BioScaffold Printer 3.3 Prime

The BS3.3 Prime is the new affordable bioprinter from GeSiM, including a set of tools for easy printing of most popular bioinks. It comes with the full software package and identical functions known from the larger GeSiM bioprinters.

Versatility Matters!

Different biopolymers require different extrusion methods:  This BioScaffold Printer BS5.1 is configured with temperature controlled pneumatic extruder, Nanolitre pipetting and mechanical piston extruder.

BioScaffold Printer 5.1/E

BS5.1/E - This bioprinter is the "working horse" for tissue engineering at production scale. It's powerful but silent motion system combines accuracy with 24/7 reliability.

BioScaffold Printer 3.2

The BioScaffolder 3.2 get's your cells in shape! This bioprinter accepts many bioinks and meltable polymers. Learn how to evolve your petri dish cell culture into an artificial piece of bone or skin.

Thin mesh printed by the MEW tool

Melt Electro Writing

High-voltage induced deposition of biopolymer fibres creates attractive growth environments for cells. (Image with courtesy from Dr. Cathal O'Connell, BioFab3D, St Vincent's Hospital Melbourne, Australia)

Algae Laden Scaffolds

Greetings from the plant kingdom: Chlamydomonas reinhardtii (Algae cells) peaceful coexists with human cells printed into an alginate/hydrogel scaffold. Courtesy of Centre for Translational Bone, Joint and Soft Tissue Research

Struts and Capsules

Fluorescence image of cell loaded Alginate capsules after printing with the GeSiM Bioscaffold printer. Courtesy of Institute of Biomaterials, Erlangen.

Development of Artifical Womens Breast

based on PCL-scaffolds. Tissue is regenerated by injecting patient-derived fat cells into the scaffold pores (Bellaseno GmbH).

The New Egg Style for Breakfast?

A thin mesh from PCL 50.000 was printed on a solid support. After detaching the flexible mesh can be moved. It adheres easily to the curved surface of the egg.

3D Bioprinters – The Modular Solution for Tissue Engineering from GeSiM

Customizable 3D-Bioprinters

Additive manufacturing (AM) for tissue engineering (TE) and regenerative medicine combines biocompatible/biodegradable polymers with living cells. GeSiM 3D bioprinters can create bioscaffolds for cell growth or deposit layers of bioinks on implants or microfluidic objects. However, artificial bones, skin, organoids still not reach their originals made by nature. Research on this subject requires flexible lab equipment, compatible even with new tools and accessories still to be developed.

Our bioprinters offer this level of versatility. Pneumatic extrusion is on board of each GeSiM bioprinter, numerous tools can be configured with your order or added years later. The user interface fits into this philosophy and adapts to each individual combination of tools.

Most extrusion tools fit to all GeSiM instruments. The combination of 3D printed scaffolds with Nanolitre pipetting and microfluidics extends the field of TE to drug discovery, cell biology and more.

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Applications at a Glance:

  • 3D Printing of very different (Bio-)polymers (can contain cells)
  • Design and print of porous – multi material – 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
  • Co-printing of two polymer melts through a gradient mixer

Don’t miss the tab CUSTOMER REFERENCES outlining selected customer projects.

Instruments

BS3.3 Prime

The choice for beginners with potentially growing demands.

It is an attractively priced but complete bioprinter package and offers temperature-controlled printing heads plus UV-light for printing of a wide range of Hydrogels, Alginates and low-melting polymers like certain PCLs.

BS3.2

BS3.2 and it’s predecessors are in use for years in labs which paved the way for tissue engineering.

It is an individually configurable bioprinter for cutting-edge research. The range of available extrusion tools accepts almost any polymer of potential use in the field of TE.  It combines highest flexibility with compact dimensions and fits in many popular biosafety cabinets.

BS5.1

It’s the perfect companion for large research teams, core facilities and service providers with 24/7 operation.

