At a Glance
The BioScaffolder (BS) printer family comprises BS3.2 with focus on research and BS4.2 offering production capabilities. Main applications are:
|- 3D Printing of very different (Bio-)polymers|
- Creation of porous structures for tissue engineering
- Co-printing of hard (E.g. PCL/PLA) and soft (E.g. Alginate) biopolymers (Sequential printing, co-axial extrusion, Nanolitre pipetting)
|- Functionalization of metal implants with soft polymer layers
- Printing of micro structures from biopolymers by melt electro spinning/writing
Printing from cartrigde enables the use of home made polymer blends. BS printers are not limited to commercially available filaments as known from the many FDM printers. Further very different printing methods for different materials can be combined on a single instrument.
Each BS printer comes with three pneumatic extruders but adapts to a wide range of materials by numerous add-on modules. Both printing tools and software are almost identical for most BS printers.
BS4.2 offers approx. double space for print targets than BS3.2.
- Three pneumatic extruders (Optionally heated) for dispensing of high-viscous/ pasty materials. Individual print parameters for each axes
- Automatic alignment for tools and building platform
- Nozzle cleaner
OPTIONS (Next Tab): GeSiM enhances the range of add-on modules for combination with the basic pneumatic extruders. At a glance:
- Heatable (190°C), coolable cartridge holders
- Pipetting unit (Low-viscous liquid handling with pipet tips, solenoid dispense valve or piezoelectric Nanoliter dispenser)
- Twin-Tip pipetting unit for mixing up tiny droplets (Low-viscous liquids)
- Piezoelectric dispense valves for high-viscous materials (Third party)
- Pneumatic Core/Shell extruders for doing hollow fibres
- Melt Electrospinning, -writing (MES)
- High-Power Syringe Extruders for melts up to 250°C
- Exposition Lamps for Curing and UV-Hardening
- FDM extruder printing commercial filaments
- Trays for target objects, e.g. cell culture plates
- Heatable (150°C), coolable (4°C, with external chiller bath) object trays
- Software for Import of STL data
BS printers adapt to your application as well as to your budget! Don’t miss the documents on the right side of this article.
Computers, Cartridges and Pressurized Air: The heart of each BS printer
The BS comprises an external “F-Box” (Right picture) managing the media supply and removal for the instrument. Furthermore it contains the control computer of the instrument.
The basic pneumatic extruder allows individual pressure settings. BS printers support 10 ml and 30ml cartridges (Left picture). Pressure ranges from 0 to 6 bar.
Many Tools, Different Materials: Click and Go!
Biomaterials of very different consistencies require very different tools (sizes) but need to be printed into one structure.
On top of this, any 3D printer works only when the first layer is well aligned to the building platform. The BS printer come with optical and mechanical calibration tools for the automatic offset compensation between the tools and the ground level. The only things you have to do is load your material, find the right pressure for extrusion and create your model. Then you are just a few mouse clicks away from your ready printed scaffold.
How to talk to your BS printer
The BioScaffolder printer receives G-code data from the control software. It is a commonly used programming language for NC (numeric control) machines. G-code is always hardware dependent, therefore a lof of different G-code flavors exist.
A reference of the GeSiM BS gcode commands is available on request.
Disclaimer: GeSiM isn’t responsible for damages of the hardware due to wrong and/or unappropriated G-code sequences sent to the machine. If you are not sure contact GeSiM in advance.
Printing of Biomaterials at defined Temperature
Different temperature control units are available for the pneumatic extruders of the BS printers. They fit to 10 Milliliter cartridges but 30 Milliliter cartridges can be used at room temperature.
A) Peltier chiller, range: 4°C to 80°C
B) Shell heater, range: RT to 100°C (190°C)
Disposable plastic cartridges with Luer lock adapter are fine for prints close to room temperature. Nozzle insulators are available for particular Aluminum needles.
Thermoplasts like PCL print better with a set comprising the proprietary GeSiM stainless steel cartridge (10 mL) and stainless steel nozzle, available at different size.
