Development of printable bacterial nanocellulose bioinks for bioprinting applications

Abstract

This research has developed a printable bioink formulated with bacterial nanocellulose (BNC) and sodium alginate. The bioink composition offers a unique combination of printability, mechanical strength, and high BNC content. A low-cost 3D bioprinting platform, with in-situ crosslinking capabilities, was designed and fabricated to facilitate the creation of complex constructs. Rheological analysis revealed shear-thinning behaviour, a critical property for efficient 3D bioprinting. The printed constructs exhibited exceptional resolution, printability, and self-supporting features at BNC loadings up to 72 wt.% in dried structures. The optimal ink composition was found, containing 7.8 wt.% BNC and 3 wt.% sodium alginate. These findings highlight the potential for this BNC-alginate bioink for bio-fabrication applications demanding both printability and high biomaterial content.

Graphical Abstract

Introduction

Cellulose is one of the most abundant natural polymers on Earth, although isolating it can be challenging because natural cellulose in plants and trees is intercalated with other polymers [1, 2]. An alternative method to produce pure cellulose is via bacterial nanocellulose (BNC), a biodegradable polymer, which is a key material for sustainable development [3, 4].

When certain bacteria, such as Komagataeibacter xylinus [3] are fermented, a separate solid phase is formed on the surface of the liquid culture, consisting of an entangled matrix of BNC nanofibrils. This nanocellulose pellicle exhibits excellent mechanical properties. Nanocrystalline cellulose has a tensile strength of 7.5–7.7 GPa, twice that of Kevlar, and an elastic modulus of 110–220 GPa, which is comparable to steel [2, 4, 5]. The pellicle serves as protection from unfavourable external environments [6].

BNC has been used for various applications in biomedical, cosmetics, food packaging, pharmaceuticals and electronics industrial sectors. Some example applications include use in oral drugs, nanocarriers, biosensors, tissue engineering, wound healing, implants, emulsions and creams [5]. There is robust evidence demonstrating in vivo biocompatibility and the antibacterial efficiency of BNC. This, together with excellent water absorption, retention, non-allergenic, non-toxic, and mechanical properties make it a desirable material for many applications, including tissue engineering [4, 7,8,9,10,11,12,13,14]. However, there are quality and manufacturing issues that need to be addressed to meet the demands of users. The conditions used to cultivate BNC and the choice of feedstock used, influence the characteristics of the BNC generated [15]. Ensuring repeatable, high-quality growth of BNC is essential for widespread use in industry. Furthermore, cellulose is held together by hydrogen bonds which create a complex crystalline structure, resulting in insolubility and lack of formability [7]. This represents a significant technical challenge for manufacturing that limits potential uses. Efficient production methods must be developed if BNC is to become a widely used material in industry [5, 15].

When dried, forces between nanocellulose hydroxyl groups cause an irreversible agglomeration and co-crystallization of the material [16]. This poses an interesting opportunity to solve the problem relating to the lack of formability of the material. By forming the BNC into an ink with suitable properties, it could be used as a material for 3D bioprinting, enabling the creation of complex geometries leading to new applications.

Bioprinting is an additive manufacturing technology that creates structures containing living cells, by depositing material layer-by-layer from a digital design, enabling users to create complex, custom geometries. Various technologies can be used to achieve this, including extrusion-based, droplet-based, and laser-based deposition methods. Extrusion-based bioprinting is a technique where bioinks are precisely deposited through a nozzle to create 3D structures layer-by-layer. Fluid bioinks can be extruded using piston-based, pneumatic-based and screw-driven pumping mechanisms. Solid thermoplastic materials are typically stored in a wire spool, and mechanically driven through heated nozzle, melting immediately before extrusion [17]. Droplet-based bioprinting creates 3D structures by ejecting tiny droplets of bioink, from a nozzle or printhead to form precise patterns. Typically, the printhead will use a piezoelectric actuator or a heating element to create these small droplets. Laser- jetting is a technique that uses focused laser pulses to precisely deposit droplets of bioink from a donor substrate, in a layer-by-layer fashion to create 3D tissue structures [18]. Another laser-based bioprinting technology is called stereolithography. This is a vat photopolymerization technique, which uses a focused light source, to selectively solidify a photosensitive bioink layer-by-layer [19, 20].

