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Technical Forum

The Effects of Mechanical Stimulation on Controlling and Maintaining Marrow Stromal Cell Differentiation Into Vascular Smooth Muscle Cells

[+] Author and Article Information
Raphael Yao

Department of Biomedical Engineering,
Boston University,
44 Cummington Mall,
Boston, MA 02215

Joyce Y. Wong

Department of Biomedical Engineering,
Boston University,
44 Cummington Mall,
Boston, MA 02215
e-mail: jywong@bu.edu

1Corresponding author.

Manuscript received August 7, 2014; final manuscript received November 24, 2014; published online January 26, 2015. Editor: Victor H. Barocas.

J Biomech Eng 137(2), 020907 (Feb 01, 2015) (7 pages) Paper No: BIO-14-1376; doi: 10.1115/1.4029255 History: Received August 07, 2014; Revised November 24, 2014; Online January 26, 2015

For patients suffering from severe coronary heart disease (CHD), the development of a cell-based tissue engineered blood vessel (TEBV) has great potential to overcome current issues with synthetic graft materials. While marrow stromal cells (MSCs) are a promising source of vascular smooth muscle cells (VSMCs) for TEBV construction, they have been shown to differentiate into both the VSMC and osteoblast lineages under different rates of dynamic strain. Determining the permanence of strain-induced MSC differentiation into VSMCs is therefore a significant step toward successful TEBV development. In this study, initial experiments where a cyclic 10% strain was imposed on MSCs for 24 h at 0.1 Hz, 0.5 Hz, and 1 Hz determined that cells stretched at 1 Hz expressed significantly higher levels of VSMC-specific genetic and protein markers compared to samples stretched at 0.1 Hz. Conversely, samples stretched at 0.1 Hz expressed higher levels of osteoblast-specific genetic and protein markers compared to the samples stretched at 1 Hz. More importantly, sequential application of 24–48 h periods of 0.1 Hz and 1 Hz strain-induced genetic and protein marker expression levels similar to the VSMC profile seen with 1 Hz alone. This effect was observed regardless of whether the cells were first strained at 0.1 Hz followed by strain at 1 Hz, or vice versa. Our results suggest that the strain-induced VSMC phenotype is a more terminally differentiated state than the strain-induced osteoblast phenotype, and as result, VSMC obtained from strain-induced differentiation would have potential uses in TEBV construction.

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Figures

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Fig. 1

Uniaxial mechanical stimulation. Single- and dual-stage mechanical stimulation were delivered at 10% strain under the specified conditions. Dual-stage mechanical stimulation consists of two distinct conditions delivered sequentially.

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Fig. 2

Measurement of cell orientation relative to the direction of mechanical strain. Cellular orientation relative to the direction of strain was measured from the angle α.

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Fig. 3

MSC alignment increased as a function of stretch frequency. MSC alignment distribution after 24 h of mechanical stimulation at (a) 0 Hz, (b) 0.1 Hz, (c) 0.5 Hz, and (d) 1 Hz frequencies shows an alignment shift toward the direction perpendicular to the axis of strain, as the strain frequency increases from 0 to 1 Hz. The dotted line depicts the frequency that would be expected at each angle grouping with random MSC alignment.

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Fig. 4

Expression of lineage-specific genes and protein shows MSC differentiation into VSMCs and osteoblasts under distinct frequencies. (a) At 1 Hz stretch frequency, VSMC-specific genes (SM-α actin, SM-MHC, and calponin) were highly upregulated compared to osteoblast-specific genes (osteocalcin and osteopontin). Conversely, osteoblast-specific genes were relatively upregulated at the 0.1 Hz frequency. Each bar represents the mean ± SD, n = 3, * p < 0.05. (b) Bright field and fluorescent images of MSCs show highly expressed SM-α actin under the 1 Hz condition and a positive expression of osteocalcin under 0.1 Hz condition. Scale bar is 100 μm.

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Fig. 5

MSC alignment distributions after dual-stage mechanical stimulation indicate MSC transdifferentiation to a VSMC phenotype. (a) MSC alignment distribution for all dual-stage combinations shows a profile similar to that observed with single-stage 1 Hz stretching, expressing a pronounced shift toward the direction perpendicular to the axis of strain.

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Fig. 6

MSC lineage-specific gene expression after dual-stage mechanical stimulation indicates MSC transdifferentiation to a VSMC phenotype. (b) Expression of VSMC-specific genes was upregulated compared to osteoblast-specific genes when transitioned from either 0.1 Hz to 1 Hz condition or 1 Hz–0.1 Hz. The gene marker distribution was similar to that seen with single-stage 1 Hz condition. The same observation was made for both the 24 and 48 h duration conditions, suggesting the capacity of transdifferentiation in only one direction regardless of duration. Each bar represents the mean ± SD, n = 3.

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Fig. 7

SM-α actin and osteocalcin staining of MSCs showed evidence of VSMC lineage commitment in all dual-stage mechanical stimulation conditions. A four two-stage protocols resulted in the same increased level of SM-α actin expression and low level of osteocalcin expression, which would be indicative of MSC commitment into the VSMC lineage. (a)–(d): 24 h stages, (e)–(h): 48 h stages. Scale bar is 100 μm.

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