3D Bioprinting in Disease Prevention & Treatment

Biological 3D printing is a technology based on the idea of additive manufacturing, which uses living cells, extracellular matrix, biological factors and biological materials as raw materials to produce biological products with life or inlife. Compared with 3D printing of metals, ceramics, plastics and other materials, the biggest difference in 3D bioprinting is the processing of living materials (such as cells and other biofunctional components) and the creation of living products

In order to deal with new pathogens and infectious diseases and accelerate drug discovery, people use advanced technologies to develop new therapeutic drugs and find effective treatment methods. 3D bioprinting is one of them. It is a versatile tool that not only enables the construction of highly biomimetic in vitro models of infectious diseases, but also offers special advantages in the preparation of vaccines, drugs, and related delivery systems.

In May 2021, an article titled "Application of 3D bioprinting in the prevention and the therapy for human diseases" was published in journal of Signal Transduction and Targeted Therapy, which is a division of Nature. Learn more about the materials and technology of 3D bioprinting and its promising applications in disease treatment from this article.

3D Bioprinting

3D Bioprinting Materials

In 3D bioprinting, the research of materials focuses on whether they have good mechanical properties, how to improve the applicability and viscosity of materials, achieve rapid crosslinking, and how to better simulate natural tissue structure to provide geometric support for 3D structures. Also, how to avoid cell damage during the printing process.

1. Bioink for 3D Bioprinting

Bioinks must be versatile under a wide range of printing conditions without clogging, ensuring a stable 3D structure and a consistent shape from batch to batch. Commonly used bioink materials include polycaprolactone (PCL), polydimethylsiloxane (PDMS) and their derivatives. This type of material has good biocompatibility, has little impact on cells, and can provide good physical support for in vitro models. These characteristics temporarily or permanently support living cells to facilitate their adhesion, proliferation and differentiation during maturation.

2. Hydrogel Bioink

Hydrogels have good adjustability, biodegradability and bioactivity. Generally, hydrogels used in bioinks have the following characteristics: moderate fluidity, fast curing, and sufficient integrity after molding. During the sol-gel transition, fibers in solution can be physically or chemically cross-linked by external stimuli such as temperature, light source, or ion concentration, and the cross-linking occurs through covalent bonds without cytotoxicity during the cross-linking process.

Hydrogels of natural origin have been widely used as bioinks, among them: alginate, collagen, gelatin, cellulose, silk fibroin, and Decellularized extracellular matrix (dECM).

3D Bioprinting Technology

Like other 3D printing technologies, 3D bioprinting is also divided into three steps, which are design model, printing model and test model. The three most critical elements in 3D bioprinting are: bioink, printing technology, and tissue reconstruction or organ functionalization in vitro. With the advancement and maturity of technology, in vitro models, surgical guides, bone/cartilage, teeth, trachea, blood vessels, eyeballs, sweat glands, and even liver, kidney and heart units have been successfully printed in the biomedical field.

The technical conditions of 3D bioprinting are relatively harsh and there are many influencing factors. The main printing technologies are divided into three categories: inkjet bioprinting, micro-extrusion bioprinting and laser-assisted bioprinting. In addition, there are some more advanced ones, such as suspension printing, coaxial printing and projection printing.

3D Bioprinting Technology

1. Inkjet Bioprinting

Inkjet printers are the most common type of printer used in abiotic and biological applications. It is derived from the inkjet printing technology of the office and is also the first bioprinting. The first attempt at bioprinting utilized a commercial 2D inkjet printer modified to print bioinks in layers. Inkjet bioprinting uses electrical heating to generate air pulses, or pulses through piezoelectric, ultrasonic, etc., and then forms droplets at the nozzle. The advantages of this method are high speed, low cost, wide range of applications, and adjustable cell and material concentrations.

However, the risk of exposure of cells and materials to thermal and mechanical stress, low droplet orientation, non-uniform droplet size, frequent nozzle clogging, and unreliable cell encapsulation are all disadvantages of inkjet printers.

