Biofabrication of 3D constructs: fabrication technologies and spider silk proteins as bioinks

Biofabrication

In 1907, a protocol was first described for maintaining the viability of isolated tissue outside of an organism [1]. This technique, called in vitro tissue culture, catalyzed a boom of biologically-based technology and debate over the possibilities and implications of this development. One of the most exciting technologies which emerged is tissue engineering. Traditionally, tissue engineering is the modular assembly of biomaterials, cells and biochemical factors into tissue-like constructs [2]. Most accept the premise that in order to do this successfully one must, to some degree, mimic the properties of the target tissue. These constructs are immediately implanted or incubated in vitro prior to implantation. Relevant applications of tissue engineering include, but are not limited to: implants for regenerative medicine [3], in vitro models [4], biobots [5], and alternative food-sources [6]. Although tissue engineering has shown promise towards these applications, few have been approved for consumer use.

The high attrition rates of tissue engineered products are often hypothesized to be due to the modularity of the approach. It results in high variability in the spatial arrangement of the different components (biomaterials, cells, soluble and insoluble biochemical entities). This is problematic for presentation of factors to cells, which direct their behavior, as well as the architecture-dependent mechanical properties of these materials [7]. Due to the intimate relationship between structure and function in biological systems, which is observed across size scales, the success of tissue engineering is thereby limited by this poor control over hierarchical structures and their assembly [8, 9]. To overcome these limits, novel technologies have been established: cell-sheet technology, embedding or molding, centrifuge casting, dielectrophoresis, magnetic-force driven cell-motion, micro-fluidics, biospraying and 3D bioprinting (Table 1). Of these, perhaps the most interesting is the process of 3D bioprinting (3DBP). In this context, biofabrication can be defined as the automated, additive assembly of a biological construct by 3D patterning of cells and biomaterials in one processing step [23, 24]. Although each of the named methods has unique advantages, 3DBP is often considered the most valuable technique for tissue engineering/biofabrication due to it having the best spatial control over specific components of the system.

The purpose of this review is to give the theoretical framework of 3DBP, and based on this framework to critically evaluate the recent success of the technology with a particular focus on its use in printing silk-based bioinks; bioinks are materials which are compatible with the 3DBP process.

3D bioprinting (3DBP)

3D printing, first patented by Charles W. Hull in 1986, is rapid fabrication of physical, three-dimensional morphologies [25]. The process can be divided into five steps: (1) generation of a 3D image, (2) re-definition of the 3D image into a stack of 2D layers by an user-demarcated thickness, (3) interfacing this data with the printer, (4) printing a layer of the previously defined thickness one-by-one until the construct is complete, and (5) any necessary post-processing of the material [26–29]. The last step, post-processing, will be discussed in greater detail in later sections, as this is dependent on the material which is used. Although this process applies to most of the existing 3D printers, it should be said that this is a general description: there are many types of 3D printing. As such, the nomenclature for this field is broad, and there is great variety depending on the subfield. For example, some are based on the use of solid or liquid materials in the printing process, while others are based on how the 3D object is created, for example, by adding material a layer at a time (additive manufacturing) [30].

3D printing fitting the definition of biofabrication is referred to as 3D bioprinting (3DBP). Its anatomical elements include: the print head, the material cartridge, the actuator, the nozzle, the working area, and the print stage. The print head is the part which connects precise, motor-controlled movement with actuation of the material. The material cartridge holds the biomaterial and the cells to be printed under user-specified conditions. The actuator is some element which applies pressure to cause material deposition. The nozzle is the orifice, frequently a blunt needle, from which material is ejected. The print stage is the surface which the 3D scaffold is printed onto, and in many set-ups also provides further motor-control. The working area is the volume of space available for the construct. 3D bioprinters are most commonly classified based on their mechanism of material deposition: extrusion, inkjet, or laser-assisted bioprinting (LAB).