BS5.1 comes with extremely powerful linear motors. This bioprinter can run unrivalled systems for gradient mixing of thermoplastic biopolymers during the print, in addition to all other extrusion tools.

BS5.1/E

The largest GeSiM bioprinter tops our  portfolio.

BS5.1/E offers almost double print bed size than all other bioprinters. The X/Y printing range is probably the largest on the market of commercial bioprinters. Customized object holders for batch production are available, please ask.

Smart Technology for Efficient Use

Internal design of a scaffold structure of two different material: Calciumphosphate and Alginate.

Internal design of a scaffold structure of two different material: Calciumphosphate and Alginate.

  • Silent but smart XYZ-drives with Micrometer precision, individually Z-axes for print tools and optical components
  • Decentralized units for printing, media control and computing save your precious space in biosafety cabinets and ensure superb heat dissipation
  • Same software for all GeSiM bioprinters with easy to use CAD import
  • UV-light sources of different intensity/ costs on the print head; camera systems
  • Standard plastic cartridges as well as all Luer- lock nozzles can be used on all GeSiM bioprinters for 3d prints at room temperature
  • Automatic alignment of all nozzle sizes to the building platform
  • Print beds for SBS well plates with easy click fixation, temperature control and customized layouts available on request

GeSiM bioprinters are easy to operate in routine use:

Fully automatic adjustment of nozzles to the print bed (Left), the print bed size of BS5.1 (Right) fits to at least four SBS well plates or equivalent objects.

Nanolitre Pipetting

Tissue Engineering often raises interface and surface questions: How do cells adhere to artificial bones, implants and similar objects? What is the optimum scaffold geometry/ scaffold material to achieve appropriated cell adherence? Which cell line works, which doesn’t? Just throwing your ready-printed 3D object into a petri dish with cell suspension? – The Nanolitre pipetting module applies cells with spatial resolution during printing. Design and realization of complex artificial tissue with several cell types – partially applied to different parts of your 3D structure – becomes feasible.

The GeSiM Nanolitre Pipetting module allows up to 96 different cell suspensions or protein solutions for embedding into 3D structures during printing.

  • CAD – based tethering of cells/proteins to parts of a 3D structure during printing,
  • Volumes <100 Microlitres per species save resources when using rare and expensive samples,
  • Onboard washing allows subsequent liquid handling of different species with one tool,
  • Dispense volume ranges from 0.1 Nanolitre to Microlitres,
  • Twin adapter for micro pipets enables parallel aspiration of two species with subsequent droplet mixing at the target position.

Refer to the tab PRINT TOOLS for technical information on available dispensers.

The pipetting module transfers up to 96 samples on 3D structures during printing

The pipetting module transfers up to 96 samples on 3D structures during printing

 

Sterile Working

Tissue Engineering assembles living cells to biopolymers. 3D printing shall be done quickly, safe and in a sterile environment. Physiological conditions have to be maintained to protect your particular cell lines which often incorporate a lot of labour and resources.

GeSiM Bioprinters always come without lid/ HEPA filters. Our instruments are designed to be placed in a professional Biosafety Cabinet (BSC) or at least in a  laminar flow hood. The usual containments offer bench space for the manual preparation of bioinks immediately before 3D printing. It eliminates contamination risks when transferring your “living bioink” from another place to your 3D printer.

GeSiM collaborates with manufacturers of appropriated devices if you don’t have one in your lab.

BS5.1/E in a Kojair Biosafety Cabinet, setup in a “yellow room” (Courtesy of IBA Heiligenstadt)

BS5.1/E in a Kojair Biosafety Cabinet (Class-II), setup in a “yellow room” (Courtesy of IBA Heiligenstadt)

The BS3.2 bioprinter in a TELSTAR biosafety cabinet

The BS3.2 bioprinter in a TELSTAR Aeolus laminar flow hood


Always on board: Pneumatic Extruders

Each GeSiM bioprinter comes with at least two pneumatic extruders. The GeSiM BioScaffold printers can be extended by numerous tools for additional extrusion principles as well as optical components, like cameras and UV lamps. For optimum configuration of tools for your particular application we offer free pre-tests with your material.