The materials used for this layer-by-layer deposition are called bioinks, and typically consist of cytocompatible hydrogels containing natural and/or synthetic polymers. Bioinks are typically solutions of biomaterials, or mixtures of biomaterials in a hydrogel form, often containing live cells desired for growth of specific tissues [21]. The properties of a bioink before, during, and after drying are crucial to ensure printability, and cell viability [22]. Printability can have varying definitions depending on the printing technology and bioink used. The focus of this work is on extrusion-based printing techniques, as this is the most used 3D printing method, especially for printing with hydrogel bioinks. A key reason for this is that the other methods are impractical for use with inks containing live cells. Droplet-based printing can involve high temperatures and high shear-forces in the droplet-creation process, which could impact cell viability negatively. Meanwhile, vat polymerisation and laser-assisted printing techniques often can’t be used as the high energy radiation used for curing or droplet creation will also be damaging to live cell cultures. In the case of extrusion-based printing, the key factors for measuring printability include extrudability, print accuracy, and shape fidelity [23]. The most important factors affecting the printability of a bioink are viscosity, gelation (crosslinking-ability), and structural fidelity [19]. Shear thinning is often an important property for bioinks allowing them to be easily extruded at a high shear rate, whilst still returning to a higher viscosity after deposition [13, 24].

To make extrudable hydrogel, BNC can be hydrolysed using sulphuric acid [7]. A greener process uses enzymatic hydrolysis to make cellulose nanocrystals (CNCs) from BNC [25]. Alternatively, mechanical methods can be used. Fluidised BNC was produced by grinding with a blender and colloidal mill [26]. Extensive work has used sodium alginate as an additive to improve nanocellulose bioink rheology. Nanocellulose-alginate bioinks exhibit thixotropic properties [9, 27], which is useful for extrusion-based printing. Alginate offers a fast-gelling capacity when mixed with divalent cation cross-linkers such as Ca²⁺, enabling the printing of more complex scaffolds. This also reduces the porosity of scaffolds [1, 9]. Hydrogel rheology is typically characterised by viscosity, storage modulus and loss modulus, which can be measured using an oscillatory-sweep test [28] using a rheometer [29]. A two-step screening process for the development of printable bioinks was proposed by Paxton, et al. The initial screening step evaluates whether an ink forms droplets or fibres upon extrusion, as well as layer stacking or merging properties. Surface tension effects will affect drop or fibre formation, which are desirable for droplet-based and extrusion-based printing techniques respectively. It is also desirable that layers of deposited material will stack well, and not merge, as this will enable the construction of high-resolution, complex structure. The second stage of screening is a rheological evaluation. For 3D printing, a low and defined yield stress is desired, so that flow of the ink can be initiated and halted promptly. It is also desirable for an ink to exhibit shear thinning properties, that is, a drop in viscosity at high shear rates (i.e., during extrusion). An ink must also exhibit a fast post-extrusion recovery of viscosity, so that it may form a self-supporting structure [24]. Furthermore, a set of generic design rules for bioink formulation has also been developed by Gatenholm, et al., which have been used commercially to produce CELLINK. The design process involves six stages evaluating rheological properties, printability, crosslinkability and long-term stability, sterility, cell viability post-printing, and cell proliferation and survival [30].

Creating bioinks with a high nanocellulose content is difficult due to high viscosity, resulting in blockages and the need for high extrusion pressures [9]. Cross-linking polysaccharide-based bioinks using Ca²⁺ cations, is an effective way to achieve rapid gelation [31, 32]. Tabriz et al. printed alginate structures directly into a CaCl₂ bath, lowering the print bed as layers were deposited. The prototype printer was built using a modified, open-source Fab@Home printer [33,34,35]. This success poses the question of whether a BNC-alginate composite bioink could be printed using a similar method, to achieve high accuracy and shape fidelity.

Existing bioinks have several key limitations. The first issue is ink viscosity; high viscosity inks hinder extrusion, requiring higher extrusion pressures and resulting in greater shear forces which could lead to cell death. Low-viscosity inks cannot retain their shape in the printed form. This issue can be mitigated by using shear-thinning materials, that exhibit a drop in viscosity at higher shear rates. Many existing bioinks demonstrate this property, but few works aim to maximise this. Finally, the issue of shrinkage is a significant challenge in 3D printing bioinks which are predominantly composed of water. Upon drying, these inks shrink significantly, often also leading to deformation as well as a reduction in mechanical performance. To reduce shrinkage, a greater proportion of solids must be added to the bioink to begin with. This work investigates whether the addition of high quantities of BNC into a bioink can improve its shear thinning and viscosity properties, while increasing the solid bulk of the material, resulting in reduced shrinkage and improved mechanical properties. Furthermore, the highly entangled nature of BNC fibres, could also give rise to improved mechanical properties.

