Functional hydrogel bioink, a key challenge of 3D cellular bioprinting

INTRODUCTION

Three-dimensional (3D) printing, also known as additive manufacturing, was first introduced in 1986.¹ Unlike the techniques that can only be used for mass-produced goods in classical manufacturing, 3D printing can produce custom products by employing standardized materials.² In recent years, 3D bioprinting has emerged as a promising new technology to create biomimetic living constructs to emulate tissues and organs, such as livers, bones, cartilage, and hearts.³ The key challenges and opportunities in this important research direction have been recently discussed in several publications.^4–6 For example, Birla and Williams emphasized the high potential of bioprinting for creation of cardiac tissue microphysiological systems and provision of an opportunity to evaluate and explore the assembly of specific parts of the heart.⁵ Placone, Mahadik, and Fisher discussed the necessity for standardization of characterization, which is important with regard to the translation potential of bioprinting.⁶ Engler and Cooper-White further presented the different viewpoints on bioprinting from academic and industry perspectives in their editorial.⁴

Following the discussions presented in these previous publications, this editorial aims to address a key challenge of 3D bioprinting, namely, the development of functional bioink material. Bioink is critical for the success of 3D bioprinting, particularly when cells are printed together using the bioink and eventually encapsulated in the printed structure—termed cellular bioprinting.^7,8 Hydrogels are a natural choice of bioink materials for cellular printing because they can provide a highly hydrated and permeable 3D polymeric structure conducive to cellular anchorage and metabolic activities. Hydrogels have been widely used as 3D biomaterials in various biomedical fields and applications including tissue engineering,^9–11 tissue/disease modeling,^12,13 drug delivery,¹⁴ and biomedical devices.¹⁵ However, owing to the stringent cellular bioprinting requirements regarding aspects such as temperature, pH, pressure, physical forces, and osmolarity for safeguarding the vitality of printed cells and bioactivity of the printed structure, a widely accepted optimal bioink hydrogel for cellular bioprinting has not yet been developed. This editorial discusses the stringent requirements to be satisfied for a functional bioink material to be deemed ideal for cellular bioprinting.

THE MULTIFACETED AND MULTISCALE REQUIREMENTS OF BIOINKS FOR CELLULAR BIOPRINTING

Ideally, printed bioink materials should emulate the biophysical and biochemical complexities of the extracellular matrix (ECM). However, this can be very challenging to achieve given the current technical capabilities of existing biomaterials. Nevertheless, to address the need to mimic diverse tissue types, bioink materials based on different printing technologies should be designed to exhibit some basic properties such that the various complex requirements can be met, and the desired functions can be executed.^16–18

Printability

The bioink materials should be precisely deposited by the printers with good spatiotemporal control and resolution. To achieve this, the type (chemical and physical) and density of cross-linking and rheological properties of bioinks are determinant factors that vary depending on the types of printing employed. For example, inkjet printing requires the property of rapid cross-linking for the formation and maintenance of the printed multi-layer structures. On the contrary, extrusion printing requires bioink materials exhibiting the shear-thinning property.^19,20

Biocompatibility and biodegradability

The printed scaffolds should be non-toxic to endogenous tissues and should, to the best possible extent, not elicit the foreign body reaction after implantation. In addition, the degradation rates should be controlled to match those of the nascent ECM deposition.²¹

Biofunctionalization

The incorporation of biomimetic components such as proteins, peptides, and growth factors to the biomaterial scaffold is known to be essential for the regulation of cell behaviors.^22,23 Such biofunctionalization of printed hydrogel structures can be achieved by including the bioactive motifs within the functional hydrogel bioinks.

