Printing bone : the application of 3D fiber deposition for bone tissue engineering

Bone chips are used by orthopaedic surgeons for treating spinal trauma and to augment large bone defects. A potential alternative to autologous bone is regeneration of bone tissue in the lab by developing hybrid implants consisting of osteogenic (stem) cells seeded on supportive matrices. Application of large bone grafts in the operation room is not a clinical reality yet due to, among other factors, cell death in de core of cm-scale implants. One of the reasons for this is the time needed for vascularisation of the grafts after in vivo implantation. Also, current hybrid grafts are rather simplistic often comprising of one cell type seeded on one type of a biomatrix, while native bone tissue consists of multiple cells, matrix molecules and growth factors each with own spatiotemporal pattern of presentation. For development of functional, large-size grafts it would be attractive to employ techniques that approach the complex organization of bone tissue. Organ- or tissue printing is a novel approach of regenerative medicine, which enables mimicking anatomical organization of tissues by developing 3D-structured cell-laden multimaterial scaffolds. In this thesis we studied the application of 3D fiber deposition (an organ printing technique) for development of (vascularized) bone grafts. We first present a literature overview addressing studies that describe a positive effect biomimicking approaches the functionality of tissue-engineered grafts, and define parameters necessary for printing of cell-laden constructs in their application as bone grafts. Hydrogel matrices are highly hydrated polymer networks used as scaffolding materials in organ printing. Of all synthetic hydrogels, photopolymerizable hydrogels formed by UV-exposure of photosensitive polymers in the presence of photoinitiators, are currently one of the most promising materials for skeletal TE due to their mechanical stability. The influence of photoexposure on cell-cycle progression, which is often cautioned for, is well tolerated by encapsulated cells in hydrogels. One of the other principal printing components is the 3D fiber deposition machine, and we tested its applicability to print cell-laden hydrogels, demonstrating that osteogenic progenitors and hydrogels can be deposited simultaneously. We showed that osteogenic progenitors survive the deposition process and retain the ability to differentiate after printing. Based on our findings we conclude that 3DF is a well-suited tool for printing of viable osteogenic grafts. Tailoring of dispensing parameters such as fiber distance, orientation, pressure, and speed was employed to modulate the characteristics of the printed scaffolds including their porosity and mechanical properties. Porous scaffold design is vital for functionality of embedded cells in vitro and supports tissue development in vivo, while addition of calcium phosphate microparticles is useful to further enhance bone formation. The second part of this thesis addressed the development of multicellular grafts whereby heterogeneous grafts demonstrate retention of cell organization introduced by the printing and result in heterogeneous matrix formation, both for vascularised bone grafts and osteochondral implants. Cumulatively, these results demonstrate the possibility of manufacturing viable and functional heterogeneous tissue constructs by a 3D fiber deposition technique, which could potentially be used for the repair of bone defects and osteochondral lesions

The osteoinductive potential of printable, cell-laden hydrogel-ceramic composites.

Hydrogels used as injectables or in organ printing often lack the appropriate stimuli to direct osteogenic differentiation of embedded multipotent stromal cells (MSCs), resulting in limited bone formation in these matrices. Addition of calcium phosphate (CaP) particles to the printing mixture is hypothesized to overcome this drawback. In this study we have investigated the effect of CaP particles on the osteoinductive potential of cell-laden hydrogel-CaP composite matrices. To this end, apatitic nanoparticles have been included in Matrigel constructs where after the viability of embedded progenitor cells was assessed in vitro. In addition, the osteoinductive potential of cell-laden Matrigel containing apatitic nanoparticles was investigated in vivo and compared with composites containing osteoinductive biphasic calcium phosphate (BCP) microparticles after subcutaneous implantation in immunodeficient mice. Histological and immunohistochemical analysis of the tissue response as well as in vivo bone formation revealed that apatitic nanoparticles were osteoinductive and induced osteoclast activation, but without bone formation. The BCP particles were more effective in inducing elaborate bone formation at the ectopic location. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A: 2412-2420, 2012

Genetic marking with the Delta LNGFR-gene for tracing goat cells in bone tissue engineering

The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (DeltaLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40-60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with DeltaLNGFR is applicable to trace goat BMSC's in bone tissue engineering researc

Tungsten melting and erosion under plasma heat load in tokamak discharges with disruptions

Full tungsten poloidal and mushroom limiters were tested in series of experiments with disruptions in the T-10 tokamak. Significant melting, formation of small craters and erosion of the tungsten limiter have been observed after ∼400 discharges with disruption. A theoretical description of the tungsten erosion at disruption in tokamak plasma is presented. The proposed model was verified by comparison with experimental observations in the T-10. The results are used for the erosion prediction of the ITER tungsten divertor. Keywords: Fusion reactor materials, Tungsten, Combining heat and particle load, Disruption, Tokamak, ITE