Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

The overall goal of the following procedure is to use a rodent model to evaluate tissue engineered grafts for anterior cruciate ligament replacement. This is accomplished by first fabricating a biodegradable polymer scaffold. In the second step, the scaffold is implanted into a rat knee.

After 16 weeks, the neo ligaments formed from the scaffold are harvested for histological analysis. Ultimately, the biomechanical strength of the collagen is tested to assess the quality of these tissue engineered grafts for use in ACL reconstruction. The main advantages of this technique over other methods of scaffold evaluation are that this model exposes the scaffold to the complex in vivo environment, while remaining cost-effective and easier to handle than larger animal models.

This model can help answer key questions in the field of tissue engineering, such as how will an engineered scaffold integrate with the in vivo environment to form a neo ligament? This technique can lead to new treatments and ACL reconstruction. As small animal models are the first step to clinical translation, Though this method can be used to evaluate our particular scaffold.

It can also be applied to other tissue engineered contracts such as those with cells and growth factors. We optimize the surgical technique by modifying the methods employed by Dr.Scott Rodeo and colleagues at the hospital for special surgery in New York. Visual demonstration of this method is critical as the electro spinning and surgical steps can be difficult to learn from written instruction alone.

When electro spinning polycaprolactone scaffolds a large acrylic box with entry points for the source polycaprolactone solution. A motor powered collecting mandrel and a vacuum port driven by a voltage source placed within a chemical fume hood will be needed to serve as an isolated vacuum medium for the electro spinning process. Before beginning the procedure, clean the box thoroughly with 70%ethanol and cover all of the surfaces with para film sheets to remove any impurities that may compromise the quality of the electros spun product.

Then turn on the fan and load three milliliters of freshly prepared polycaprolactone polymer into a 10 milliliter syringe with a blunt ended 18 gauge needle. After removing any air bubbles, lock the solution into a programmable syringe pump and insert the needle into the acrylic box through the entry port, leaving about a half inch of the needle outside of the box. Next, cover the rotating lath mandrel tightly with a thin strip of aluminum foil and lock the mandrel into the motor on the opposite side of the box, about 15 centimeters away from the syringe needle to act as the collector insert a plastic hose into the vacuum port.

Then connect the hose to the fume hood vacuum source and turn it on. Now cover the acrylic box with the lid and set the infusion rate of the programmable syringe pump to 2.5 milliliters per hour. Then ground the negative lead turn on the motor to operate the mandrel at 3, 450 RPM and use an alligator clip to attach the positive lead of the voltage source to the needle tip outside of the box.

Once infusion of the polycaprolactone solution has begun to turn on the voltage source and set it to a 20 kilovolt operating voltage. After 12 minutes of infusion, a homogenous cuff will have formed from 0.5 milliliters of the Polycaprolactone solution. Turn off all equipment including voltage, source, motor, vacuum, and syringe pump.

Remove the mandrel from the motor, remove the polymer cuff from the mandrel, and make a single transverse cut yielding a rectangular electro spun mat. Then laser cut the mat to form multiple small sheets, which are later cut into strips stacked four layers thick and sutured together to form scaffolds before starting the surgery. First, confirm adequate anesthetization by lack of a response to hind foot pressure.

Then make a two centimeter long vertical incision medial to the knee centered at the level of the patella, retracting the skin laterally as necessary. Next, use a scalpel to make a medial para patella arthrotomy just medial to the patella, extending proximally to the level of the musculo tendonous junction of the quadriceps and distally to the level of the patella tendon insertion on the tibial tubercle. Now make a one centimeter vertical incision through the knee capsule just lateral to the patella tendon to release the patella laterally.

Then ensuring that the knee is extended, pass a pair of fine scissors under the patella from the lateral to the medial side, and spread the scissors a couple of times so that the extensor mechanism can be translated to either side while flexing the knee. Translate the patella laterally to expose inside of the knee joint, confirming a clear visualization of the intercondylar notch and femoral condyles. Then transect the ACL and PCL in the notch.

Now place the tip of a 1.6 millimeter K wire attached to a power drill on the ACL origin in the intercondylar notch and drill sup laterally while an assistant stabilizes the knee. Then pass the K-wire in and out a few times to ensure clear passage for the graft. Visualize the exit point on the lateral aspect of the femur, removing any soft tissue as necessary with a scalpel.

When the tunnel is clear, place the K-wire on the ACL footprint on the tibial plateau and drill an laterally while an assistant stabilizes the knee. Then visualize the exit point on the anterolateral proximal tibia using the scalpel to clear the soft tissue as necessary so that the point where the K-wire exits the tibia is fully visible and pass a shortened Keith needle no more than two inches long through the femoral bone tunnel. Now thread the two suture ends from one end of the graft through the eye of a needle and use the needle to pull one end of the graft through the femoral tunnel.

Then use the needle to pass the other end of the graft through the tibial tunnel using a four zero Vicryl suture. Next, affix the femoral end of the graft to the surrounding soft tissue with a figure of eight stitch while keeping the knee in extension to create manual tension in the graft. Use another figure of eight stitch to affix the tibial end of the graft to the surrounding soft tissue, cutting off the excess graft on both ends with a pair of scissors and leaving one to two millimeters on each end past the stitch.

Finally extend the knee and reduce the patella. Then using a four zero Vicryl suture place a single figure of eight stitch to close the medial joint capsule, preventing lateral subluxation of the patella and close the skin with a running subcuticular five zero Monocryl or Vicryl suture. At 16 weeks following the reconstruction, histological analysis of the sectioned knee demonstrates that the scaffold matrix becomes largely infiltrated by fibroblasts, secreting eosinophilic collagen with good integration into the bone tunnels.

In this experiment, the biomechanical properties of the joint were assessed immediately after sacrifice. All tested samples failed at the mid substance using low displacement curves generated from tensile testing. Failure load and stiffness were computed for each group.

At 16 weeks post implantation, the electro spon polymer graft had approximately doubled the peak load and stiffness of the graft tested immediately post implantation. Although these values were still lower than the native ACL Once mastered, the animal surgery can be performed in approximately 15 minutes. While attempting this procedure, it's important to remember to perform a good closure in which no suture is visible from the outside.

Following this procedure, other endpoint analyses such as additional staining and biochemical assays can be performed. This technique paved the way for researchers in the field of tissue engineering to evaluate multiple graft options for ACL reconstruction action. After watching this video, you should have a good understanding of how to fabricate a polymer scaffold for implantation in a rodent model of ACL reconstruction.

Don't forget that working with live animals can be hazardous, and that precautions such as abiding by veterinary recommendations and wearing personal protective equipment should be taken at all times while performing this procedure.