Theme 5: Tissue Engineering for Regenerative Medicine

Enabling technologies for Regenerative Medicine

3D printing/additive manufacturing

3D printing and 3D bioprinting have the potential to revolutionise healthcare. 3D printing, also called additive manufacturing, is the process of creating a three-dimensional [3D] model by laying down successive layers of material that build upon each other. Printing materials or ‘ink’ can be a variety of substances including plastics, polymers, metal alloys, among others. When the ink being used is living cells, the 3D printing is called 3D bioprinting. Both 3D printing and 3D bioprinting are increasingly being used to advance healthcare.

Go-to person for additive manufacturing

Dr Tim Woodfield, University of Otago

tim.woodfield@otago.ac.nz

Examples of bioprinting are:

  • A procedure that takes a scan of a burn wound and then the 3D bioprinter directly prints skin cells onto the burn wound in layers, which replace the damaged skin. Using the scan as a map, the printer is able to fill in the burn wound until it is covered with the new skin cells. Wake Forest School of Medicine is a leader in this work.
  • The use of modified ink jet printers, with bio-ink made up chondrocytes and PEG-DMA, to print cartilage. Cartilage is a good fit for 3D bioprinting because chondrocytes secrete their own matrix, do not need many nutrients and overall require only low maintenance.
  • A process using three bio-inks; one containing only ECM; the second ECM plus living cells; and the third that melts as it cools [!] rather than as it warms. An interconnected network of filaments is printed, and then melted by chilling the material and suctioning the liquid out to create a network of hollow tubes, or vessel precursors. Injection of human endothelial cells into the latter expedites the formation of the blood vessel lining. The end result is a construct containing blood vessels and three different types of cells, thus approaching the complexity of solid tissue.
  • An integrated system capable of printing bio-scaffold structures and automating the process of cell seeding. The key components of the system are a customised bio-scaffold printer and a spheroid seeding device. The former generates the physical bio-scaffold for cell seeding, and the latter separates and places individual cells within the porous lattice the scaffold.

Despite such anecdotes bioprinting technology still has to overcome a number of challenges to achieve its full translational potential, and to progress from lab to bedside. Probable challenges are:

Choice of biomaterials: The successful manufacture of viable self-sustained tissue means the biomaterial properties of the polymer must mimic the biomaterial properties of the native tissue.
Choice of cells: Management techniques for primary cells, multipotent stem cells and pluripotent stem cells pose their own singular problems.
Design of environment: The highly toxic chemicals and high temperatures required for 3D processing risk affecting polymer chemistry and cell viability. Achieving safe and effective means of avoiding this risk will not be easy.
Achievement of in vivo compatibility: The challenge is to ensure the stringent process of bioprinting will not affect the cells, even where organs are generated from patients’ own cells and thus avoid immune response or rejection.

4D techniques, which allow cells in suspension to self-organize autonomously and without external intervention, are now firmly part of an extensive additive manufacturing mileu. Printing combined with self-assembly constitutes an exciting new tool. One of the best examples of self-assembly in humans/animals is organ formation and morphogenesis in the embryo. Self-assembly in TE could be applied to (i) biomaterial [scaffold] and (ii) cells. Self-assembly could be accomplished in vitro – before implantation of a TE construct and/or in vivo.

Microfluidics

Microfluidics constitute a key platform providing capabilities ranging from high-content analysis and drug screening to TE, and promise advanced insights into cellular dynamics generally and stem cell biology and ‘management’ in particular.

Go-to persons for microfluidics

Dr Volker Nock, University of Canterbury

volker.nock@canterbury.ac.nz

Dr Mike Arnold, Callaghan Innovation

Mike.Arnold@callaghaninnovation.govt.nz

In TE the technology is expected to make a major contribution in two areas in particular:

  • facilitating the growth of complex tissues, where microfluidic structures ensure a steady blood supply, so circumventing the problem of providing larger tissue constructs with a continuous flow of O2 and nutrients, and the egress of waste products
  • developing, when combined with micro- and nano-technology, in vitro physiological systems for studying fundamental in vivo biological phenomena. Microfluidics can be used to generate, for example, shear stresses, growth factor gradients, co-cultures and migration assays. Considerable work has been done on the vascular and neural systems, the liver, cancerous tissue and stem cells.

Also, because the microenvironment can be controlled in microfluidic platforms, the technology has much to offer stem cell biology. The super-sensitivity of stem cells to environmental clues is able to be 'managed' in a way that traditional cell culturing techniques do not allow.

Vascularisation of engineered tissue continues to be a challenge. Tien et al2 developed methods to form vascular scaffolds based on removal of a sacrificial material or on the bonding of gels. Long-term stable vascularisation of microfluidic gels appears to require leakproof vessels. Perfusion introduces chemical and mechanical signals that play complementary roles in controlling the stability of the vessel wall. Computational models enable optimization of microfluidic designs for which perfusion through the vessels is stable and sufficient to oxygenate surrounding tissue. Fig 2 is an icon from this work, illustrating the flow of red blood cells [RBCs]:

A diagram showing red blood cells flowing through a blood vessel.

Fig 2 The use of microfluidics in vascularisation.

Multiaxial-loading bioreactors

Multiaxial-loading bioreactors, which constitute another flow cytometry-based array of tools, enable investigations into cell and tissue growth, extracellular matrix secretion and their variation in vivo by introducing, concomitantly and variably, dynamic mechanical signals such as compression, hydrostatic pressure and pulsatile flow of medium to cell-seeded scaffolds. In such systems the operator can intervene to apply a sequence of different conditions to the developing tissue, including making sequential adaptations to changing scaffold properties.

