Alteration of cell cytoskeleton and functions of cell recovery of normal human osteoblast cells caused by factors associated with real space flight

Experiments involving short-term space flight have shown an adverse effect on the physiology, morphology and functions of cells investigated. The causes for this effect on cells are: microgravity, temperature fluctuations, mechanical stress, hypergravity, nutrient restriction and others. However, the extent to which these adverse effects can be repaired by short-term space flown cells when recultured in conditions of normal gravity remains unclear. Therefore this study aimed to investigate the effect of short-term spaceflight on cytoskeleton distribution and recovery of cell functions of normal human osteoblast cells. The ultrastructure was evaluated using ESEM. Fluorescent staining was done using Hoechst, Mito Tracker CMXRos and Tubulin Tracker Green for cytoskeleton. Gene expression of cell functions was quantified using qPCR. As a result, recovered cells did not show any apoptotic markers when compared with control. Tubulin volume density (p<0.001) was decreased significantly when compared to control, while mitochondria volume density was insignificantly elevated. Gene expression for IL-6 (p<0.05) and sVCAM-1 (p<0.001) was significantly decreased while alkaline phosphatase (p<0.001), osteocalcin and sICAM (p<0.05) were significantly increased in the recovered cells compared to the control ones. The changes in gene and protein expression of collagen 1A, osteonectin, osteoprotegerin and beta-actin, caused by short-term spaceflight, were statistically not significant. These data indicate that short term space flight causes morphological changes in osteoblast cells which are consistent with hypertrophy, reduced cell differentiation and increased release of monocyte attracting proteins. The long-term effect of these changes on bone density and remodeling requires more detailed studies.

Hypoparathyroidism in adult patients with beta-thalassemia major

To evaluate the prevalence of hypoparathyroidism in adult transfusion-dependent patients with beta-thalassemia major in a teaching referral hospital in Oman. All adult [>13 years] patients with beta-thalassemia major seen at Royal Hospital in Oman between 2004 and 2006 were studied. Demographic, pharmaceutical, clinical and biochemical data were collected for all the subjects. Analyses were performed using both descriptive and univariate statistics. A total of 31 patients were included into the study with an overall mean age of 19 +/- 3 years ranging from 14 to 30 years. Just over half of the subjects were males [n=16; 52%]. All the patients were on hypertransfusion and combined chelation therapy with desferrioxamine 40-60 mg/kg 5 days per week and deferiprone 75 mg/kg/day. Three of the patients had low levels of parathyroid hormone [<1.6 pmol/l]. A further three patients had normal levels of parathyroid hormone [1.6 - 9.3 pmol/l] in the presence of low serum calcium levels [<2.1 mmol/l]. These patients [with normal hypoparathyroid hormone levels, but lower calcium levels] were also defined to have hypoparathyroidism bringing the total prevalence of hypoparathyroidism in this cohort of adult patients with Beta-thalassemia major to 19% [6 out of 31]. The patients with hypoparathyroidism had statistically significantly lower levels of parathyroid hormone [2.7 versus 5.3 pmol/l; p=0.031] and serum calcium [1.7 versus 2.3 pmol/l; p=0.004] compared to those without hypoparathyroidism. The prevalence of hypoparathyroidism in adult beta-thalassemia major patients at this referral center was significantly higher [19%] than those reported elsewhere [2.5 and 10.7%]

Cell death induced by hydroxyapatite on L929 fibroblast cells

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.