Shear Stress in 3D-Bioprinting strongly impacts Human MSC Survival and Proliferation Potential
*A. Blaeser1, D. F. Duarte Campos1, U. Puster1, H. Fischer1
1Universitätsklinikum RWTH Aachen, Zahnärztliche Werkstoffkunde und Biomaterialforschung, Aachen, Deutschland
Cell damage caused by shear stress during 3D-bioprinting is a common problem. Using a micro-valve based bioprinter we conducted a broad study on how shear stress during printing affects cells. We identified three key factors that directly influence shear stress and investigated their impact on cell behavior.
Materials and Methods:
Experiments with varying printing pressure (0.25-3 bar), alginate-hydrogel viscosity (30-300 mPas) and nozzle diameter (150, 300 and 600 μm) were conducted. Their effect on cell viability, proliferation and expression of mesenchymal makers (5 independent donors) was studied for up to seven days. The shear stress occurring in the nozzle was calculated using a fluid dynamical model and correlated to the cell-study results.
Results and Discussion:
Varying the above mentioned parameters we were able to investigate a wide shear stress range (700-11,000 Pa). For shear stress up to 3,000 Pa the post-printing cell viability was 97±1 %. With increasing shear stress cell survival significantly decreased [90±7 % (3,000-6,000 Pa) and 74±21 % (> 6000 Pa)]. Furthermore, MSC printed at a shear stress of 2,500 Pa exhibited significantly higher proliferation rates than those printed at shear stress higher than 3,000 Pa (4,000-10,000 Pa). Instead, the proliferation potential of MSC printed at high shear stress was significantly lower than the non-printed control group.
The results indicate that the level of shear stress is the main factor that impacts cell survival and proliferation potential regardless of how the shear stress was obtained e.g. by high pressure, a highly viscous material or geometric restrictions. Thus, to achieve high cell survival and proliferation all printing parameters need to be outbalanced.
Optimale Auslegung eines künstlichen Adersystems
*R. Jaeger1, J. Courseau1
1Fraunhofer-Institut für Werkstoffmechanik IWM, Polymertribologie, biomedizinische Materialien, Freiburg, Deutschland
Im Labor gezüchtete Gewebemodelle können prinzipiell zur Untersuchung der Verträglichkeit von chemischen Substanzen oder der Wirksamkeit von Medikamenten herangezogen werden. Hierfür ist es notwendig, dass die Zellen des Gewebemodells ausreichend mit Sauerstoff und Nährstoffen versorgt werden können. Generative Fertigungsverfahren ermöglichen es, ein künstliches Gefäßsystem zu fertigen, das für eine ausreichende Versorgung der Zellen des Gewebemodells eingesetzt werden kann. Bei der optimalen Auslegung eines generativ gefertigten Adersystems sollten sowohl die Versorgung der Zellen als auch die Komplexität des Adersystems berücksichtigt werden. Einfacher strukturierte Aderbäume sollten wegen ihrer Robustheit und leichteren Herstellung gegenüber komplexeren Strukturen bevorzugt werden, wenn sie eine vergleichbare Leistungsfähigkeit zeigen.
Materialien und Methoden:
Bei der rechnerischen und experimentellen Ermittlung von Nährstoffkonzentration wurde von einem quadratischen Hydrogelsubstrat ausgegangen, in dem Zellen eingebettet sind und das durch ein Adersystem mit Nährstoffen versorgt wird. Durch die Diffusion der Nährstoffe aus dem Adersystem durch das Hydrogel und deren Verbrauch durch die Zellen stellt sich eine Gleichgewichtskonzentration der Nährstoffe ein. Diese Gleichgewichtskonzentration wurde mit einem finite-Differenzen-Verfahren berechnet. Die räumliche Verteilung der Nährstoffkonzentration hängt unter anderem von der Topologie des Adernetzwerks ab. Die Leistungsfähigkeit des Adersystems wurde mit dem Verhältnis der ausreichend versorgten Fläche zur Gesamtfläche des Hydrogels beschrieben. Experimentell wurde die Leistungsfähigkeit verschiedener Adersysteme anhand der Diffusion eines Farbstoffs in ein unbesiedeltes Poly(vinylalkohol) Cryogel verglichen. Die Färbung des Hydrogels, die durch das partielle Eindringen des Farbstoffs (Indigokarmin) zu einem geeignet gewählten, festen Zeitpunkt gemessen wird, beschreibt näherungsweise die Verteilung der Gleichgewichtskonzentration von Nährstoffen, die sich durch den Stoffwechsel von Zellen im Hydrogel einstellen würde.
