The Relationship Between the Substrate Strain and Cell Strain
Beth A. Kirkpatrick, James H-C. Wang, Ph.D.

Musculoskeletal Research Center
Department of Orthopaedic Surgery
University of Pittsburgh Medical Center, PA

Introduction
Previous studies have shown that mechanical stretching of cells affect many cellular functions, such as DNA and protein syntheses (1,3,4,5). In these studies, cells were grown on a deformable substrate such as silicone materials (6). It is clear that when stretching, the substrate deforms and hence the cells are strained. However, it is unclear whether the substrate strain is completely transmitted to the cells.

The objective of this study is to determine the substrate strain, cell strain, and their relationship. Because cells attach to the substrate by discrete focal adhesion contacts (2), we hypothesize that substrate strain is not completely transmitted to the cells.

Methods and Materials

Materials

Custom-made silicone dishes, ProNectin F, a bioengineered polymer for promoting cell attachment, from BioSystems (CA). Florescent microbeads, 1 mm in diameter, purchased from Molecular Probes (Eugene, OR). Human aortic endothelial cells (HAEC), essential basal medium (EBM) and supplements, as well as Hepes buffered saline (HBSS), trypsin-EDTA, and trypsin neutralizing solution (TNS) were obtained from Clonetics (Walkersville, MD).

Methods

Cell Culture
HAEC were cultured in EBM with supplements according to the manufacture's instructions. The cells were grown in plastic dishes and were incubated at 37ūC in a humidified atmosphere at 95% air and 5% carbon dioxide. The medium was changed every three days.

Plating to Silicone Dish
Cells were then plated on the custom-made smooth. The dishes were autoclaved then thoroughly washed with 70% ethanol and rinsed twice with phosphate buffered saline (PBS). They were coated with ProNectinF 10 _g/ml for 10-20 minutes. The ProNectin F was then extracted from the dishes, and the dishes were once again washed twice with PBS. Before adding the trypsin to detach the cells, HBSS was added to wash the cells. After extracting the HBSS, trypsin-was added to detach the cells from the culture dish. After a short incubation time, TNS was added. The solution was then spun down at 1000 rpm for 5 minutes. The solution was then extracted and fresh EBM was added to re-suspend the cells in the medium. After the HAEC were plated to the coated silicone dishes, the cells in the dishes were incubated overnight.

Cell Strain Measurement
After incubation, the silicone dishes were statically stretched at 15% with a custom-made stretching device. Phase contrast microphotographs were taken of the cells before and after stretching using Kodak 100 chrome slide film. The slides were then scanned into the computer using a Nikon slide scanner and Nikon slide scanning software. The cell area measurements were then taken using Scion Image. This was accomplished by using a free hand line tool to trace the outline of the cell. The cell outline was traced three times, and the mean of the three measurements was obtained.

In order to determine the extent of strain transmitted to the cell, the following method was used. A florescent microbead solution was made using a 1:500 mix of florescent microbead solution to EBM. The cell medium in the silicone dish was removed and 5 ml of medium containing 10 ml of florescent microbeads was added. The cells were then covered and incubated for 30 minutes to allow the florescent microbeads to settle down to the cells and the silicone dish. Then, the florescent microbead solution was removed and the cells were gently washed twice with EBM-2 to remove any unattached microbeads. Florescent microphotographs (Fig. 1) were then taken of the individual groups of HAEC before and after stretching using Kodak 400 color slide film. Image analysis was done using Scion Image and the substrate strain and strain transmitted to the cell from the substrate was determined using the custom-designed strain calculation software, the Excel program.

A B
Figure 1. Example of a cell before (A) and after (B) with florescent microbeads attached.

Statistical Analysis
An unpaired t-test was used to compare the substrate strain on the smooth silicone surface and the cell strain. A difference is considered to be significant if p < 0.05.

Results
We found that the axial strain on the substrate is 8.3 ± 1.3% (Mean ± SD), and the axial cell strain is 6.2 ± 0.9% (Fig. 2). There is a significant difference between these two strains (p = 0.004). The transverse strain on the substrate is 3.7 ± 0.67% in compression, and the transverse cell strain is 2.9 ± 1.1% in compression (Fig. 3). There is no significant difference between the transverse strains (p = 0.31).


Figure 2. Comparison of the axial strain of the substrate (8.3%) and the cell (6.2%).
Statistically, a significant difference was found (p = 0.004).



Figure 3. Comparison of the tranverse strain of the substrate (3.7%) and the cell (2.9%).
Statistically, a significant difference was found (p = 0.31).

Dicussion
The major finding of the study is that the cell strain is significantly lower than the substrate strain, which corresponds with our initial hypothesis. This is due to the fact that the cells do not completely attach to the substrate surface, but rather, the cells attach by discrete focal points. Therefore, the substrate strain cannot be completely transmitted to the cell surface. Another reason for the incomplete transmission of substrate strain to a cell is that the cell has a thickness which separates the cell surface from the substrate. Some strain is most likely lost when passing through the thickness of the cell to the cell surface.

In summary, this study showed that the applied substrate strain is not completely transmitted to cells. Further studies will be determination of whether there is a regional variation in cell strain from one region to another (e.g., the region around a nucleus versus other regions). Also, since actin cytoskeleton maintains integrity of a cell, we will examine its role in the transmission of substrate strain to the cell.

References
1. Banes, A.J., M. Tsuzaki, J. Yamamoto, T. Fischer, B. Brigman, T. Brown, and L. Miller. Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals. Biochem Cell Biol 73: 349-65, 1995.

2. Burridge, K. and K. Fath. Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton. Bioessays 10(4): 104-8, 1989.

3. Davies, P.F., and S.C. Tripathi. Mechanical stress mechanisms and the cell: An endothelial paradigm. Circ Res 72: 239-45, 1993.

4. Leung, D.Y.M., S. Glagov, and M.B. Mathews. Cyclic stretching stimulates synthesis of matrix components by arterial smooth muscle cells in vitro. Science 191: 475-477, 1976.

5. Shirinsky, V.P., A.S. Antonov, K.G. Birukov, A.V. Sobolevsky, Y.A. Romanov, N.V. Kabaeva, G.N. Antonova, and V.N. Smirnov. Mechano-chemical control of human endothelium orientation and size. J Cell Biol 109: 331-9, 1989.

6. Wang, H.C., W. Ip, R. Boissy, E.S. Grood. Cell Orientation response to cyclically deformed substrates: Experimental validation of a cell model. Journal of Biomechanics 28: 1543-1552, 1995.

Acknowledgments
I would like to thank Dr. Wang for his instruction and guidance. I would also like to thank Dr. Woo and Dr. Gilbertson for the opportunity to work at the MSRC for yet another summer, Darrick Chyu for his invaluable assistance in doing the data analysis, and Fengyan Jia for helping me along the way.