Mechanical Piston Extruders

Piston Extruders on BS5.1: Pair of two HT-Extruders with gradient mixer (left); 50 mL extruder (right)

Piston Extruders on BS5.1: Pair of two HT-Extruders with gradient mixer (left); 50 mL extruder (right)

Pneumatic extruders work well for most cell friendly bioinks. Limitations often occur when particular material properties (Stiffness, viscoelasticity) challenge the pressure control system and cause fading accuracy.

 Powerful motor driven piston extruders extend the capability of the pneumatic extrusion system:

  • High pressure (Virtually > 100 bar) prints viscoelastic and high-viscous materials, even through tiny nozzles.
  • For very low-viscous bioinks constant piston strokes ensure constant material flow, independent on the material level inside the cartridge.

The High-Temperature Piston Extruder (HT-Extruder) prints thermoplastics like PLA (Polylactic acid) and ABS 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. Particular metal nozzles with diameter ranging from 100 Micrometers to 1,500 Micrometers are available for the HT-Extruder.

The Gradient Mixer (Available for BS5.1 only, left above) combines the outlets of two HT-Extruders with a special mixing head. It allows varying mixing ratios of two thermoplastic melts during one print.

3D printing of larger batches becomes available by the 50 mL piston extruder (right above). Adapters for smaller cartridges are available on request.

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Core/Shell Extruder for coaxial printing of two materials

Core/Shell extruder for two materials

Core/Shell extruder for two materials

Schematic function

Schematic function

SEM image of C/S strand with empty core lane

SEM image of C/S strand with empty core lane

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 the BioScaffold printers allows simultaneous printing of two materials with different properties in a coaxial arrangement. The tool features a pair of nozzles – an inner core nozzle, surrounded by the outer shell nozzle. The print parameters can be set individually for both channels in order to match properties of the respective bioinks. Nozzle diameters range from 100 Micrometre to 5000 Micrometre.

Core/Shell strands with cells inside after 7 days cultivation (Bright field image) [1]

Core/Shell strands with cells inside after 7 days cultivation (Bright field image) [1]

Nuclei stained blue [1]

Nuclei stained blue [1]

[1]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

Nanolitre Pipetting Tools

The GeSiM bioprinters BS3.2 and BS5.x are available with the optional Nanolitre pipetting module. It transfers small liquid sample amounts from a 96 well micro titer plate to a defined locations on the target object during 3D printing.

Piezo pipet on a 3D print

Piezo pipet on a 3D print

Source plater heater with 96 well plate; Drop camera (left)

Source plater heater with 96 well plate; Drop camera (left)

Pair of piezo pipets on a swivel (Twin tip)

Pair of piezo pipets on a swivel (Twin tip)

Pipets for different volume range are available:

  • Passive needles for syringe supported displacement dispensing. The lowest dispense volume might be half a Microliter.
  • The volume range between fifty Nanolitres and a few Microlitres is covered best by the solenoid dispense valve.
  • Piezoelectric nozzles for free-flight micro drop dispensing (Viscosity limit around 10 mPa*s). The lowest dispense volume is in the range of 0.1 Nanolitres.

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 each other and mix up on the target surface.

Heatable versions of the piezoelectric nozzles are available for liquids with higher but temperature dependent viscosity.

Curing/ Photopolymerization

Omnicure S1500 lamp on BS3.1; Optical fibre on the print head

Omnicure S1500 lamp on BS3.1; Optical fibre on the print head

Among others the GeSiM BioScaffold printers support the Omnicure S1500 UV lamp. GeSiM assembles the necessary optical fibre to the XYZ gantry. A special lens head attaches to one of the three cartridge holders.

