxyapatites (Mx (Maxgraft), Cb (Cerabone), Ap (Apatos), Go (Gen-Os), respectively for graphs (b–e)), p < 0.05.