Various works have reported success in 3D bioprinting with BNC. Wu, et al. demonstrated that the addition of BNC enhanced overall nerve cell growth, adhesion and provided orientational guidance within printed scaffolds, demonstrating potential for neural tissue engineering [36]. Zeng, et al. successfully printed high-fidelity ear and nose-shaped scaffolds using a composite bioink of gelatine methacryloyl and BNC, finding that the addition of the BNC significantly improved the mechanical properties of the hydrogel, and promoted cell migration [37]. Wang, et al. used maleic acid and TEMPO ((2,2,6,6-Tetramethylpiperidin-1-yl)oxyl) modified BNC to improve the performance of printed gelatine scaffolds for in vivo bone generation [38]. Finally, Wei, et al. combined TEMPO oxidised BNC with sodium alginate, and laponite nanoclay, resulting in an ink with excellent structural stability and cell proliferation [39]. These works demonstrate the benefits of incorporating BNC into 3D printing bioinks, however, all of them used extremely low proportions of BNC in the ink’s compositions, typically only around 0.3 wt.%. In these works, BNC is used as an additive, rather than the predominant material. As previously mentioned, it is challenging to further increase the BNC content, due to the great rise in viscosity. However this poses the question, of whether further increasing the BNC content of a bioink will reap further benefits in relation to the resulting mechanical and biological properties of the scaffolds.

While bioprinting appears to be one of the most promising technologies within the medical, pharmaceutical, and animal-free food production industries [19], there are many technical challenges which need to be resolved. Part of the problem is the vast range of materials, as well as potential printing technologies available, some of which are better matched than others. Creating an ‘all-in-one’ printer is challenging. Existing state-of-the-art bioprinters are expensive (10,000 – 1,000,000 USD) [40], and this represents a high barrier to entry for research and development into bioprinting applications. Commercially available bioprinters lack customizability, which is important due to the large variability in the types of materials which can be used. Furthermore, they have limited print volumes, often < 5 ml, limiting potential applications to small build sizes. Additionally, is clear that post-print gelation using crosslinking is key to printing more stable scaffolds. Currently, there are not any commercially available bioprinters that offer the functionality of printing directly into a crosslinking bath. Many attempts have been made to create an ultra-low cost bioprinter, although none address all these issues [41,42,43,44,45,46,47].

The key aims of this work aim to answer the questions posed here, relating to whether a BNC-based bioink could be formulated, with a high BNC load-factor, such that BNC is the predominant material making up most of the the ink’s composition. This work takes advantage of novel techniques such as in-situ crosslinking, to improve printability and print performance of the newly developed BNC bioinks. These new bioink formulations could offer improved performance in the tissue engineering industry, offering improved mechanical properties, long-term stability and excellent biocompatibility [7, 8, 10, 13]. Additionally, this work aims to address the shortcomings of existing commercial bioprinters; by developing an open-source, extremely low-cost printer incorporating novel features including significantly increased print volume and in-situ crosslinking. This will enable the creation of larger, more complex structures at low-cost, with the outlook of accelerating research and development of bioprinting technology for industrial applications.

Results

Bioprinting platform

The prototype bioprinting platform was successfully used to evaluate the printability of different bioink compositions, successfully building tube-shaped structures over 3 cm high. Prints over 5 cm high could be achieved with a 2.5 mm printhead nozzle. With a 0.85 mm nozzle, print heights of up to 3 cm were possible. This indicates a reasonable print-resolution. It is worth noting that the prints were limited by the high viscosity of the ink rather than the capabilities of the printer. The total cost of the prototype setup was less than £100, demonstrating the possibility of significantly reducing the cost of bioprinting equipment from the extremely high prices of machines currently on the market. A method of in-situ crosslinking was demonstrated successfully, by manually pouring the crosslinking solution into the print-bowl. This could very easily be automated by adding an additional syringe-pump to pour the crosslinking fluid into the print-bed as layers of material are deposited. This rudimentary setup was able to print some of the more challenging inks, such as pure alginate, which is not naturally self-supporting, demonstrating the effectiveness of the in-situ crosslinking technique. This prototype was capable of printing ink volumes up to 60 ml. Because this prototype had limited motion capabilities with only a z-axis and a rotating print-bed for creating tube-shape structures, it was not possible to quantify the position accuracy or repeatability of the machine. The prototype had no temperature control or sterility features, both of which are essential for maintaining live cell viability during printing, thus making live cell printing difficult.

BNC-loaded bioink characterisation

Table 1 shows the different hydrogel bioinks tested and assessed printability. The hydrogel samples generated were compared using previously defined metrics [24, 30], as shown in Table 2.

