General Perspective on the Function, Material, Development, and Applicability of Bio-Printers

Authors: Arda Bulut — Mehmet Eren Erken — Sarp Emre Turan

A. INTRODUCTION

The lack of compatible organs and tissues to use in transplantations and laboratory research has always been an issue in biological sciences. In organ and tissue transplantations, it is crucial to facilitate a rate of similarity between the donated material and the recipient’s original components.² Antigens are substances which grant cells identity and can create an immune response through their interactions with the receptors found on the surfaces of white blood cells.¹ A moderate to over activation of the immune response in the recipient vessel during transplantation procedures may result in acute organ or tissue rejection.² Because of this, any transferred solid organ or tissue should match with all six primary histocompatibility agents.² It has been shown by Smith et al. that an increased number of correlations within human leukocyte antigen (HLA) matching, between the donor and the recipient, reduced the magnitude of the immune response greatly.² However, finding the ideal match of HLA among donors for a given recipient may prove to be difficult as antigens can vary among different subjects.¹ To combat this many immunosuppressant agents such as corticosteroids, azathioprine, mycophenolate, cyclosporine, tacrolimus, antilymphocyte sera and rapamycin have been used widely in the treatment of transplant patients.³ However, long term use of calcineurin inhibitors and these immunosuppressants showed threatening side effects, one of which is nephrotoxicity.³ Similarly, in laboratory research, it is very crucial to obtain vessels of pristine condition to conduct experiments on. However, this may not always be the case as desired material or quality may not be acquirable at a given time. When faced with such insufficiencies, researchers have found the solution in creating the desired material from artificial 3D constructs. In this paper, the historical path of bio-printers, their fundamental working principles, the types of biomaterial they use, their limitations and possible improvements to the techniques have been discussed and summarized.

B. HISTORICAL DEVELOPMENT OF BIO-PRINTERS

To combat the issues of tissue individualization, and direct adaptation of tissue and organs, the first bioprinters were introduced in 1983 by Charles Hull who was the researcher to patent the stereolithographic method of 3D printing.⁴ After Charles Hull, Emmanuel Sachs was the first researcher to ever use a 3D printer with materials such as plastic, ceramic and metal.⁵ Following these improvements, in 1996, the first ever biomaterial was used in a printer to assist in tissue regeneration.⁴ It was considered to be a breakthrough when in 2001, the first bladder constructs and cell seedings were generated through the scaffold based printing technology.⁴ In the upcoming years of 2002 and 2003, researchers came up with a new method of bioprinting which was capable of characterizing cells through their viability.⁴ It was at this time that researcher Thomas Boland came up with the method of inkjet printing and elevated the pre-existing printing technology significantly.⁶ Using this newly acquired technology, professionals were able to synthesize tissue directly without any scaffold intermediates.⁷ The bio-printing technology was constantly worked and developed to points at which laboratories can print scaffold-free vascular constructs, skin, hepatocytes, articular cartilage, liver tissue, pancreas tissue and functional heart valves.⁷ Bioprinters were also tested to print and construct stem cells. However, as Ong et al. describes in their perspectives, a completely successful method as to the printing of mesenchymal stem cells and their seeding has not yet been established.⁸ The use of bio-printers in stem cell research is still experimented on as to the technique that is to be applied and the type of required biomaterial to carry out the printing process.⁸

C. BASIC PRINCIPLES OF BIOPRINTERS AND INTRODUCTION TO VARIOUS TECHNIQUES

The working principle of bioprinters is quite similar to that of the conventional 3D printers. Conventional 3D printing and Bioprinting resemble each other in two key concepts which concern the type of materials used in the application of the technique and the building phase of the product. Bioprinting is an additive manufacturing process where biomaterials such as cells and growth factors are combined to create tissue-like structures that imitate natural tissues or organs.⁹ This method forms organs and tissues in a layer-wise fashion by using a distinct reagent named “bio-ink” and combining it with the pre-existing 3D printing technology. Bio-ink can be described as filaments of bioprinters.¹⁰ Just as some objects are created by filament layers in the 3D technique, bio-inks are the main structures of layers that will turn into biologic structures such as tissues. It can be composed of just cells, but in most cases, an additional carrier material that envelops the cells is also added. Bioprinters’ working process can be divided into 3 key steps: Pre-bioprinting, bioprinting, and post bioprinting.¹¹

Pre-bioprinting, which is the initial step of the procedure, includes the composition of a draft which has to be analyzed by the printer before any complex structures are synthesized. This process involves developing a digital file which is mostly generated through computed tomography (CT) and magnetic resonance imaging (MRI) scans that the printer can recognize.¹² Through this document, the printer analyzes the complex and obtains a biopsy of the structure and uses these data to decide on the type of raw materials which are to be used in the synthesis. After an image is created, cells mixed with bio-ink are incubated and the number of cells is checked to conclude whether it is enough for the whole process or not.