Melt Electro Spinning-Writing (MEW)

The optional MEW module for the BioScaffold printers BS3.2 and BS5.x combines pneumatic extrusion and high-voltage induced fibre deposition for particular thermoplastic materials. Strand size and layer thickness come down below 100 Micrometres. Strand thicknesses of 10…20 Micrometres can be achieved from materials like PCL with a molecular weight of up to 50,000.

Micro-Mesh from PCL 50,000l 100 layers. The strand distance is 500 Microns (Left side) and 200 Microns (Right side)

Micro-Mesh from PCL 50,000l 100 layers. The strand distance is 500 Microns (Left side) and 200 Microns (Right side)

In contradiction to Melt Electrospinning (MES) Melt Electrospinning Writing deposits each fibre in a regular manner. CAD geometries can be printed to a certain extent. The MEW module extends the standard configuration of the instrument by a special tray with embedded electrode, a high-voltage generator and security measures.

Powder Pipet (Patent pending)

Tiny amounts of granular solid materials can be added to 3D prints by the new powder pipet: It’s kind of a vacuum tweezer with a defined pore chip or micro basket on the foot side. It aspirates either single grains or a few micrograms from a reservoir and dispenses it on a target. The foot area of the powder pipet is customized for a particular granule and application.

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A particular biopolymer is usually available with very different compositions, molecular weight and physical properties (melting points, viscoelasticity). Please download the Tools at a Glance brochure for more information. GeSiM is ready for print tests with your own bioink/ biopolymer. Please contact us for arrangements.

Plasma  Pen

The Plasma Pen (developed and manufactured by Nadir S.r.l.) is an atmospheric plasma jet that allows the ionization of argon gas by applying high voltage (HV) near the channel where the gas is flowing. It can be configured to each GeSiM bioprinter as individual tool.

Plasma Pen on BS5.1

Plasma Pen on BS5.1

Plasma treatment of a bone biopolymer scaffold during printing

Plasma treatment of a bone biopolymer scaffold during printing

Schematic of Nadir Plasma Jet Module: Argon Channel (1), carrier gas or reactive gas capillary (2), cooling or shielding gas channel (3), plasma plume (4)

Schematic of Nadir Plasma Jet Module: Argon Channel (1), carrier gas or reactive gas capillary (2), cooling or shielding gas channel (3), plasma plume (4)

Applications:

  • Activating and cleaning the whole object porosity
  • Sterilization with oxidative plasma during polymer printing
  • Functionalization with a proper chemical precursor without solvents

Benefits:

  • Reductive or oxidative plasma can be used to clean surfaces, remove organic dirt or polymeric layers
  • Surfaces can be activated to improve adhesion of cells, printed material or different melted materials
  • Inlet capillary for the introduction of vapour and aerosol precursors allows deposition of functionalities, coatings and nanocomposites

The Plasma Pen was developed and adapted to GeSiM products within the frame of the European FAST project. Please download the FAST brochure for more information.


Please download the BioScaffolder brochure for more details on available tools or just contact us for full catalogue with all technical details and specs.


Highest Flexibility for a Changing World

GeSiM instruments incorporate highest modularity. Even when acquired for a particular project, additonal tools for upcoming projects are available at affordable costs.

Our budget line BS3.3 Prime comes with a fix combination of tools, whereas BS3.2 and BS5.1 can be configured almost arbitrarily. Here we show popular setups, please visit the preceding tab “Print Tools” for more information or download the brochure below.

Hardware upgrades mostly can be done without a service visit of GeSiM engineers (Limited for BS3.3 Prime). GeSiM permanently developes new tools/modules, probably fitting to your personal BioScaffold printer.  The user software GeSiM Robotics reconfigures easily and reflects always the actual status when adding new hardware. Tool-related pieces of software can be added similar to the download of apps for your phone.

The GeSiM factory service is available for older BioScaffold printers as well as for retrofitting of BS3.3 Primes with fix configuration.

