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Running head: A REVIEW OF 3D BIOPRINTING TECHNOLOGY, MATERIALS,

CHALLENGES, AND FUTURE OUTLOOKS

A Review of 3D Bioprinting Technology,

Materials, Challenges, and Future Outlooks
Kristine Suritis

Gifted and Talented Mentorship

April 24, 2020

Advisor: Nathan Boggs

James Anthony

Instructor: E. Leila Chawkat

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A REVIEW OF 3D BIOPRINTING TECHNOLOGY, MATERIALS, CHALLENGES, AND
FUTURE OUTLOOKS

Abstract
This paper discusses the principles of regenerative medicine and its applications to tissue
engineering. This analysis describes the requirements for a successful bioink, elementary technologies
used in three-dimensional (3D) bioprinting, commonly used crosslinking methods, and various materials
used in the formulation of bioinks. The review then discusses an emerging technology, four-dimensional
(4D) bioprinting which incorporates a fourth dimension, change over time in response to external stimuli,
in the construction of a biological scaffold. Various external stimuli are mentioned and possible cellular
and construct responses explained. Finally, the review describes current challenges encountered in
bioprinting and addresses future prospects of such technology.
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A REVIEW OF 3D BIOPRINTING TECHNOLOGY, MATERIALS, CHALLENGES, AND
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Table of Contents

Title Page…………………………………………………………………………………………... 1

Abstract……………………………………………………………………………………………. 2

Table of Contents……………………………………………………………………………………

Introduction………………………………………………………………………………………... 4

Literature Review………………………………………………………………………….............. 5

Conclusion………………………………………………………………………………………...

References………………………………………………………………………………………...
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Introduction
3D printing is an additive manufacturing process that creates an object through successive layering

of material (“What is 3D printing?”). 3D printing stands to have a wide variety of applications including

construction, industrial manufacturing, prototyping, education, and healthcare. In the healthcare field, 3D

bioprinting is a technology which stands to revolutionize tissue engineering and regenerative medicine.

Regenerative medicine and tissue engineering are broad terms which refer to fields whose goal it is to

research and utilize the body’s own healing processes to restore function to damaged tissue (“What are

tissue engineering and regenerative medicine”). 3D bioprinting involves the deposition of biological inks

containing live cells into a predetermined structure to create functional biological tissues or organs

(Gopinathan et al., 2018). The structure of the printed construct is determined in the creation of a

computer aided design (CAD) which is then exported to and read by a 3D printer. There are numerous

printing methods including extrusion bioprinting, inkjet bioprinting, stereolithography, and laser assisted

bioprinting. Along with various printing methods, the bioinks that are used to print are composed of a

variety of diverse materials including agarose, alginate, collagen, fibrin, polymers, and silk.
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Literature Review

Bioink Requirements

Throughout the 3D bioprinting process the encapsulated cells must remain in conditions that allow

for their survival and proliferation and continued biological activity post printing. During the

bioprinting process the bioink and cells are exposed to mechanical stress which can cause cell

death and lower cell viability. In order to accomodate living cells within the bioink all components

must be biocompatible and nontoxic. Printability of bioink largely relies on the viscosity of the

solution and the printing parameters. The cells in the 3D printed construct should retain their shape

even after printing. The construct should support and promote cell attachment, growth, and

proliferation while allowing for oxygen permeability and the development of a vascular system to

transport nutrients and metabolic waste (Gopinathan et al., 2018; Mandrycky et al., 2016).

Bioprinting Methods

There are several 3D printing methods that can be utilized to develop a construct including

extrusion bioprinting, inkjet bioprinting, thermal transfer, stereolithography, and laser-assisted

(Collins et al., 2014; Mandrycky et al., 2016).

Extrusion Based

Extrusion based bioprinting uses a pneumatic pump or mechanical screw to force the bioink

through a nozzle in a steady stream onto the substrate below. One of the benefits of extrusion

bioprinting is that through the application of steady pressure a diverse range of materials and

viscosities can be used. However, extrusion bioprinting can expose cells to a greater amount of

mechanical stress which can result in harm to the cells within the construct (Gopinathan et al.,

2018; Huang et al., 2017; Mandrycky et al., 2016).

Inkjet Bioprinting
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Inkjet bioprinting, unlike extrusion bioprinting, dispenses bioinks in droplets as opposed to a

steady cylindrical stream. Inkjets printers have a similar structure to commercially available

printers and are a relatively low cost option. They also have a high print speed and have the

potential for multiple print heads working simultaneously while maintaining high cell viability

(appr. 80% - 90%). However, the bioinks used must have a lower viscosity in order to form the

droplets needed to print (Gopinathan et al., 2018; Mandrycky et al., 2016).

