Groupe PERSEUS, Faculté de Génie Département de génie mécanique, Université de Sherbrooke
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Bruneau, A., Champagne, N., Cousineau-Pelletier, P., Parent, G., Langelier, E. Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies. J. Vis. Exp. (41), e2176, doi:10.3791/2176 (2010).
Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.
Prior to any manipulation, you must identify the group of tendons to be used depending on the experiment you are conducting and the apparatus at your disposition. For our purposes, ventral tendons were chosen because they are smaller and thus easier to manipulate when measuring the cross-sectional area and fitting them into the bioreactor chamber.
Please note that all instruments are autoclaved or sterilized with 70% ethanol. Moreover, a spray bottle containing 70% ethanol is placed beside each work station to sterilize the experimenterís gloves before each operation.
Part 1: Extraction
After resection, the tail is carefully manipulated by its extremities to avoid damaging the tissues. Also, to conserve cell viability, all manipulations are carried out in cold saline solution.
1B) Work station:
Part 2: Cross-sectional area measurement7
When the tissue undergoes mechanical characterization or stimulation, its mechanical properties are described by normalizing the force inside the tendon to stress. This is why we evaluate cross-sectional area.
2B) Work station:
Part 3: Rinsing
To remove contamination that may have occurred during the previous manipulations, the tissues are rinsed under biosafety cabinet.
3B) Work station:
Part 4: Loading into bioreactor chamber
To avoid further contamination, the following manipulations are also conducted in the biosafety cabinet.
4B) Work station:
Part 5: Representative Results:
The outcome of the protocol shows that when performed correctly, our tissue rigorous isolation and preparation procedure make it possible to maintain tissue sterility, viability and integrity.
First, using the simple and repetitive extraction method, we are able to extract tail tendons without damaging the collagen network as it can be observed by a microscopic analysis of H&E tainted sections realized after the extraction.
Figure 6. Tendon collagen network after extraction (5μm longitudinal section stained with H&E).
We then cultured tendons for up to ten days and conducted sterility tests. Each day, we plated used culture solution on agar and incubated it for 24 hours. Since no bacterial growth was observed, we concluded that our manipulations do not result in contamination.
With the profile reconstruction algorithm and optic micrometer, we are able to estimate the cross-sectional area within a 2% margin of error7.
Figure 7. Profile reconstruction of a RTT.
Finally, we assessed viability using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells after the rinsing step and after a twelve-day culture period. Since a large majority of green fluorescent live cells were evident, we can confirm that our isolation procedures are successful in preserving live tissue. The same test was performed two hours after attaching the tendon into the bioreactor chamber. We verified that the dehydration and glue at the anchor had not spread in the tissue between both anchors.
Figure 8. Tissue viability at the anchor (green = live cells, red = dead cells)
By applying those experimental procedures, we can conduct a wide variety of in vitro studies on this kind of tissues. As for example, a study on tissue degeneration was carried out by the application of under-stimulation to RTTs for a period of ten days. Each day, we evaluated tissue mechanical properties in non-destructive stress relaxation tests. At the end, we were able to observe RTT stress variation and thus analyze the progression of mechanical properties.
Figure 9. RTT's stress variation evaluated trough relaxation test (day 1 to 10).
No conflicts of interest declared.
This article was supported by NSERC Grant # 299280, IRSST as a scholarship to G. Parent and FRSQ as an undergraduate student research award to M. Cyr. We thank Yoan Lemieux-Laneville for performing the manipulations recorded on video.
|D-PBS||Reagent||Wisent Inc.||311-410-CL||Saline solution|
|Glucose||Reagent||Wisent Inc.||609-037-EL||Saline solution
|Sodium Bicarbonate||Reagent||Wisent Inc.||600-105-CG||Culture solution
|FBS||Reagent||Wisent Inc.||090150||Culture solution
|Optic Micrometer||Tool||Custom Made|
|Manipulation plate||Tool||Custom Made|
|Bioreactor chamber||Tool||Custom Made|