2x 10mL cartridge with heaters; Camera; 30 mL cartridge, Piezo Pipet (From left)

2x 10mL cartridge with heaters; Camera; 30 mL cartridge, Piezo Pipet (From left)

10mL cartridge with heater and UV head; Piezo valve (High viscosity); 30mL cartridge; Piezo Pipet (From Left)

10mL cartridge with heater and UV head; Piezo valve (High viscosity); 30mL cartridge; Piezo Pipet (From Left)

HT-Extruder; 2x 30mL cartridges; Piezo Pipet (From left)

HT-Extruder; 2x 30mL cartridges; Piezo Pipet (From left)

Particular applications may require tools for high pressure, high temperature, extremely thin strands and others:

HT-Extruder; 10mL Peltier chiller; Z-sensor; Twin Piezopipet (From left)

HT-Extruder; 10mL Peltier chiller; Z-sensor; Twin Piezopipet (From left)

HT-Extruder 2x with gradient mixer; 50mL Extruder (From left)

HT-Extruder 2x with gradient mixer; 50mL Extruder (From left)

50mL Extruder 3x

50mL Extruder 3x, for printing of large models and batch production

GeSiM Robotics

The user software of all our Bioprinters is called GeSiM Robotics. It is a flexible but intuitive software:

  • Configuration and automatic alignment of print tools and print targets
  • Quick test prints considering varying print parameters give best settings for a particular combination of cartridges, nozzles and biopolymers
  • Optical monitoring of drop-on-demand pipets
  • Replacement of cartridges/ nozzles on demand during a 3D print eliminates the risk of unsuccessful runs due to clogged nozzles and run out cartridges
  • Planning and execution of batch processes (sequences) allows copies of a 3D structure or different separate 3D structures unattendedly.

Processing of External CAD Data

GeSiM Robotics supports STL and 3mf formats. 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.

3D models printed from STL files: Women breast model (left), bulbasaur (right)

3D models printed from STL files: Women breast model (left), bulbasaur (right)

The BioScaffold printer software defines internal scaffold- structures in between surfaces imported from a STL file. The 3mf format works similar but supports different objects in one file. It is the preferred data standard for multi-material scaffolds.


Printing Life to Shape the Future

Beside of instrumentation, 3D printable materials are the next key to success in tissue engineering. Over the past decade research teams worldwide focused on tuning quite common materials like gelatine into 3D printable bioinks. Opposing specs like cell friendliness and mechanical stiffness have to combine for one product.

GeSiM bioprinters accompanies several of these groups, therefore contributing to the scientific progress. Like most bioprinters, GeSiM BioScaffold printers accept all commercially available bioinks, granules, powders, pastes, liquids and filaments.


InnoRegen, Inc.

In order to offer all components for tissue engineering from one source we collaborate with InnoRegen, Inc., based in Daegu, Korea. GeSiM is selling InnoRegen Products and will be happy to discuss your particular application. Please contact us for price quotation on InnoRegen Bioinks.

InnoRegen Hydrogels

Prints best at temperature between 4°C and room temperature, e.g. with the GeSiM Peltier module.

Gel4Cell

Bioink for 3D bioprinting with gelatine hydrogel for increased biocompatibility and structural safety applied throughout the study of regenerative medicine.

  • Cell compatible material
  • High printability and uniformity
  • UV-cross linkable for structural stability

Gel4Cell – Peptides

Peptide conjugated hydrogels supporting cell growth with particular tissue types.

  • Gel4Cell – BMP: Bioink, which can be applied to bone regeneration research by combining BMP-2 peptide, a bone regeneration growth factor.
  • Gel4Cell – VEGF: Bioink, which can be applied to vessel and skin regeneration research by combining VEGF peptide, an angiogenesis factor.
  • Gel4Cell – TGF: Bioink, which can be applied to cartilage regeneration research by combining the cartilage regeneration growth factor TGF-β1 peptide.

Gel4Tissue

Bioink, which can be applied to artificial organ/tissue regeneration studies by combining pig’s intestinal membrane extracellular vagina (SIS-ECM).