Table 1 Bioink development

Table 2 Bioink evaluation and benchmarking. The generated bioinks have been qualitatively benchmarked on ten metrics, based on the requirements established by Paxton and Gatenholm [24, 30]. Each key ink composition was ranked from 0–10 on each metric, based on experimental observations; 10 being ‘perfect’ and 0 being ‘bad’. Metrics were included to evaluate BNC content of the formulations, and flexibility of the resulting dried material, which was assessed by creating a thin strip, and manually bending

In addition to establishing which ink compositions worked best, it was also found that in cases where pre-print partial crosslinking was used, the order of combining ingredients made a difference to the ink rheology. Adding alginate to 0.25% CaCl₂ results in the alginate forming very small, crosslinked pieces in a soup of CaCl₂ solution. Adding solid CaCl₂ to 5% alginate produced a homogeneous ink but it was not self-supporting. Adding a small quantity of dilute CaCl₂ solution to pre-mixed alginate solution and quickly blending produced an easily printable, self-supporting ink.

The water content of the generated bioinks was determined and is reported in Table 1. The most successful bioink had a BNC loading of 7.8 wt.% and alginate loading of 3 wt.%, meaning that the resulting prints were ~ 72 wt.% BNC when they were dried.

Figure 1 shows the results from the flow sweep tests. All the inks exhibit shear-thinning properties. The raw BNC paste also shows some shear thinning, but because it has a ‘gritty’ texture, resulting from solid fibres suspended in water, the results become distorted at higher shear rates, until the test fails. The viscosity of the ink increases as the concentration of CaCl₂ increases, up to a concentration of 0.25 wt.%, after which, no further increase is seen. BNC almost supplements the CaCl₂, as this ink had extremely similar properties to the 0.25/0.45 wt.% CaCl₂ inks, without the addition of any crosslinking agent.

Fig. 1

Rheological testing results from flow-sweep tests. It is evident that all the inks exhibit shear thinning properties, ideal for bioprinting. As the degree of pre-crosslinking increases, the viscosity and shear thinning property increases, up until 0.45 wt% CaCl₂, after which no further improvement is seen. The BNC ink has the greatest shear thinning property, with the highest viscosity at low shear rates, and a very low viscosity at high shear rates. Pure BNC paste does not behave as a fluid and so the test fails at higher shear rates. Key: alg 0.05 = 5 wt.% alginate + 0.05wt.% CaCl₂, BNC ink = see Table 1.24/5, BNC = 7.8 wt.% BNC

Discussion

A low-cost bioprinting platform has been developed that allows larger print volumes than any currently available bioprinter and incorporates new features such as in-situ crosslinking. Existing state-of-the-art printers typically only offer print volumes of up to 10 ml, for example, the CELLINK line of bioprinters [48, 49]. This work forms a proof-of-concept study which could be developed to create a state-of-the-art bioprinter. Core parts of the printer’s design have been made open source, which will make bioprinting more accessible to researchers and students, giving a lower barrier to entry for those interested in research in this space. There are several improvements needed to create a market-ready, scalable product.

Overall, the robustness of the entire printing platform needs to be improved, by using more resistant materials that will not flex or vibrate. This prototype, whilst able to print with considerable accuracy, would benefit from a stronger frame. This would significantly increase the consistency of printing. Accuracy and repeatability are two of the most important criteria for 3D printers, as these determine the dimensional accuracy and tolerances of the printed forms relative to the prescribed digital designs.

Another often-cited metric for state-of-the-art bioprinters is the attainable minimum layer height, which is often determined by the nozzle size. In this work, an 18-gauge needle, with a 0.85 mm internal diameter, was used as a printhead to extrude newly developed bioinks. A finer needle could not be used because of the high viscosity of the bioinks. A greater extrusion pressure would be required to extrude this ink through a finer nozzle. Printing with the 18-gauge needle was challenging and required precise tuning of the ink viscosity to ensure it was soft enough to pass through the nozzle without clogging. Many state-of-the-art bioprinters use up to 27-guage nozzles [48, 49], with a 0.2 mm internal diameter, achieving much greater print fidelity, although most traditional bioinks are significantly less viscous than these alginate-BNC composites.

An x–y gantry must be added to move the nozzle in the horizontal plane. The print-bed would house a container for crosslinking solution and would only move in the z-direction. Additionally, a method of controlling the z-height of crosslinking-solution must be added, either by means of changing the fluid level or lowering the print-bed into the solution. The developed extrusion system had some inconsistencies in extrusion rate, this could be improved by using more robust parts, such as a stronger, geared, motor. A stronger extrusion system would need to be developed, to allow for printing of such viscous bioinks with greater accuracy and control. This may also offer the possibility of faster printing speeds, making use of the printing platform more scalable. This extrusion system could also be further developed to enable even greater print volumes for larger-sized prints. Finally, the addition of a sterile housing would be essential, as well as a temperature-controlled environment to enable successful printing with live cell-loaded inks.

