3D BioPrinting

Bioscaffolder -  Bio printing - Bioprinting - Tissue Engineering- 3D Printing

3D bio printing of human tissue has been around since the early two-thousands. Nowadays scientists are in the midst of moving from printing tiny sheets of tissue to entire 3D organs.

BioScaffoldes - What is it ?
A Biocompatible/ Biodegradable Cell Growth Environment

Tissue engineering and tissue regeneration is becoming a promising approach e.g. to cure severe bone injurie.

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,enabling uninterrupted layers of artificially grown tissue. 

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…)

Multi-Matirial Scaffolds
BioScaffolder 3.1: Just another 3D Printer?

Up to four independent Z-axes is a unique feature for a benchtop 3D printer. BS3.1 supports up to three pneumatic channels for various configurations including multiple different dispense needles. An optical system aligns all tools and does an offset correction. This approach allows generation of 3D structures fabricated from materials with completely different properties, for example a thermoplast in one cartridge and a hydrogele in another cartridge.

Shrink the Scaffold
Miniaturized PCL Scaffolds

Some applications require downscaling of 3D printing to get into the range of small organs like blood vessels. BS3.1 offers tools for optimizing/ accomplishing standard pneumatic printing.

Aluminum nozzles (See Options) fit well to the BS3.1 cartridge heater. As the nozzle temperature approaches the melting point, tiny PCL struts can be printed more easily. The cube consists of 200 layers, 0.05 mm hight each. The strut width is about 0.33 mm.

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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 (Picture above) has a width of about 0.04 mm. PCL 50,000 was printed at a voltage of +30 kV and a pressure of 0.6 bar.

The MES module for BS3.1 is currently under development and will be launched 2016.

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APPLICATIONS

General Overview :
What is this instrument used for?
  • Production of multi-material bioscaffolds with defined inner structure
  • Printing of 3D bodies from thermoplastics
  • Development of implant replacements
  • Printing of live cells ("Organ printing"), either embedded in the scaffold material or seeded by a piezoelectric microdispenser
  • Deposition of coatings on surfaces
  • Application of rare samples onto 3D structures during printing

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

Struts and Capsules
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.

Light microscopy images of cell-loaded alginate capsules in ALP-loaded alginate struts immediately after fabrication. The free
space in the center of the image is a macropore.

 

Fluorescence microscopy image after 10 days of culture and OsteoImage®, DAPI and
Vybrant staining capsules loaded with ALP. Green: calcium phosphate. Blue: cell nuclei. Red: cell body. Scale bars: a = 200 µm, b = 500 µm

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

Fabrication of photosynthetic Algea-laden Hydrogel Scaffolds
Green Bioprinting

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. [1]

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.

                                        

 Algae loaden scaffold after 1 day of culture [1]                         Algae loaden scaffold after 12 days of culture [1]

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. [1] The immobilization of microalgae in the plotted structures resulted in an enhanced viability and stable growth rates even under suboptimal culture conditions. [2]

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[1] 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

 
[2] 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

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Centre for Translational Bone, Joint and Soft Tissue Research
Technische Universität Dresden

 

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Alginate / Methylcellulose Blands for 3D printing
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

3D plotting of alginate-based hydrogel scaffolds: (A) 3 wt% alginate without methylcellulose, four layers; (B) 3 wt% alginate + 9 wt% methylcellulose (alg/MC), four layers; (C) alg/MC, 20 layers; (D) alg/MC, 50 layers; (insert) top view. [1]

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.

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[1] 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

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Centre for Translational Bone, Joint and Soft Tissue Research
Technische Universität Dresden

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Artifical Tissues from the Inkjet
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)

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[1] 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).

[2] 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).

[3] 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)

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 Fraunhofer Institute for Interfacial Engineering and Biotechnology
Nobelstrasse 12
70569 Stuttgart

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As Fast As Possible
Highly Customized and Affordable Implants by a new Hybrid 3D Printing Technology

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 proce

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