The overall goal of the following experiment is to observe the effects of perivascular stem cell implantation in ectopic and orthotopic bone differentiation models. This is achieved by first isolating stem cells from adipose tissue using fax technology. Then custom scaffolds are fabricated to provide a surface for cell differentiation in either an ectopic or orthotopic environment.
To test PSC mediated bone formation skeletal defects are created using a skid mouse muscle pouch model, skid mouse calvarium model, or athymic rat femoral segmental defect model and the engrafted scaffold is pleased within the defect. Radiographic histologic and biochemical analysis reveal that perivascular stem cell differentiation leads to significant bone formation in vivo. The main advantage of using paravascular stem cells over other more traditional stem cell sources is that PSCs or paravascular stem cells are a more homogenous population, which increases stem cell purity, enhances trophic factor production, and potentially increases bone formation.
Begin by creating a scaffold as described in the accompanying manuscript. Prepare the scaffold for implantation by adding cell suspension to it. For the mouse muscle pouch or calvarial defect models, use a density of 250, 000 cells per a hundred microliters after anesthetizing.
Six week old skid mice with isoflurane and buprenorphine. Make a standard Betadine preparation in the hind limb. Next, make a two millimeter longitudinal bilateral incision and use blunt dissection to cut four millimeter deep pockets in the biceps femoris muscles.
Parallel to the muscle fiber only minimal bleeding should be encountered. Insert the implant engrafted with PSCs. Now suture the fascia overlying the muscle with a simple continuous pattern with five oh Vicryl.
Next, close the skin with five oh Vicryl in a subcuticular pattern.Postoperatively. Treat the animals with buprenorphine for 48 hours and T-M-P-S-M-X for 10 days. The mouse should be walking with ease on postoperative day one.
After isof fluorine, anesthesia of a 12 to 14 week old skid mouse. Make a standard Betadine preparation of the calvarial region. Begin with an eight millimeter long skin incision on the mid sagittal suture of the mouse calvarium.
Next, gently remove the calvarial periosteum with a cotton tipped applicator and use an applicator to soak up the bleeding. Now using a diamond coated trephine bit attached to a high speed dental drill, make a four millimeter parietal bone defect. Make the defect full thickness, such as all the way through the bone, but take extreme care not to injure the underlying dura mater as this will interfere with healing.
Now gently place the implant and grafted with PSCs in the defect site. Finally, suture the skin with six oh Vicryl postoperatively. Treat the animals with buprenorphine for 48 hours and T-M-P-S-M-X for 10 days.
Begin by scrubbing and preparing the femur of a 12 to 14 week old amic rat, which has been anesthetized with Isof fluorine. Used the standard Betadine protocol. Make a 27 to 30 millimeter longitudinal incision over the anterior lateral aspect of the femur.
Next, expose the femoral shaft by separating the vast lateralis and biceps femoris muscles to avoid excessive bleeding. Be careful around the major blood vessels. To maximize the consistency of bone regeneration, completely.
Remove the periosteum overlying the femoral defect, along with the resected femoral segment. Avoid damaging the femoral nerve to prevent neurologic damage. Now place a polyethylene plate on the anterior lateral surface of the femur.
Six pre-drilled holes in the plate, accommodate 0.9 millimeter diameter threaded kirschner wires. Using the plate as a template drilled six threaded kirschner wires through the plate, and both cortices. Using a small oscillating saw blade, make a six millimeter mid dile defect.
Then implant the PSC and grafted scaffold into the defect. Close up the overlying muscle and fascia with four oh Vicryl absorbable sutures. To secure the implant in place, give the rat analgesia for two days postoperatively and monitor it for wound dehiscence over four weeks post muscle pouch implantation.
There was a gradual increase in bone volume and bone mineral density as observed with PSC engraftment by micro CT and XR analysis over the course of eight weeks after introducing a calvarial defect, no bone healing was observed without a scaffold due to the critical size of the defect with a scaffold but no cells. There was minor healing estimated around 20%as expected. A gradual increase in ossification of the defect was seen with a seeded scaffold.
Major ossification occurred within two weeks. Complete ossification occurred by eight weeks after introducing a femoral defect. A gradual increase in ossification occurred when the defect was treated with BMP two as early as four weeks union is observed in some animals, and by eight weeks all animals were healed.
In contrast, no bone union was observed without a scaffold or with a scaffold lacking cells or recombinant protein. After watching this video, you should have a good idea of how to isolate paravascular stem cells, how to then utilize paravascular stem cells in combination with PLGA osteoinductive scaffolds, and then finally, how to use these combination scaffold and sell products for three different methods of tissue engineering while attempting to perform each procedure as presented here, there's a few things that's important to keep in mind. First, with the calvarial defect model, precise preservation of the dura matter ensures that proper healing will ensue.
Secondly, for the rat femoral segmental defect model, it's important to minimize trauma to the surrounding nerves and blood vessels. Once an individual masters a certain surgical technique as presented here, you can expect that each muscle pouch implantation takes approximately 20 minutes to execute, and each calvarial defect and femoral segmental defect may take up to 30 minutes to properly execute.