Ergebnisse und Diskussion:
Die Leistungsfähigkeit von Adersystemen wurde rechnerisch in Abhängigkeit von der Komplexität des Adersystems (der Anzahl der Verzweigungen), der Kantenlänge des Substrats und der der Eindringtiefe der Nährstoffe (die sich aus der Diffusivität des Hydrogels und dem Nährstoffverbrauch der Zellen ergibt) untersucht. Die Simulationen zeigen, dass Adipocyten in einem PVA-Hydrogel mit 5 mm Kantenlänge schon mit einem einfach verzweigten Adersystem versorgt werden können. Größere Substrate oder Zellen, die einen höheren Nährstoffverbrauch haben, erfordern jedoch Aderbäume mit mehr Verzweigungen. Das entwickelte Modell ermöglicht es, das optimale Adersystem für ein spezifisches Gewebemodell zu finden. Eindringversuche mit Farbstoffen, die eine ähnliche Diffusionskonstante wie die Nährstoffe haben, können als sondierende Messungen an unbesiedelten Hydrogelen dienen. Mit diesen Messungen kann die Leistungsfähigkeit verschiedener Adersysteme verglichen werden, bevor aufwändige Zellkulturtests ausgeführt werden.
Die Ergebnisse wurden im Artivasc 3D Projekt erzielt, das vom 7. Rahmenprogramm der EU gefördert wird (grant agreement n° 263416)
3D plotting of fine structured scaffolds consisting of a calcium phosphate cement and a growth factor loaded alginate-gellan gum hydrogel: fabrication and in vitro characterization
*T. Ahlfeld1, A. Lode1, Y. Förster1,2, A. R. Akkineni1, F. Schuster1, S. Brüggemeier1, S. Knaack1, M. Quade1, S. Rammelt1,2, M. Gelinsky1
1University Hospital Carl Gustav Carus and Faculty of Medicine, Technische Universität Dresden, Centre for Translational Bone, Joint and Soft Tissue Research, Dresden, Deutschland
2University Hospital Carl Gustav Carus Dresden, University Centre for Orthopaedics and Trauma Surgery, Dresden, Deutschland
3D plotting is an extrusion-based method of additive manufacturing (AM) with high potential for tissue repair and regeneration. A broad range of materials can be processed, including calcium phosphate cements (CPC)1 or hydrogels like alginate or gellan gum2. Outstanding advantage of 3D plotting, compared to other AM techniques, is the possibility of processing those materials under mild conditions and therefore, controlled integration of sensitive growth factors into scaffolds during fabrication.
CPC has good mechanical properties and is established as bone replacement material, however, hydrogels are favored for drug loading and allow even cell encapsulation. Multichannel 3D plotting gives the opportunity to have both, CPC and hydrogel strands, in one scaffold design. Based on our former study3, the aim of this work was to produce fine structured, growth factor loaded scaffolds consisting of CPC with some strands replaced by a hydrogel containing VEGF. The fabrication process was optimized and the scaffolds were characterized with respect to the material properties (pore structure, porosity, swelling behavior and mechanical strength) and with respect to release of VEGF in biologically active form. Furthermore, in vitro cell culture experiments with mesenchymal stromal cells were performed to study the cytocompatibility of the scaffolds.
Materials and Methods:
Plotting materials were a gamma-sterilized CPC paste (InnoTERE GmbH, Radebeul), autoclaved 16 % alginate paste (alginic acid sodium salt, Sigma-Aldrich), and autoclaved 2 % gellan Gum (Gelzan, Sigma-Aldrich). Alginate-gellan gum hydrogel blend was prepared by mixing in a beaker in a mass ratio of 85 to 15. While mixing, VEGF stock solution as well as - for stabilization of VEGF - heparin solution was added to the hydrogel paste to achieve a final concentration of 1 μg VEGF and 0.1 mg heparin per ml hydrogel.
Materials were processed with a multichannel 3D plotter (BioScaffolder 2.1, GeSiM, Großerkmannsdorf). After setting in 1 M CaCl2 solution (to crosslink the alginate-gellan gum hydrogel) for 10 min, CPC was additionally set in humidity for three days.
Nondestructive characterization of the biphasic scaffolds were performed with μCT; mechanical behavior was measured by uniaxial pressure test using a Zwick testing machine. Swelling of the hydrogel strands in the CPC matrix was observed microscopically.
To study the release of VEGF, scaffolds were incubated in cell culture medium; at different time points, supernatants were collected and used for VEGF quantification by ELISA. The bioactivity of the released VEGF was studied in an endothelial cell proliferation assay as well as in an in vitro angiogenesis assay, respectively. Cell attachment and proliferation on the scaffolds was tested by in vitro cell culture with mesenchymal stromal cells (MSC).
