Three-Dimensional Bioprinting for Tissue Engineering and Regenerative Medicine in Down Under: 2020 Australian Workshop Summary

Bioprinting technology is a fast-evolving multidisciplinary field, harnessing the benefits and capabilities of three-dimensional printing and regenerative medicine approaches to interface the fields of biology and engineering.¹ It allows the incorporation of traditional additive manufacturing approaches toward the biologic fabrication of microscale tissues through the spatiotemporal deposition of cell-laden bioinks (Figure 1).² Bioassembly is the coordinated assembly of cells, spheroids, and biologic factors into desired geometries within in vitro microsystems and biofabricated constructs.⁴ To date, the most common bioprinting approaches employed include extrusion-based, inkjet, and microvalve; alongside light-based approaches of stereolithography, direct laser writing, digital light processing, and volumetric techniques.^5,6 The ability to incorporate living cells, biomaterials, and biologic factors within biomimetically engineered tissue constructs holds great potential to recapitulate complex elements of natural tissues and organs.⁷

Figure 1.: Side-by-side comparison of tissue architecture to commonly used bioprinting techniques resolution. Modified with permission from Ref. ³.

The Australian Bioprinting Workshop for Tissue Engineering and Regenerative Medicine was founded to create a community of scientists, clinicians, and engineers to work in synergy with industry, government, and regulatory agencies to tackle key challenges in the bioprinting field and facilitate the clinical translation of this promising technology.⁸ Annual workshops are hosted by the University of Technology Sydney (UTS) alongside company sponsorship, with support from professional three-dimensional printing networks such as 3D Heals. In recent years, Australia has seen considerable growth and investment dedicated toward establishing collaborative research centers to maximize research impact through industry-orientated projects and the clinical translation. This is supported by the several bioprinting laboratories located within a hospital, such as the Herston Biofabrication Institute at Metro North Hospital, Brisbane, and BioFab3D located within St Vincent’s Hospital, Melbourne. These workshops seek to build on existing collaborative partnerships to assist in publications of international scientific consensus on key developmental areas including biomaterials for bioprinting, organ-on-a-chip studies, and the bioprinting of tissue-specific models for drug development and oncology applications.⁹ Key elements in developing these events have been the principle that all member publications should reflect a consensus of those engaged in its continued discussions.

The meeting was opened by Prof. Joanne Tipper (the Head of the School of Biomedical Engineering at UTS), and Dr. Carmine Gentile (workshop organizer and leader of the Cardiovascular Regeneration Group both at UTS and at the Kolling Institute/University of Sydney), followed by introducing the program for the day and chairing the opening session focusing on industry innovation, as detailed in the following.

Industry Innovation

The purpose of this session was to provide the opportunity to hear directly from world leaders in the field of bioprinting how commercially available bioprinting platforms are helping researchers in tackling several of the problems characteristic of bioprinted tissues. This session was opened by Dr. Manuel Figueruela García (Regemat3D) with a focus on the customization of bioprinting and bioreactor platforms that can be applied through tissue specificity for regenerative medicine applications. Dr. Matija Rojnik (Fluicell) followed by presenting the capabilities of achieving multicellular models with single cell bioprinting precision through harnessing power of microfluidics-based approaches. Derek Mathers (Advanced Solutions Life Sciences) and Dr Martin Engel alongside Dr. Jeremy Dobrowolski (Inventia Life Science) discussed the benefits of establishing automated workcell operations for biofabrication processes and recent advances in three-dimensional cell culture using digital bioprinting platforms, respectively. Dr. Haruka Yoshie (CELLINK) closed the session by detailing a range of tissue-specific applications, spanning from extrusion-based and light-activated polymers, with a focus on their limitations and possibilities associated with each fabrication modality.

Biomaterials for Bioprinting

Identifying the optimal biomaterial for bioprinting tissues and organs is one of the greatest challenges in defining the microenvironment in which for cells to grow.¹⁰ Prof. Gordon Wallace and Dr. Zhilian Yue (University of Wollongong, UOW) introduced their recent progression on biopolymers for bioinks for bioprinting, raising issues associated with the sourcing of biomaterials, processing requirements, and logistical considerations required when planning for the future clinical translation of bioprinted tissues and organs.^11,12 Dr. Khoon Lim (University of Otago) opened his presentation on the use of light-activated polymers for bioprinting applications by giving a definition of commonly used bioprinting techniques and their respective advantages and disadvantages according to their resolution, speed, and complexity. Dr Lim’s presentation covered aspects of photo-crosslinking approaches (Figure 2, A),¹³ an example of a bioprinted cartilage construct (Figure 2, B),¹⁴ routinely used photoinitiators (Figure 2, C) and a bioprinted distal lung subunit demonstrating the oxygenation of red blood cells within a biocompatible hydrogel (Figure 2, D).¹⁵