To initiate the printing process, the previously prepared mixture of cells and bio-ink are placed in a printer cartridge and deposited using the patients’ medical scans.¹³ Then the bioprinter starts to create structures by applying strands of material on the provided schematic. Developing different types of tissue requires researchers to use different types of cells, bio-ink, and methodologies. Most preferred techniques are inkjet-based printing, which is a non-contact process that the image reconstruction is based on creating and precisely positioning picolitre volume of “bio-ink” on a substrate under computer control ,laser-assisted bioprinting, which uses laser-induced forward transfer (LIFT) effect to print different living cells and biomaterials with precision and micrometer resolution, magnetic bioprinting which functions through the basic principle of magnetic levitation, and extrusion-based bioprinting which shines through as the most popular approach in research and commercial fields and aims to dispense low viscous bio-inks that contain air bubbles.¹⁴

In post-bioprinting, the final products are crosslinked to create a stable end-structure. Crosslinking is usually done by treating the construct with either ionic solutions or UV light — the constructs composition helps researchers determine what kind of crosslinking to use.¹⁵ Once the process of crosslinking is brought to an end, these organs or tissues are put in an incubator for cultivation. After the completion of their full development, the ultimate products can be used in their respective fields.

D. INTRODUCTION TO TRENDS IN BIO-MATERIALS

Materials used in bioprinters differ in a very large scale from types of collagens to artificial silks.¹⁶ These reagents are collectively named as bioink and the bioprinters may require different types of bioink depending on the composition of the extracellular matrix.

Collagen is one of the main components of the extracellular matrix and is obtained from natural biomaterials.¹⁶ Appearing in all connective tissues and extracellular matrix, collagen makes approximately 25% of the dry weight of the human body and it is used as a bioink constituent in 3D bioprinting either by itself or in combination with other groups with regard to its excellent bio-compatible properties.²¹ “ Because of both their abundance and wide range of use, collagens are the primary material of choice for bioink.¹⁶ The main challenges in using collagen as bioink for extrusion printing are long gelation time, swelling, and limited printability.

Collagen at 3 mg/ml concentration cannot be printed in free standing threads due to its decreased stiffness. Additionally, when printed with a nozzle of small diameter (30 mm), a strong variability in the line dimensions was found with changing printing parameters, such as nozzle speed, humidity and stage heat.²¹ For these reasons other bioinks constructed with different chemicals and/or processes may be used. These types of bioinks include Hyaluronic acid-based, fibrin-based or silk-based reagents.

Hyaluronic acid (HA) is a natural extracellular matrix which is abundantly seen in cartilages and connective tissues. Hyaluronic acid-based bioinks are the most promising bioinks among the listed inks as it has proven itself to be a very prominent biomaterial in printing and developing 3D biological structures. Ouyang et al. reported a HA-based 3D printed construct that was synthesized by using a secondary crosslinking methodology. They demonstrated the capability of HA-based dual crosslinked bioinks for 3D bioprinting, where it displayed no loss in mechanical properties after printing as well as revealed good cellular adhesion properties.¹⁶ ’ ¹⁷

There are also researches focused on optimizing Fibrin-based bioinks. Fibrin is a type of protein which is active within the bloodstream and allows for blood clots. Enzymatic treatment of thrombin allows for the synthesis of a fibrin hydrogel, from fibrinogen, which shows remarkable biocompatibility and biodegradation capabilities but lacks strong mechanical foundations. “Fibrin is a protein which is seen in the blood and helps in clotting. Fibrin hydrogel can be made from fibrinogen by enzymatic treatment of thrombin. This hydrogel has excellent biocompatibility and biodegradation properties, but it has weak mechanical properties.”¹⁶ These weak mechanical properties are not limited to Fibrin-based bioinks. The dense movements of the hydrogel restrain the cellular network along with the functional integration of the synthesized scaffold. Functionality of a moderately sized scaffold is highly affected through vascularization and it is not yet possible to provide such an infrastructure with the current 3D printing technology. However, it is still discussed to use sacrificial reagents during the construction of the scaffold to provide mechanical strength. These sacrificial compounds fill empty spaces and provide mechanical support to the printing reagents throughout the procedure. These compounds are later removed from the end-product through post-processing techniques. Carbohydrate glass, pluronic glass and gelatin microparticles are the most popular sacrificial compounds that are currently experimented on as to their applicability. “Dense hydrogel environments limit the cellular network and functional integration of the constructed scaffold. For any moderate sized biological scaffold to be functional, vascularization is of utmost importance, and is not possible with the current 3D printing technology. To address this problem, incorporation of sacrificial materials during the scaffold fabrication has been used by many researchers. These materials fill up the void spaces, providing mechanical support to the printing materials, and once constructs are fabricated, they are removed by post-processing methods. Many sacrificial/fugitive materials including carbohydrate glass, pluronic glass, and gelatin microparticles are currently under investigation.” ¹⁷ Zhang et al. reported to have used fibrin hydrogels along with PCL/PLCL to render 3D models of urethra and seeded a number of cell variations to investigate the in vitro effects of the said reagent.