Alternative Methods

There are other bioprinting methods such as stereolithography where light is used to

“selectively solidify a bioink in a layer-by-layer process that additively builds up objects.”

Laser assisted bioprinting utilizes a laser to precisely create droplets from a donor layer which

then land on a substrate below and create a construct in a layer by layer additive process

(Mandrycky et al., 2016).

Figure 1: The process of bioprinting, illustrates several printing methods listed and described above
(Mandrycky et al., 2016)

Crosslinking methods
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To improve the structural retention of bioinks after printing they can be physically or

chemically crosslinked. Crosslinking methods vary by material used and need to accommodate

differences in material viscosity and cell density (Mandrycky et al., 2016; Stanton et al., 2015).

Bioink Materials

Agarose

Agarose, derived from seaweed, is a widely used biopolymer in the biomedical field due to its

excellent gel formation properties which can have diverse applications. A potential drawback

to the use of agarose is its limited ability to support cell growth (Gopinathan et al., 2018).

Alginate

Alginate, derived from brown algae, is an inexpensive biopolymer. The viscosity of an alginate

bioink can be controlled by changing the concentration of alginate; a higher concentration

increases viscosity and printing resolution while a lower concentration makes the bioink less

viscous and decreases print resolution. In addition to a controlled viscosity, alginate is highly

biocompatible, does not evoke an inflammatory response, and can use capillary forces to trap

water and other molecules in the construct while still allowing for diffusion from the interior

out. However, printed constructs do not support high levels of cell adhesion or proliferation

without the addition of supplemental agents. The construct should be exposed to a crosslinking

agent (such as CaCl2 solution) to improve structural stability (Aljohani et al., 2018;

Gopinathan et al., 2018; Mandrycky et al., 2016; Panwar et al., 2016).

Collagen

Collagen is an abundant protein that can be found in bones, muscles, skin, and tendons. A

benefit of collagen is that during the printing process it can better protect cells from harm. On

the other hand, one of the drawbacks to using pure collagen is its low viscosity making it
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difficult to work with. However in combination with other natural or synthetic materials it can

increase printability and benefit the mechanical properties of construct (Mandrycky et al.,

2016; Ozbolat et al., 2016).

Fibrin

The combining of fibrinogen and thrombin creates fibrin which is used in tissue engineering

for its cell adhesion properties and easy crosslinking. Fibrin is a good bioink because its

properties can be easily modified by altering component proportionality (Mandrycky et al.,

2016).

Silk

Silk proteins have been used in bioinks for their non-toxicity, slow degradation rate,

polymorphic features, good mechanical properties, aqueous solubility, and ability to protect

cells during printing (Mandrycky et al., 2016; Tao et al., 2015).

Prokaryotes v. Eukaryotes

To create a cell laden construct the bioink that is used needs to contain cells. A hydrogel is a

polymeric network that is designed to contain cells for 3D bioprinting, once live cells are

added to a hydrogel it is then considered a bioink. The cells that are added to the hydrogel can

be either prokaryotes (bacteria) or eukaryotes (animal and plant cells). One of the key

differences between the two is that prokaryotic cells are far more adaptive and have “diverse

metabolic activity” and can therefore live in virtually whatever ecological niche they are

exposed to. The prokaryotic cells are “much more hearty and not as easy to kill as mammalian

cells” which would allow them to sustain higher mechanical stresses during printing while

retaining high cell viability. Despite these factors, the majority of 3D bioprinting research has

been conducted with eukaryotic cells (Lehner et al., 2017; Schaffner et al., 2017).

4D Bioprinting and External Stimuli

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Based off of 3D bioprinting discussed earlier in this review, 4D bioprinting utilizes 3D bioprinted

structures while adding a fourth dimension—time. Prints are exposed to external stimuli triggering

a response in the construct changing its shape or behavior to mimic functionality and structure of

tissues. External stimuli could include: humidity, temperature, electric field, magnetic field, light,

pH, and acoustics (Ashammakhi et al., 2019; Gao et al., 2016; Li et al., 2016; Yang et al., 2019).

Humidity

In response to humidity, the construct should swell or shrink changing its size and structure.

The development of an agarose-PCAD composite material and a cellulose based material

allows for quick actuation in response to humidity change. This is possible because of the

combination of materials some with “rapid absorption of water but low desorption” and some

with lower absorption but faster desorption (Ashammakhi et al., 2019; Li et al., 2016)

Temperature

When exposed to significant temperature changes the construct can expand, contract, or fold.

