Characterization of the osteoblast-like cell phenotype under microgravity conditions in the NASA-approved Rotating Wall Vessel bioreactor (RWV).
Weightlessness induces bone loss in humans and animal models. We employed the NASA-approved Rotating Wall Vessel bioreactor (RWV) to develop osteoblast-like cell cultures under microgravity and evaluate osteoblast phenotype and cell function. Rat osteoblast-like cell line (ROS.SMER#14) was grown in the RWV at a calculated gravity of 0.008g. For comparison, aliquots of cells were grown in conventional tissue culture dishes or in Non-Rotating Wall Vessels (N-RWV) maintained at unit gravity. In RWV, osteoblasts showed high levels of alkaline phosphatase expression and activity, and elevated expression of osteopontin, osteocalcin, and bone morphogenetic protein 4 (BMP-4). In contrast, the expression of osteonectin, bone sialoprotein II and BMP-2 were unaltered compared to cells in conventional culture conditions. These observations are consistent with a marked osteoblast phenotype. However, we observed that in RWV osteoblasts showed reduced proliferation. Furthermore, DNA nucleosome-size fragmentation was revealed both morphologically, by in situ staining with the Thymine-Adenine binding dye bis-benzimide, and electrophoretically, by DNA laddering. Surprisingly, no p53, nor bcl-2/bax, nor caspase 8 pathways were activated by microgravity, therefore the intracellular cascade leading to programmed cell death remains to be elucidated. Finally, consistent with an osteoclast-stimulating effect by microgravity, osteoblasts cultured in RWV showed upregulation of interleukin-6 (IL-6) mRNA, and IL-6 proved to be active at stimulating osteoclast formation and resorbing activity in vitro. We conclude that under microgravity, reduced osteoblast life span and enhanced IL-6 expression may result in inefficient osteoblast- and increased osteoclast-activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness.
Rucci
Nadia
N
Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy.
Migliaccio
Silvia
S
Zani
Bianca Maria
BM
Taranta
Anna
A
Teti
Anna
A
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019149:Bioreactors; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000235:genetics; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D003599:Cytoskeleton / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D015676:Osteonectin / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D016212:Transforming Growth Factor beta; D014481:United States; D018557:United States National Aeronautics and Space Administration / Q000331:legislation & jurisprudence; D014893:Weightlessness
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016207:Cytokines; D015676:Osteonectin; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D000469:Alkaline Phosphatase