Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro.
Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.
Cartmell
Sarah H
SH
School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Porter
Blaise D
BD
García
Andrés J
AJ
Guldberg
Robert E
RE
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019149:Bioreactors; D001842:Bone and Bones / Q000737:chemistry; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D050659:Core Binding Factor Alpha 1 Subunit; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008567:Membranes, Artificial; D051379:Mice; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D009363:Neoplasm Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology*; D015675:Osteocalcin / Q000378:metabolism; D010477:Perfusion / Q000295:instrumentation* / Q000379:methods; D010812:Physical Stimulation / Q000295:instrumentation / Q000379:methods; D016062:Porosity; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D000469:Alkaline Phosphatase