A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine. We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

Various therapeutic approaches, e.g. in case of trauma or cancer require the transplantation of autologous tissue. Depending on the size and the origin of the harvested tissue, these therapies can lead to iatrogenic complications and donor site morbidities. In the future, these side effects could be avoided by transplanting artificially generated tissue consisting of different cells types and matrix components derived from the host body. Tissue that is grown in the patient could be advantageous compared to the more simply structured in vitro grown alternatives. In order to overcome the limitations of graft vascularisation the arteriovenous (AV) loop technique has been established for different tissues in the last years and was adapted for lymphatic tissue engineering in the present study. We utilized the AV loop technique to grow human lymphatic vasculature in vivo in the RNU nude rat. A combination of human lymphatic endothelial cells (LEC) and bone marrow-derived mesenchymal stem cells was implanted in a fibrin matrix surrounding the AV loop. After 2 or 4 weeks of implantation the animals were perfused and the tissue was harvested. It could be demonstrated by immunohistochemistry for human LYVE1, human CD31 and murine podoplanin, that the implanted cells formed human lymphatic vasculature in the AV loop chamber. Beside development of murine podoplanin positive vasculature in the AV loop tissue, vasculature positive for human marker proteins developed in comparable numbers. This suggests that implanted LEC are able to improve the lymphatic vascularisation of the newly engineered tissue. Thus, we were able to establish an in vivo tissue engineering method to generate lymphatic vascularized soft tissue. An axially vascularized transplantable lymphatic vessel network was engineered without requiring advanced cell culture equipment, rendering the lymphatic AV loop highly suitable for applied regenerative medicine.

Animal models are important tools to investigate the pathogenesis and develop treatment strategies for breast cancer in humans. In this study, we developed a new three-dimensional in vivo arteriovenous loop model of human breast cancer with the aid of biodegradable materials, including fibrin, alginate, and polycaprolactone. We examined the in vivo effects of various matrices on the growth of breast cancer cells by imaging and immunohistochemistry evaluation. Our findings clearly demonstrate that vascularized breast cancer microtissues could be engineered and recapitulate the in vivo situation and tumor-stromal interaction within an isolated environment in an in vivo organism. Alginate–fibrin hybrid matrices were considered as a highly powerful material for breast tumor engineering based on its stability and biocompatibility. We propose that the novel tumor model may not only serve as an invaluable platform for analyzing and understanding the molecular mechanisms and pattern of oncologic diseases, but also be tailored for individual therapy via transplantation of breast cancer patient-derived tumors.