Historical Perspective
Bioprintable tough hydrogels for tissue engineering applications

https://doi.org/10.1016/j.cis.2020.102163Get rights and content

Highlights

  • State-of-the-art in bioink formulations for tough hydrogel printing.

  • Integration of different crosslinking and reinforcing mechanisms during 3D printing.

  • Important characteristics of bioiks from both rheological and biological perspective.

  • Microstructural perspective of 3D printing and its transition to 4D printing.

  • Applications of bioprinting in tissue engineering and bioelectronics with future prospects.

Abstract

Bioprinting is an advanced fabrication approach to engineer complex living structures as the conventional fabrication methods are incapable of integrating structural and biological complexities. It offers the versatility of printing different cell incorporated hydrogels (bioink) layer by layer; offering control over spatial resolution and cell distribution to mimic native tissue architectures. However, the bioprinting of tough hydrogels involve additional complexities, such as employing complex crosslinking or reinforcing mechanisms during printing and pre/post printing cellular activities. Solving this complexity requires attention from engineering, material science and cell biology perspectives. In this review, we discuss different types of bioprinting techniques with focus on current state-of-the-art in bioink formulations and pivotal characteristics of bioinks for tough hydrogel printing. We discuss the scope of transition from 3D to 4D bioprinting and some of the advanced characterization techniques for in-depth understanding of the 3D printing process from the microstructural perspective, along with few specific applications and conclude with the future perspectives in biofabrication of hydrogels for tissue engineering applications.

Introduction

Biofabrication is an advanced and emerging platform of the 21st century which has the potential to revolutionize the current medical research [1]. It is otherwise known as additive manufacturing technique in which instead of polymer melts and metals, live materials (hydrogels and cells) are used to construct a functional living tissue [2]. According to the Health and Human services department of USA, over 0.1 million patients seek for an organ transplant and 20 people died each day while awaiting a transplant in 2017 [3]. The limited number of organ donors and high demand for organ transplantation leads to an increased interest in constructing scaffolds, which mimics the natural tissues [4]. Natural tissues such as ligaments and tendons exhibit anisotropic structural properties, which are extremely difficult to accomplish by conventional fabrication approaches [5]. On the other hand bioprinting technique overcomes the geometrical limitations offered by the conventional methods and enables assembling cells, biomaterials and biomolecules in a spatially controlled manner to mimic the nano, micro and macro architectures of the native tissues [6]. It is estimated that the global market size of 3D bioprinting in 2012 was $ 2.2 billion, and this is predicted to reach $ 10.8 billion by 2021 [7].

The major bioprinting techniques used in current research include extrusion printing, inkjet printing, laser-assisted bioprinting [[7], [8], [9]]. The schematic of the different printing techniques is shown in Fig. 1. Briefly, inkjet bioprinting is a process of dispensing low viscosity bioinks onto the substrate to form multilayered droplets and small-scale 3D constructs as shown in Fig. 1A [10]. The general functioning of inkjet bioprinter is in two modes, namely continuous inkjet and drop on demand [8]. The former category is the most commonly used inkjet bioprinting by using thermal or piezoelectric actuator to dispense the bioink [11,12]. It offers many advantages including high printing speed, excellent spatial resolution and low cost [3]. However, it can only adopt low viscosity bioink, which affects the use of bioink with high cell density, thereby limiting the fabrication of thick 3D structures [13]. Extrusion printing/robotic dispensing is a layer by layer deposition of moderate to highly viscous bioinks on the substrate by extrusion from nozzle and solidifying subsequently to generate a 3D structure (Fig. 1B). The rate of extrusion can be controlled by pneumatic, piston and screw, which facilitates the optimization of printing speed to match the crosslinking time, thereby achieving the required spatial resolution. It offers the advantage over inkjet bioprinting in terms of handling large amount of bioink, high viscosity materials (high cell densities), balancing the printing quality and printer's cost [14]. However, in some cases the narrow nozzles require large shear forces to dispense the bioink which has negative impact on the cell viability [15]. On the other hand, laser-assisted bioprinting is a process in which the bioink droplets are expelled by using a laser pulse, thereby forming a small scale bio construct (Fig. 1C) [16]. It offers the advantage of printing moderate viscosity bioinks with excellent spatial resolution and excludes any contamination which directly affects the cell compatibility [17,18]. However, the long exposure of laser during gelation leads to the decrease in cell viability [19].