Figure 2.: Fundamentals and applications of light-based bioprinting techniques. A: Comparison of photo-crosslinking approaches, adapted with permission from Ref. ¹³. B: One-step photoactivation of dual-functionalized bioinks for chondral regeneration (I) GelMA-Tyr. (II) Schematic of intraoperative administration of GelMA-Tyr to the chondral defect. (III) Setup of the pushout assay to determine bond-strength. (IV) Cartilage biopsies adhered together using GelMA-Tyr, adapted with permission from Ref. ¹⁴. C: Absorption spectra and molar extinction coefficients of commonly used photoinitiators for light-based bioprinting. D: Architectural design of an alveolar model topology and distal lung subunit (top) and corresponding photographs of printed hydrogels with RBC perfusion while the air sac was ventilated with O₂ (below) scale bar = 1 mm, adapted with permission from Ref. ¹⁵. RBC, red blood cell.

Bioprinting of Organoids and Tissues

Next-generation organogenesis has the potential to revolutionize many biofabrication processes. These include cosmetics testing and drug development by improving clinical trial speeds and model efficacy while reducing associated costs and the dependence for early-stage animal studies.¹⁶ Prof. Anthony Weiss (University of Sydney) summarized his group’s recent efforts in the generation and use of human recombinant elastin-based bioinks for bioprinting vascularized soft tissues.¹⁷ Central to developing the next generation of human tissues and organs is the need to address the fields most prominent hurdle of achieving perfusable vascularization within bioprinted constructs and in vitro systems.^18,19 A/Prof. Jeremy Crook (UOW), Dr. Carmine Gentile (UTS), and Dr. Anita Quigley (RMIT) reported their recent progression in reproducing neuronal models (Figure 3), cardiac and skeletal muscle models (Figure 4), respectively.

Figure 3.: Bioprinting neuronal models. A: Cornea stroma showing schematics of (I) human cornea (II) fabrication of EC film (III) electro compaction to form orthogonally arranged collagen fibrils (IV) fabricated 3D-CSM (V) orthogonally arranged layers of hCSCs within construct (VI). Comparison of transparency, glucose permeability, and degradability between bioprinted cornea and human cornea, adapted with permission from Ref. ²⁰. B: Human neural tissues from neural stem cells using a conductive biogel and printed polymer microelectrode arrays for three-dimensional Electrical Stimulation, adapted with permission from Ref. ²¹. C: Artificial nerve conduit (I) interaction model (II) integrated and regenerated ANC at day 30, adapted with permission from Ref. ²². ANC, artificial nerve conduit; CSM, corneal stroma model; EC, electro-compacted collagen; hCSCs, primary human corneal stromal cells.

Figure 4.: Bioprinting of skeletal muscle and cardiac models. A: Neural cell integration of bioprinted skeletal muscle construct accelerates restoration of muscle function at defect site (I) design concept (II) vascularization (III) myofiber formation and maturation (IV) histological examination after 8 weeks, adapted with permission from Ref. ²³. (V) Wet spun biosynthetic fiber with myoblasts (longitudinal and cross sections inset) adapted with permission from Ref. ²⁴. (VI) Confocal three-dimensional rendering of bioprinted myogenic progenitors subjected to differentiation conditions for 7 days (top) and depth coding of extensive myotube formation (bottom), adapted with permission from Ref. ²⁵. B: Cardiac spheroids form microvasculature with cardiomyocytes, endothelial cells and fibroblasts (I) and ECM deposition of collagen type IV (II), adapted with permission from Ref. ²⁶. C: Perfusable cardiac tissue fabricated by SWIFT (I). Three-dimensional CAD model of a normal human heart. (II) SWIFT process (III). Viability staining of cross sectioned cardiac tissues after 24 hours of perfusion, adapted with permission from Ref. ²⁷. D: Full-size model of an adult human heart (I) using the FRESH bioprinting technique, (II–III) perfusable segment of the coronary artery (IV) bioprinted heart model, adapted with permission from Ref. ²⁸. CAD, computer-aided design; ECM, extracellular matrix; SWIFT, sacrificial writing into functional tissue.

