Initial methods for the engineering of thick vascularised tissues

Breast reconstruction is typically required after mastectomy in patients suffering from breast cancer. Standard methods comprise autologous grafting, thus replacing the tissue with tissue from another part of the body from the host or replacement through tissue from a donor, which is denoted as allograft transplantation. However, if the host has no superfluous tissue available, an autologous replacement is not possible. Also, donor site morbidity represents a negative aspect of this approach. Allograft transplantation is known for an immune response from the host and subsequent rejection of the tissue which would imply another operation. Both of these approaches are costly and risky.
In tissue engineering, the objective is to replace, repair or improve a tissue or even an organ. Thereby synthetic or natural materials can be used. An engineered tissue has to be composed of an extracellular matrix which is responsible for stability and hosting of the cells. The goal of an engineered tissue is that after implantation in vivo it is able to maintain cell density and cell survival and to fulfill the required biological function of the tissue. For tissues bigger than 400 _m, a vasculature is needed to avoid graft necrosis by delivering nutrients and removing cell waste products. Thus, the challenge nowadays is to generate and engineered tissue in vitro with embedded vasculature which can then do anastomosis with the graft vascularisation ones implanted in the patient. Here we attempted to simplify the production of the engineered tissue with cost-effective and time-effective reproducible methods to have a basis for further studies. Gelatin enzymatically crosslinked with Transglutaminase was chosen as a scaffold for the cells and its biocompatibility for adipose derived stromal cells was investigated for four different gelatin concentrations (6%, 8%, 10%, 12%) and the effect of the different stiffness of the gelatin on the cells. Also the diffusional characteristic of this hydrogel were tested with FITC dextran and ultimately a design for a multichannel gelatin was made for the further perfusion experimentations in future.
It was shown, that every gelatin concentration was biocompatible to the ASC’s and that there was no change in morphology of the cells. Also with 3D construction of z-stacks it could be made visible, that after 7 days of seeding of ASC on top of the gelatin, some cells migrated through the gelatins and this for all concentrations. Thus, for stability reasons of the gelatin, the 12 % gelatin can be used for further experiments. The fluorophore FITCdextran was shown to diffuse through the gelatin and lastly, a multichannel mold and a personalised perfusion set up for channel perfusion could be planned.
These first experiments can be the basis for trying to engineer a multiple channel gelatin which will be seeded with ASCs and growthfactors and afterwards perfused with culture medium in a long term test where the hypothetical endothelization of the channels by differentiation of ASCs could be observed with the Confocal laser scanning microscope. Once this result has been obtained, complex vasculature geometry may be produced by using 3D printing techniques which could represent a basis for an in vivo transplantation of a thick engineered tissue in the body of a human.