3D Structures from Melted Thermoplastics
Our advanced motor driven piston extruders extend the capability of the pneumatic basic set:
- High pressure (Virtually > 100 bar) prints viscoelastic and high-viscous materials, even with tiny nozzle diameters
- Constant piston moves ensure constant material flow, independent on the material level inside the cartridge
The High-Temperature Piston Extruder prints thermoplastics like PLA (Polylactic acid) at temperatures up to 250°C. It comes with the stainless steel cartridge as well as stainless steel nozzle. A two zone heater compensates the temperature drop from the cartridge to the nozzle.
The Gradient Mixer (Available for BS4.2 only) combines the outlet of two HT-Extruders with a special mixing head. It allows varying mixing ratios of two thermoplasts during one print.
Printing of larger volumes (50 mL) is available by the piston extruder for hydrogels.
Tablets for Object Fixation
The standard tray for BS printers accomodates two well plates (BS3.2 three well plates, BS4.2 six plates), e.g. cell culture plates. Trays for glass slides or other targets are available on request. The trays come with a snap-in fixation for quick replacement on the instrument main deck.
Temperature controlled well plate holders are available on request.
Low-Volume Sample Handling
Optionally BS printers can be equipped with a pipetting unit. Diverse pipetting needles applies small volumes (0.1 Nanolitre to a few Microlitres) of low-viscous liquids to previously printed scaffold struts. These liquids may contain drugs, cells or particular proteins.
There are two tools available:
- Passive needle for syringe supported displacement dispensing. The lowest dispense volume might be half a Microliter.
- Piezoelectric nozzles for free-flight microdrop dispensing. The lowest dispense volume is in the range of 0.1 Nanolitres:
Piezoelectric GeSiM dispensers emit single drops in the range of 250 Pikolitre (Drop diameter about 50 Microns). Small amounts of proteins or cell suspensions can be applied to single or multiple layers of a bioscaffold structure during the printing process.
- The innovative Twin-Tip-Pipettor operates two separate GeSiM piezoelectric nozzles on a swivel. For aspiration both nozzles dip into separate wells of a 96-well-plate. The droplets of both nozzles than hit together and mix up on the dispense target.
- The volume range between fifty Nanolitres and a few Microlitres is covered best by the solenoid dispense valve (Not for living cells.)
Contradictionary to the cartridge system the pipetting unit aspirates from 96 well plates. Only a few Microliters of each species are required. Heaters are available both for the sample plate and the piezo nozzle for producing microdrops of melted materials.
Piezoelectric Pipettor on BS3.1; Click here for full size BS3.1 video on YouTube
Artificial Tissue from Maccaroni Strands
Soft matrices like Hydrogels are cell friendly materials but hard to print into stable 3D structures. The combination with more stiff thermoplastic scaffolds may cause biocompatibility issues.The pneumatic C/S-extruder (Core/Shell extruder) for BS printers allow to combine two materials with different properties in a coaxial manner. The tool is equipped with a kind of double nozzle with different but corresponding inner and outer diameter, respectively. The print parameters can be set individually for both channels in order to match properties of the respective bioinks.
A. R. Akkineni, T. Ahlfeld, A. Lode and M. Gelinsky: A versatile method for combining different biopolymers in a core/shell fashion by 3D plotting to achieve mechanically robust constructs, IOP Publishing Ltd., Oct 2016
Centre for Translational Bone, Joint and Soft Tissue Research
Technische Universität Dresden
Hardening of Scaffold Struts
There are lamp heads with different excitation wave lengths and different wave length available for the exposition of printed scaffold layers (Third party manufacturers). The lense head goes on one of the tool axes of BS3.1. One application is the hardening of UV curable materials.
For high power exposition we recommend the Omnicure 1500. Alternatively, LED-lamps from THORLABS are available with many different wave lengths but at limited power. The max. LED output power depends on the wavelength and is in the range of 50 mW to 210 mW.
Processing of External CAD Data
This extension just comprises software – A dedicated input filter reads in STL files. An STL file describes triangulated surfaces in a three-dimensional Cartesian coordinate system. It is widely used for rapid prototyping, 3D printing and computer aided manufacturing.
The BioScaffolder 3.1 software allows to define inner “scaffold-” structures whereas the STL file brings in just surfaces. Like with the built-in scaffold generator, up to three different materials and different pore sizes can be assigned to the CAD model.
Melt Electrowriting (MEW)
The optional MEW module of BS3.1 combines pneumatic extrusion and high-voltage induced fibre deposition for particular thermoplastic materials. So far PCL 50,000 can be printed.
