A Multidisciplinary Analysis of Green-Bioprinting Methods & Design Principles

A Multidisciplinary Analysis of Green-Bioprinting Methods & Design Principles

Video summary of research

Aitch M. Hunt

Table of Contents

Abstract 4

A Multidisciplinary Analysis of Green-Bioprinting Methods & Design Principles 5

Background 9

Primary Research Article 11

Discussion 14

Figures 17

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Abstract

Plant biofabrication can be defined as the construction of materials from or with that of plants or plant-derived products (Groll et al., 2016). The recent and expansive emergence of biofabrication is largely due to its broad scale of application and relevancy to important trades such as medicine and industrial design. The continuation of advancement in this field requires a cross-examination of the principles of design to enhance process and production of biofabrication as a utility for various applications. This paper will focus on the 3D bioprinting techniques of green- bioprinting, the additive measures and development of components needed to run these processes, and the integrations of design methodologies to initiate an improved interdisciplinary approach to process and products of biofabrication.

Keywords: green bioprinting, biofabrication, design principles, plant cells, 3D plotting, alginate, agarose, methylcellulose, dispensing, bioink.

A Multidisciplinary Analysis of Green-Bioprinting Methods & Design Principles Biofabrication is the construction of products of, from, or with raw materials found in nature using modern tools of technology (Mironov et al., 2009). It serves as a technological platform for various methods and technologies developed to manufacture biologically derived and inspired materials. As a core technology in biofabrication, three-dimensional (3D) bioprinting is an additive measure of biofabrication that utilizes cells, proteins and biomaterials as building blocks for 3D-printed biological models, biological systems and therapeutic products (Sun et al., 2020). This technology uses cell-by-cell fabrication techniques to build pre-programmed models out of living cells. There are three main types of 3D bioprinting: extrusion-based bioprinting (EBB), droplet-based bioprinting (DBB), and laser-based bioprinting (LBB) (Kundu, Pati, Hun Jeong, & Cho, 2013).

The fabrication of advanced biological models is a relatively new field of research, but interest and innovation in the field is entering a phase of great exponential growth and organization consolidation (Guillemot et al 2010). The Biofabrication Journal scored an impact factor of 8.213 for the year 2019. This ranking is a significant value when considering its young age at only 11 years old (Reuters 2019).

The potential applications of emerging biofabrication technologies are not only broad in scope, but also geared towards addressing world-wide priorities. The applications of this technology spread across the vast spectrum of domains in which material science carries any importance. It extends from the production of food and energy, to that of industrial manifestations in architectural infrastructure, to that of medicinal integrations for regeneration of tissues, or as a transportation device for drug-testing in pharmaceutical research (Seol, Kang, Lee, Atala, & Yoo, 2014). Although the applications of this technology reach extensively over seemingly unrelated communities of science and design, the potential benefits of successful integration between these various fields of research will ensure its continued success.

Appropriate implementation of plant biofabrication technologies between the fields of science and design will render its affordances under three major advantages: economical, environmental and improved repercussions associated with the manufacturing of all basic commodities. When taking in these factors of global opportunity, growth, and impact, recent analysis of data seen in Figure 1 suggests that biofabrication could become a dominant technological platform and new paradigm for 21st century manufacturing (Mironov et al., 2009).

If this area of research is to continue to rest on the cutting-edge of science, consideration of major allegations involved in process and product design are essential for the continued sustainability of biofabrication technology in today’s world. The current limitations of biofabrication circumvent the gaps between overlaps of multidisciplinary action. Conflicting constraints often appear in interdisciplinary fields because they require highly specialized knowledge bases from various fields of study in addition to a way in which to connect them (Kundu et al., 2013). Persuasion of this necessity in the field might start with the examination of the word itself. “Bio-fabrication” denotes a multi-faceted nature. Even within this nature, “biology” and “fabrication”, exists a finer division of intrinsically related domains of science and design. Biofabrication is the intersection of developmental biology, mechanical engineering, and material science, and therefore requires an interdisciplinary process to serve as a foundation for cross-communication across platforms of science and design. This research will examine the specific tools and techniques involved in tissue engineering coupled with the foundation in the core design rules and principles of “building with biology” by surveying strengths and weakness of these domains in isolation from each other. A critical analysis of a research paper on “Green Bioprinting” will be used to then demonstrate the necessity and future applications of this integration in the field of biofabrication.

