title: Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens.
abstract: 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.
authors.affiliation: Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Bucaro
authors.forename: J
authors.initials: J
authors.lastname: Fertala
authors.forename: C S
authors.initials: CS
authors.lastname: Adams
authors.forename: M
authors.initials: M
authors.lastname: Steinbeck
authors.forename: P
authors.initials: P
authors.lastname: Ayyaswamy
authors.forename: K
authors.initials: K
authors.lastname: Mukundakrishnan
authors.forename: I M
authors.initials: IM
authors.lastname: Shapiro
authors.forename: M V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D016475:3T3 Cells; D000464:Alginates / Q000737:chemistry; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D001703:Biophysics / Q000379:methods; D015153:Blotting, Western; D001842:Bone and Bones / Q000166:cytology*; D048429:Cell Size; D002470:Cell Survival; D005434:Flow Cytometry; D020723:Glucuronic Acid / Q000737:chemistry; D018470:Gravity, Altered; D006603:Hexuronic Acids / Q000737:chemistry; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D008928:Mitochondria / Q000473:pathology; D009141:Musculoskeletal System; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology*; D010641:Phenotype; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013026:Space Flight; D019311:Staurosporine / Q000494:pharmacology; D013314:Stress, Mechanical; D013997:Time Factors; D014893:Weightlessness; D018474:Weightlessness Simulation
chemical_list: D000464:Alginates; D017304:Annexin A5; D006603:Hexuronic Acids; D019253:Proto-Oncogene Proteins c-bcl-2; D012313:RNA; D020723:Glucuronic Acid; D019311:Staurosporine