In contradiction to Melt Electrospinning (MES) Melt Electrospinning Writing deposits each fibre in a regular manner accordingly to the CAD model.
The Melt electrowriting module extends the standard configuration of the instrument by a special tray with embedded electrode, a high-voltage generator and security measures. The minimum strut width is in the range of 10…20 Micrometers. Left side picture shows the print setup with nozzle electrode, the right side shows the high-voltage generator.
A Biocompatible/ Biodegradable Cell Growth Environment
Tissue engineering and tissue regeneration is becoming a promising approach e.g. to cure severe bone injuries [Learn more…].
Artificial tissue grown from differentiable cells often needs to be in a particular 3-dimensional shape for implantation. Bioscaffolds can serve as a cell growth environment for artificial tissues, by supporting supply of the cells and removal of the metabolites. Usually bio-scaffolds consist of a porous material to be seeded with differentiable cells. After implantation in the host organism the scaffold material is designed to degrade,
Right image: A scaffold structure 36 mm by 36 mm with inner pitch of 1mm.
Up to now bioscaffolds of basic geometries have served as research platforms for cell biologists and material researchers. As complex 3D-geometry becomes increasingly important the BS3.1 supports this through the data filter for external STL files (E.g. CT data, external CAD software…)
BioInks fill the gap between a 3D printer and tissue engineering
Biopolymers for cell cultivation usually show a low viscosity. 3D structures of defined shape, however, require stiff materials with low cell viability potential. An contradiction in terms?
The picture presents one possible approach: Composites of two or three materials give home to cells in a “stiff” 3D environment.
Combined printing of very different polymers can be challenging for the printer: Different needle sizes, different print parameters for each needle. All this is available with BS3.1
Up to now commercial bioinks are expensive and not always working in a suitable manner. Our RESOURCES section lists publications of GeSiM customers/ partners working on biomaterials. Examples:
- Researchers at Technical Universität Dresden show how low-viscous alginate turns into a valuable bioink by adding Methylcellulose. (More…)
- A blend of PCL-PEG with ADA (Dialdehyde gelatine hydrogel) was investigated by the Biomaterial Resarch Group at FAU Erlangen. (More…)
- VELOX(R) bone substitute cement paste is based on calcium-phosphate and now commercially available from INNOTERE GmbH, Radebeul, Germany.
BS3.1 now is probably the worlds only bioprinter with built-in two-component extruder: A so called Core/Shell extruder produces “maccaroni” structures, e.g. for direct combination of a soft, cell-friendly polymer with an alginate of much higher viscosity.
Miniaturized Scaffolds from Biopolymers
Some applications require downscaling of 3D printing to get into the range of small organoids like blood vessels. BS3.1 offers tools for optimizing/ accomplishing standard pneumatic printing.
Solid biopolymers like PCL and PLA offer better 3D support than low-viscous bioinks and offer higher 3D resolution, respectively. The easiest way is to use the cartridge heater along with stainless steel cartridges and aluminum/steel needles.
Very thin needles plus high power: The High-Power-Syringe Extruder extends the range of the pneumatic system in terms of pressure (> 100 bar) and temperature (< 250°C).
Melt Electrospinning (MES) provides much smaller structures than pneumatic printing. It requires high electrical voltage (15…30 kV) between the dispense nozzle and the building platform.
The struts of the MES mesh have sizes down to 0.01 mm. PCL 50,000 was printed at a voltage of +30 kV and a pressure of 0.6 bar.
What is the GeSiM BioScaffold printer used for?
|- Production of support structures (Scaffolds...) for tissue engineering|
- Printing of 3D bodies from biopolymers
- Research and development of biopolymers for sustainability and environmental protection
|- Application of living cells and bacteria, either embedded in a biopolymer or seeded by the optional pipetting unit
- 3D Printing of ceramic pastes and bone replacement materials
- Melt electro spinning writing for research on lymph nodes and eardrums,
This section presents highlights of the work of our customers with GeSiM instruments for bioprinting. It is neither comprehensive nor can GeSiM be responsible for content and correctness.
Sequential Bioprinting with GeSiM Instruments
PCL (PolyCaproLactone) is a popular hard-phase biopolymer for tissue engineering and 3D printing. It is biocompatible and – to a certain extent – biodegradable. Multi-printhead instruments like the GeSiM BS31 easily combine PCL struts with cell friendly alginate/hydrogels.