Extrusion 3DBP, sometimes referred to as direct-write printing, is a set-up where the mechanical or pneumatic pressure is applied to a cartridge of material to extrude a continuous solution [31]. In the case of inkjet 3DBP, heat or acoustic energy is used to propel droplets of solution; the pressure in the cartridge is kept constant with compressed gas [32]. In LAB, a high energy density beam is directed through a glass slide onto an energy absorbing layer, typically gold or titanium, and the focused energy causes the formation of a concave pocket in the material layer, and subsequently droplets or a jet being propelled towards a collector [33, 34]. Generally, the final printed volume is composed of single droplets; therefore they are correspondingly depicted in Fig. 1. Each of these actuation mechanisms has direct and indirect effects. The direct, downstream effects are on the materials which can be printed and on the shape of the volume which is printed (Fig. 1).

Fig. 1:

The different types of 3D bioprinting set-ups. They are defined based on their mechanism of material deposition, the viscosity range of printable materials, and the morphology of the printed volume (i.e. fiber or droplet). These definitions are given in relation to a representation of a print head which shows the actuator, the housing, the material cartridge, and the nozzle (needle) [26, 32, 35, 36].

In order to be suitable for biofabrication, the most critical characteristics of a 3DBP process are that it is (1) cell-friendly, (2) reproducible and practical, (3) it allows for printing complex physical and chemical gradients, and (4) geometric structures. The performance of printers is typically evaluated by cell density and viability (fulfills requirement 1), process speed, resolution and accuracy (fulfills requirement 2), and the range of printable materials (fulfills requirement 3 and 4). How well these different printer set-ups generally perform will now be discussed based on these requirements.

Bioinks

In 3D bioprinting (3DBP) the term “bioinks” is used to describe cell-encapsulating material-matrices which combine printability with cytocompatibility. These demands are quite high and often result in contradicting requirements, making bioinks one great challenge in biofabrication. An ideal bioink can be printed, has high shape-fidelity upon printing, is cytocompatible, and is tailored to its target tissue. Amongst studied bioinks, hydrogels have had the greatest tendency towards success [29].

The major physicochemical parameters determining the printability of a hydrogel are their viscosity and their rheological properties. During 3DBP, the bioink should extrude smoothly and undergo a rapid gelation after printing. If the bioink is already pre-gelled, then the printing process should not result in irreversible damage of the polymer network. Adequate mechanical properties, which can be tailored by polymer concentration or crosslinking of the hydrogel, are necessary to retain the designed and fabricated shape up to clinically-relevant sizes [41]. As previously stated, the requirements imposed by the technique for the bioinks tend to conflict with the biological requirements imposed by the cellular components. The final constructs should allow migration, proliferation and support targeted differentiation of encapsulated cells, which typically calls for a soft substrate. Additionally, the gelation process should be mild and cell friendly [20]. Finally, once the hydrogel precursors have been printed and the cells have survived, the scaffold must degrade at a pre-determined rate when exposed to physiological conditions found in the target tissue [42]. Refer to Fig. 2 for representation of these requirements.

Fig. 2:

Physicochemical and physiological requirements of the bioink. Physicochemical properties are related to the printability by the viscosity and macromolecular structure of the material. The printed construct should also allow for diffusion, relating the printed architecture to the cytocompatibility. The physiological activity is related to cytocompatibility by the degradation products, the behavior under physiological conditions, and the biological activity (e.g. cell binding motifs). The final product, the cell-loaded construct, should seamlessly combine these qualities.

3D bioprinting with recombinant spider silk proteins

Recently, the recombinant spider silk protein eADF4(C16) and a variant containing an RGD-motif were established as bioinks. eADF4(C16) consists of 16 repeats of a module C mimicking the repetitive core sequence of dragline silk Araneus diadematus fibroin 4 (ADF4) of the European garden spider (Fig. 3a) [81, 82]. The recombinant spider silk proteins were assessed regarding their printability [40], and spider silk constructs could be printed by robotic dispensing using a print head with an electromagnetic valve. The hydrogels were process-compatible and had high shape fidelity (Fig. 3b). The printability is based on the β-sheet transformation of the proteins during gelation and shear thinning behavior of the hydrogels (Fig. 4).

