生体力学とMechanobiological研究ラット尾腱の準備

The video you're about to see contains the experimental procedures used to isolate and prepare rat tail tendon for biomechanical and mechanical biological studies. The purpose of this article is to demonstrate the methods and instruments that were developed and optimized in order to maintain tissue viability, sterility, and integrity to the preparation procedure. We'll discuss several features concerning the main steps of the preparation, beginning with extraction, followed by cross-sectional area measurement, rinsing, and finally the loading into the bioreactor chamber.

Let's get started. The extraction is the first step of the preparation in order to conserve the cell viability. All its manipulations are performed in cold seline solution.

After the ratti ized, the tail is resected rinse with 70%ethanol and transferred into a dish containing cell and solution and kept on ice. To facilitate the tendon extraction, the skin has to be removed. Taken into account that we work with ventral tendons.

Since they are smaller and as easier to manipulate with our equipment, an incision is made all along the dorsal side of the tail. Proper tail orientation is then verified by observing the anatomy at the proximal end. As we can see, the ventral side shows a larger number of tendons and the presence of a blood vessel can be verified by the drops of blood obtained.

When gently squeezing the tail, the incision is realized. Starting from proximal end and using a pair of surgical scissors. Care is taken to manipulate the tail by Its extremities.

When the Incision is completed, the skin is removed, starting also from the proximal end using fingers or forceps. Once again, care is taken to manipulate the tail by its extremities. Afterwards, the tail is rinsed into The solution to remove hairs and blood.

The specimen is then transferred into a dish containing fresh solution and kept on ice. At this point, the experimenter changes gloves to expose the tendon. The distal tail end is resected two to three vertebra shorter.

The vertebras are the visible bundles between the tendon bundles and they also can be felt when gliding the fingers all along the tail. A view on the light microscopy of a transversal section realized along the vertebra. Skoal end shows that there are six different tendon groups since the tendon from each of them is attached to the vertebra and that we are using only the ventral tissues.

Four tendons are pulled out at each cut, two from the lower groups and two from the lateral groups. To realize the resection, you may use surgical shear to break the intervertebral disc and a scalpel on a cutting board for the soft Tissues. At this point, the ventral side is recognized by the presence of bigger tendon spon.

Once the orientation is verified, the tendon are gently extracted from the distal end using forcep when no more tissues can be teased out, the tail is resected on vertebral shoulder like Shown previously. After The extraction, the tissue is immediately immersed into the solution and by holding both extremities, the tendon is transferred into a manipulation plate containing also the cell solution and on which tape is used to identify the proximal end. A microscopic analysis of h and e tainted longitudinal section shows that by using this simple and repetitive method, the extracted tissue seal has a well lined and densely packed collision network.

After manipulation, when tissues undergo mechanical stimulation, we describe their mechanical properties by normalizing the force inside to stress. Therefore, we must evaluate their cross-sectional area. In order to do this, the tenant profile is reconstructed by using a stereo microscopy equipped with a digital camera.

With this method, the tissues integrity is preserved since snow load or stress are directly applied to it. The digital camera takes pictures of tenant projection over 180 view. Within a reference frame, a vision assistant algorithm localizes the upper and lower specimen edges.

Then an algorithm with MATLAB software is used to carry out the profile reconstruction. An optic micrometer was designed to keep the tissue in the focal zone. Since outside this region, the projection is fuzzy and refocusing would shift the local reference frame.

The micrometer includes a measuring compartment to prevent tissue dehydration. Two positioning systems with rotating stage, allowing specimen rotation and two anchoring devices made with silicone tubes compressed against shaft colors. First, the measuring compartment is filled with cold cell solution until a silicone tubes entrance are Submerged.

Then the spaceman is transferred into the measuring compartment using forceps and by only holding its Extremities. By Using a micro volume ppe, the tendon extremities are drawn via section into the silicone tubes. Aspiration is then performed until the end extends beyond the shaft colors.

