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Research Papers

Applying Physiologically Relevant Strains to Tenocytes in an In Vitro Cell Device Induces In Vivo Like Behaviors

[+] Author and Article Information
Jung Joo Kim

Auckland Bioengineering Institute,
University of Auckland,
Auckland 1010, New Zealand

David S. Musson, Jillian Cornish

Department of Medicine,
University of Auckland,
Auckland 1010, New Zealand

Brya G. Matthews

Department of Medicine,
University of Auckland,
Auckland 1010, New Zealand;
Department of Reconstructive Sciences,
University of Connecticut Health Center,
Farmington, CT 06030

Iain A. Anderson

Auckland Bioengineering Institute,
University of Auckland,
Auckland 1010, New Zealand;
Department of Engineering Science,
University of Auckland,
Auckland 1010, New Zealand

Vickie B. Shim

Auckland Bioengineering Institute,
University of Auckland,
70 Symonds Street,
Auckland Central,
Auckland 1010, New Zealand
e-mail: v.shim@auckland.ac.nz

1Corresponding author.

Manuscript received December 1, 2015; final manuscript received June 12, 2016; published online November 3, 2016. Assoc. Editor: Nathan Sniadecki.

J Biomech Eng 138(12), 121003 (Nov 03, 2016) (9 pages) Paper No: BIO-15-1617; doi: 10.1115/1.4034031 History: Received December 01, 2015; Revised June 12, 2016

We have developed a novel cell stretching device (called Cell Gym) capable of applying physiologically relevant low magnitude strains to tenocytes on a collagen type I coated membrane. We validated our device thoroughly on two levels: (1) substrate strains, (2) cell level strains. Our cell level strain results showed that the applied stretches were transferred to cells accurately (∼90%). Our gene expression data showed that mechanically stimulated tenocytes (4%) expressed a lower level of COL I gene. COX2 gene was increased but did not reach statistical significance. Our device was then tested to see if it could reproduce results from an in vivo study that measured time-dependent changes in collagen synthesis. Our results showed that collagen synthesis peaked at 24 hrs after exercise and then decreased, which matched the results from the in vivo study. Our study demonstrated that it is important to incorporate physiologically relevant low strain magnitudes in in vitro cell mechanical studies and the need to validate the device thoroughly to operate the device at small strains. This device will be used in designing novel tendon tissue engineering scaffolds in the future.

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Figures

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Fig. 1

Overall structure of the Cell Gym. The detailed structure of the control box made up of linear actuator and eight silicon baths are shown. This is connected to a computer for precisely applying mechanical strain.

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Fig. 2

Design process of the silicon bath. The bath is made up of the silicon part (a) which also has microgrooves (c) on the bottom of the surface and attached with a clamp system (b) for accurate application of mechanical strains (a) customized silicon bath with plastic pieces, (b) silicon bath assembled with clamps and filled with cell culture media, and (c) SEM images showing tenocytes attached to a microgrooved silicon bath.

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Fig. 3

Validation process for substrate and cell level strains. The substrate level strain was validated by cross correlation technique that tracked the movement of speckle patterns shown in (a). The cell level strains were measured first by dividing the region into three (b) and separately measuring engineering strain of the cells within these three regions (a) silicon bath with speckle pattern for computing cross correlation for surface strain and (b) three regions within the silicon bath used in strain measurements.

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Fig. 4

Measuring cell level strains from images during cyclic stretch. First single cell images were extracted (a) and then the major axis through the cell was drawn to measure 1D engineering strain after mechanical stimulation (b) (a) original cell image from Cell Gym and (b) measuring engineering strain from cell images.

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Fig. 5

Validation results for (a) substrate level strain, (b) cell level strains. (a) Substrate level strain results showed that when 8% strain is applied, the average transferred strain to the substrate is 6.85% and 90% of the area in the surface are within one standard deviation of this average value. (b) Cell level strain results showed that cell level strains are similar to the substrate level strains. The x-axis indicates the absolute magnitude of stretching in mm (0–2 mm) which corresponds to 0–8% in our case. Error bars indicate SD (a) strain map for the substrate level strains when 8% of strain was applied and (b) measurement of cell level strains measured at 2,4,6,8% applied strains.

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Fig. 6

Gene expression results after mechanical stimulation. When 2 and 4% strains were applied, Collagen type I showed a significant decrease in the expression level. Cox-2 increased upon mechanical stimulation too but did not reach statistical significance.

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Fig. 7

Collagen synthesis and gene expression patterns after application of in vivo like mechanical stimulation reported in Ref. [28]. The top row shows the comparison between our results and those reported in Miller's study. The time-dependent patterns of collagen synthesis level changes over 3-day period from our study matches the one reported in Miller's study shown on the top right-hand corner (modified from Ref. [28] to show the time periods of interest). The gene expression level changes of mechanosensitive genes are shown in the next three rows (1–4) (a) comparison of collagen synthesis between in vitro and in vivo experiments and (b) gene expression changes over 3-day period after mechanical stimulation mimicking Miller's study.

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