Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.

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In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

Generation of axially vascularized bioartificial bone might be performed using matrix neovascularization in connection with osteoblast injection. We sought to evaluate whether prevascularization of porous hard matrices using an arteriovenous (AV) loop promotes survival of transplanted osteoblasts. A processed bovine cancellous bone matrix was inserted into the AV loop. Six weeks later, 5 x 10(6) carboxyfluorescein diacetate-stained osteoblasts were injected into the matrix (group A, n = 34). Osteoblast-seeded matrices without prevascularization were implanted subcutaneously as controls (group B, n = 32). Specimens were subjected to histologic, morphometric, and molecular-biological analysis after 1, 4, 8, and 16 weeks. Upon cell injection, matrices were completely vascularized. An intense foreign body reaction was observed in matrices from both groups. Group A was significantly superior to group B in terms of osteoblast survival at any time point. Expression of bone-specific genes was detected in the AV loop group but not in the subcutaneous control. Bone formation was only detectable in 1 long-term animal of group A. This study demonstrates for the first time that axial prevascularization increases the survival of implanted osteoblasts in porous matrices. Matrices with optimized biocompatibility might eventually facilitate generation of axially vascularized bone tissue after injection of osteogenic cells in the AV loop model.

Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor-site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β-tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto-transplanted bone marrow aspirate from the iliac crest. The following post-operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio-venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain-free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor-site defect utilizing TE and RM techniques in human patients with long-term stability.

In addition to good mechanical properties needed for three-dimensional tissue engineering, the combination of alginate dialdehyde, gelatin and nano-scaled bioactive glass (45S5) is supposed to combine excellent cellular adhesion, proliferation and differentiation properties, good biocompatibility and predictable degradation rates. The goal of this study was to evaluate thein vitro and in vivo biocompatibility as a first step on the way to its use as a scaffold in bone tissue engineering. In vitro evaluation showed good cell adherence and proliferation of bone marrow derived mesenchymal stem cells seeded on covalently crosslinked alginate dialdehyde-gelatin (ADA-GEL) hydrogel films with and without 0.1% nano-Bioglass®(nBG). Lactate dehydrogenase (LDH)- and mitochondrial activity significantly increased in both ADA-GEL and ADA-GEL-nBG groups compared to alginate. However, addition of 0.1% nBG seemed to have slight cytotoxic effect compared to ADA-GEL. In vivo implantation did not produce a significant inflammatory reaction, and ongoing degradation could be seen after four weeks. Ongoing vascularization was detected after four weeks. The good biocompatibility encourages future studies using ADA-GEL and nBG for bone tissue engineering application.

The VRAM flap is a reliable and safe method for pelvic reconstruction in patients with advanced disease requiring pelvic exenteration and irradiation, with a relatively low rate of donor and recipient site complications. In this first study, to compare a large number of patients with VRAM flap reconstruction to patients without pelvic VRAM flap reconstruction, a clear advantage of simultaneous pelvic reconstruction is demonstrated.

Vascularization still remains an obstacle to engineering of bone tissue with clinically relevant dimensions. Our aim was to induce axial vascularization in a large volume of a clinically approved biphasic calcium phosphate ceramic by transferring the arteriovenous (AV) loop approach to a large animal model. HA/β-TCP granula were mixed with fibrin gel for a total volume of 16 cm 3 , followed by incorporation into an isolation chamber together with an AV loop. The chambers were implanted into the groins of merino sheep and the development of vascularization was monitored by sequential non-invasive magnetic resonance imaging (MRI). The chambers were explanted after 6 and 12 weeks, the pedicle was perfused with contrast agent and specimens were subjected to micro-computed tomography (µ-CT) scan and histological analysis. Sequential MRI demonstrated a significantly increased perfusion in the HA/β-TCP matrices over time. Micro-CT scans and histology confirmed successful axial vascularization of HA/β-TCP constructs. This study demonstrates, for the first time, successful axial vascularization of a clinically approved bone substitute with a significant volume in a large animal model by means of a microsurgically created AV loop, thus paving the way for the first microsurgical transplantation of a tissue-engineered, axially vascularized bone with clinically relevant dimensions.