Although there has been a tremendous development in 3D printing technologies and significant progress in hydrogel research over the last few years, the number of successful outcomes in bioprinting is limited when compared to 3D printing of hydrogels (Fig. 2A) [21]. However, most hydrogels are brittle, having fracture energy of the order of 10 J/m2. By comparison, the fracture energy for cartilage is ~1000 J/m2. The printing of hydrogels with novel microstructure, toughness and excellent mechanical properties is a highly challenging topic. Thus, bioprinting a tough hydrogel which can absorb the applied energy and deform without fracturing involves additional complexities of integrating toughening/reinforcing mechanisms during the printing process. In general, hydrogels are reinforced by introducing the dissipation mechanisms into the polymer networks and maintaining high elasticity during deformation [22]. The dissipation mechanisms may include pullout of fibers/fillers, reversible crosslinks, domain transformation and chain fracture, whereas the elasticity can be maintained by using hybrid/high functionality crosslinkers, interpenetrating long chain networks and meso/macro composites [23]. Significant efforts have been devoted to bioprint tough hydrogels by integrating the expertise from material scientists and biological sciences along with the engineering techniques as shown in Fig. 2B. However, integrating those complex toughening/reinforcing mechanisms into the printing process without compromising the cellular activities is a bottleneck in current research and still at the initial stage.

The reinforcing mechanisms not only determine the mechanical characteristics of the final construct but also influences the flow, strength and recovery behavior of the precursor solution (bioink), which determines the print resolution/precision and structural integrity of the hydrogel [24]. Several reviews are out there on development of bioink [13,21] and printing of hydrogels [2,6,20]. However, to our knowledge this report is one of its first kind in bioprinting gel, which focuses on the reinforcing mechanisms to engineer tough hydrogels with different microenvironments and understanding the 3D printing from the intrinsic microstructural perspective. In this review, we guide the reader through the selected biomaterial based bioink formulations for tough and stable hydrogel printing, mainly focusing on the mechanism of crosslinking/reinforcing, printability & cell viability, followed by their applications.

Section snippets

Bioink formulation for tough hydrogel printing

Bioink formulation is the most critical component in the bioprinting process as it should satisfy both rheological/flow and biological properties to achieve desired printability and biological activities respectively [25]. Hydrogel forming polymers are the most satisfactory materials in the bioink formulation because they are generally cell friendly and properties like flow tunability, stimuli responsiveness, easy functionalization for 3D bioprinting [[26], [27], [28]]. Besides, the polymer

Transition from 3D printing to 4D printing

Over past two decades there has been a significant advance in additive manufacturing industry and 3D-printing has found many applications in various engineering fields and in biomedicine [96]. With the increase in hydrogel research with focus on tunable properties and cell-hydrogel interactions, it has been possible to mimic the natural ECM by 3D printing the cell-laden hydrogels which often termed as 3D bioprinting [97]. Although 3D bioprinting made it possible to mimic the complex

Advanced characterization of bioinks and printed constructs

In several studies, the bulk flow properties of the inks were systematically investigated using rheology to establish the suitability of a material for 3D printing. However, their micro-structural orientation during flow and transformation from fluid state to gel state during 3D printing is not well understood. Ideally there are three stages involved in the process of hydrogel 3D printing. The first one is the structural organization of the ink during extrusion process and the structural

Applications

The bioprinting is a fascinating technology, which offers the holistic control over physical and biological properties and found many applications in tissue engineering. It offers the versatility of printing complex architectures from soft to hard/rigid scaffolds along with electronics. Some of the applications that have been possible due to advancements in bioprinting are highlighted below.

Conclusions and future prospects

In this communication the recent developments on bioink formulations to print tough hydrogels with specific emphasis on integrating reinforcing or toughening mechanisms during printing is reviewed. It is evident that the hydrogels derived from the natural polymers and their hybrids are highly biocompatible and potential candidates in bioink formulations. However, there are other materials like supramolecular hydrogels (host-guest), stimuli responsive materials need to be considered in bioink

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

The authors acknowledge RMIT University for providing the PhD scholarship for Pramod Dorishetty. This work was supported by Australian Research Council (ARC) funding through discovery project.

Pramod Dorishetty obtained his bachelor's (2014) and master's (2016) degree in Chemical Engineering from Osmania University and IIT Madras respectively, India. Mr. Pramod is currently a PhD student at the School of Engineering, RMIT University, Australia. His research interests include engineering tough hydrogels from protein polymers to mimic natural tissue analogues and development of protein inks for 3D printing complex architectures for load bearing tissue engineering applications.

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  • Cited by (0)

    Pramod Dorishetty obtained his bachelor's (2014) and master's (2016) degree in Chemical Engineering from Osmania University and IIT Madras respectively, India. Mr. Pramod is currently a PhD student at the School of Engineering, RMIT University, Australia. His research interests include engineering tough hydrogels from protein polymers to mimic natural tissue analogues and development of protein inks for 3D printing complex architectures for load bearing tissue engineering applications.

    Naba K. Dutta is currently a Professor at the Chemical & Environmental Engineering Discipline of RMIT University. Upon receiving his PhD from IIT, Kgp, and post-doctoral research in France, he joined Monash University and later University of South Australia. His research interest is in nanobioconjugates and smart biopolymers for catalytic and biomedical applications.

    Namita Roy Choudhury is a Professor at the at the Chemical & Environmental Engineering Discipline, School of Engineering, RMIT University. She received her PhD from IIT, Kharagpur and subsequently did her post-doctoral research at CNRS, Mulhouse, France. Choudhury's research interest spans from Hydrogels to Biomimetic polymers to printable inks for biomedical and advanced manufacturing.

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