Col4Cell

Collagen hydrogel for increased biocompatibility and structural safety.

InnoRegen Thermoplastic Biopolymers

Meltable biopolymers with good fit to the GeSiM cartridge heater and HT-Extruder.

  • Polyinks – PCL: Caprolactone Synthetic Polymer with excellent biodegradability. For use with cells Gel4Cell bioink can be used within the same print. The printing temperature is between 65°C and 100°C.
  • Polyinks – PLA: L-lactide synthetic polymer with excellent biodegradability, printing temperature is between 200°C and 250°C.
  • Polyinks – PLCL: A blend of PCL and PLA with controlled biodegradability, printing temperature between 70°C and 250°C.

Biodegradability:
PLCL(W): 2….6 weeks                    PLCL(M): 6…10 weeks                   PLCL(Y): 10…16 weeks

What are the GeSiM BioScaffold printers 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.


Melt Deposition Modelling of Bioactive Organic/Inorganic Hybrid Scaffolds with HT-Extruder

Research at the Laboratoire de Physique de Clermont, France is focused on the development of biomaterials with tailored bioactive, osteostimulative and other therapeutic properties for bone repair and regeneration. In particular, key studies focus on the design of an organic/inorganic platform technology inspired by the complex physiological structure of the bone: hybrids materials combining polymers and inorganic species at the molecular scale. The components of such materials can be modified to obtain a wide range of formulations with diverse physicochemical and biological properties. This, in turn, highly influences the processing conditions of each hybrid formulation into its final form.

Investigations are being carried out to understand and tailor the processing into purposefully designed  3D implantable scaffolds with microporosity and nanotopographical features that can aid  cell adhesion and in-growth of tissues and vessels. After successful results using conventional scaffold fabrication techniques (Bossard et al., 2018, Biomedical Glasses), our current studies focus on the possibility to apply additive manufacturing (AM) to organic/inorganic hybrid. The optimization of AM for hybrids can open up to new shapes and structures, helping to achieve topographical features, and possibly clinical applications, that are currently unattainable with other techniques.

The Bioscaffolder 3.2 by GeSiM proved itself to be an adequate platform for our purpose. Thanks to its modularity, it is easy to quickly perform the testing of several candidate materials with different set-ups, often in parallel. It is  possible to run proof-of-concept small batch trials for several newly synthesized formulations, optimizing time and resources. In particular, current results, although at their early stages, confirmed that with careful setting of the HT extruder module it is possible to obtain thrust forces that are sufficient for the extrusion of bioactive hybrids of polycaprolactone and bioactive glass, through both 500 and 400 μm nozzles. The temperatures and flow rates used ranged between 80-120 °C and 3-5 μm/s, respectively, depending on the molecular weight of the polymer used. With these parameters the successful direct printing of a bioactive glass hybrid was performed for the first time.

PCL/ Bioglass scaffold in a tweezer

PCL/ Bioglass scaffold in a tweezer

The test object at 10mm size

The test object at 10mm size

Internal porous structure of a hybrid scaffold

Internal porous structure of a hybrid scaffold

Courtesy of: Dr. Lukas Gritsch, Prof. Jonathan Lao

Universite Clermont Auvergne,
Laboratoire de Physique de Clermont,
CNRS/IN2P3, Biomaterials group,
France

References:

[1]            H. Granel, C. Bossard,, J Lao, Y. Wittrant, et al – Bioactive Glass/Polycaprolactone Hybrid with a Dual Cortical/Trabecular Structure for Bone Regeneration – ACS App. Biomater. 2(8), 3473-3483, 2019.

[2]            Cédric Bossard, Henri Granel, Édouard Jallot, Christophe Vial, Hanna Tiainen, Yohann Wittrant, J. Lao – Polycaprolactone / bioactive glass hybrid scaffolds for bone regeneration, Biomedical Glasses 4(1), 108-122, 2018.


