Practice of a testing bench to study the effects of cyclic stretching on osteoblast-orthopaedic ceramic interactions.

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.

Synergy between ascorbate and alpha-tocopherol on fibroblasts in culture.

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and alpha-tocopherol in cells is difficult to detect as both ascorbate and alpha-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and alpha-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and alpha-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and alpha-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of alpha-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected alpha-tocopherol but only in presence of cells. Cell viability was preserved by alpha-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and alpha-tocopherol corresponds to a consumption of ascorbate which spares alpha-tocopherol but only in presence of cells.

Production of matrix metalloproteinase 2 in fibroblast reaction to mechanical stress in a collagen gel.

The reaction of fibroblasts to mechanical forces generated by fibroblasts themselves in anchored collagen lattices was studied. Fibroblasts were cast in collagen gels. Free retracted gels (RG) were compared with stressed gels (SG) in 3-day and 14-day experiments. As previously described, SG showed an increase in protein, mainly collagen, biosynthesis. Matrix metalloproteinases (pro-MMP-2 and MMP-2) were studied by zymography. Certain cell membrane components, integrin alpha(2) and phosphatidylserine, were studied by flow cytometry with antibodies against the integrin alpha(2) subunit and with annexin V binding. Mechanical stress stimulated production of pro-MMP-2 both in the short-term (3-day) and the longer term (14-day) cultures. However, the pro-enzyme was not more activated and there was no difference in the amount of MMP-2 between RG and SG. There was only an increase with time under both conditions. The stressed fibroblasts reacted early with an increase in the integrin alpha(2) subunit, but the stimulated cells disappeared from the 14-day cultures. The number of cells measured in terms of the amount of DNA decreased between day 3 and day 14, mainly in the SG due to cytolysis. This cell stress was related to an alteration in the plasma membrane detected by the annexin V marker.

Monitoring of ascorbate at a constant rate in cell culture: effect on cell growth.

Ascorbic acid (vitamin C) is a primary antioxidant for cells. But, ascorbic acid added to culture medium is not readily available to cells in culture, because it is unstable in aqueous media. We determined the conditions required to obtain and maintain a constant concentration of ascorbate in the culture medium using ascorbate and ascorbate-phosphate. The study was carried out with human fibroblasts and the amounts of ascorbate in the culture medium were determined by high performance liquid chromatography. A mixture of 0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a constant concentration of ascorbate in the culture medium. This constant ascorbate concentration proved to be nontoxic for cells and stimulated cell growth in the short term and long term.