Another important property to analysed in more depth, is the flow initiation and fibre formation of the ink using this printing platform [24]. Once the extruder was turned on, it took a while for flow to start, forcing the ink through the fine nozzle. Often the ink would suddenly begin flowing very rapidly, before returning to a stable flow rate. This demonstrates that flow cannot be easily stopped and started, making more complex prints challenging, if they cannot be printed with a single continuous fibre extrusion. Similarly, flow does not stop immediately when the extrusion motor stops, because it takes some time for the built-up pressure to release. Fine-tuning of the flow-start and stop functions may be able to remedy this. The implementation of other technologies such as smart-vision could also help to ensure the material is behaving as the program expects, whilst also providing in-situ monitoring of the print in progress.

For the first time, BNC has successfully been printed into a multi-layered, self-supporting structure, by creating a composite with sodium alginate. Excellent print-quality was achieved, with BNC forming over 70 wt.% of the dry-weight material composition. Additional research could further increase the BNC content. Rheological testing has validated the properties of these bioinks, quantitatively demonstrating appropriateness for bioprinting.

Table 1 shows the development of the bioink. Originally, attempts were made to add BNC to the ink [33], creating a bioink containing both BNC and partially crosslinked sodium alginate. While this composition could be successfully printed, the BNC concentration was very low. The rheological study shown in Fig. 1 indicated that adding BNC paste to alginate solution had the same effect as partially-crosslinking, but to a greater extent, outperforming the partially crosslinked ink. The BNC-alginate ink had a higher viscosity and showed greater shear thinning than any of the partially crosslinked alginate inks. By eliminating the partial crosslinking step, an ink with a higher BNC content could be created with improved rheological properties. Furthermore, if we compare the rheological testing results from other bioinks developed in previous studies, we find that our ink outperforms previous works with a higher viscosity at low shear rates, and greater shear thinning at high shear rates. For example, at a low shear rate of 1 s⁻¹, the viscosity of our BNC ink is over 10 times that presented in the work by Wang, et al. [38]. Meanwhile, at a high shear rate of 100 s⁻¹, the viscosity of our ink is approximately the same as Wang’s. This means it becomes just as fluid during the high shear of extrusion, while behaving more solid-like, after extrusion, providing better support and stability for bioprinting.

The key requirement of bioprinting is the ability to print bioinks with sufficiently high viscosity to generate self-supporting structures, while still forming an interface layer, which can bond to subsequent layers. The original ink formulation, using partially crosslinked alginate, was problematic because the ink was very inconsistent, containing fully crosslinked particles. When this ink was dried in a sheet, it fell apart due to the lack of adhesion between these small particles as shown in Fig. 2. The BNC-alginate composite developed in this work overcomes this, as no pre-print partial crosslinking is needed to achieve adequate viscosity, and the fibrous nature of BNC increases the strength and flexibility of the generated samples. Furthermore, because the BNC-alginate composite has not been partially crosslinked, the layers have excellent adhesion. Further mechanical testing is necessary to determine if the layer-adhesion is improved compared to the original partially crosslinked alginate ink.

Fig. 2

a dried sheet of pure alginate solution, and (b) dried sheet of partially crosslinked alginate solution, showing that the crosslinked sample contains many cracks, while the pure alginate sample forms a smooth, continuous sheet

Table 2 shows that the BNC-alginate composite bioink outperformed all the other tested compositions on the accepted metrics [24, 30]. The images in Tables 1.4 and 1.5 show impressive resolution, the first showing a single-layer-walled tubular tower several centimetres tall, with a 0.85 mm wall/layer thickness, and the second showing a tubular tower with a diameter of around 4 cm, with a multi-layered wall, several centimetres tall. Repeatability was tested by creating multiple prints with the optimised BNC-bioink. Two examples are shown in Table 1.4 and 1.5, and more than ten prints were made in total to ensure that the results were repeatable, generating tall structures, with overhangs, and checking the layer adhesion manually each time.