MEW Milk Protein/PCL Blends for Skin Regeneration

The GeSiM bioprinter BS3.1 is an important tool at the Department of Chemistry at the University of Otago, New Zealand.

Demand for skin replacements is rapidly increasing as burn and full-thickness wounds are difficult to repair due to the low regeneration capability of innate tissues, as well as the physical drawbacks associated with currently available substitutes.

Polycaprolactone (PCL), a bioresorbable and biocompatible, synthetic polymer was selected as scaffold material due to its mechanical stability, flexibility, and superior melt processing properties. In order to increase PCL’s biological functionality bioactive milk proteins (MPs) were blended with PCL.  To date (August 2019, GeSiM), this is the first study of its kind detailing the tissue regenerative capacity of PCL containing MPs as bioactive additives for skin regeneration using MEW.  The aim of this study was to MEW MP/PCL tissue engineered constructs (TEC) and assess their suitability for generating tissue in vitro.

The MPs, lactoferrin (LF) and whey protein (WP), were mixed with PCL individually at varying concentrations (0.05%, 0.1%, 0.25%), and in combination (COMB) at concentrations of 0.25% e ach. TECs were characterized chemically, physically, and their biological activity assessed in vitro.   Physical characterisation of MEW MP/PCL scaffolds showed that reproducible, layered micron range scaffolds could be fabricated; displaying high porosity, low degradation, and rapid protein release. Biological activity, determined via an in vitro skin model using human keratinocytes (HaCaTs) and normal human dermal fibroblasts (Nhdf) cells, showed significantly increased cell growth, spreading, and infiltration into LF (0.25%) containing scaffolds and COMB scaffolds when compared to PCL alone (p≤0.05). These findings demonstrated that the combined addition of LF and WP increased the biological activity of MEW PCL scaffolds and could be potentially used as a TEC for deep tissue dermal regeneration.

Visual comparison of scaffolds. Scaffolds generated as a result of setting pore size parameters on the GeSiM 3.1 Bioscaffolder. software. From left, the pore size parameters were set to 500 x 500µm, 400 x 400µm, and 300 x 300µm.

Visual comparison of scaffolds. Scaffolds generated as a result of setting pore size parameters on the GeSiM BioScaffolder 3.1 software. From left, the pore size parameters were set to 500 x 500µm, 400 x 400µm, and 300 x 300µm.

SEM micrographs of scaffolds cultured at 14, 21, 28, 35, and 42 days. Scale bar = 200 µm

SEM micrographs of scaffolds cultured at 14, 21, 28, 35, and 42 days. Scale bar = 200 µm

References:

A/P Azam Ali,  Director – Bioengineering Programme & Centre for Bioengineering & Nanomedicine (Dunedin Hub), University of Otago, Dunedin, NZ

Dr. Jaydee Cabral, Senior Lecturer, Centre for Bioengineering & Nanomedicine (Dunedin Hub), University of Otago, Dunedin, NZ


PCL-PEG Blends for Tissue Engineering

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 [1] 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.

Scheme of a hard-soft phase scaffold with the hard thermoplastic phase (grey) and the soft hydrogel phase (yellow) containing the cells [1]

Scheme of a hard-soft phase scaffold with the hard thermoplastic phase (grey) and the soft hydrogel phase (yellow) containing the cells [1]

Stereomicroscope images of a plotted PCL-PEG (7030) scaffold as fabricated: topview (a); and side view (b) (scale bar = 2 mm) [1]

Stereomicroscope images of a plotted PCL-PEG (7030) scaffold as fabricated: topview (a); and side view (b) (scale bar = 2 mm) [1]

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.
Fluorescence microscope images (a–f) of the actin cytoskeleton (red) and the cell nuclei (green) of ST2 cells in a PCL-PEG ADA-GEL construct after 28 days of incubation of different magnification: (a,b) overview images; (c) densely packed area of the cells covering both materials; (d) cell morphology on the hard phase; (e) cell agglomerate and spread single cells in hydrogel; and (f) densely packed area of cells (hydrogel phase) [1]

Fluorescence microscope images (a–f) of the actin cytoskeleton (red) and the cell nuclei (green) of ST2 cells in a PCL-PEG ADA-GEL construct after 28 days of incubation of different magnification: (a,b) overview images; (c) densely packed area of the cells covering both materials; (d) cell morphology on the hard phase; (e) cell agglomerate and spread single cells in hydrogel; and (f) densely packed area of cells (hydrogel phase) [1]

References:

[1] 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 at 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.