In vitro characterizations and ability of the bioink to support nerve regeneration were also investigated by England et al. who have tried to apply fibrin with HA hydrogels to accomplish encapsulation of cells.¹⁶ With these ongoing researches, Fibrin-based bioinks were successfully applied to print skin, heart, and neural constructs. Particularly, Cubo et al. managed to fabricate a bioprinted skin substitute that resembled native skin in vivo, from plasma-derived fibrin, primary fibroblasts and keratinocytes.¹⁸

While aiming to reduce costs and make bioprinting more practical, silk-based bioinks gained popularity. The reason as to why silk drew the researchers’ attention is that by simply controlling the shear force, such as applying pneumatic pressure, it is possible to induce sol-to-gel transition by changing the secondary conformations and allowing random coils of silk fibroin polymers to undergo β-sheet crystallization; hence eluding the requirement of high temperature or toxic organic solvents into the material. This feature of silk fibroin has encouraged several scientists worldwide to explore the applications of silk in 3D bioprinting.¹⁹ Silk fibroin is a natural protein obtained from silkworm and the silk-based scaffolds are more frequently used in regenerative medicine and tissue engineering. Using silk-gelatin based bioink is noted to enhance the biocompatibility of the final compound and the silk fibroin protein with alginate bioink that contain gelatin-silk based ink and free-standing silk-based bioinks consisting of PEG in their composition have been reported by various researchers to show excellent printability with high resolution in when used in Inkjet printing techniques.

Recently, spider silk is also getting more attention because of its mechanical properties. In a related work, DeSimone et al. used recombinant spider silk proteins in developing 3D printing bioinks. The spider silk protein was thermally gelled along with mouse fibroblast cell lines. Even though printed constructs showed less cell viability in spider silk protein based bioinks, when it was combined with gelatin, the results were promising.

Hence, to further improve and enhance the cell viability properties, addition of biocompatible materials in silk are discussed to increase the quality of the printed materials.²¹ Also a research group in the Department of Biomedical Engineering at Tufts University reported that they were able to demonstrate in vitro that the material was stable under physiological conditions and could be adjusted to compensate for the soft tissue mechanical properties. They were also able to show in vivo that the material was biocompatible and could be altered to maintain shape and volume up to three months while promoting cellular infiltration and tissue integration.²⁰ They also mention that to meet this need they have developed various silk based bioinks by using gelatin as a bulking agent and glycerol as a non-toxic additive to induce physical crosslinking. They have concocted these inks to optimize printing efficacy and resolution for patient specific geometries that are to be used for soft tissue reconstruction protocols.²⁰

E. CRITIQUE OF TECHNICAL LIMITATIONS AND POSSIBLE IMPROVEMENTS

Although various developments and breakthroughs were achieved in the last twenty years, the field of bioprinting is still in search of new approaches that are capable of optimizing different aspects of the procedure to yield better quality compounds and negate any possible risks. These reconceptions mainly aim to minimize cell damage throughout their cultivation and improve the tissue manufacturing process.²²

In essence, lowering the number of cell devastations is one of the most effective ways to improve the quality of the process, as damaged cells might cause grievous problems in printed tissues or organs such as histo-incompatibility and necrosis, which are serious problems that necessitate immediate action and utmost attention. To counter such outcomes, materials used in bioprinting should be well organized. These materials should support cell metabolism and provide a similar environment to that which is found in vivo. The biomaterials used should display excellent biocompatibility and tunable mechanical strength to achieve an intended and fully functional result.²³ Moreover, printing sensibility could be enhanced through mechanical implementations. In 3D bioprinting, a long-term goal is to achieve controlled single cell deposition. Especially for complex multi-cellular organs, single-cell control can simulate the structure of the human body and allow it closely mimic human organs. Unfortunately, existing bioprinting technologies cannot yet achieve single-cell control. Improving the accuracy of the printers, coupled with a reasonable control algorithm, is an important way to enhance the function of the fabricated tissues and organs.

Other attempts aim to increase the quality of the fabricated tissue. Recent researches are directed on the enhancement of sensors, omittance of scaffolds during printing, and robotic solutions which can improve the outcome of the general process greatly.

Sensors require to be developed in terms of data collection and analyzing abilities. Sensors could be integrated within the bioprinters for enhanced data gathering and analysis. During the actual printing process, it would be of real value to gauge real-time measurements of parameters such as the temperature of the printing chamber, humidity within the printing chamber, pressure in the extruder, speed, and fidelity of the printing process, etc.²⁴ As the bioprinting process usually takes hours to complete, sensors and other regulatory components could prove to be beneficial in maintaining and controlling the environment in which the process takes place.

The notion of scaffold-free tissue engineering, which utilizes the self-organization and self-assembly based approaches, has gained massive attraction. Because of their wide range of applications and the general increase in productivity that they provide, spheroid printing technologies are expected to be the future of tissue engineering.²⁵

Finally, particular robotic systems like BioAssemblyBot aim to negate any technical errors of bioprinters that support the use of vertical bioprinting technologies.²⁶ Unlike traditional 3D bioprinters, BioAssemblyBot utilizes a freely moving robotic arm to print in multiple axes and perform additional tasks which include bioprinting, object placing, pipetting, calibrating, injecting, and provide further assistance in the processes of tissue cultivation, modeling, and soft device fabrication.²⁷

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