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Research Papers

# Mechanisms of Strain-Mediated Mesenchymal Stem Cell Apoptosis

[+] Author and Article Information
E. M. Kearney, P. J. Prendergast

Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland

V. A. Campbell1

Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland; Department of Physiology, Trinity College Dublin, Dublin 2, Irelandvacmpbll@tcd.ie

1

Corresponding author.

J Biomech Eng 130(6), 061004 (Oct 09, 2008) (7 pages) doi:10.1115/1.2979870 History: Received September 18, 2007; Revised May 14, 2008; Published October 09, 2008

## Abstract

Mechanical conditioning of mesenchymal stem cells (MSCs) has been adopted widely as a biophysical signal to aid tissue engineering applications. The replication of in vivo mechanical signaling has been used in in vitro environments to regulate cell differentiation, and extracellular matrix synthesis, so that both the chemical and mechanical properties of the tissue-engineered construct are compatible with the implant site. While research in these areas contributes to tissue engineering, the effects of mechanical strain on MSC apoptosis remain poorly defined. To evaluate the effects of uniaxial cyclic tensile strain on MSC apoptosis and to investigate mechanotransduction associated with strain-mediated cell death, MSCs seeded on a 2D silicone membrane were stimulated by a range of strain magnitudes for $3days$. Mechanotransduction was investigated using the stretch-activated cation channel blocker gadolinium chloride, the $L$-type voltage-activated calcium channel blocker nicardipine, the c-jun $NH2$-terminal kinase (JNK) blocker D-JNK inhibitor 1, and the calpain inhibitor MDL 28170. Apoptosis was assessed through DNA fragmentation using the terminal deoxynucleotidyl transferase mediated-UTP-end nick labeling method. Results demonstrated that tensile strains of 7.5% or greater induce apoptosis in MSCs. $L$-type voltage-activated calcium channels coupled mechanical stress to activation of calpain and JNK, which lead to apoptosis through DNA fragmentation. The definition of the in vitro boundary conditions for tensile strain and MSCs along with a proposed mechanism for apoptosis induced by mechanical events positively contributes to the development of MSC biology, bioreactor design for tissue engineering, and development of computational methods for mechanobiology.

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## Figures

Figure 1

FACS analysis on mesenchymal stem cells after 21days in culture. 97% of the population express the positive marker CD90 while only 3% of the population express the negative marker CD45.

Figure 4

Effect of the SACC blocker, gadolinium chloride (10μM) and an L-type calcium channel blocker, nicardipine (0.5μM) on strain-induced increase in DNA fragmentation. Mechanical strain of 10% for 3days significantly increases DNA fragmentation. In the presence of gadolinium chloride, 10% strain significantly increases DNA fragmentation. In contrast, in the presence of Nicardipine, the strain-induced increase in DNA fragmentation is abolished. The results are expressed as a mean±SEM for four randomly selected fields in five to eight independent experiments. The asterisks indicate the difference from nonstrained controls (one-way ANOVA, Tukey’s post hoc; p<0.05).

Figure 5

Effect of mechanical strain on JNK activation in MSCs. MSCs exposed to 10% strain for 3days (ii) displayed more intense phospho-JNK immunoreactivity within the cytosol and nucleus compared to unstrained cells (i). In the presence of gadolinium chloride (iii) and (iv), MSCs exposed to 10% strain (iv) also displayed more intense phospho-JNK immunoreactivity compared to unstrained cells (iii). Images are representative of five independent experiments. The arrows indicate cells displaying phospho-JNK immunoreactivity. The scale bar is 50μm.

Figure 6

Effect of MDL 28170 and JNK inhibitor 1 on strain-induced apoptosis. Mechanical strain of 10% for 3days significantly increases DNA fragmentation in MSCs. The strain-induced increase in DNA fragmentation is abolished when cells are mechanically stimulated in the presence of MDL 28170 (10μM) or JNK inhibitor 1 (1μM). The results are expressed as a mean±SEM for four randomly selected fields in 5–13 independent experiments (one-way ANOVA, Tukey’s post hoc; p<0.05).

Figure 7

Mechanical strain of 10% for 2days increases calpain activity in MSCs. Exposure of MSCs to 10% strain for 2days(0.17Hz) increased calpain activity and this was abolished by the calpain inhibitor MDL 28170 (10μM). The results are expressed as a mean±SEM for six independent observations.

Figure 8

Flowchart demonstrating strain-induced apoptotic signaling. Exposure of MSCs to 7.5% and 10% strain leads to apoptosis via L-type VACCs. During exposure to strain of 7.5% or greater, JNK becomes phosphorylated and calpain becomes activated, leading to downstream DNA fragmentation.

Figure 2

Single station stretching device. Strain magnitude is controlled by an eccentric cam that delivers displacement to a linear slide assembly to which the silicone strip is attached. The insertion points for the cell-seeded strip correspond to positions A and B in the diagram. The amount of displacement can be controlled by the size of the cam. Frequency is controlled by the voltage supplied to the motor.

Figure 3

Effect of uniaxial cyclic strain magnitude on MSC viability. (a) Exposure of MSCs to 2.5% and 5% strain for 3days does not influence cell viability as assessed by analysis of DNA fragmentation by TUNEL staining. However, strain of 7.5% and 10% for 3days(0.17Hz) significantly increased DNA fragmentation. The results are expressed as a mean±SEM for four randomly selected fields in five independent experiments. The asterisks indicate the difference from nonstrained controls (Student’s paired t-test; p<0.05). (b) Representative images of cell viability analyzed by the TUNEL histological method. In both unstrained control (i) and (iii) and following 2.5% strain conditions (ii), histological evidence of apoptosis is not detected. However, following 10% strain (iv), cells that have undergone apoptosis have a dark nucleus representing DNA fragmentation. The arrows indicate cells displaying representative positive TUNEL staining of DNA fragmentation. The scale bar is 150μm.

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