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

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.

References:

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 Algae-laden Hydrogel Scaffold

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 1 day of culture [1]

Algae loaden scaffold after 12 days 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]

References:

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

Alginate/Methylcellulose Blends for 3D printing

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]

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.

References:

[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

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.

1a) CT data set of human hand

1a) CT data set of human hand

1b) Bone extraction

1b) Bone extraction

1c) Segmentation of the scaphoid bone

1c) Segmentation of the scaphoid bone

1d) 3D model of scaphoid bone

1d) 3D model of scaphoid bone

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.

2) Scaphoid bone printed from bone cement [1], [2]

2) Scaphoid bone printed from bone cement [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).

References:

[1] T. Ahlfeld, T. Köhler, Ch. Czichy, A. Lode, M. Gelinsky: A Methylcellulose Hydrogel as Support for 3D Plotting of Complex Shaped Calcium Phosphate Scaffolds. Gels 2018, 4, 68 (14 pages)

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 process that is even capable to produce gradients in the bulk or surface properties of the individual scaffold. Furthermore, the incorporation of smart fillers with bio-active properties into the scaffold material will reduce the risk of post-surgery infections.
Printing of PEOT/PBT mixture with varying ratio

Printing of PEOT/PBT mixture with varying ratio

GeSiM has been engaged for the development of high-performance syringe extruders for printing of polymer gradients within a single AM step. We developed a specialized mixing device to combine two separate HT syringe extruders. The new mixer fits onto the BioScaffold printer BS5.1.

Further GeSiM adapts the NADIR plasma pen onboard of the BioScaffold printer, it now synchronizes with all other BS tools. The plasma pen is a highly flexible tool to modify surfaces layer-by-layer during the 3D print. Depending on its configuration the plasma pen sterilizes, activates surfaces or deposits reactants. This atmospheric plasma jet is realised starting from the unique design developed by Nadir S.r.l. (Patent no. WO / 2015/071746) that ensures an efficient, cold and clean plasma for the surface treatment of the most delicate and heat sensitive materials. Nadir plasma is based on a 27MHz RF (radio frequency) generation system that allow to obtain one of the coldest plasma present in the market, able to preserve a high efficiency in the desired surface treatment of materials

In particular, Nadir torch allows:.

  • The surface etching to increase the roughness at nanoscale and to expose the fillers to the surface
  • The coating with nucleophilic groups
  • The coating with electrophilic groups
  • The control of the surface wettability.
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Materials for Bioprinting

Several partners developed new biopolymers and fillers. New materials e.g. to mimick artificial bones are now commercially available:

  • PEOT/PBT polymer composites (master batches) (Polyvation BV, The Netherlands)
  • Fillers as drug delivery systems for bioactive molecules (Prolabin & Tefarn S.r.l., Italy
  • Polymer compounds with active and bioactive properties (Nadir S.r.l., Italy)
  • Dosable Reduced Graphene Oxide (Abalonyx AS, Norway)

For more information please download the FAST brochure.


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:

Press contact via the project coordinators:

Dr. Lorenzo Moroni, Maastricht University,
Dr. Alessandro Patelli, Nadir S.r.l.


Brochure BioScaffolder

Introduction to GeSiM Bioprinters

BioInks available from GeSiM

Made by InnoRegen, Inc., Korea

Tools & Materials

Tools at a Glance

Which tool for which material? A quick guide.

European Project FAST

Functionally graded additive manufacturing scaffolds by hybrid manufacturing