Clinical Perspectives

Cross-talk with clinical partners is fundamental for speedy translation of findings from the bench to the bedside. Prof. Peter Choong (University of Melbourne) described his clinical perspectives for bone tissue bioprinting to open the session and how surgeons can work together with engineers and biologists to look for practical solutions to key clinical problems that typically arise from trauma and osteoarthritis. Prof. Choong outlined the regenerative potential of combing stem cells with bioprinting techniques to fill bone defects and by developing a handheld device for surgical applications (Figure 5, A).^29,30 Dr. Liudmila Polonchuk (Hoffman La-Roche) discussed the potential use of bioprinted tissues for drug development, with an application in cardiac safety.

Figure 5.: Biofabrication integrating three-dimensional bioprinting and other technologies. A: Handheld co-axial bioprinting for in situ surgical cartilage repair, (I) design model with core-shell extrusion (II) intraoperative photograph of device repairing chondral defect site, adapted with permission from Ref. ³¹ (III–V) display handheld skin printing device model, post in situ deposition and after 28 days healing with MSCs containing Fibrin-HA biomaterials, respectively, adapted with permission from Ref. ³². B: Thermofluidic heat exchangers for actuation of transcription in artificial tissues (I), bioprinting model (II), Infrared thermography of heat perfused hydrogels (left) and live/dead stain (right), adapted with permission from Ref. ³³. (III) Lung-on-a-chip cancer model, adapted with permission from Ref. ³⁴. C: Modeling cell proliferation and migration to explain (I) pore bridging dynamics in three-dimensional printed scaffolds, adapted with permission from Ref. ³⁵ and (II) three-dimensional spatial heterogeneity image analysis, reproduced with permission from Ref. ³⁶. D: Magnetic levitational bioassembly of tissue constructs consisting of human chondrocytes in microgravity, showing (I) fabrication process, (II) histological examination, and (III) photograph of assembled three-dimensional structure once returned to earth, modified with permission from Ref. ³⁷. HA, hyaluronic acid; MSCs, mesenchymal stromal cells.

Commercial Pathways

The four major driving factors of bioprinting development can be attributed from increasing public and private investments in drug discovery, cosmetics testing, tissue regeneration, and medical device development.³⁸ To safeguard patient safety whilst aiding the successful translation of bioprinting technologies, Michelle Knight (Hydrix) discussed the regulatory requirements to ensure maximum benefit and minimum risk to the patient. Prof. Dianne Nicol (University of Tasmania) followed by detailing the patentability of bioprinting technologies, highlighting that there are currently approximately more than 700 patents and applications worldwide with only a small percentage abandoned, suggesting an active growth phase.³⁹

Stepping into the Future

A better understanding of where the field is potentially going and where new technologies may benefit from the cross-talk between experts in different fields was the focus of this session. Dr. Alfredo Martinez-Coll (UTS) opened the session with a presentation on market trends in bioprinting technologies, in which organ-on-a-chip models were described as playing a key role in the development of the field. Dr. Jesus Shrestha discussed three-dimensional printed microfluidic lung-on-a-chip models for respiratory diseases and drug studies shown in Figure 5, B⁹ followed by, Dr. Mark Allenby (Queensland University of Technology) describing the potential of harnessing computational modeling to enhance biofabrication processes, as shown below in Figure 5, C.³⁵ Next, Dr. Irina Kabakova (UTS) outlined the capabilities of three-dimensional noncontact micromechanical characterization for bioprinted structures and materials,⁴⁰ followed by the final keynote presentation for the day on the magnetic levitational bioassembly of three-dimensional tissue constructs in space by Dr. Vladimir Mironov (3D Bioprinting Solutions) as shown in (Figure 5, D).³⁷ The decision to conduct bioassembly experiments of cartilage generation in space further warrants a growing vested interest in biofabrication technologies and in the evaluation of the effects of microgravity on human intervertebral discs and articular cartilages during long-term spaceflights.

Conclusions

Recent advances in bioprinting technologies for tissue engineering and regenerative medicine approaches show great potential for the strategic arrangement of multiple materials, cells, and extracellular matrix components at different length scales to replicate aspects of the heterogenous compositions and complex hierarchal organization commensurate to native tissues and organs. For this reason, bioprinting techniques are well placed to recreate physiologically relevant microenvironments to further the clinical translation of this promising technology from the bench to bedside.

Acknowledgment

A particular thanks to 3D Heals for their kind support as well.