Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.
The overall goal of the following procedure is to transplant autologous tissue for its expansion during reconstructive surgery. This is accomplished by first harvesting a biopsy from the tissue of interest. Next, the biopsy is minced and a plastic compressed polycapro-lactone, or PCL, collagen autograft is constructed.
The PCL and otologous minced tissues are then integrated to create the 3D scaffold. We have used a one to six expansion rate. Next, the scaffold is transplanted back into the subject.
Ultimately, the regeneration of the autografted tissue can be assessed by immunohistochemical analysis of the proliferation and reorganization of the cells within the scaffolds and minced tissue particles. This method will be demonstrated in an animal model, on skin, and on urothelium. The implication of this technique extends toward the treatment of severely malformed tissues or after trauma in instances where there's severe lack of tissue for the surgical repair.
The main advantage of this technique over existing methods, like in vitro culturing, is that this method can be performed as a one-stage surgical procedure. Visual demonstration of this method is critical, as the steps from the tissue harvest to the transplantation need to be performed under sterile conditions. To obtain a porcine bladder biopsy specimen, first, grasp the bladder with the forceps, and use a sterilized measuring tape to determine the dimensions of the tissue.
Next, use a sterilized pen to mark an ellipsis shaped biopsy two by one centimeter, aiming for a one to six expansion rate. Then, excise the marked tissue with a scalpel, and place the bladder biopsy specimen in DMEM under sterile conditions. To harvest a skin biopsy specimen, use a dermatome to harvest a 0.3 millimeter partial thickness specimen.
Then, place the tissue in DMEM and cover the wound area with a dressing. Next, wash the bladder biopsy twice in DMEM and transfer the tissue onto a sterilized dissecting plate, with the mucosa facing upwards. Using dissection pins, fix one side of the specimen to the plate and use fine scissors and forceps to separate the mucosal tissue from the detrusor muscle.
Then, place a mincing device on top of the mucosa and pass it vertically and horizontally from one end of the tissue to the other to obtain 0.8 by 0.8 millimeter pieces of minced tissue. Note that the mincing device needs to be pressed on the tissue while running over it. To prepare a plastic compressed collagen autograft, first add one normal sodium hydroxide drop by drop into 10X DMEM supplemented with collagen type I until the medium turns from intense yellow to pink.
After confirming a pH of 7.4 to 8, carefully mix two milliliters of DMEM into the solution. Then, plate approximately two milliliters of collagen into a well of a 20x30x10 millimeter steel rectangular mold and incubate the collagen for ten minutes. When the collagen has set, plate the appropriate volume of PCL biomaterial on top of the gelled collagen and pour the remaining six milliliters of collagen on top of the PCL.
Place the mold back into the incubator for another 20 minutes. Next, stack a thick layer of gauze pads onto a sterile surface. Then, place a 400 micron thick stainless steel mesh on top of the gauze, followed by a 110 micron thick sheet of nylon mesh.
Gently transfer the collagen gel onto the nylon mesh and carefully remove the rectangular steel mold. Transfer the minced tissue onto the collagen. Here, we aim at a one to six expansion rate.
Then, place a new layer of nylon mesh on top of the collagen gel and tissue, then place a second steel mesh on top of the nylon mesh, followed by a loading plate weighing a minimum of 120 grams. After five minutes, remove the weight, steel meshes, and nylon from the autograft. For in vitro culture of the autograft, first cut the thinned construct into pieces small enough to fit into 12 well plates.
Then, add one milliliter of keritinocyte medium to each well and incubate the tissues for three to six weeks. To transplant an autograft with minced bladder mucosa to a pig bladder, moisten the graft with DMEM. Then, graft the construct to the healthy tissue with fine running monofilament sutures.
Confirm that the graft is water tight by filling the bladder with saline through the indwelling urinary catheter. To transplant an autograft with minced skin epidermis to a full thickness wound, moisten the graft with DMEM. Then, using interrupted sutures in the corners and middle of the autograft, attach the construct to the bottom of the skin full thickness wound.
Finally, cover the wound with a plastic dressing to keep it moist. As observed in this image of a minced skin autotransplant, the composite biograft can sustain grasping, suturing, and surgical handling. Immunohistochemistry at different time points during the in vitro culture reveals that the cells proliferate, migrate, and reorganize into a continuous epithelium with a microarchitecture typical for the cell phenotype.
For example, at two weeks, the cell layer on the surface of the skin autograft is approximately two layers thick. To the right, bladder epithelium is about eight layers thick after the same culture period. After five to six weeks, the continuous epithelium is approximately eight to ten cell layers thick in both minced skin epithelium and minced bladder mucosa autografts.
Compared to tissue expansion in vitro, this method is quick, less labor intensive, and does not require sophisticated laboratory resources. In addition, all parts are FDA approved, allowing their use in standard surgical setting. While attempting this procedure, it's important to remember to prepare the gel, biotransplants, and autologous tissues under sterile conditions.
Following this procedure and other methods like in vitro culture of the cells can be performed to answer additional questions about cell proliferation and differentiation. After its development, this technique may pave the way for researchers in the field of regenerative medicine. By these means, we could treat severe malformations in the skin or urinary system with autologous tissue engineered transplants.