Hard-phase biopolymers shall be optimized towards a quick degradation/mass loss when getting in contact with body fluid. An inherent drawback of pure PCL is the relatively high stability under physiological conditions. Here we present a recent study  addressing this problem. It was conducted using the predecessor of BS3.1, BS2.1.
PCL and polyethylene glycol (PEG) blends (PCL-PEG) together with alginate dialdehyde gelatine hydrogel (ADA-GEL) loaded with stromal cell line (ST2) were investigated.
The PCL-PEG blends showed a much faster degradation and a mass loss tending to be almost equal with the corresponding content of PEG being ~14% for the PCL-PEG 8020 and ~23% for the PCL-PEG 7030 compositions. The wetting behaviour and the cell behaviour were improved in comparison to pure PCL. Blends showed improved hydrophilicity and cell response with PEG blending increasing the degradation and decreasing the mechanical properties of the scaffolds.
 Tobias Zehnder, Tim Freund, Merve Demir, Rainer Detsch and Aldo R. Boccaccini: Fabrication of Cell-Loaded Two-Phase 3D Constructs for Tissue Engineering, Materials 2016, 9(11), 887
3D-printing of Cell-loaded Alginate Capsules suspended in Hydrogel
Printable biomaterials can benefit from complex compositions: The release of drugs or cell growth have to be controlled after printing. A group from the Friedrich-Alexander University in Erlangen added prefabricated capsules enriched with cells to hydrogel before printing.
The GeSiM BioScaffolder was part of this study. It presents a novel method to produce macroporous hydrogel scaffolds in combination with cell-loaded capsule-containing struts by 3D bioplotting.
This approach enables loading of the capsules and strut phases with different cells and/or bioactive substances and hence makes compartmentalization within a scaffold possible.
The goal was to produce scaffolds for possible applications in bone tissue engineering consisting of alginate struts containing alginate capsules enriched with MG-63 osteoblast-like cells and ALP (alkaline phosphatase). Two combinations were compared, namely ALP in the struts and cells in the capsules and vice-versa. Both combinations were cytocompatible for cells and mineralization of scaffolds could be detected in both cases, according to an OsteoImage staining. ALP had no adverse effect on cytocompatibility and enhanced mitochondrial activity.
Different components desirable for bone regeneration, e.g., cells and bioactive proteins, can be incorporated both in the capsules and struts. This enables compartmentalization of components, which facilitates greater flexibility in modification of the scaffold.
Rainer Detsch, Bapi Sarker, Tobias Zehnder, Aldo R. Boccaccini and Timothy E.L. Douglas:
Additive manufacturing of cell-loaded alginate enriched with alkaline phosphatase for bone tissue engineering application. De Gruyter, BioNanoMat 2014; 15(3-4): 79–87
The “Green Bioprinting” approach is expected to bring an advantage for existing applications of microalgae in the biotechnological field as, e.g. harvesting and separation procedures could be simplified and the co-immobilization of microalgae with (e.g. plant growth promoting) bacteria could be conducted in a spatially organized manner. In addition, this novel approach opens further possibilities for new, future-oriented applications such as the usage of microalgae or other plant cells in the medical field. The cocultivation of algae in close vicinity to human cells could enable a sustained delivery of oxygen or secondary metabolites with therapeutic potential to human cells without the need of external supply. The fabrication of patterned coculture scaffolds can be easily realized by two-channel plotting. 
In this study, conducted by GeSiM customers at the Centre for Translational Bone, Joint and Soft Tissue Research at the Technische Universität Dresden in collaboration with partners from the Institute of Bioprocess Engineering at the TU Dresden, a simple geometry was chosen to demonstrate embedding of microalgae in an alginate hydrogel scaffold by 3D plotting.
It was demonstrated that microalgae can be immobilized in 3D alginate-based scaffolds with predesigned geometry. The alginate matrix has proven its suitability for cultivation of the embedded algae—as indicated by cell growth and photosynthetic activity.  The immobilization of microalgae in the plotted structures resulted in an enhanced viability and stable growth rates even under suboptimal culture conditions. 
