ABSTRACT
Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non-rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3-E1 osteoblast-like cells for 1-14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T-1/2 stem cells, MC3T3-E1 osteoblast-like cells, or MLO-A5 osteocyte-like cells to the action of these agents. RT-PCR analysis indicated that MC3T3-E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector-averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death.
Subject(s)
Apoptosis , Osteoblasts/cytology , Weightlessness Simulation , Animals , Cell Differentiation , Cell Line , Cell Survival , Cells, Cultured , Gene Expression , Humans , Nitroprusside/pharmacology , Osteoblasts/drug effects , Osteoblasts/metabolism , Staurosporine/pharmacologyABSTRACT
Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3-E1 osteoblast-like cells were cultured in alginate carriers in the NASA-approved high aspect ratio vessel (HARV). This system subjects the cells to a time-averaged gravitational field (vector-averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl-2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector-averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector-averaged gravity. When challenged with staurosporine, cells subjected to vector-averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface-seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector-averaged gravity.
Subject(s)
Bone and Bones/cytology , Osteoblasts/pathology , Weightlessness , 3T3 Cells , Alginates/chemistry , Animals , Annexin A5/pharmacology , Apoptosis , Biophysics/methods , Blotting, Western , Cell Size , Cell Survival , Flow Cytometry , Glucuronic Acid/chemistry , Gravity, Altered , Hexuronic Acids/chemistry , Humans , Membrane Potentials , Mice , Mitochondria/pathology , Musculoskeletal System , Osteoblasts/metabolism , Phenotype , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Space Flight , Staurosporine/pharmacology , Stress, Mechanical , Time Factors , Weightlessness SimulationABSTRACT
We have generated mesoscopic patterns of viable Escherichia coli on Si(1 1 1), glass, and nutrient agar plates by using a novel laser-based transfer process termed matrix assisted pulsed laser evaporation direct write (MAPLE DW). We observe no alterations to the E. coli induced by the laser-material interaction or the shear forces during the transfer. Transferred E. coli patterns were observed by optical and electron microscopes, and cell viability was shown through green fluorescent protein (GFP) expression and cell culturing experiments. The transfer mechanism for our approach appears remarkably gentle and suggests that active biomaterials such as proteins, DNA and antibodies could be serially deposited adjacent to viable cells. Furthermore, this technique is a direct write technology and therefore does not involve the use of masks, etching, or other lithographic tools.
