Microgravity environments cause aggregate formation and accelerated mineralization of human preosteoblastic cells. Objectives: The hypothesis of this study was that aggregate formation and mineralization would occur with less than 10 million cells as previously described. Methods: Human embryonic palatal mesenchymal preosteoblastic cells (HEPM, ATCC 1486) were cultured at 1, 2.5, 5, or 10 million cells in a rotary wall vessel to simulate a microgravity environment for 7 days. Cultures were supplemented with EMEM medium (10% FBS), 5mM beta-glycerophosphate, 50ug/ml ascorbate. Aggregate size was measured using a ruler or micrometer, and mineralization and collagen expression was detected using Von Kossa and Masson Trichrome histological stain. Scanning electron microscopy (SEM) was used for structural comparison and elemental analysis for calcium and phosphorous detection. Immunohistochemistry was used to detect the expression of the osteogenic markers BSPII and osteopontin. Results: Aggregate formation was detected for all cell concentrations studied. Size and level of calcium expressed appeared to correlate to cell number (P<0.01). As revealed by Von Kossa stain, calcium expression was detected throughout the aggregate in areas of dense to sparse expression, but presence was independent of cell number. The central area of the aggregates contained cells and did not appear necrotic suggesting that cells in the center were viable and nutrient and oxygen diffusion was satisfactory even in the absence of scaffolding structures. Masson's trichrome stain showed collagen throughout all areas of the aggregates. SEM aggregates had similar microscopic structural patterns demonstrating their organized formation. Positive staining for BSPII and OP showed that aggregates share common differentiation proteins with in vivo bone formation. Conclusions: It has been demonstrated that mineralized macroscopic aggregates can be formed with as few as 1 million cells. These results may translate into use of microgravity environments for tissue engineering of bone to graft craniofacial osseous defects. NIH/NIDCR R21-DE016677-01.

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