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. 2020 Feb 15;21(4):1313.
doi: 10.3390/ijms21041313.

In Vivo and In Vitro Mechanical Loading of Mouse Achilles Tendons and Tenocytes-A Pilot Study

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Free PMC article

In Vivo and In Vitro Mechanical Loading of Mouse Achilles Tendons and Tenocytes-A Pilot Study

Viviane Fleischhacker et al. Int J Mol Sci. .
Free PMC article

Abstract

Mechanical force is a key factor for the maintenance, adaptation, and function of tendons. Investigating the impact of mechanical loading in tenocytes and tendons might provide important information on in vivo tendon mechanobiology. Therefore, the study aimed at understanding if an in vitro loading set up of tenocytes leads to similar regulations of cell shape and gene expression, as loading of the Achilles tendon in an in vivo mouse model. In vivo: The left tibiae of mice (n = 12) were subject to axial cyclic compressive loading for 3 weeks, and the Achilles tendons were harvested. The right tibiae served as the internal non-loaded control. In vitro: tenocytes were isolated from mice Achilles tendons and were loaded for 4 h or 5 days (n = 6 per group) based on the in vivo protocol. Histology showed significant differences in the cell shape between in vivo and in vitro loading. On the molecular level, quantitative real-time PCR revealed significant differences in the gene expression of collagen type I and III and of the matrix metalloproteinases (MMP). Tendon-associated markers showed a similar expression profile. This study showed that the gene expression of tendon markers was similar, whereas significant changes in the expression of extracellular matrix (ECM) related genes were detected between in vivo and in vitro loading. This first pilot study is important for understanding to which extent in vitro stimulation set-ups of tenocytes can mimic in vivo characteristics.

Keywords: cell culture; extracellular matrix; mechanical loading; tendon.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mechanical loading did not induce histological alterations in the in vivo loaded Achilles tendon. The loaded and non-loaded Achilles tendons were dissected and further processed for histological analysis. (A) Representative Hematoxylin and Eosin images of the loaded and non-loaded Achilles tendon. Scale bar: 200 µm. (B) Collagen fiber distribution. (C) Variation of fiber orientation (n = 6).
Figure 2
Figure 2
Cell morphology is partially influenced by mechanical loading. (A) The nuclear size is not significantly affected. (B) Cell nuclei in vitro were significantly rounder compared to in vivo (* p < 0.05, n = 6). (C) Phalloidin/ 4′,6-Diamidin-2-phenylindol (DAPI) staining of loaded murine tenocytes in vitro. Scale bar: 100 µm.
Figure 3
Figure 3
Differential expression of tissue-specific markers. (A,B) Tenogenic, (CE) osteogenic, chondrogenic, and adipogenic markers. Gene expression is given as fold change to non-loaded controls (horizontal line). Inter-group differences are marked with an asterisk (* p < 0.05, ** p < 0.01, n = 6).
Figure 4
Figure 4
Mechanical loading influences the expression of ECM components. (A,B) Collagens, (C,D) matrix metalloproteinases (MMPs), (E,F) Tissue inhibitor of metalloproteinase (TIMPs), (G,H) Integrins. Gene expression is given as fold change to non-loaded controls (horizontal line). Significant difference to non-loaded control is marked with a pound sign and inter-group differences with an asterisk (#, *: p < 0.05; ##, **: p < 0.01, n = 6).

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