Oliver Bleiziffer

Translating tissue engineering technology platforms into cancer research

•  Introduction •  History of tissue engineering •  Physiological and structural aspects of 2D versus 3D culture in cancer research •  State of the art of 3D culture systems in cancer research •  New tissue engineering‐routed scaffolds for 3D culture •  Endothelial progenitor cells and tumour vasculature •  In vivo models •  Arteriovenous loop isolation chamber for tumour angiogenesis research •  Conclusion

Gene transfer strategies in tissue engineering

•  Introduction •  Methods of gene delivery ‐  Non‐viral techniques ‐  Naked DNA ‐  Cationic liposomes ‐  Viral techniques ‐  Retrovirus ‐  Adenovirus ‐  Adeno‐associated virus ‐  Herpes simplex virus •  Gene delivery from scaffolds for tissue engineering •  Gene therapy applications in tissue engineering ‐  Skin and wound healing ‐  Cartilage ‐  Bone ‐  Nerve ‐  Liver and endocrine pancreas ‐  Blood vessels •  Outlook

Impaired wound healing in an acute diabetic pig model and the effects of local hyperglycemia.

Diabetic wounds result in significant morbidity, prolonged hospitalization, and enormous health‐care expenses. Pigs have been shown to have wound healing resembling that in humans. The aim of this study was to develop a large‐animal model for diabetic wound healing. Diabetes was induced by streptozotocin injection in Yorkshire pigs. Full‐thickness wounds were created and dressed with a sealed chamber. Nondiabetic pigs with or without high glucose wound fluid concentration served as controls. Glucose concentration in serum and wound fluid was measured and collected. Wound contraction was monitored, and biopsies were obtained for measurement of reepithelialization. Wound fluid was analyzed for insulin‐like growth factor‐1 (IGF‐1), platelet‐derived growth factor, and transforming growth factor. Glucose concentration in wound fluid initially followed serum levels and then decreased to undetectable on day 9. Reepithelialization was significantly delayed in diabetic pigs. In nondiabetic pigs, wounds treated in a local hyperglycemic environment, and thus excluding the effects of systemic hyperglycemia, showed no difference in wound closure compared with controls. This suggests that delayed wound healing in diabetes is not induced by local high‐glucose concentration itself. Analysis of growth factor expression showed a marked reduction in IGF‐1 in the diabetic wounds. Diabetic pigs have impaired healing that is accompanied by a reduction of IGF‐1 in the healing wound and is not due to the local hyperglycemia condition itself.

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle

BackgroundTissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA®-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin.ResultsWhereas rapid lysis and contraction of pure collagen I- or fibrin-matrices have been great problems in the past, the latter could be overcome by combining both materials. Significant proliferation of cultivated myoblasts was detected in collagen-I-fibrin matrices and collagen nanofibers. Seeding cells on parallel orientated nanofibers resulted in strongly aligned myoblasts. In contrast, common collagen sponges and OPLA®-scaffolds showed less cell proliferation and in collagen sponges an increased apoptosis rate was evident. The application of genipin caused deleterious effects on primary myoblasts.ConclusionCollagen I-fibrin mixtures as well as collagen nanofibers yield good proliferation rates and myogenic differentiation of primary rat myoblasts in vitro In addition, parallel orientated nanofibers enable the generation of aligned cell layers and therefore represent the most promising step towards successful engineering of skeletal muscle tissue.

Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model.

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 cm3, 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. Copyright

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto‐transplanted versus in vitro expanded MSC with or without bone morphogenetic protein‐2 (BMP‐2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto‐transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT‐PCR analysis before subcutaneous implantation in combination with BMP‐2 and β‐tricalcium phosphate/hydroxyapatite (β‐TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT‐PCR evaluation. Sheep MSC were CD29+, CD44+ and CD166+ after selection by Ficoll gradient centrifugation, while directly auto‐transplanted MSC‐populations expressed CD29 and CD166 at lower levels. Both, directly auto‐transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto‐transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β‐TCP/HA matrix comparable to the application of 60 μg/ml BMP‐2 only or implantation of expanded MSC. Bone matrix proteins were up‐regulated in constructs following direct auto‐transplantation and in expanded MSC as well as in BMP‐2 constructs. Up‐regulation was detected using immunohistology methods and RT‐PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto‐transplanted or expanded MSC with β‐TCP/HA granules alone. Hence BMP‐2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.

