An in vitro study of osteoblast vitality influenced by the vitamins C and E

Abstract

Vitamin C and vitamin E are known as important cellular antioxidants and are involved in several other non-antioxidant processes. Generally vitamin C and vitamin E are not synthesized by humans and therefore have to be applied by nutrition. The absence or deficiency of the vitamins can lead to several dysfunctions and even diseases (e.g. scurvy). The main interest in this study is that vitamin C and E are known to influence bone formation, e.g. vitamin C plays the key role in the synthesis of collagen, the major component of the extracellular bone matrix.In the present study we evaluate the effect of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) on the proliferation and differentiation of primary bovine osteoblasts in vitro. Starting from standard growth medium we minimized the foetal calf serum to reduce their stimulatory effect on proliferation.An improved growth and an increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin was observed while increasing the ascorbic acid concentration up to 200 μg/ml. Furthermore the effects of α-tocopherol on cell growth and cell differentiation were examined, whereby neither improved growth nor increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin were detected.Further investigations are necessary to target at better supportive effect of vitamins on bone regeneration, and healing.

Figure 1

Influence of different FCS-concentrations (2% up to 10% FCS) on the cell proliferation of osteoblast like cells cultivated in media M over 5 days.

Figure 2

Cell morphology of osteoblast like cells (M x100) with 4% FCS (a = after 1 day and b = after 5 days in culture, medium M).

Figure 3

Cell proliferation of osteoblast like cells in medium with different ascorbic acid concentrations and medium S as reference (without vitamin C).

Figure 4

Osteoblast like cells (M x100) after 5 days in media with various ascorbic acid concentrations (a = 0.0 μg/ml, b = 25 μg/ml, c = 100 μg/ml, d = 200 μg/ml, e = 300 μg/ml, f = 400 μg/ml, g = 500 μg/ml).

Figure 5

Immunohistochemical analyses (a-f) of collagen I (a, b), osteonectin (c, d), osteocalcin (e, f) of osteoblast like cells (staining toluidine blue) (g, h) in media with 200 μg/ml ascorbic acid (left column) and without ascorbic acid (right column) after 14 days.

Figure 6

Cell proliferation of osteoblast like cells in media S and media M (S without and M with 25 μg/ml ascorbic acid) with and without 0,16 μg/ml vitamin E.

Figure 7

Immunohistochemical analyses of characteristic extracellular bone proteins: collagen I (a, d, g), osteonectin (b, e, h), osteocalcin (c, f, i) in media S without vitamin C and E (a-c), with 0,16 μg/ml vitamin E (d-f) and media M with 25 μg/ml vitamin C and 0,16 μg/ml vit. E (g-i) after 14 days.