 A. Lode, F. Krujatz, S. Brüggemeier, M. Quade, K. Schütz, S. Knaack, J. Weber, T. Bley, M. Gelinsky: “Green bioprinting: Fabrication of photosynthetic algae-laden hydrogel scaffolds for biotechnological and medical applications”, Engineering in Life Sciences, Volume 15, Issue 2, pages 177–183, March 2015
 F. Krujatz, A. Lode, S. Brüggemeier, K. Schütz, J. Kramer, T. Bley, M. Gelinsky, J. Weber: „Green Bioprinting: Viability and growth analysis of microalgae immobilized in 3D-plotted hydrogels versus suspension cultures“, Engineering in Life Sciences, Volume 15, Issue 7, pages 678–688, October 2015
Centre for Translational Bone, Joint and Soft Tissue Research
Technische Universität Dresden
The Instant Recipe for Tissue Engineering?
The group of Prof. Michael Gelinsky at the Technische Universität Dresden conducted a study to overcome the limitations of biofabrication with cell-friendly TE Materials. The aim of the study was to develop a plotting material that is based on alginate, the probably most popular substrate material for biological 3D printing. The goal was to find a composition optimized both for printing and for cell embedding.
Basically a rather easy approach was used: Addition of Methylcellulose (MC) to low concentrated alginate. That leads to an enhanced viscosity and therefore improved printing conditions. The MC did not contribute to the gelation and was released from the scaffolds during the following cultivation. Mesenchymal stem cells were added to the alginate-MC blend and showed high viability after several weeks of cultivation within the plotted scaffolds.In this work both cytocompatibility and mechanical properties of the alg/MC material were investigated. The developed plotting material allows to print 3D objects in the centimetre range and even complex geometries.
 Kathleen Schütz, Anna-Maria Placht, Birgit Paul, Sophie Brüggemeier, Michael Gelinsky and Anja Lode: Three-dimensional plotting of a cell-laden alginate/methylcellulose blend: towards biofabrication of tissue engineering constructs with clinically relevant dimensions, Journal of Tissue Engineering and Regenerative Medicine, Article first published online: 22 JUL 2015
Centre for Translational Bone, Joint and Soft Tissue Research
Technische Universität Dresden
Development of Bioinks for TE
Pneumatic extrusion allows printing of biocompatible materials in a wide viscosity range. However, the minimum feature size is somewhat larger than 100 µm due to the high fluidic resistance of pasty materials inside of narrow nozzles. Piezoelectric printing allows much finer drops but valve less dispensers are usually limited to an upper viscosity of about 10 mPa*s. Piezoelectric valve dispensers basically allow higher viscosities but apply high shear stress to embedded cells leading to a low viability rate.
The Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik is working on „bioinks“, that means printable material systems made from biomolecules of the extracellular matrix (ECM).
The developed material systems are based on water-soluble collagen. The chemical composition can be modified in order to adjust the viscosity in a range from 3….120 mPa*s. These bioinks are therefore printable even through valve less piezoelectric nozzles (Left: GeSiM micro dispenser with reservoir on top on a modified Nano-Plotter).
The variation of the matrix composition allows the adjustment of the following properties:
- Viscosity and gelling properties of the non-linked bioink
- Mechanical properties of the cross-linked hydrogels
- Composition and biological function of the cross-linked hydrogels (ECM)
 Eva Hoch, Thomas Hirth, Günter Tovar, Kirsten Borchers: Chemical tailoring of gelatin to adjust its chemical and physical properties for functional bioprinting. Journal of Materials Chemistry B 1, 41, 5675-5685 (2013).
 Eva Hoch, Christian Schuh, Thomas Hirth, Günter E. M. Tovar, Kirsten Borchers: Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation; Journal of Materials Science: Materials in Medicine 23, 11, 2607-2617 (2012).
 Sascha Engelhardt, Eva Hoch, Kirsten Borchers, Wolfdietrich Meyer, Hartmut Krüger, Günter E. M. Tovar and Arnold Gillner: Fabrication of 2D protein microstructures and 3D polymer-protein hybrid microstructures by two-photon polymerization. Biofabrication 3, 2, 025003 (2011)
Fraunhofer Institute for Interfacial Engineering and Biotechnology
Highly Customized and Affordable Implants by a new Hybrid 3D Printing Technology
FAST press release, January 29, 2016:
Eight European companies and research institutes have teamed up in the EU-funded research and innovation project “FAST”, which stands for “Functionally graded Additive Manufacturing (AM) Scaffolds by hybrid manufacturing”, to make a new 3D printing technology available for the manufacture of implants customized to the patient at affordable cost. Specific patient implants can promote effective preoperative planning, shortening the time of surgery and improving the lifetime of the implant.
