The overall goal of this microsurgical approach is to generate an arteriovenous loop for engineering axioli, vascularized, and translatable tissues, and analyzing vascularization in vivo and in isolated and well characterized environment. With this method, we can use our own bodies as living bio reactors for the generation of different kinds of axiolly vascularized tissue tailored to the individual patient's requirements. The main advantage of this technique is that the generated tissue can be translated with its vascular access and connected to local vessels at the recipient's side for immediate supplying with oxygen and nutrients.
In addition to tissue engineering studies, this method can provide insight into the angiostenosis processes in a living organism. It can also be applied to pathological situations, such as cancer angiogenesis. Generally, individuals new to this method will struggle because of the high complexity of the surgery, such as the micro anastomosis of the submillimeter vessel.
We first had the idea for this method when we were confronted with clinical cases with large tissue defects, which required the transplantation of atholo vascularized tissue creating a significant donor side mobility. After confirming proper anesthesia, weigh the rat to calculate the drug doses. Then, apply eye ointment to prevent dryness while under anesthesia.
After administrating pain medications and antibiotics, place the rat over a heating pad and administer continuous isoflurane. Then, reconfirm the level of anesthesia using a toe pinch. If possible, also use pulse oximetry to check the rat's anesthesia level.
Proceed by shaving the inner sides of the hind limbs. And then disinfecting the surgical area with alternative scrubs of betadine and alcohol. Then, spread the hind limbs and secure them with adhesive tape.
Finally, position the rat under a surgical microscope and drape it. Proceed with the surgery using steril procedures. Open the skin in the middle of the left thigh with a longitudinal incision from the upper knee to the groin using a scalpel.
Next, using dissecting scissors and micro forceps, cut the subcutaneous tissue and fascia in layers that are about three centimeters long. Stop when the femoral vascular bundle is exposed from the pelvic artery in the groin to the knee. Next, using admin tissue scissors and micro forceps separate the vessels and remove the admin tissue.
As needed, coagulate the side branches using electric coagulation. Once completed, cover the operated field with a damp compress and repeat the procedure on the right side of the rat. Now, with the first procedure completed symmetrically, commence with harvesting the venus graft.
First, ligate the right femoral vein using electric coagulation on the proximal and distal ends to obtain a graft of about one to 1.5 centimeters. Next, remove the venus graft with micro forceps. For dilating the opening of the venus graft, use the vessel dilator.
Then, flush the venus graft with a haperin solution using an irrigation cannula. Transfer the fleshed graft to the left thigh and cover the field at the right side with a damp compress. Next, ligate the femoral vein on the left side proximately at the inguinal region with a mico vessel clamp.
Then coagulate the femoral vein distally at the upper knee before the branching. For the anastomosis, connect the proximal end of the venus graft with the proximal end of the vein using an end to end technique with an 11-0 suture. Begin with placing the first two sutures at the 12 o'clock and the six o'clock positions.
Then, put in two to three more sutures between these points on the front side. And then put two to three more sutures into the back side. The anastomosis of the loop vessels is the most critical step of the protocol.
It is important to make sure that the sutures are performed as accurately as possible to reduce the risk of and closure of the loop vessels. Next, ligate the femoral artery with a micro vessel clamp and use electronic coagulation, similarly to ligating the femoral vein. Before proceeding, make sure the looped vessels are not twisted.
Then, anastomose the distal end of the venus graft with the proximal end of the artery, using the same suture technique as demonstrated previously. After the second anastomosis, administer 25 iu of heparan intravenously. Now, double check that loop vessels are not twisted and then remove the clamps.
Observe the loop for about five minutes, checking for leakage and patency. If there is patency, the loop will expand and the pulse of the artery will be visible. During the observation, dripple papaverine on the vessels to prevent vascular spasms.
Next, pre-fill the implantation chamber with the first half of the matrix mixture, about half a millileter. Then, embed the loop into the implantation chamber. Close the chamber entrance with two clots of fibrin and fill the chamber with the second half of the matrix mixture, for a total volume of one millileter.
Now, prefix the chamber on the thigh, seal the chamber with its lid, and secure the lid with a nonabsorbable 6-0 suture. Then, secure the implantation chamber to the thigh with an additional nonabsorbable 6-0 suture. Stop any bleeding using electric coagulation.
Finally, close the skin with absorbable 4-0 sutures and cover the wound with aluminum spray. For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat A-V loop model. Vascularization was observed using 3D micro computer tomography.
Pre-vascularization of a processed bovine cancellus bone matrix for six weeks led to superior survival of the injected osteoblasts. Loops were harvested, sectioned, and stained at various time points post implantation. For example, H and E staining showed that use of a beta tricalcium phosphate hydroxyapatite bone substituent with mesenchymal stem cells resulted in many vessel connections within six weeks.
Once mastered, this technique can be done in five to six hours, if it is performed properly. Following this procedure, a large variety of different The various cells factors or anti can be implanted to study a wide area of conditions. This technique also works in a large animal sheep venus loop model.
So it should allow researchers to generate vascularized tissues in large clinically relevant amounts. This model may provide the opportunity to study vasularization under pathological conditions. For example, the influences of various growth factors and cells during tumor vessel network formation could be investigated
