The aim of the study was to investigate the effect of cyclic mechanical strain on differentiation markers in the presence or absence of dexamethasone. Human bone marrow stromal cells (BMSC) from seven donors (32.5±6.2 years) were cultivated with (D+) or without (D-) dexamethasone. A cyclic mechanical strain with an elongation of 2% (D+2; D-2) or 8% (D+8; D-8) was applied for three days with a stimulation time of three times two hours each day. Levels of alkaline phosphatase (ALP) and osteocalcin (OC) were compared after time intervals of four and seven days. mRNA expression of Collagen I, III and Cbfa1 was investigated after one, four, and seven days. ALP levels were significantly increased in the D+8 group after four and seven days (147.1±6.3%; p<0.05 and 168.6±6,5%; p<0.03) and in the D-8 group after 7 days (197.4±10.4; p<0.04). Cyclic strain had a significant influence on ALP-secretion (F=7.5; p<0.01). In the D-8 group there was a significant increase in OC secretion after 4 days (140.9±12.5%; p<0.05).; p<0.01). The effect of stretching was significantly stronger than that of dexamethasone (F=17.2 vs. 1.8). Collagen I (Col I) expression was upregulated in D+8 cultures after 4 days (215.0±53.3 p<0.04) and after seven days (166.7±55.7; p<0.04). Collagen III (Col III) expression was upregulated in D+2 and D+8 cultures after 4 days (200.7±16.3 and 185.9±12.7; p<0.04) and after seven days (154.4±10.1 and 118.8±16.4; p<0.04). There was a significant increase of Cbfa1 expression in D+8 cultures at all investigated time intervals (day 1: 105.5±3.7%; day 4: 104.7±3.0%; day 7: 104.4±2.1%; p<0.03). Stretching (F=20.0; p<0.01) was a stronger contributor to Cbfa-1 expression than dexamethasone (F=12.1; p<0.01) Cyclical mechanical stimulation with 8% elongation increases ALP and OC levels and upregulates Col I and III synthesis and Cbfa1 expression. In the short term, cyclical stretching is a stronger differentiation factor than dexamethasone. Cyclical stretching and dexamethasone both enhance the osteogenic commitment of hBMSC.

No abstract

Most investigations of the sensitivity of cultured mammalian cells to fluoride have been concerncd with a determination of the levels of fluoride which inhibit growth. While some studies (1, 2 ) have suggested that the growth of mammalian cells can be inhibited by from 0.045 to 2 ppm F, most reports (3)(4)(5)(6)(7)(8) have indicated that a minimum of 5-15 ppm F (0.3-0.8 mM) is required to decrease the rate of cell division or accumulation of cell protein. At higher levels of fluoride, there appears to be a sigmoidal dose-response relationship with slight inhibition a t 10 to 20 ppm and compkte inhibition of growth a t 40 to 50 ppm (6, 7 ) . Although i t has been observed that cellular ATP levels are decreased (9), and glucose uptake per milligram of cellular protein is increased (8,9) in fluoride-treated cells, the mechanism by which fluoride inhibits growth is not understood. To further characterize the toxicity of fluoride in mammalian cell cultures, the distribution of fluoride between cells and medium was determined. A comparison of intracellular fluoride concentrations with the concentrations of fluoride reported (10) to inhibit various enzymes in vitro may allow a prediction as LO enzyme inhibitions of physiological significance.Methods. Suspension cultures of HeLa CCL 2 cells and L cells (mouse fibroblasts) with generation times of 36 and 19 hr, respectively, were utilized for these experiments. The L cells were grown in medium B of Medappa et al. (1 1) and the HeLa cells were grown in the medium described by Carlson and Suttie (9) modified by the substitution of a Ca-Mg-free salt mixture (12) and the addition of 0.1 % Pluronic F-68 detergent. The cells were resuspended in fresh medium at a cellular concentration of 4 x 10" cells/ml for an equilibration period of 1 hr before measuring fluoride distribution. A mixture of carrier-free ISF prepared by neutron activation (13) and NaF in a total volume of 3 ml was added to 200 ml of cell suspension in a 500 ml Erlenmeyer flask, and the cells were incubated for 1 or 2 hr at 37" on a rotatory shaker. The cell suspensions were then centriiuged a t 5OOg for 1-2 min; and the cell pellets were resuspended in 10 ml of supernatant. After the addition of 10 pCi of 14C-inulin, 2.5 ml aliquots of cell suspension were injected into specially designed glass centrifuge tubes (14) having a reservoir of 2.5 ml capacity connected with a capillary 6 CM long and 1 mm in diameter and centrifuged at 2000g for 20 min a t 4 ' . The cell pellets and supernatant medium recovered from centrifugation were analyzed for lSF and 14C-inulin according to standard procedures. Since inulin cannot penetrate mammalian cells, the I4C in the cell pellets was used to calculate the amount of entrapped medium. Intracellular fluoride concentrations were calculated from the amounts of 18F in cell pellets and entrapped medium, the specific activity of lXF in the medium, and the amount of intracellular water in pellets. Intracellular water values were calculated from the wet and dry weights of cell pelle...

The influence of autolysis on the immunhistological detectability and the immunological properties of tissue-bound basement membrane antibodies were investigated in the rat kidney. The immunhistological demonstration of antibodies was not affected up to 3 days post mortem. The disturbing autofluorescence of the autolytic tissue can be avoided by using peroxydase labeled antibodies. Basement-membrane antibodies that were eluted from the kidneys after an autolysis period of 2 days showed a homogenous linear deposition in kidney basement membrane after reinjection into normal rats. Their antibody properties were not affected.

In the short-term over seven days, cyclic stretching is a stronger differentiation factor than dexamethasone.