Results and Discussion:
Fine structures of CPC were plotted successfully. The strands had a thickness of 200 μm and the pores the predefined, complex arrangement. Replacement of selected CPC strands by hydrogel was achieved using multichannel plotting. Different types of hydrogels were tested: cell adhesion was best for an alginate-gellan gum blend. In addition, the VEGF release as well as maintenance of its biological activity was better for this hydrogel combination compared to a pure alginate hydrogel.
Micro-CT analysis of the biphasic CPC/alginate-gellan gum scaffolds revealed open porosity, even after partial replacement by the alginate-gellan gum hydrogel. In addition, microscopic images of the swollen hydrogels showed that the pores remained open, even if the maximum swelling of the alginate-gellan gum was reached. Porosity and mechanical strength of the scaffolds including the polymer blend strands were the same compared to pure CPC scaffolds up to an exchange rate of 24 %. The biphasic scaffold released VEGF in a sustained manner, the bioactivity was proven in an in vitro angiogenesis assay.
In conclusion, the combination of a blend consisting of alginate and gellan gum is a strong alternative to commonly used hydrogels. Selective replacement of CPC strands with alginate-gellan gum strands is an option to gain from advantages of this hydrogel combination without loss of the main characteristics of a pure CPC scaffold.
1Lode et al., J Tissue Eng Regen Med, 2014, 682-693
2Luo et al., Adv Healthcare Mater, 2013, 777-783
3Luo et al., J Mater Chem B, 2013, 1, 4088-4098
Strontium Substitution of 3D Powder Printed Magnesium Phosphate Scaffolds
*S. Christ1, S. Mandal2, A. Kumar2, B. Basu2, J. Groll1, U. Gbureck1
1Universitätsklinikum Würzburg, Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde, Würzburg, Deutschland
2Indian Institute of Science, Laboratory for Biomaterials, Materials Research Centre, Bangalore, Deutschland
Magnesium phosphate cements (MPC) are fast degrading synthetic bone implant materials enabling replacement of bone grafts by autologous bone tissue . Substitution of magnesium ions by strontium seems to be a promising way to promote proliferation and differentiation of osteoblast precursor cells  and therefore support tissue ingrowth. This study applied such Sr2+-modified MPC to 3D powder printing technology for a free form fabrication of patient individual implants.
Materials and Methods:
3DP was performed with Mg3-xSrx(PO4)2 (x=0, 0.5, 1) powder supplemented with 5 % (hydroxypropyl)methylcellulose and water as binder followed by sintering at 1100 °C. Sintered samples were compared with post-hardened ones which converted into struvite (MgNH4PO4 · 6H2O) after immersion in 3.5 M ammonium phosphate solution. Materials were characterized by means of XRD, SEM, μCT and mechanical testing. Additionally, degradation products were detected by inductively coupled plasma - mass spectroscopy. Biological evaluation was performed with osteoblast cell lines MG63 and hFOB.
Results and Discussion:
Strontium substitution and phase conversion after post-hardening was proved by XRD, SEM and μCT (Fig. 1). Mechanical properties of post-hardened samples were in the range of those for cancellous bone with a maximum compressive strength of 36.7 MPa with a Weibull modulus of 4.3 - 8.8. Passive degradation was high for all cements and strontium release of approx. of 127 mg/l was detected over 10 days. The high degradation rate, however, reduced cell attachment during in vitro tests especially for strontium substituted cements.
Material properties could be maintained after strontium substitution leading to good printing results with an adequate reliability of sintered and post-hardened samples. Even though a cell promoting effect of strontium could not directly be proved, a high degradation rate paves the way for a resorbable implant material that can be replaced by newly formed bone tissue.
The authors would like to acknowledge financial support from Bayer Fellowship Program.
 B. Kanter et al., Acta Biomater. 2014, DOI 10.1016/j.actbio.2014.04.020.
 T. Brennan et al., Br. J. Pharmacol. 2009, 157, 1291-1300.
Indirect Inkjet 3D Printing of Porous beta-TCP Scaffolds for Bone Tissue Regeneration Applications
*I. Arhire1, R. Gadow1, A. Bernstein2
1Institut für Fertigungstechnologie keramischer Bauteile IFKB, Stuttgart, Deutschland
2Klinik für Orthopädie und Unfallchirurgie, Muskuloskelettale Forschung, Freiburg, Deutschland
Additive manufacturing of calcium phosphate powders into porous scaffolds holds great promise in the field of tissue engineering for patients with bone defects due to traumas or natural illnesses, such as osteoporosis. With respect to the more conventional technologies, 3D Printing has the great advantage of yielding highly-porous, patient-customized implants based on medical data sets. Calcium phosphates are a primary focus in bone reconstruction due to their biodegradability and osteoconductive properties.
