Biophysics of biofabrication.

AFFILIATIONS Christchurch Regenerative Medicine & Tissue Engineering (CReaTE) Group, Department of Orthopaedic Surgery, Centre for Bioengineering & Nanomedicine, University of Otago, Christchurch 8011, New Zealand Complex Tissue Regeneration Department, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, 6229 ER Maastricht, The Netherlands Department of Bioengineering, Rice University, Houston, Texas 77005, USA


INTRODUCTION
In the special topic, "Biophysics of Biofabrication," published in APL Bioengineering, we are pleased to present a special collection of papers that provide a window into the biophysics of biofabrication and showcase some of the latest studies that have investigated or incorporated these principles into the technological development of new biofabrication lines. The special topic is also complemented by challenges and opportunities from multidisciplinary contributors in the field.
Biofabrication is revolutionizing the way automated fabrication of complex living tissues and hierarchical scaffolds are additively manufactured and offers the potential to synergistically enhance broad healthcare and biotechnology challenges. Biofabrication, 3D bioprinting, and bioinks have more recently been defined within the context of tissue engineering and regenerative medicine (TERM) 1-3 and biofabrication technologies and approaches described in a series of extensive reviews. [4][5][6][7][8][9][10] As an extension to classic tissue engineering, biofabrication enables high levels of control over the spatial deposition of cells, materials, and other factors. Rapid technological evolution in the field of biofabrication has seen the development of new bioprinting and bioassembly techniques convergent with additive manufacturing technologies to generate a large library of potential hybrid approaches and hybrid constructs.
There have been rapid advancements in the development of hybrid biofabrication technologies and associated bioinks and biomaterials inks. 1-3 These individual biofabrication techniques (or combinations thereof) include traditional extrusion-based bioprinting that has been widely adopted as well as cell-jetting and the more recent lithography-based bioprinting, embedded bioprinting, and volumetric bioprinting approaches, 5,6,8-10 all of which place significantly different demands and processing criteria on bioinks containing cells and/or bioactive molecules required to successfully and accurately fabricate complex, high resolution 3D tissues, or hybrid constructs. 4,6,7,10 Cellladen bioinks are a formulation of cells suitable for processing by an automated biofabrication technology that may also biologically contain active components, whereas non-cell-laden biomaterial inks are printable (bio)materials that can act as support scaffolds and sacrificial templates, or for delivery of bioactive factors for which cells are added post-fabrication. Therefore, it is also critical that the field understands and considers the fundamental biophysical and rheological requirements as well as the underlying processing and manufacturing challenges in the development of new bioinks and biofabrication lines in order the advance the field further.
As summarized below, this special topic on Biophysics of Biofabrication provides a collection of articles describing challenges and opportunities in bioink development, fundamental parameters for controlling bioink rheology and cross-linking, and their relationship in emerging hybrid biofabrication technologies to fabricate complex functional 3D tissues as well as alternative biofabrication strategies for modulating cell function and cell-material interactions.

SUMMARY OF THE AREAS COVERED Challenges and opportunities for clinical and commercial translation
Placone et al. 11 review the challenges facing the field of biofabrication and opportunities or development of new bioinks materials helped through standardized characterization techniques to better understand necessary physical properties as well as improved education and training opportunities to better communicate and fill knowledge gaps in the biofabrication community.
Birla and Williams 12 then describe a potential roadmap for 3D bioprinting human hearts from an industry perspective, discussing the complexity of biofabricating microcirculation, macrovascular structures, and cardiac conduction system as well as heart valves and cardiac muscle, with the recent development and adoptions of embedded bioprinting technologies, such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH).

Academic vs industry perspectives in 3D bioprinting
APL Bioengineering Editors Engler and Cooper-White 13 present their inaugural viewpoint perspective on "academic vs industry perspectives in 3D bioprinting" by presenting a point/counterpoint summary highlighting differing arguments from review articles by Placone et al. 11 and Birla and Williams 12 on the status of bioinks for 3D biofabrication and their potential for clinical and commercial translation.

Fundamental parameters for controlling bioink rheology and cross-linking
Cooke and Rosenzweig 14 provide an overview of the fundamental rheological parameters required for bioinks and characterization methods available to assess printability and print fidelity as well as the effect of bioink rheology on cell viability. The latest developments and future opportunities in bioink formulations and embedded bioprinting approaches to overcome rheological limitations of bioinks are discussed.
Furthermore, Nieto et al. 15 review the fundamentals in biophysics of cell and light interactions in photo-cross-linking based biofabrication. The development of flexible new photocurable resins and photoinitiators that are biocompatible heavily relies on understanding basic principles and interactions inherent to various light-based biofabrication techniques. These are reviewed, and perspectives on how linear and non-linear mechanisms using light influence cell and biomaterial properties during and after bioprinting and ultimately the eventual biofabrication of functional tissues are discussed.

Emerging biofabrication and bioink technologies
Shiwarski et al. 16 describe the fundamentals of how embedded bioprinting approaches, such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH), can reduce the high yield-stresses on traditional extrusion bioprinting of hydrogel bioinks by extruding within a support bath, particularly advantageous for low viscosity bioinks with soft mechanics for better replicating microenvironments for improved cell function and bioactivity. Examples describing the customizability of the FRESH printing technique by tailoring the chemical composition of the support bath and cross-linking chemistry for biofabrication of a wider range of bioinks in complex 3D structures are presented.
Ji and Guvendiren 17 then review emerging complex or hybrid 3D biofabrication techniques to achieve the necessary complexity for fully functional tissues and patient-specific disease models. These include omnidirectional strategies, such as magnetic or acoustic levitation bioprinting, volumetric bioprinting, microfluidic and 4D bioprinting, and in situ bioprinting approaches directly in surgery.
The article by Alberto and Melchels 18 then introduces novel shape-memory materials that respond to temperature over extended periods of time in vitro obtained through unique thermomechanical properties. Using lithography-based printing, complex 3D examples of photo-crosslinked biomaterials [poly(D,L-lactide) dimethacrylate] are provided that can retain prolonged recovery of shape memory capacity up to 2 weeks.

Modulating cell-material interactions via biofabrication
In addition to 3D biofabrication strategies, complementary methods to control key cellular processes that occur across a wide range of tissues and environments are also important considerations for future developments of the field. In this regard, Schneider et al. 19 describe an alternative new microfluidic organ-on-a-disk system, which adopts centrifugal forces to control microphysiological conditions on cells and automate high throughput fabrication of multicellular aggregates. The simple centrifuge-based process offers perfusion capability without complex pump systems and reduced potential for cell damage due to fluid flow of microphysiological bioreactor systems.
Finally, Bannerman et al. 20 review epithelial-to-mesenchymal transition (EMT) processes that are critical in tissue development, disease, and regeneration. Specifically, they introduce biological, biomaterial, biochemical, and physical properties that have been utilized to control or modulate EMT in tissue engineering and regenerative approaches in vivo, with a specific focus on the heart and cardiac development. The review further describes new perspectives on bioengineering methods to control cell function and ultimately lead to the development of new therapies.

CONCLUSIONS
In summary, the special topic Biophysics of Biofabrication provides a glimpse into the rapidly expanding field of bioink development and the biophysics and bioengineering criteria that need to be considered across a range of biofabrication technologies. This collection provides new directions for current researchers wishing to keep abreast of new developments in an exciting field as well as a useful introduction for those just entering the biofabrication field. We believe that in the future, biophysical principles that form the foundation for the development of new biofabrication technologies and bioink materials should become a focus of more active research in order to better understand and control the cellular and biological processes governing the biofabrication of complex biological tissue constructs.