(P 132) Dynamic Cell Culture Prevents Core Necrosis and Creates Meaningful Osteoblastic Networks in Thick 3D Tissue-Engineered Constructs

• Published in: Tissue engineering. Part A Vol. 14; no. 5; p. 840
• Main Authors: Allori, A C, Sailon, A M, Clark, E, Cretiu-Vasiliu, C, Smay, J, Ricci, J L, Warren, S M
• Format: Journal Article
• Language: English
• Published: 01.05.2008
• ISSN: 1937-3341; 1937-335X

Introduction: Bone lacuno-canalicular fluid flow ensures chemo-transportation and provides a mechanical stimulus to cells. A novel flow-perfusion bioreactor was used to provide chemotranspor-tation through a thick 3D scaffold in vitro. We hypothesize that flow perfusion will improve cellular distribution and viability throughout the construct and that osteoblasts will form meaningful osteoblastic networks. Methods: 3D bioprinted cylindrical hydroxyapatite-tricalcium-phosphate scaffolds (24x6 mm, 200x200 mu m pore size) were seeded with MC3T3-E1 cells (4x10 super(6) cells/cm super(3)) and subjected to dynamic cell culture (0.4 dyn/cm super(2)) in osteogenic medium. Control scaffolds remained in static culture. A dye-transfer technique using diI and calcein AM was used to label cells to check for gap junction formation. Samples were harvested at 2, 4, 6, 10, 14, and 21 days, plastic-embedded, and analyzed by confocal microscopy and scanning electron microscopy. Results: Histologic analysis confirmed uniform cell distribution after seeding and that >90% of cells survived the time allotted for cellular adherence. By 6 days in static culture, 80% of cells in the core of the scaffolds were non-viable, while 90% of those at the peripheral crust survived. In contrast, scaffolds subjected to flow perfusion demonstrated uniform cell density and 95% viability throughout the construct at all time points. Osteoblasts assumed characteristic dendritic phenotype and extended podocytes toward other cells, with evidence of gap junction formation. Conclusion: Flow perfusion ensured adequate chemo-transportation to all regions of a thick 3D scaffold, such that cellular viability and distribution were preserved. The 3D environment was conducive to the formation of meaningful osteoblastic networks.