Learn about 3D bioprinting in disease prevention and treatment from 3D bioprinting materials, 3D bioprinting technology and 3D bioprinted vaccines, therapeutics and delivery systems.

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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.

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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.

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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.

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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.

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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.
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

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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.

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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.

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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

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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.

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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

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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).
Related articles:
[1]. Strategies Of Oral Drug Delivery: From Prodrug, Nanoparticles to 3D Printing
[2]. Polyethylene Glycol (PEG) Hydrogel Based 3D Bioprinting
[3]. The Role of PEGylated Materials In 3D Bioprinting