Andreas Arkudas

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

A new approach to tissue engineering of vascularized skeletal muscle.

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo‐tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous‐loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantation of carboxy‐fluorescine diacetate‐succinimidyl ester (CFDA)‐labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin‐matrices without AV‐loops. Intra‐arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA‐fluorescence, still showing positive MyoD staining. RT‐PCR revealed gene expression of MEF‐2 and desmin after 4 weeks on the AV loop side, whereas expression analysis of myogenin and MHCembryo was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre‐vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle‐like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.

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.

Successful human long-term application of in situ bone tissue engineering

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.

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

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.

Tissue engineering and regenerative medicine -where do we stand?

Introduction Intrinsic and extrinsic vascularization of TE constructs – the AV‐loop model Gene Transfer Techniques Combining mesenchymal stem cells with the AV‐loop model of intrinsic vascularization TE and RM in the context of Cancer Research Newly discovered cells of potential benefit for RM Summary

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.