Biofabrication in Biomedical Engineering. Already, advances in 3D bioprinting have begun to percolate the fabric of science and design through the field of medicine. Biofabrication advances are traditionally embedded in the medical refinement for applications in tissue regeneration and drug delivery assays (Iravani & Varma, 2019). A famous example of this introduction is the study, Bioreactivity of decellularized animal, plant, and fungal scaffolds: perspectives for medical applications, conducted by scientists at the Worcester Polytechnic Institute (Figure 2). In this experiment, researchers were able to replicate the complexities of cardiovascular tissue in the vascular network of decellularized spinach leaf tissue. This study demonstrates the necessity of cross- discipline integration for continual growth of biofabrication techniques, particularly related to the field of medicine where tissue engineering research faces many design challenges (Predeina, Dukhinova, & Vinogradov, 2020). A researcher, Glenn Gaudette, from the same department of this research project commented in a press statement on the importance of cross-disciplinary action within this field. He said, “Adapting abundant plants that farmers have been cultivating for thousands of years for use in tissue engineering could solve a host of problems limiting the field” (Bhattacharya, 2017).

Biofabrication in Design. There have been numerous attempts from designers to incorporate biofabrication technologies in the development of industrial products such as building materials for infrastructure, fashion textiles, and human-computer interactions (Figure 3). SymbioticA’s Tissue Culture and Art Project from 1996 demonstrates preliminary attempts to bridge the gap between abstract concepts in science with observable events in nature (Catts & Zurr, 2002). Since then, many designers have conducted top-down explorations outlining speculative applications of biotechnology and ethical implications of these futuristic technologies. This cross- discipline approach enables direct manipulation of material properties on a molecular scale, ultimately granting more control over the final product than is possible with other traditional materials (Joachim, 2014).

Although it is important to consider the humanistic effects of certain advances in science, these speculations lack purpose if there is no method of scientific research or experimental data to solidify these claims for actual consideration.

In summary, design integration of biofabrication technologies proves to assess the “why” of its relevance to the future but lacks the vehicle of “how”. Scientific methods enable the development of products through understanding the mechanism but lack the characteristics of versatility of biofabrication applications extrapolated from core principles of design. Scientific analysis provides the “how” yet lacks the “why” in relation to physical manifestation and consumerist ideals. The practice of innovative 3D -bioprinting practices lack substance in the field of creative application and fabrication methods for material science (Jakab et al., 2019). This evaluation of cross-pollination between scientific method and design principles will proceed through the vehicle of biofabrication as a technological connection between both domains.

In order to further address this gap, an examination of the inner workings of previously established processes of bioprinting have been pulled from significant technological advances in the traditional application of medicine. This will be used as a cross-disciplinary tool for production of industrial materials that enable rapid prototyping of the technology to further the advancement of biological fabrication and implementation through the process of design.

Background

In the various methods of 3D-bioprinting, biomaterials are produced in spatially predefined locations with confined three-dimensional structures called scaffolds (Sun et al., 2020). Cells are seeded onto solid and biodegradable scaffolds, and tissue formation is typically induced by biomolecules such as growth factors (Seol et al., 2014). Traditionally, tissue scaffolds have been assembled from synthetic and animal materials. However, the utilization of these types of materials for tissue engineering and 3D-bioprinting have encountered many obstacles such as the scarcity of the material and the high cost (Iravani & Varma, 2019). In their paper on Green Bioprinting, Seidel et al. proposes a creative solution as an alternative to these materials that would avoid many of these issues alongside a list of additional benefits to come (Seidel, Julia et al., 2017).

The porous composition and malleable physiology of plants make them ideal candidates for the building blocks of scaffold assembly in biofabrication (Ovsianikov, Yoo, & Mironov, 2018). The appeal of plant-derived proteins for three-dimensional constructs extends even further when examining their large-scale effects on production. For one, accessibility to the plant material required for such undertakings is available to all researchers (Iravani & Varma, 2019). The basic manufacturing of plant material is simply just growing the plant from seed. It is therefore more environmentally sound than that of traditional production methods where large amounts of fossil fuels are consumed and released in transportation, machine operation, and biproducts of synthetic or animal- based materials (Forgacs & Sun, 2013). Plants are also renewable, and the environment would benefit just from having more of them around.

The physical properties of plant machinery is another highly desirable trait for large-scale production and maintenance. Some of these characteristics include the optimization of surface area in plant tissues, interconnected porosity, their ability to efficiently retain and transport fluids, and the modular network of their vascular tissues (Iravani & Varma, 2019).