In comparing the above results, to those found in previous literature, we find that certain characteristics are improved. In the images and videos included in the work by Wei, et al., [39] we can see excellent flow properties of the bioink, including good fibre formation and flow initiation, excellent layer stacking and printability. While the attained print quality looks very good, it is worth noting that this was achieved with a modified, commercially available 3D printing platform. It is also important to note that the BNC content in their bioink was significantly lower than that in the ink presented in this work. While no mechanical testing data is available, it is highly likely that the increased BNC content would yield improved mechanical properties, as well as better long-term in-medium stability for 3D printed scaffolds. Furthermore, while Wei’s bioink had a significantly higher viscosity at low shear rates, its viscosity at high shear rates also was very high, one thousand times the viscosity of our bioink at a shear rate of 1000 s⁻¹. This means that the shear forces within the bioink will be extremely high during printing of their bioink, likely resulting in poor cell survival rates when printing with live cultures, as well as creating a requirement for very high extrusion pressures. Our bioink mitigates these issues due to its excellent shear-thinning properties.

The final three metrics of success, sterility, cell survival, and cell proliferation, were not included, although evaluating the inks on those metrics would be an essential next step for use in biomedical applications. The use of plastic materials could lead to difficulties in sterilisation of the equipment, resulting in cross-contamination between bioinks and cell-types. A future design would need to consider disassembly and choosing different materials for effective sterilisation of the printing platform. Furthermore, the lack of environmental control would be highly problematic in the cultivation of live cells – additional features would be needed for maintaining sterility and consistent environmental conditions for the successful growth of live cell cultures. There is the added implication, that even if sterility and temperature control features are added to this platform, the crosslinking salt solution could have an adverse effect on live cell cultures. Research has shown that addition of salts to bacterial cell cultures, can lead to reduced growth rates and maximum cell densities, although this is dependent on the salt used, and bacterial strain [50]. However, other salts have also been found to accelerate bacterial growth [51]. This is possibly due to the disruption of bacterial cell osmotic balance, leading to water leaving the cells, resulting in cell shrinkage and potential cell death. An in-depth, culture-specific investigation would be needed to determine the effect of cross-linking in salt solutions. One solution could be to only use post-print crosslinking (as with the final BNC-alginate ink), preventing inner areas from exposure to the salt solution. Furthermore, encasing living cells in microcapsules, which dissolve after the printing process could protect them during the printing process. Genetic modification could also enable development of cells which are resistant to degradation on exposure to salt solutions. A more in-depth investigation into the potential toxicity of the materials used and the viability of cell growth in the platform would be needed for this new bioprinter and bioink to be used at larger scales, for biological applications.

The composition of 7.8 wt.% BNC paste with 3 wt.% sodium alginate, created the most successful prints. Higher concentrations of BNC generated a hydrogel that was too viscous to be extruded with the prototype printing platform, and lower concentrations had lower viscosity so printed structures were less rigid and did not self-support. Furthermore, maximising the dry weight minimises shrinkage of the resulting structures when dried. Lower concentrations of alginate resulted in an inhomogeneous bioink that fell apart. Lower concentrations did not provide enough material for crosslinking during the printing process, resulting in print failure as shown in Table 1.3.

All the inks generated exhibited shear thinning properties as seen in Fig. 1. This property is incredibly useful for 3D printing because it means the viscosity will drop within the nozzle, making it easier to extrude, but will be rapidly regained enabling the formation of self-supporting structures. This is especially useful in bioprinting, as it can reduce cell death due to shear forces [9].

The broader implications of this work relate to making bioprinting more accessible, as well as creating more robust tissue scaffolds. By developing this new, low-cost bioprinter, we begin to pave the way for affordable more scalable innovation via a more affordable research tool. By enabling low-cost exploration of tissue scaffolds, this tool could serve as a stepping stone for development of new materials and applications for larger-scale production. It will enable rapid prototyping and iteration of new bioink formulations, and geometries for research and development. The bioprinting platform could also make tissue-engineering education more accessible for students interested in exploring the world of bio-printing. Additionally, the new bioink formulation, with improved rheological and mechanical properties and higher BNC loading, could enable the creation of stronger, and more stable tissue scaffolds, resulting in new applications in tissue engineering and regenerative medicine.

Further work will investigate enhancement of the bioink’s formulation using of additives, for example, to improve water retention and flexibility. Additionally, a complete characterisation of the generated materials, including mechanical testing and long-term stability, is essential to completely understand the potential applications of the generated bioinks. The incorporation of living cells would be a natural next step in the development of this bioink for tissue-engineering and biomedical applications. Cell viability studies would also be needed to investigate the effects of different bioink compositions on the viability and proliferation of embedded cells, particularly for tissue engineering applications. A viability study could also look more closely into the effects of different crosslinking agents. Another direction for further research could be into multi-material printing, for deposition of multiple different kinds of bioinks simultaneously, potentially unlocking new applications. A more complete 3D bioprinter would need to be developed with multiple additional features discussed in this section, including full three-dimensional printing for production of high-fidelity models, as well as features for environmental control and sterility, which may be challenging for large material quantities and print volumes. Careful material selection and design-for-disassembly would be key to enabling easy sterilisation of the equipment. It is important to note that all these further developments would come at an increased cost and complexity for the bioprinter, so it will be important to recognise this trade-off and look for innovative ways to keep the platform economically viable for industrial use.