The operation of these bioreactors showcases the large number of sensitive interrelationships that exist between scaffolds, cells, developing tissue and externally imposed mechanical forces. See fig 3. The understanding of, and the ability to manage, these interrelationships is a key requisite in controlled manufacture of targeted clinic-ready tissue.

A diagram of inter-relationships. The title is \

Fig 3 Some of the interrelationships that exist between scaffolds, cells, developing tissue and externally imposed mechanical forces

Electrospinning

Electrospinning shares characteristics of both electrospraying and conventional solution dry-spinning of fibers. It uses an electrical charge to draw very fine [typically on the micro- or nano- scale] fibres from a liquid and offers capability in building biological [both animal and plant] and synthetic nanofibre matrices for cell culturing.

Go-to persons for electrospinning

Dr Samaneh Karimi ("Sammi"), NZ Institute of Plant and Food Research

samaneh.karimi@plantandfood.co.nz

Dr Nick Tucker, University of Lincoln

NTucker@lincoln.ac.uk

Dr Joel Segal, University of Nottingham

joel.segal@nottingham.ac.uk

Fig 4 shows the basics of electrospinning:

A schematic diagram showing elements of electrospinning. The elements are ground earth, collector (shaped like a plate), high voltage power supply, and syringe. The syringe contains polymer solution, extruding a liquid jet in a spinning fashion with a spinneret onto the collector. The spinneret is connected to the the power supply, which in turn is connected to the ground earth and collector. The liquid jet forms a Taylor cone, a cone formed by a jet of charged particles that emanates above a threshold voltage.

Fig 4 The essential elements of electrospinning

Electrospinning lends itself to combination with other technologies in the pursuit of 'better' outcomes e.g. in combination with Fused Deposition Modelling [FDM] and using advanced scaffold materials it offers possibilities for engineering blood vessels that are not compromised by factors common to traditional tissue engineering [TE] approaches such as poor mechanical properties, thrombogenicity and cell overgrowth inside the construct. Centola et al3

…..developed a poly-L-lactide [PLLA]/poly-epsilon-caprolactone [PCL] scaffold releasing heparin by a combination of electrospinning and FDM. PLLA/heparin scaffolds were produced by electrospinning in tubular shape and then FDM was used to armour the tube with a single coil of PCL on the outer layer to improve mechanical properties. Scaffolds were then seeded with human mesenchymal stem cells [hMSCs] and assayed in terms of morphology, mechanical tensile strength, cell viability and differentiation. This scaffold design allowed the generation of both a drug delivery system to counter thrombogenic issues and a microenvironment able to induce endothelial differentiation. Concomitantly, the PCL external coiling improved mechanical resistance of the microfibrous scaffold. By the combination of electrospinning and FDM and exploiting the biological effects of heparin, they developed an ad hoc differentiating device for hMSCs seeding, able to induce differentiation into vascular endothelium.

Another example is the combination of electrospinning and Ink Jet Printing technologies. Wake Forest Institute of Regenerative Medicine in North Carolina, looking to manufacture cartilage tissue at an injury site, subjects a synthetic polymer solution to the electrospinning process to create a porous nanostructure which serves as a ‘nesting ground’ for cartilage cells, making it easier for cartilage adjacent to the injury site to grow back into it. Flexible mats of this electrospun polymer then have a solution of cartilage cells deposited on them using traditional ink jet printing. A series of these ‘seeded’ mats are layered together, until they form a structure measuring 10 cm diagonally by 4 mm thick. This material is the finished implantable cartilage.

Fig 5 illustrates the process:

A diagram showing combination of electrospinning and ink jet printing. A custom controller is connected through fibre optics or Bluetooth to an inkjet printhead. It is also connected to a high voltage power supply, which is connected to the collector plate and electrospinning printhead. Colour cells and electrospun fibres are extruded from the printheads onto the collector, with the diagram showing a printed layer over an electrospun layer on the collector.

Fig 5 Electrospinning and Ink jet printing combination

There is growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. An investigation into a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined 3D micro-topography at the surface using patterned resin formers produced by rapid prototyping [RP] has recently been conducted. Researchers say future work will investigate the use of such patterned fibre scaffolds to replicate tissue geometries further for use in the development of in vitro models for studying disease, toxicity studies and the screening of potential therapeutics.

On a final note, it has been demonstrated in recent years that the development and production engineering principles used for many mechanical products can be adapted to the requirements of biotechnology products. Assuming a 'logical trajectory of thinking' that builds on such knowledge this portends the increasingly successful integration of mechatronics [mechanical-electrical constructions] and biotechnology. Three areas of promise are stem cell manufacture, artificial organs and bioreactors.

In the production of stem cell-derived tissue and organ cells, for instance, biomechatronics can be used to address in an ordered way a number of challenges in the design of manufacturing systems, including: the expansion stage of production; the control of cell differentiation; analysis and process control; and automation and scale-up.


2 Tien J, Wong KHK, Truslow JG. Vascularization of microfluidic hydrogels In: Bettinger C, Borenstein JT, Tao SL. Microfluidic Cell Culture Systems. Oxford: Elsevier; 2013.

3 Combining electrospinning and fused deposition modelling for the fabrication of a hybrid vascular graft. Centola M, Rainer A, Spadaccio C, De Porcellinis S, Genovese JA, Trombetta M. Biofabrication: 2010 Mar; 2(1):014102. Epub 2010 Mar 10.