There are various technologies developed for 3D bioprinting. This paper will focus specifically on extrusion-based process of bioprinting due to its wide use in research as a result of affordability, versatility in mechanical modifications, wide range of biomaterial that can be experimented with, and its ability to produce high densities of cells (Kyle et al., 2017; Jovic et al., 2019; Leberfinger et al., 2019). The range of biomaterials tested to work with this technology include hydrogels, biocompatible copolymers, and cell spheroids. These are important to the versatility of scaffold design because they each have unique printable properties such as viscosity, density, or shear-thinning behavior (Hölzl et al., 2016).

The biofabrication research on extrusion-based method of 3D bioprinting has potential to benefit from the cross-application of design principles and the scientific methods at a scale of two-fold: extraction and application. The extraction of certain design principles such as rapid prototyping and iterative thinking will lend itself to the efficiency of modular stages of bioink production, plotting resolution and scaffold stability. The process will also need a consistent method to effectively communicate results with other experimenters. This can take influence from the standardized properties of the scientific process. From here, the resulting product will need a mechanism of transportation into the world of industrial consumerism. The requirements for successful adaption of this new technology into real-world usage relies on the core considerations of every design initiated product or process: affordances of scalability, cost, environmental impact, and public acceptance. To assess firsthand, these principles in action, the focus will now shift to the methodologies involved in that of Green Bioprinting, a strategy of extrusion-based bioprinting using plant polymer scaffolds (Seidel, J. et al., 2017).

Primary Research Article

A further investigation into the technological mechanics and products of 3D bioprinting will be done through the examination of a research paper written by the inventors of the word “Green Bioprinting”. The scientific research article is titled, “Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds” and is written by Julia Seidel, Tilman Ahfeld, Max Adolph, Sibvlle Kümmritz, Juliane Steingrower, Felix Krugatz, Thomas Bley, Michael Gelinsky, and Anja Lode (Seidel, Julia et al., 2017).

The aim of their research was to further the evolution of bioprinting fabrication technologies through the application of 3D-bioprinting advances in the medical field to the 3D bioprinting of plant cells. They called this alginate-based hydrogel blend suitable for 3D plotting of hydrogel constructs “Green Bioprinting”. The successful utilization of green bioprinting directed research towards improvements in the overall process of Green Bioprinting. Improvement was defined by the balanced relationship between high-shape fidelity of the scaffold and the ability to embed plant cells and their cultivation within the gel matrix.

Extrusion-based cell laden plant bio-polymers were substituted for traditional material used in scaffold production to overcome barriers such as biocompatibility, accessibility of material, and clinical testing. Successful implementation of this technology would also enhance scientific procedures and research surrounding tissue culture in plant biotechnology such as callus-derived cell suspension cultures and immobilization techniques (Figure 5).

Methods and Procedures. The researchers executed a series of modular development and testing within three overarching stages of 3D bioprinting (Figure 4): the first stage was the synthesis of bioink, second was the optimization of technological parameters of printing, and lastly was the evaluation of the 3D bioprinted products (Hölzl et al., 2016). In this experiment, the bioink is synthesized from plant cell-laden plant biopolymers and hydrogels. The hydrogels were developed containing alginate (2.8 wt%), agarose (0.9 wt%) and methylcellulose (3 wt%), a unique mixture (alg/aga/mc) evaluated through rheological characterization using a rotary rheometer to measure viscosity and shear-thinning performance of the hydrogel blend. Next, the bioink was put to use in the extrusion-based bioprinting phase. The researchers adjusted the speed of the Bioscaffolder 3.1 bioprinter and dosing pressure of the hydrogel from the nozzle of the machine. Each cell sheet was rotated 90

degrees after each printed line to form a waffle-like matrix (Figure 7). Successful cross-linking of alginate was achieved after these scaffolds were plotted into six-well plates in air, incubated, and washed with MS medium. From here, structure, swelling, and various mechanical properties of the scaffold were assessed through a series of testing (Figure 8). After determining the adeptness for printing, the researchers moved to the fabrication of plant cell-laden hydrogel scaffolds using in vitro cell culture of basil for the plant cell bioprinting and plotting. Finally, successful implementation of the hydrogel scaffold was assessed by characteristics of cell viability and metabolic activity. The results were summed up in a statistical analysis of the data.