Bulk structural properties

The printed structure should possess and exhibit suitable biophysical properties including stiffness, swelling, and permeability, which will significantly impact the structural stability of the printed constructs, biotransport, and the biological behaviors of seeded cells.^24–26

Microscopic structural properties and cell adaptability

The degradation and remodeling of natural ECM is indispensable to many key cellular events such as spreading, proliferation, migration, and differentiation.^27,28 In the case of the printed structures with encapsulated cells, the microscopic structural dynamics can be essential for allowing the adaptation of the printed bioink structures to cells, which would enable effective cell–material interactions and the associated intracellular signaling and, therefore, facilitate unimpeded growth and development of cells in 3D.^29,30 Existing photocurable or ionically crosslinkable bioinks give rise to highly stable matrix structures after the printing process owing to the lack of sufficiently dynamic crosslinks.³¹ The degradation of the printed structures can allow for increased microscopic structure dynamics to be exhibited over a long term; however, it cannot address the demands of the rapid cellular growth events during the early stage, which can be critical for the maturation of printed biological tissues.³² Nevertheless, the usage of degradable bioink hydrogels is necessary to ensure long-term integration of the printed structures with host tissues in the context of in vivo implantation.

Batch-to-batch consistency

For scaled-up bioprinting and translational applications, the bioink materials also need to exhibit low batch-to-batch variations to ensure quality control. Because most existing bioink materials used for cellular bioprinting are based on natural polymers, which can show large variations in terms of molecular weights and compositions, achieving consistent quality in the bioink is particularly challenging. The development of new biosynthetic technologies facilitating enhanced quality control is necessary to improve the batch-to-batch consistency of bioinks.

COMMONLY USED HYDROGELS FOR CELLULAR BIOPRINTING

Despite the significant progress made in cellular bioprinting, the choice of bioink hydrogels remains limited. Bioink hydrogels currently used for 3D cellular bioprinting are predominantly based on natural and naturally derived polymers, synthetic polymers, and hybrid polymers.^16,33 Natural and naturally derived polymers demonstrate properties similar to those of native ECM molecules and also show inherent bioactivities; however, batch-to-batch variations may arise with respect to the composition and quality in this case.^34,35 Collagen-based bioink was used to rebuild the components of the human heart with precise control at various scales. The printed heart enabled cell migration and vascularization and reproduced a specific anatomical structure.³⁶ Gelatin, derived from collagen and modified with methacrylamide (GelMA), was used as a bioink to fabricate 3D printed cell-laden constructs, enabling high cell viability of hepatocarcinoma cells.³⁷ On the other hand, synthetic polymers are more reproducible and can be tailored to demonstrate specific physical properties but may lack the bioactivity for supporting cell growth.^38,39 Warner et al. reported a fractal geometry device composed of poly(ethylene glycol) diacrylate.⁴⁰ This hybrid polymer is also a promising material for 3D printing.^41,42 For instance, a two-component bioink composed of poly(ethylene oxide) and cell/gelatin methacryloyl was photocrosslinked to construct porous hydrogel, which promoted cell spreading and proliferation.⁴³ Moreover, a robust but stretchable hydrogel with a cellularized structure was fabricated using a nanocomposite ink composed of nanoclay, alginate, and poly(ethylene glycol).⁴⁴

PERSPECTIVE

Thus far, most bioink materials employed for bioprinting have been selected on the basis of either biocompatibility with cells and tissues or printability and physical properties. However, no bioink materials have been able to satisfy the multifaceted chemical, biological, and biophysical property requirements, as delineated in the previous section. Particularly, in the case of the biophysical properties, no biomaterial scaffold can simultaneously exhibit excellent bulk mechanical performance and still provide the highly dynamic structural environment at a cellular scale, which is found in natural soft tissue ECM. It is even more challenging to achieve these multiscale differential properties in the printed biomaterials. Therefore, much research needs to be dedicated in basic and translational biomedical studies toward the development of novel and advanced bioink biomaterials in the coming years before the full potential of 3D cellular bioprinting can be realized. APL Bioengineering has recently introduced a new special topic on “Functional Biomaterials” and highly encourages submissions related to critical challenges faced in the research field of biomaterials, such as those highlighted in this editorial.