Conclusions

A new composite bioink has been developed containing BNC and sodium alginate. This bioink has been printed using a novel bioprinting platform which enables in-situ crosslinking to solidify hydrogel bioinks. The rheological properties of the bioinks were determined, and the viscous properties of the inks can be altered by changing the bioinks’ composition. The best-performing bioink composition contained 7.8 wt.% BNC and 3 wt.% sodium alginate, resulting in an overall 72 wt.% BNC when fully dried. This new bioink contained a significantly greater quantity of BNC compared to any of the previous works attempting to 3D print with this material, achieving a load factor over 20 times greater. The resulting printed structures were stable and could be formed in multilayered structures with high fidelity. The rheological properties of the new bioink surpassed any of the inks found in previous literature, demonstrating an excellent shear-thinning property with very high viscosity at low shear rates and very low viscosity at high shear rates. This implies a reduction in potential cell death during the bioprinting of inks containing live cell cultures, due to reduced shear forces during extrusion. Furthermore, the increased quantity of BNC in the ink has the potential to improve the mechanical properties and long-term stability of printed scaffolds. The properties of this new composite post printing and drying, were impressive in terms of flexibility and strength. This is a promising new biomaterial composite, and the bioink has potential to be used in many applications, including tissue engineering and regenerative medicine. Further development of the bioink would involve exploring additional additives, conducting in-depth assessments of material properties, and cell viability studies, followed by in-vitro and in-vivo testing of live cell-containing scaffolds.

The prototype bioprinter successfully demonstrates the feasibility of building an extremely low-cost bioprinting platform with the capability of printing large volumes of high-viscosity materials, while also crosslinking printed structures in-situ. These combined capabilities have yet to be seen in the bioprinting world and are beginning to pave the way for scalable developments in the field. Further development of the bioprinter would involve the addition of features for full three-dimensional printing, as well as taking into consideration sterility and environmental control requirements.

Methods

Development of a low-cost bioprinting platform

A low-cost 2.5 axis hydrogel 3D printer was developed, with the capability of printing tube-shaped structures. This design could be upgraded to a full 3-axis printing platform, although this is beyond the scope of this work. The high viscosity of the bioink and high shear forces of the extrusion process result in high extrusion pressures. A custom syringe extruder was assembled using a NEMA 17 stepper motor (42A02C-XH2.54, Shenzhen Rtelligent Mechanical Electrical Technology Co.), a 60 ml plastic syringe (CoKeeSun) and a 2 mm leadscrew (150 mm T8-2 Lead Screw, Sourcingmap), using a housing (3D printed, Original Prusa Mini + , Prusa3D) designed by Constantijn C [52, 53] for 3D printing with clay. The extruder was controlled using an A4988 stepper-motor driver (Haljia) and Arduino (Elegoo Mega 2560, Elegoo Official). A z-axis was built using a single NEMA 17 stepper motor, and a scissor-lift mechanism was constructed using laser-cut acrylic parts. Rather than assembling an x–y gantry, a simple turntable was attached to the z-axis, powered by another NEMA 17. This enabled the printing of tube-shaped structures required to validate the printing platform design, as well as the generated bioink.

Layer-by-layer crosslinking was achieved by manually adding the crosslinking agent to the print-bed as the print progressed, similar to the method employed by Tabriz, et al. [33]. Layers of the cross-linkable biopolymer are extruded onto the print-bed. As layers are deposited, they are submerged into a crosslinking bath, solidifying them, and enabling further layers to be deposited on top. Due to diffusion of crosslinking ions, the layers immediately above the surface of the bath also become partially crosslinked. As such, a few millimetres of the build are kept above the fluid-line of the crosslinking bath, so that the newly printed layer can successfully adhere to the layer beneath. Optimal print speeds were determined visually by gradually increasing the material extrusion rate by approximately 0.005 mm³/s at a time, until a continuous flow was achieved. The feed-rate was increased to maximise print-speed until the syringe handles began to bend, as the maximum pressure was achieved at this point. The printer was controlled via the Arduino command line interface on a connected computer. Extrusion rates could be changed by varying the delay time between sending step pulses to the stepper motor controllers. The same technique was also used to vary the turntable speed to match the extrusion rate, although this could also be altered by moving the turntable to change the diameter of the tube, therefore changing the speed of the print-bed relative to the nozzle. The Arduino was programmed to autonomously lower the z-height of the print-bed after the completion of each revolution of the turntable. Figure 3 shows a CAD model of the prototype printing platform.