Results. There were three categories of results measured: hydrogel suitability for printing with plant cells, scaffold integrity, and success rate of plant cells embedded into the hydrogel scaffolds. The hydrogel mixture successfully printed an evenly porous three-dimensional construct compatible with the plant cells and was stable under cultured conditions. Most of the embedded cells survived plotting and crosslinking (Figure 9). Over several weeks, the agglomeration of basil cells increased in size in a relatively even dispersal throughout the matrix. These results indicated high viability of the embedded cells. These results in combination with the metabolic activity test results (Fig. 10), which revealed an accumulation of energy input over time, indicated sustained survival and growth of the cell-embedded agglomerates. The stable growth of cell tissue culture embedded in a 3D bioprinted scaffold warrants further research into the use of this method for future advances in immobilization and monitorization techniques in plant biotechnology research. This successful translation of a technology originally intended for medical use, renders Green Bioprinting a primary example of a biofabrication invention that furthers the industry’s success through a cross-analysis of the scientific method and the principles of design. The modular breakdown of technology, segmented areas of focus, and rapid testing are great examples of the integration of problem-based methodologies of design used to further the abilities of scientific research (Taura, 2016). The relevancy of unified practices between mechanical engineering and developmental biology are observed in the important mechanical and chemical properties of the bioink and scaffold structure. Improved formulation of future applications of this novel tool can be furthered through a better understanding of material science and design implications using speculative design principles.

Conclusion. The researchers concluded a reasonable application of green bioprinting technology for improvements in plant biotechnology procedures and abilities. The authors suggest improved opportunities in this field as a result of improved access to standardized research on metabolic interactions and process optimization. This would be achieved through the properties of the defined matrix developed by green bioprinting that allow for single-cell isolation experiments, time and space resolved analysis, and control over diffused substance paths for observations of metabolic activity. Furthermore, they promptly suggest applications of this research outside of biotechnology on an industrial scale for building materials or food production.

Discussion

Biofabrication rests under the umbrella category of biotechnology. In contrast to the general sciences, which strive to gather information about nature through experimentation, the aim of biofabrication reflects more accurately the definition and role of technological innovation. Biofabrication is the engineering of a novel material through the understandings gained from science. This is an important differentiation to make because unlike the scientific process, the methodology used in biofabrication research focuses on the mechanisms of the process itself and the affordances of the end result. These distinct priorities in the field are in closer alignment with procedures utilized within the field of design. Important elements of the design process (Figure 6) worthy of employment for biofabrication pursuits are the principles of rapid prototyping, translation, implementation, and generation of predefined optimization in accordance with future applications of the technology (Taura, 2016).

Many recent advances in biofabrications have been with the intent of medical application. Bioengineered tissues purposed for medicinal use attempt to replicate the characteristics of a human organ. Therefore, requiring these models to emulate the complexity of the human body at a microscopic level and with absolute accuracy and precision. To be of any success in these attempts, the bioproducts from scaffold degradation and the biomaterial itself must not be toxic and coexist naturally with the complexity of a living multicellular organism (Iravani & Varma, 2019). Not only are the requirements of incredibly high standard, but the aftermath of integrating the tool into real practice would take a long time due to clinical testing of a long-lasting material.

What if, however, the technology advances made in 3D bio-printing started at a more basic level of design aspiration. Instead of trying to replicate intricacies of human life using life, research efforts were placed into replicating basic commodities and processes of human interaction using the building blocks of life to further benefit the environment and the organisms living inside of it.

The research paper by Seidel et al. is a good example of how a shift in traditional intention of application of biofabrication can largely benefit our understanding of the field as a whole. Through the examination of the components that make up the large area of bioprinting, organized subunits of known problems can begin to be addressed, fine-tuned, and ultimately connected back into the larger picture. In design, they call this structural breakdown of a whole into its pieces, the Theory of Gestalt (Michalek & Papalambros, 2002) .

Many barriers encountered in the medical applications of this technology, and the gaps that result as a translational error, require a shift towards a revolutionized recap on the process and products in design. Gearing the direction of research towards goals that are more feasible, testable, easier to control and manipulate, development of this technology for non-medical applications such as animal free leather, bio-reactive clothing, sustainable building material, would be a much more reliable goal for biofabrication research (Jakab et al., 2019) . Acknowledging the famous industrial design and architectural principle, “form follows function”, would further benefit cohesion of priorities in scientific research and design.