Fig. 3:

(a) eADF4(C16) and eADF4(C16)-RGD are made of 16 C modules. The C-module reflects the consensus sequence of the repetitive core sequence of Araneus diadematus fibroin 4 (ADF4), one of the main components of the dragline silk of the European garden spider (A. diadematus). Dragline silk is the best characterized spider silk, constituting the outer frame of orb webs and serving as a lifeline for the spider [80]. (b) Going from a CAD template (left) to a 3DBP recombinant spider silk construct (right). Recombinant eADF4(C16) was printed by robotic dispensing. In the CAD template, the different shades of gray represent thickness with darker shades representing multiple layers. In the image of the construct it can be qualitatively observed that the construct has the same shape as the CAD file, and the printed strands made of spider silk also have high shape fidelity without the use of post-processing, crosslinking or thickeners.

Fig. 4:

Printing process for a physically crosslinked recombinant spider silk bioink. Cell-loaded spider silk constructs were printed by robotic dispensing, as mentioned in Fig. 3. The process begins with preparation of the hydrogel from a cell-loaded solution. The corresponding Fourier-transformed infrared spectroscopy (FTIR) structure data shows a peak shift corresponding to β-sheet formation which occurred during self-assembly of the hydrogel. The next step in the process represents the printing of the hydrogel accompanied by alignment of β-sheets under shear-stress, and this corresponds to the given rheological behavior with increasing angular frequency leading to a decrease in complex viscosity, which is called shear-thinning. The final construct is represented by a stereoscope image of the layered structure. The right-hand image represents the presence of viable cells (redlines reflect auto-fluorescence of spider silk; red stained cells are dead and green stained ones viable).

It was shown that recombinant spider silk proteins can be used as bioink for 3D printing without the need of additional components or post-processing [40]. In contrast, alginate and fibroin need post-processing with crosslinkers or thickeners added to the solution to increase the printing fidelity [20, 58]. For more detailed information of other bioinks, refer to [29] and Table 2.

To produce cell-loaded 3D hydrogel constructs, cells were encapsulated within a highly concentrated silk solution before gelation. The addition of cells to the bioink did not influence the self-assembly into a hydrogel or the printability of the material [40]. The cells survived the printing process and were viable at least 7 days in situ. The viability within the spider silk hydrogel could be quantified with 70.1 ± 7.6 %. Although the cell viability in the spider silk constructs is lower when compared to established bioinks such as alginate (∼90 %) and gelatin (∼98 %), it could be shown that the printing procedure did not significantly affect viability, since after printing 97 % of the cells survived [40, 42, 83].

Conclusion and future perspectives

In conclusion, 3D bioprinting (3DBP) techniques hold potential to overcome the current, process-based challenges faced in tissue engineering: high variability and low control over the placement of different scaffold components. Of the different types of 3DBP, it seems as though extrusion printing will be one excellent option for the future of biofabrication, despite some of its drawbacks (nozzle clogging, resolution). Extrusion printing allows for fabrication of clinically-relevant constructs (size, cell density) and greater ease in bioink development. Additionally, these advantages outweigh the disadvantages. For example, bioinks are critical in cell viability after printing (physicochemical properties) and cell behavior throughout construct maturation (physiological properties). Current bioinks tend to be better in either the “cell friendliness” (e.g. fibrin, gelatin) or printability (recombinant spider silk protein). Future work will most likely focus on polymer blends such that advantages are conserved or enhanced, and the disadvantages minimized or eliminated.

In terms of cell viability after printing, it is reasonable to hypothesize that cell viability is directly correlated with the mechanical stress that the cells are exposed to. In the optimal viscosity range for extrusion printing, there seems to be some protection to shear stress which is absent in inkjet printing; in LAB there are virtually no shear forces on the bioink, due to the nozzle-free set-up. However, LAB is incompatible with higher viscosity ranges, due to the incompability of cells with certain wavelengths and energy densities. Thereby, due to the greater flexibility in bioink development, it seems as though extrusion bioprinting will be the technology that shows the greatest potential in the future. However, it is also possible to imagine future developments will also focus on combining the different types of 3D bioprinting in order to further optimize the process.