Also, a common disadvantage is that the biomaterial must be liquid in order to form water droplets. Therefore, the printed liquid must form a three-dimensional structure with structural organization and function. Another limitation of using inkjet bioprinting technology is the difficulty in achieving biologically relevant cell densities. Typically, low cell concentrations (less than 10 million cells/mL) are used to promote droplet formation, avoid nozzle clogging and reduce shear stress. Higher cell concentrations may also inhibit some hydrogel cross-linking mechanisms.

However, inkjet printing technology has great potential in the regeneration of human functional structures, and is currently mainly used for in situ regeneration of skin and cartilage.

2. Laser Assisted Bioprinting

Laser-assisted bioprinters work by using focused laser pulses to transfer material from a supporting "target" to a "receiving" substrate. An aqueous solution of bioink (cells or other bioactive ingredient) is spread on top of the absorbing layer, and a laser beam is focused to the interface of the target substrate and the absorbing metal. Once pulsed, the laser causes thermal volatilization of picoliter volumes of bioink near the interface, creating microbubbles. The creation and expansion of such microbubbles results in the ejection of microliter volume droplets of the bioink towards the receiving substrate. By using a computer-controlled translation stage, droplets can be deposited onto a receiving substrate at specific locations where they land. The entire process does not result in loss of viability or DNA damage in bacteria or mammalian cells.

Laser Assisted Bioprinting

The advantage of this technology is that the nozzle is open, and the absence of traditional printing nozzles means that bio-inks with higher viscosity can be printed. Bio-inks with different material properties or cell components can be easily changed. Lasers are used to drive the deposition of microliter sized droplets for very high precision printing. The disadvantage is the complexity of the system and operation. It must be set in a sterile environment to ensure the sterility of the final product. The price is high and the volume of deposited material is limited. Over time, the overall volume deposition is poor and the receiving substrate environment needs to be maintained.

Currently, the most notable applications of laser-assisted bioprinting are skin and bone. Its high-resolution patterns have been used to print multi-layered skin structures, including keratinocytes and fibroblasts, as a therapeutic approach in the treatment of burns.

3. Extrusion Bioprinting

Extrusion bioprinting is one of the most widely used 3D bioprinting technologies. The material in the sample pool can be extruded continuously by mechanical force, and then the three-dimensional spatial structure can be obtained by controlling in the X, Y and Z axes. The method is applicable to a wide range of material viscosities, high viscosity materials are usually used for support structures, and low viscosity materials are often used to provide an extracellular environment to maintain cell viability.

extrusion-based-bioprinting

Extrusion bioprinting builds structures by extruding bioinks to form continuous fibers, which are divided into three types: pneumatic extrusion, piston extrusion, and screw extrusion. The resolution of this printing method is not high, about 200μm. The main advantage of extrusion bioprinting is that it can deposit high cell density, which is beneficial to meet the needs of tissue engineering. The disadvantages are slow fabrication, lower cell viability, and limited resolution. Although this technology takes a long time to print high-resolution complex structures, it can print more types of products. Currently, this technique has been used to create a variety of tissues and models, including aortic valves, in vitro drug metabolism, and tumor models.

Alternatively, there is a FRESH printing technology (or, free-form reversible embedding of suspended hydrogels), which distributes low-viscosity bioinks into a bath of particle gels, and 3D printing and crosslinking are done simultaneously, accelerating ink deposition. FRESH printing has a higher resolution and can use any soft gel biomaterial for bioprinting. It is currently the preferred bioprinting method for tissue engineers and is widely used in the construction of complex geometric tissues such as muscles, hearts, and blood vessels.

3D Bioprinted Vaccines, Therapeutics and Delivery Systems

The high flexibility and versatility of 3D bioprinting offers advantages for the efficient production of vaccines, therapeutics, and related delivery systems.

3D Bioprinting System

1. RNA printing

CureVac, a pioneer in mRNA printing, developed "RNA printing" for a rabies vaccine. mRNA vaccines are currently being developed for: yellow fever, Lassa fever, MERS, and COVID-19. The latest news shows that the company is cooperating with Tesla to establish a new company for the "RNA printing" business.