Automatic quantitative micro-computed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct.

INTRODUCTION We invented an automatic observer-independent quantitative method to analyze vascularization using micro-computed tomography (CT) along with three-dimensional (3D) reconstruction in a tissue engineering model. MATERIALS AND METHODS An arteriovenous loop was created in the medial thigh of 30 rats and was placed in a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix, filled with fibrin (10 mg/mL fibrinogen and 2 IU/mL thrombin) without (group A) or with (group B) application of fibrin-gel-immobilized angiogenetic growth factors vascular endothelial growth factor (VEGF¹⁶⁵) and basic fibroblast growth factor (bFGF). The explantation intervals were 2, 4, and 8 weeks. Specimens were investigated by means of micro-CT followed by an automatic 3D analysis, which was correlated to histomorphometrical findings. RESULTS In both groups, the arteriovenous loop led to generation of dense vascularized connective tissue with differentiated and functional vessels inside the matrix. Quantitative analysis of vascularization using micro-CT showed to be superior to histological analysis. The micro-CT analysis also allows the assessment of different other, more complex vascularization parameters within 3D constructs, demonstrating an early improvement of vascularization by application of fibrin-gel-immobilized VEGF¹⁶⁵ and bFGF. CONCLUSIONS In this study quantitative analysis of vascularization using micro-CT along with 3D reconstruction and automatic analysis exhibit to be a powerful method superior to histological evaluation of cross sections.

Dose-Finding Study of Fibrin Gel-Immobilized Vascular Endothelial Growth Factor 165 and Basic Fibroblast Growth Factor in the Arteriovenous Loop Rat Model

The angiogenic effects of different concentrations of vascular endothelial growth factor (VEGF) 165 and basic fibroblast growth factor (bFGF) immobilized in a fibrin-based drug-delivery system were quantitatively assessed in the arteriovenous (AV) loop model. An AV loop was created in the medial thigh of 60 rats. The loop was placed in a Teflon isolation chamber and embedded in 500 microL of fibrin gel loaded with VEGF and bFGF in four different concentrations (no growth factor, 100 ng/mL of VEGF, 25 ng/mL of VEGF and bFGF, 100 ng/mL pf VEGF and bFGF). The explantation intervals were 1, 2, and 4 weeks after the initial operation for all groups. Specimens were investigated using (micro-CT) and histological and morphometrical techniques. After 2 weeks, the cross-section area and construct weight were significantly lower with the use of 100 ng/mL of VEGF and bFGF. Micro-CT and histology showed significantly greater vascular density and number of vessels of the constructs at 2 and 4 weeks when 100 ng/mL of VEGF165 and bFGF were applied than in the growth factor-free specimens. The angioinductive effects were dose-dependent, with best results when using 100 ng/mL of VEGF165 and bFGF. The greater tissue formation was accompanied by faster resorption of the fibrin matrix.

Engineering axially vascularized bone in the sheep arteriovenous‐loop model

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)‐loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2), were harvested and directly autotransplanted in combination with β‐tricalcium phosphate–hydroxyapatite (β‐TCP–HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP‐2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β‐TCP–HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro‐computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV‐loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP‐2 stimulation in about 12 weeks in the sheep AV‐loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies. Copyright

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix.

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue‐engineered matrices. The aim of our study was to investigate the impact of a well‐characterized murine embryonal EPC line (T17b‐EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time‐points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β‐galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell‐free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.