Fig. 3

CAD schematic of the prototype bioprinting platform

Preparation of BNC-loaded hydrogel bioinks

BNC pellicles were grown using kombucha cultures. Kombucha culture starter medium was purchased in vacuum-sealed packages (Etsy). Containers were sterilised using iso-propanol (99.8%, APC Pure), and then rinsed three times with distilled water. They were then filled with distilled water, 6% v/v kombucha starter, and 6% w/v glucose powder (Glucose, Dextrose Powder, Thornton and Roass). Excess CaCO₃ (98.3% Calcium carbonate, Intra Laboratories) was added to maintain the pH of the culture (typically 5 g/L). The cultures were briefly stirred to dissolve all the solids, and then covered with fabric or parafilm (Laboquip-Bemis Parafilm M 996, LaboQuip). The cultures were then left to incubate in a temperature-controlled environment between 27–30 °C, for 2–4 weeks, until biofilm growth was complete.

Alginate is a viscous fluid with low storage modulus and high loss modulus. This means that it flows easily and does not retain its shape [54]. Printing pure alginate directly into a crosslinking bath is problematic, as the material shrinks due to the osmotic pressure difference between the alginate gel and crosslinking bath [55]. To overcome this, a method was used to partially crosslink the alginate hydrogel prior to printing, with fully crosslinking after extrusion [33]. Pure sodium alginate solution (Sodium Alginate, European Origin, Special Ingredients) was partially crosslinked using calcium chloride (Calcium Chloride, premium food grade di-hydrate flakes, Intra Laboratories) solution, by adding a small amount and blending for a few seconds. Initial quantities were selected based on several previous research papers as well as manual experimentation, starting at 5% w/v sodium alginate and 0.5% w/v calcium chloride. The quantities were increased 1% w/v and 0.1% w/v at a time for sodium alginate and calcium chloride respectively, until a stable, stackable bioink was produced. The crosslinking bath was prepared by adding calcium chloride to water at 10 wt.%. 0.5%w/v Tween 80 (APC Pure) was used as a surfactant, to reduce the surface tension of the solution [56, 57]. This enabled finer control over which layers became crosslinked, preventing layers above the crosslinker fluid line from solidifying prematurely.

BNC was then incorporated into the ink. BNC pellicles were vigorously blended using a handheld blender for ~ 5 min (MultiQuick 1 Hand Mixer MQ10.001P, Braun), and strained to remove lumps that could cause blockages. As a starting point, a bioink was prepared containing 50 wt.% BNC pulp, and 50 wt.% of the ink from the previous stage. The ink composition was modified to optimise printability, by altering the quantities of BNC paste, and bioink starting in 5 wt.% increments and then in 1 wt.% increments until the most stable bioink was attained (assessed visually and manually based on print appearance, layer stacking and adherence of sequential layers to one another). The solid content of the BNC paste was determined by drying a weighed sample in an oven. The resulting bioink contained 6.30 wt.% BNC.

Adding BNC paste made the ink noticeably thicker and more self-supporting, (increased storage modulus). Therefore, an alginate-BNC ink without partial crosslinking was also tested. The pellicles were dried slightly before blending to increase the BNC-loading. A paste containing 7.78 wt.% BNC was produced. Alginate powder was added to this paste in increments of 0.5 wt.% until a self-supporting ink was produced. A diagram with iterative ink-optimisation is displayed in Fig. 4. A list of key ink compositions is given in Table 1.

Fig. 4

Flow chart illustrating the process used to find the optimal BNC-alginate bioink composition

Material characterisation

The water content of the BNC paste was measured by weighing a small sample and drying it in an oven. The rheological characteristics of alginate hydrogels are well known [58,59,60]. Rheological analysis was used to characterise the developed BNC-alginate composite bioinks. This allows for validation of key bioink properties set out by Paxton and Gatenholm [24, 30] and enables quantitative explanations of composite materials performance. Flow sweep tests were completed (Discovery HR-1 Rheometer, TA Instruments), using shear rates from 0.001–1000 s⁻¹. Rough, 25 mm diameter plates were used with a 300 µm gap and tests were run at 23 °C, allowing 5 s equilibrium time and 10 s averaging time for each data point. Rheology tests were completed on partially crosslinked alginate bioinks, all containing 5 wt.% alginate, and varying percentages of CaCl₂. The same tests were performed on raw BNC paste and the optimised BNC bioink.