2. Drug printing

When 3D printing technology was first introduced into the pharmaceutical field, it was hoped to improve medication compliance by controlling drug release, reducing dosing frequency, and increasing dosing dose.

Aprecia Pharmaceuticals' anti-epileptic drug Spritam (levetiracetam) was the first 3D printed pill, approved by the FDA in 2015. The content of active ingredients in tablets prepared by 3D printing can reach 1000 mg, which is 5 times that of ordinary tablets (200 mg). Moreover, the tablet is easy for epilepsy patients to take, disintegrates in a few seconds in an aqueous solution, and releases it stably in the body.

Others have reported customizable 3D-printed tablets that can meet the diverse needs of patients and achieve more complex releases. There are three types of custom tablets: with surface-erodable polymers, without surface-erodable polymers, and non-permeable polymers with protective coatings. 3D printing designs drug chambers of various shapes, using different surface polymers to achieve constant speed, controlled release, and sustained release. It can be seen that in the manufacture of complex pills, 3D printing technology is expected to become a cheap and efficient method of custom drug preparation.

3. Cell/drug delivery system

In personalized medicine, 3D printing technology can quickly build specific-shaped stents according to the diseased part.

Aldrich et al. designed a 3D composite scaffold with antimicrobial efficacy for the treatment of post-craniotomy infection. They used 3D printing to construct polycaprolactone (PCL)/hydroxyapatite hydrogel composite scaffolds that encapsulated antibiotics and macrophages. Then, this composite scaffold was implanted into a S. aureus-infected calvarial defect model, and it was observed that the antibacterial activity of the cells was enhanced and the bacteria were transformed into metabolically sensitive ones.

4. Drug sustained release system

Yi et al. used extrusion printing technology to develop a polymeric patch, which is a mixture of PCL, PLGA and 5-fluorouracil, and its shape can be constructed according to different administration sites. In a rabbit model, they observed a sustained release of the drug for 4 weeks and significant shrinkage of pancreatic tumors.

5. Pulse drug delivery system (PDDS)

In 2017, McHugh et al. introduced a single-injection platform for pulse-release vaccines.

They used 3D printing technology to encapsulate ovalbumin in PLGA microcapsules, inject them subcutaneously into mice, and adjust the release curve and degradation rate by controlling the ratio of ethylene and lactide. The pulsatile drug delivery system contains polymer particles with various degradation rates in a single dose, and the release profile is consistent with traditional vaccination.

6. Aerosol delivery system

Aran et al. developed a system called MucJet system for oral vaccination, where they used a biocompatible and water-resistant photopolymeric plastic resin for 3D printing to deliver fluorescein-labeled ovalbumin to the buccal mucosa via a high-pressure liquid jet.

The MucJet system consists of two compartments, an inner and an outer, which contain propellant and vaccine, respectively. As the polymeric membrane of the MucJet system dissolves, the propellant in the outer compartment produces CO2 gas, which increases pressure and pushes the piston in the inner compartment containing the vaccine, ejecting the vaccine. In vitro studies showed that the delivery efficiency of MucJet was improved by nearly eight times, and in vivo studies observed that the immunogenicity of the antigen was enhanced by three orders of magnitude.

Conclusion

The combination of 3D printing technology and biomedicine can not only construct primary organs and in vitro models, but also prepare various complex structures according to the needs of vaccines, drugs and delivery systems. With the development of artificial intelligence, intelligent 3D bioprinting technology is already under research. It is believed that in the near future, human beings will use more efficient means to find more personalized treatment methods and obtain more and better medical products.

PEG based hydrogels are most used polymers in 3D bioprinting technologies for their good biocompatibility in both in vitro and in vivo conditions. As a lead PEG supplier, Biopharma PEG provides some PEG derivatives for 3D printing, such as AC-PEG-AC, AC-PEG-RGD and 8-ArmPEG-AC.

Reference:
[1]. Application of 3D bioprinting in the prevention and the therapy for human diseases. Signal Transduction and Targeted Therapy. Volume6, Article number: 177 (2021).