ラットの両側膝蓋腱損傷モデルの幹細胞療法の評価

The overall goals of this bilateral rat model are to optimize the use of animals and evaluate treatment candidates for tendon defects. This method can help answer any key questions in orthopedic field. Such as evaluation of cellular therapy on tendinopathy.

The main advantage of this model is that it controls interindividual variation, thereby, reducing the number of animals required to detect differences between treatments. Visual demonstration of the model is critical because the creation of bilateral standardized patella tendon defects in rats is difficult to learn. The model requires a good knowledge of the local anatomy, strong basic surgical skills, and precision while working on small structures.

To begin wash a harvested equine umbilical cord twice in a 50 milliliter tube using room temperature PBS containing antibiotics. Next, transfer the tissue to a 150 millimeter plate and section it into two inch long fragments. Then transfer the fragments to a 50 milliliter tube and wash them with PBS containing antibiotics to remove the blood.

Do this two or three times at room temperature. Next, cut the fragmented parts of the umbilical cord longitudinally to expose the vessels that run its length. Using forceps and scissors, remove these vessels, which include two arteries and a vein.

Now, from two or three fragments, collect the jelly-like matrix surrounding the vessels. Scrape it away with a scalpel. Then mince the jelly-like matrix, also known as Wharton's Jelley, into fine pieces.

Next, transfer the jelly into a 50 milliliter tube with 15 milliliter of 0.1%collagenase type 1A in PBS and incubate the tissue at 37 degrees Celsius with gentle shaking for three to four hours until it is completely dissolved. Next, centrifuge the digested tissue and aspirate the supernatant. Now, we suspend the digested tissue in 15 milliliters of PBS and mix by pipetting.

Then, repeat this wash with PBS three times using a slower centrifugation. After the last wash, resuspend the cells in 15 milliliters of PBS and then use a 100 micron cell strainer to filter the cells and centrifuge for five minutes at 150 times g to collect the cells. Resuspend the cells in 10 milliliters of freshly made culturing medium at 37 degrees Celsius.

Transfer the suspension into a 25 square centimeter culture flask and incubate the cells, changing the medium every third day. When the cells reach 70 to 80%confluence, passage them, aspirate the medium, and wash the cells twice with room temperature PBS without antibiotics. Then use three milliliters of 0.25%trypsin edta to detach the cells.

End the reaction with a six milliliter addition of warm medium. Collect the suspended cells and pellet them. Discard the supernatant and resuspend the pellet in one milliliter of medium.

To complete the passage, reseed a tissue culture flask with 5, 000 cells per square centimeter and continue growing the culture. To begin, prepare fresh Chitosan solution. Then load each well of a 12 well plate with 500 microliters of solution.

Swirl the plate to distribute the solution over all surfaces. Then, dry the plate under a laminar flow cabinet. After 16 to 24 hours, at thin film of chitosan will form in the plate wells.

Neutralize this film by adding one milliliter of 0.5 normal sodium hydroxide to each well. Then, incubate the plate for two hours at room temperature. Later, aspirate the sodium hydroxide from each well and then wash each well with one milliliter of distilled water.

Perform three water washes then wash each well with one milliliter of 70%ethanol once. After removing the ethanol wash, replace it with another milliliter of 70%ethanol and incubate the plate overnight in a laminar flow cabinet to sterilize the chitosan film. Then next day aspirate the remaining ethanol from each well and wash each well with PBS three times.

Remove the PBS then further sterilize the chitosan film using an overnight direct exposure to ultraviolet light without the lid. Now, to form spheroids of umbilical cord derived mesenchymal stem cells, seed the wells with 20, 000 cells and incubate the plate under normal or hypoxic conditions. Place an anesthetized rat between two 0.5 liter bottles filled with warm water and cover the bottles with a cloth to keep the rat warm while not incurring skin injuries.

To reduce risk of hypothermia, cover the body with bubble wrap. Next, to avoid skin trauma, apply hair remover cream over both stifles and use a tongue depressor to remove the cream and hair. Then, tape each limb to the table.

Now, scrub the surgical site with chlorhexidine digluconate and rinse it with 70%ethanol. Do this three times and proceed with the surgery. With a sterile number 15 scalpel, incise the skin in a proximal to distal direction on the cranial medial aspect of the stifle.

Start the incision about one centimeter proximal to the level of the patella and extend it approximately five millimeters distally to the tibial tubercle. Next, free the underlying subcutaneous tissue with the scalpel and reflect the skin to expose the patellar tendon. Now, align a 0.99 millimeter diameter kirschner wire against the patellar tendon between the distal aspect of the patella to the tibial tuberosity.

Then, while keeping the wire stable, use the scalpel to make incisions on both sides of the wire, releasing a portion of tendon about one millimeter wide. Then resect the central section proximally and distally using fine iris scissors. In parallel, have an assistant prepare the clot.

To do so, thaw the plasma sample and mix 20 microliters of it with half a million mesenchymal stem cells. Next, in a microcentrifuge tube, mix six microliters of 10%calcium chloride with 100 microliters of thawed plasma, this will activate that plasma. Then, mix the cell suspension into the activated plasma, this will cause the plasma to clot.

Now, place the prepared clot within the patellar tendon defect. Limit the manipulation of the clot to minimize fluid release from the clot. Then, using five o polyglactin nine 10 sutures, carefully close the fascia with a cruciate pattern without interfering with the clot.

Finish by closing the skin with an intradermal pattern. Cells were isolated from the umbilical cords of six mares and cultured under standard conditions or on chitosan films. The cells cultured on chitosan formed spheroids.

The patellar tendon defect model was tested on many rats and a battery of behavioral tests were used to estimate the recovery. By 28 days post operative, the rats had returned to baseline strength after an initial deficit in strength measured at seven days post operatively. Upon histological examination with masson's trichrome stain, moderate arterial and capillary infiltration was noted along with moderate to extensive collagen formation whereas cartilage formation was not supported.

Overall, the bilateral patellar tendon defect model limits interindividual variation without significant morbidity. Thus, reducing the number of animals needed for testing. After watching this video, you should have a good understanding of how to create a rat bilateral patellar tendon window defect model and how rats recover from this surgery.

Once mastered, the procedure can be done under 30 minutes with three investigators. The first investigator prepares the cell, the second performs the surgery, while the third assists with the surgery and monitors anesthesia. Following this procedure, methods such biomechanical testing, histology and immunohistochemistry can be used in order to compare the effect of treatment on tendon healing.