An advantage of using thermo-responsive hydrogels is that they are biocompatible. However,

some thermoresponsive materials are unable to support cell growth and the high temperature

changes needed to result in a significant change in construct shape or behavior often lead to

lower cell viability and diminished function (Gao et al., 2016; Li et al., 2016).

Electric Field

The construct should be able to expand, constract, fold, or erode when an electrical stimulus of

varying intensity and direction is applied. Some materials that can be used to respond to

electrical stimuli are several types of carbon-based nanomaterials and hydrogels which can be

combined with conductive polymers to allow for the effective distribution of electrical stimuli

throughout the construct (Ashammakhi et al., 2019; Li et al., 2016).

Magnetic Field
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The addition of magnetic nanoparticles to a construct can allow for effective response to

magnetic field stimulus. The development of an alginate-based construct with interconnected

pores possessing magnetic stimulus responsive capabilities allowed for the rapid activation and

compression of the pores to allow for the movement of water externally from internal pores

(Ashammakhi et al., 2019; Li et al., 2016).

Light

The use of light as an external stimulus can be more precise than previously mentioned

methods because it can be highly focused to only activate certain parts of the construct.

Depending on the photo-responsive material used, a variety of wavelengths of light can be

effective including UV, infrared (IR), and near-IR. IR in particular has a lower phototoxicity

and higher tissue penetration compared to UV light because of its lower absorbency rate

(Ashammakhi et al., 2019; Li et al., 2016)

pH

pH levels can also be used as an external stimuli and some pH-sensitive hydrogels “swell

significantly when in contact with body fluids” (Gao et al., 2016).

Acoustics

Acoustic waves, at varying energies, can be used as an external stimuli inducing “physical or

chemical changes in a materials, particularly at high energies”. The use of rapid and accurate

acoustic waves to change cell pattern requires no physical contact (Ashammakhi et al., 2019;

Yang et al., 2019).

Challenges Associated with Bioprinting

Despite the impressive technological developments that have allowed 3D and 4D

bioprinting to progress to their current states there are still challenges that limit the practicality and

applications of bioprinting. One of the major constraints for 3D bioprinted constructs is the
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development of a vascular network. To support cell growth a vascular network needs to be

established which transports nutrients and metabolic waste to and from each cell. In increasingly

large constructs without a proper vascular system the interior of the construct will see increased

levels of cell death. Another challenge is the formation of the bioinks. The cells used in the ink

need to be cultured and harvested and then mixed with a potentially highly viscous hydrogel to

attain a homogeneous bioink. More research has to be conducted to determine the relationship

between bioink viscosity and ideal printing parameters. As viscosity of a bioink increases so do

the shear forces and mechanical stressors during the printing process which can cause damage to

the cells. A primary challenge in 4D bioprinting is the ability to design smart materials that closely

mimic natural tissues and biological functions. Many of the external stimuli, if used too extremely,

can cause damage to the cells and construct (Castro et al., 2017; Haung et al., 2017; Mandrycky et

al., 2016).
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Conclusion

This review described the basis of 3D and 4D bioprinting in its applications to regenerative

medicine and tissue engineering. It outlined various technologies and materials used in 3D and 4D

bioprinting processes. The paper addressed both the advantages and challenges encountered with

bioprinting processes including technological and biological difficulties. With further development of 3D

and 4D bioprinting techniques the applications of this technology can benefit a wide variety of fields and

disciplines. The research can be applied to such goals as printing sample organ tissues for drug testing,

developing more reliable drug delivery systems, printing large, fully functioning organs to eliminate the

need for a transplant list, or printing numerous small organs that functionally match a traditional organ

while eliminating some of the challenges presented earlier in this review (Collins, 2014). With further

research and development 3D and 4D bioprinting will be the future of regenerative medicine.
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References
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Ashammakhi, N., Ahadian, S., Fengjie, F., Suthiwanich, K., Lorestani, F., Orive, G., . . . Khademhosseini,
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Castro, N. J., Meinert, C., Levett, P., & Hutmacher, D. W. (2017). Current developments in
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Collins, S. F. (2014). Bioprinting is Changing Regenerative Medicine Forever. STEM Cell Research
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Gao, B., Yang, Q., Zhao, X., Jin, G., Ma, Y., & Xu, F. (2016). 4D Bioprinting for Biomedical
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Huang, Y., Zhang, X.-F., Gao, G., Yonezawa, T., & Cui, X. (2017). 3D bioprinting and the current
applications in tissue engineering. Biotechnology Journal. https://doi.org/10.1002/biot.201600734

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Stanton, M. M., Samitier, J., & Sánchez, S. (2015). Bioprinting of 3D hydrogels. Lab on a Chip, (15).
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