Fabrication of bone scaffolds via indirect 3D Printing of beta-tricalcium phosphate (β-TCP) powder is investigated in this work. The aim is to gain a better understand of the interplay between the initial relevant powder properties (flowability, particle size distribution and morphology) and the printing parameters (layer thickness, binder saturation) on the scaffold final quality. Printability of both binder coated and binder blended powders is examined. Final scaffolds are characterized with respect to their physico-mechanical properties, microstructure and cytocompatibility. Possible steps in improving scaffold quality in powder-based 3D Printing are addressed in conclusion.
Materials and Methods:
Beta-TCP powder (Budenheim, Germany) was investigated in both spherical and anisotropic morphology. In order to produce spherical powder feedstock with appropriate particle size distribution for the printing process, spray-granulation investigations with respect to process parameters (nozzle configuration, pressure, pump flow rate) were carried out.
The role of the adhesive binder, to hold the beta-TCP granules together during the printing, was performed by dextrin polymer (Carl Roth, Germany). The binder was incorporated by two methods: blending in the powder feedstock as separate grains and powder coating.
Binder saturation and layer thickness were varied to improve scaffold accuracy and quality.
Results and Discussion
The 3D Printing powder feedstock was characterized in terms of morphology, particle size and particle size distribution. Flowability, governing the printing process, was assessed with the Hausner Ratio. Powder bed density was measured for each type of powder in order to correlate with the printed scaffolds porosity.
Printed strucutures were characterized with respect to their microstructure and porosity. Chemical composition after heat treatment was assessed with X-Ray Diffraction and Raman Spectroscopy. Mechanical properties were evaluated and cytocompatibility tests performed.
Possible steps in improving the printing processing for bioceramic powders were discussed.
Biofabrication of 3D alginate based hydrogel for cancer research: Characterisation of HCT116 tumor cells development
*R. Detsch1, J. Ivanovaka2, T. Zehnder1, P. Lennert2, B. Sarker1, R. Schneider-Stock2, A. R. Boccaccini1
1Friedrich-Alexander Universität Erlangen-Nürnberg, Lehrstuhl Biomaterialien , Erlangen, Deutschland
2Universitätsklinikum Erlangen, Arbeitsgruppe Experimentelle Tumorpathologie des Instituts für Pathologie, Erlangen, Deutschland
In general, there is an increasing interest in three-dimensional (3D) culture models for several biomedical fields such as tissue engineering, drug screening, cell therapy, and tumor growth modelling. In detail the 3D systems should mimic the structural architecture and biological functions of the extracellular matrix. Among the naturally occurring biopolymers, alginate as polysaccharide and gelatin as protein are promising hydrogels for cell encapsulation and three dimensional plotting (biofabrication) . Designing of biomaterials as exogenous matrix that can promote cell adhesion migration and proliferation starts with the understanding of cell-material interactions in 3D. Therefore, we investigated the behaviour of colon cancer cells in plotted 3-dimensional hydrogel matrices.
Materials and Methods:
3D hydrogel-based constructs were fabricated by bioplotting technique in layer-by-layer fashion using alginate solution and covalently crosslinked alginate di-aldehyde-gelatin (ADA-GEL)  hydrogels as ink-matrices followed by ionic gelation with calcium chloride solution. The fabricated constructs were extensively characterised to understand their physico-chemical features. HCT116 colon cancer cells were immobilised with different densities (1.5x106 - 5x106 cells/ml) of hydrogel solutions and subsequently plotted. The cell loaded hydrogels were cultured in DMEM medium for 8 and 14 days. Afterwards, cell viability, distribution, attachment and proliferation as well as epithelial-like phenotype expression of HCT116 were analysed.
Results and Discussion:
The obtained results indicated that after 8 days of incubation, cells embedded in the ADA-GEL matrix showed higher vitality and proliferation with enhanced cellular networks through the hydrogel matrix in comparison to pure alginate. Moreover, the analysed mRNA expression of α and ß-integrins of cells cultured showed that the integrin subunits deferred based on the cell focal adhesion characteristics. Western Blot analysis as well as high resolution microscopic studies (CSLM and SEM) showed that the tumour cells bioplotted in ADA-GEL behaved like epithelial cells after 14 days of cultivation. Thus, ADA-GEL matrix seems to simulate the physiological micro-environment of the tumour. The results of this study showed that ADA-GEL hydrogel is able to build 3D structures that closely reflect the cell-microenvironment mimicking the ECM with precise control of microarchitecture. In conclusion, we developed a 3D cancer test system that enables drug testing, studying of cancer pathogenesis and anti-cancer mechanism.
 Detsch, R., Sarker, B., Zehnder, T., Douglas, T. E. L. & Boccaccini, A. R. Additive manufacturing of cell-loaded alginate enriched with alkaline phosphatase for bone tissue engineering application. BioNanoMaterials 15 (3-4), 79-87 (2014).
 Sarker, B. et al. Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties. J. Mater. Chem. B 2, 1470 (2014).