Scaffolds production for tissue regeneration is one of the main fields where the “Design for Function” feature of AM makes the difference relative to the other production techniques, in particular if in the production process all the needed “functions” can be introduced: shape and porosity, mechanical stability and biochemical properties such as cell growth control or antibiotic function. The FAST project aims to develop a cost-efficient technology to integrate all these “functions” in a single AM process that is even capable to produce gradients in the bulk or surface properties of the individual scaffold.
Thus not only customized shapes of scaffolds can be produced, but also bulk and surface properties of the scaffold material can be tailored according to the specific needs of each individual patient.
In practice this will translate into improved tissue ingrowth and regeneration properties combined with higher structural stability of implants. Furthermore, the incorporation of smart fillers with bio-active properties into the scaffold material will reduce the risk of post-surgery infections. Thus, the FAST technology has the potential to increase the patients’ comfort at an affordable cost for them and the healthcare system. The project will demonstrate its developments in a small pilot production of scaffolds for bone regeneration to be tested in a pilot in vivo trial.
The FAST project is scheduled to run for four years from 1st December 2015 to 30th November 2019 with a budget of 4,9 MEUR, which is funded by the European Union under the H2020 Framework Programme for Research and Innovation.
FAST project partners:
- Abalonyx AS, Norway
- Fraunhofer Institute for Surface Engineering and Thin Films IST, Germany
- Fundacion Tecnalia Research & Innovation, Spain
- GeSiM Gesellschaft für Silizium-Mikrosysteme mbH, Germany
- Maastricht University, The Netherlands
- Nadir S. r. l., Italy
- Polyvation BV, The Netherlands
- Prolabin & Tefarm S. r. l., Italy
Press contact via the project coordinators:
- Dr. Lorenzo Moroni, Maastricht University, firstname.lastname@example.org
- Dr. Alessandro Patelli, Nadir S.r.l., email@example.com
Reconstruction of a Human Scaphoid Bone
The replacement of bones in course of accident treatment usually requires titanium implants. It is a widely used material for trauma surgery but shows inherent drawbacks: A mismatch of mechanical properties, interface issues to the surrounding soft tissues and no capability to grow.
A joint research project of GeSiM and Dresden University of Technology (TU Dresden) – Centre for Translational Bone, Joint and Soft Tissue Research – aimed in establishing 3D printing of patient-specific implants of a degradable biomaterial. As model the human scaphoid bone was selected and 3D data extracted from a CT scan.
In a first step the CT data of the patient were analysed to separate bones from the remaining tissue (Virtual environment/ contouring). Next the CT data was transformed into a 3D DICOM model using an Open Source software package. The scaphoid bone was isolated from the complete bone set to generate 3D STL data, a format describing surfaces by triangularization. The STL format is widely used by all kind of 3D printers, also the GeSiM BS3.1.
Finally the STL data of the scaphoid bone was loaded into the software of the GeSiM BS3.1. The bone model was printed from calcium phosphate bone cement VELOX® from InnoTERE GmbH, Radebeul.
This work is a research project without clinical background. Future research may be focusing on the settlement of osteoblasts or mesenchymal stem cells in the scaffold structure for subsequent incubation and generation of an artificial “living” bone.
We thank the BMWi for funding this work (AiF-ZIM program, project number KF2891602).
 T. Ahlfeld et al., Centre for Translational Bone, Joint and Soft Tissue Research, Technical University Dresden
 M. Heller, H.-K. Bauer, E. Goetze, M. Gielisch, I. T. Ozbolat, K. K. Moncal, E. Rizk, H. Seitz, M. Gelinsky, H. C. Schröder, X. H. Wang, W.E.G. Müller, B. Al-Nawas: Materials and Scaffolds in Medical 3D Printing and Bioprinting in the Context of Bone Regeneration. Int. J. Computerized Dent. 2016, 19, 301-321 (Figure 6)