Once both Sides are done, the shaft colors are tightened by using a neck key to compress the tube. Now that the tendon is fixed, the apparatus Is installed under the stereo micr cup. The tendon can be observed directly from the computer screen at a magnification of 105.

For consistency, the tendon is stretched until the creases are no longer perceptible, followed by an additional 0.4%deformation. Since the specimen is pictured from zero to 170 degree, initial position and focus on adjustment are required to obtain a clear picture over the entire circumference. In fact, 18 pictures are taken where specimen rotation is increased by 10 degrees between each view.

With this method, it is possible to estimate the cross-sectional area of a rat tail tendon without damaging the tissue. At the end, a profile reconstruction is obtained within a margin of 0.5 to 2%error. Following measurement, the tissues are rinsed to prevent contamination that may have occurred during the previous manipulations.

Sterile manipulation plate containing fresh setting solution are placed in the cabinet, one being used for the rinsing procedure while the other will receive the rinse tendon. The experimenter washes his glove with 70%ethanol before introducing the extracted tendon in the biosafety Cabinet. By holding only the extremities, the tendon is carefully transferred from well to well.

Finally, It is transferred into an individual manipulation plate containing also sterile cell solution. For protocol validation, we conducted viability and sterility test. First cell viability was assessed using live dead viability cytotoxicity kit for mammalian cells.

After a culture of 12 days, a great majority of green fluorescent live cells were visualized confirming that the isolation procedures are successful in preserving live tissue. Examinations were then conducted to check for contamination of used culture. Media was plated on agar and incubated for 10 days since no bacteria growth was observed.

We conducted that our manipulations are sterile. The anchoring system use exploits the advantage of tissue dehydration and glue for small specimen such as rat tail, tendons, glue penetrates the tissue. That's fix in all subunits, whereas dehydration enhance its mechanical properties to avoid further contamination.

All the loading procedure is performed under biosafety cabinet where all instrument and chamber components are sterile. For the loading operation, the bioreactor chamber is fixed on a positioning jig. Two mobile arms are set to receive the tendons anchors.

A cover closes the chamber to prevent contamination. First, the anchors are deposited on the manipulation plate extremities. Their spools are centered with the groove longitudinal axis and over the tendon.

The tendon is then wound up And around one anchor. The Anchor is rolled over for approximately three quarters of a turn while the tissue's body remains in the solution. By holding the loose extremity, we ensure the tissue's attachment To the anchor.

The same procedure is then repeated for the other extremity. Once both Sides are fixed, the anchors are rolled or unrolled equally until there is six centimeters. Between the two extremities, two drops of 2.5 microliter of ocreate are deposit on the tendons wounded part.

To enter adhesion Care is taken. To avoid putting glue in the cell solution, A minimum of five minutes is required for the glue to dry completely. To validate our protocol, we conducted tests to verify the influence of the glue and anchor point dehydration on the tissue viability.

After two hours, we observed that the extremity condition had not spread without the tissue. In fact, there's a well-defined color limit marking the boundary of the red fluorescent dead cells found at the anchor point and the majority of green life cells of the tissue body. In the meantime, the bioreactor chamber Is filled with fresh culture media.

Once the Wound of part of the tendon is dry, it is rehydrated with cell solution. Then the anchored tissue is transferred in the chamber using forcep. The cover is closed to avoid further contamination.

The positioning jig will be removed once the chamber is installed in the bioreactor. We have just demonstrated how to prepare a rat tail tendon for biomechanical and mechanical biological studies. By applying those procedures, we can conduct a wide variety of in vitro studies on these tissues.

For example, a study on tissue degeneration was carried out by the application of under stimulation for a period of 10 days. Each day, we evaluated the tissue, mechanical properties and non-destructive stress relaxation test. At the end, we were able to observe stress variation.

We have provided evidence that our rigorous isolation and preparation procedures make it possible to maintain tissue viability, ity and anti. Thank you for watching and good luck with your own experiments.