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Research Papers

Effect of Pulse Frequency on the Osteogenic Differentiation of Mesenchymal Stem Cells in a Pulsatile Perfusion Bioreactor

[+] Author and Article Information
Katherine D. Kavlock

School of Biomedical Engineering and Sciences,  Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0211

Aaron S. Goldstein1

School of Biomedical Engineering and Sciences, Department of Chemical Engineering,  Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0211, e-mail: goldst@vt.edu

1

Corresponding author.

J Biomech Eng 133(9), 091005 (Oct 11, 2011) (6 pages) doi:10.1115/1.4004919 History: Received June 28, 2011; Accepted August 18, 2011; Published October 11, 2011; Online October 11, 2011

Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E2 were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.

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Copyright © 2011 by American Society of Mechanical Engineers
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Figures

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Figure 1

Diagram of (a) a perfusion flow chamber containing a seeded scaffold and (b) schematic of perfusion flow system

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Figure 2

Flow profiles of (a) 0.017 Hz, (b) 0.050 Hz, and (c) 0.083 Hz pulsatile perfusion conditions

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Figure 3

Cell density in PLGA porous scaffolds at 15 days normalized to the no flow control. Data were combined from two replicates of n = 3 samples, and data for each replicate were normalized by the mean for the no flow control. Bars correspond to the mean ± SEM for n = 6 (or n = 5 for continuous flow) samples, and no differences between groups were statistically significant (p > 0.05).

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Figure 4

ALP activity per cell for MSCs seeded in PLGA scaffolds at day 15. Data were combined from two replicates of n = 3 samples, and data for each replicate were normalized by the mean for the no flow control. Bars correspond to the mean ± SEM for n = 6 (or n = 5 for continuous flow) samples. No differences between groups were statistically significant.

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Figure 5

Gene expression of (a) OPN and (b) OC by MSCs cultured in PLGA scaffolds for 15 days. β-actin was used as the housekeeping gene and data are normalized to the no flow condition. Data are mean ± standard error for n = 6 (or n = 5 for continuous flow) samples. An asterisk denotes a significant difference with respect to the no flow control.

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Figure 6

Accumulation of (a) OPN and (b) PGE2 per scaffold at 0, 3, 6, 9, 12, and 15 days. Data are mean of n = 2 replicates (with three scaffolds per replicate).

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