Seidel and authors of the Green Bioprinting research paper demonstrated the benefits of incorporating this principle into the development of ideal characteristics for their hydrogel blend and scaffold structure. Starting with the question enabled their research to move past the common faults of most bioprinting technology. In design, this approach is called an “inquiry-based approach” (Michalek & Papalambros, 2002). Most of bioprinting experiments start with the biomedical requirements of a scaffold design, for example. A scaffold design for medical use must be biodegradable, biocompatible, porous, durable, multi-cellular, and precise. Instead of trying to address all of the complex properties required for a suitable scaffold for biomedicine, the researchers in the Seidel et al. paper evaluated these pieces as components of a larger picture and moved away from the medical field applications as a whole. This new perspective gave rise to the discovery of a totally new and promising tool and area of research.

The shortcomings of the research conducted by Seidel et al. was its lack in resource for testing the certain parameters and properties of Green Bioprinting. The authors stated a difficulty in measuring properties of the cell agglomerations and growth due to their small size. Another setback of this extrusion-based method was the speed of the printing process itself. A tool of design that would be particularly helpful to the shortcomings of process would be the development of an interactive algorithm and software capable of interpreting behavioral changes of specified factors in scaffold design, hydrogel blend, and printing parameter (Michalek & Papalambros, 2002). In doing so, a refined formulation of the problem directed at of the material itself, might open an opportunity up for additional research and advances in green bioprinting (Michalek & Papalambros, 2002).

Much of the discussion so far has covered the reasons cross-disciplinary influence, action, and collaboration are needed in biofabrication, but actionable steps to initiate this process are just as important. In order to begin this process, a universal methodology is of the most importance. It is needed to enable further expansion and potential applications of this technology and its components (Forgacs & Sun, 2013). A set of unified criteria and a common framework are missing from bioprinting experimentation and this is largely the makeup of barriers involved in the lack of communication around the advances and movements in bio-printing across multidisciplinary research teams (Hölzl et al., 2016). Standardized units of measurement, inappropriate evaluation tests, and missing bioprinting parameters need to be reassessed for future studies of bioprinting technologies.

In conclusion, a design intervention in the field of biofabrication could strongly enhance its large-scale integration into global priorities for improved conditions of material and food production, the environment, the economy in regard to industrial and medical affiliations, and consumer goods.

Figures

Figure 1.The Scimago Journal & Country Rank of The Journal of Biofabrication: (on the left) The SJR is a size-independent prestige indicator that ranks journals by their “average prestige per article”. It measures scientific influence of journals by accounting for the number of citations received by the journal and the importance of the journals where such citations came from. It measure the scientific influence of the average article in a journal and expresses how central to the global scientific discussion an average article of the journal is; (on the right) The y-axis of this chart represents the number of times this journal was cited by entries of other journals divided by the total number of articles published in the journal, Biofabrication. The x-axis shows the evolution of the average number of times documents published in Biofabrication in the past years have been cited in the current year. The two year line is equivalent to the impact factor ™ (Thomson Reuters) Metric (SCImago, ).

Figure 2 Using decellularized plants as perfusable tissue engineering scaffolds
Figure 3. The BioShoe was grown using an interdisciplinary approach of crossing processes and material for a combinatory effect of multifactorial traits of form and function from various organisms. Photograph by Bill Waters. © Biocouture.
Figure 4. The schematic representation of three bioprinting stages: pre-printing, extrusion-based bioprinting, and post-printing analysis (Mancha Sánchez et al., 2020).
Figure 5. Green Bioprinting—fabrication of tailorable 3D plant cell cultures as a new tool to study plant cell immobilization and to optimize production processes (Seidel, J. et al., 2017)
Figure 7. Plotted hydrogel scaffolds after crosslinking and incubation. (A) Scaffolds with different set strand distances, two layers; (B) scaffold fabricated in 0°/90° configuration, 20 layers, strand distance: 2.5 mm; (C) horizontal pores in a scaffold fabricated in 0° 0°/90°90° configuration, 14 layers, strand distance 2 mm (side view with enlarged section) (Seidel et al., 2017). 
Figure 8. Scaffold properties showing the micro and macropore sizes (Seidel et al., 2017)
Figure 9. Live/dead imaging of basal cells (A) in suspension, (B) embedded in alg/aga/mc scaffolds (four layers) after 1, 6, 14 and 20 days of cultivation and (C) an overview of a scaffold from day 14; living cells (green) stained with FDA and dead cells (red) stained with PI (Seidel et al., 2017).
Figure 10. Stereo light microscopy images of one and the same plotted alg/aga/mc scaffold with embedded growing basil cells on cultivation days 1 and 9. The development of each agglomerate during cultivation can be monitored (Seidel et al., 2017).
Figure 6. The Design Cycle Model

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