The Use of Bioprinting in Regenerative

Tissue Engineering

Kristine Suritis
Independent Research I G/T
10 May 2019

Advisors:
Dr. Aldo Boccaccini
Dr. Liliana Liverani
Dr. Peter Torzilli
TABLE OF CONTENTS 2

Abstract 3

Introduction 3

Literature Review 4

Data Collection 10

Chart 10

Conclusion 13

References 14

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I. Abstract

This review paper discusses the elementary requirements of a successful bio scaffold

including mechanical as well as biological properties. This analysis describes the various

methods of treatment for severe bone injuries and highlights the successes and shortcomings of

each. It then describes the emerging field of bioprinting as a fabrication method of bio scaffolds

and the potential it holds while also mentioning the current obstacles. Through an analysis of

several sources a comparison is made between several types of bioprinting to determine the most

successful method. While there are challenges that face bioprinting as an emerging field, it holds

the potential to eliminate the use of bone grafts and with more development, be able to create

custom printed organs and tissues eliminating the need for a transplant list.

II. Introduction

Scaffolds, are not only meant for large scale construction purposes. They can also be used

on the cellular level to regenerate, regrow, and replace injured tissues and organs. Bioscaffolds

are artificially made structures that promote cell growth and development to replace tissue

deficits in the body. According to Azizeh-Mitra et al. (2016), “Every year, more than 1 million

surgical procedures involving the partial excision of bone, bone grafting, and fracture repair are

performed in the USA … A substantial percentage is for the elderly, the number of which is

expected to double in the next 25 years.” There are roughly four million surgeries that utilize

bone grafts to treat bone defects each year. With an increasing demand for bone grafts, supply is

not increasing at the same rate and other substitutes have to be developed (Turnbull et al., 2018).

As the elderly population increases, large bone deficits that do not heal on their own will also

increase, therefore requiring alternative tissue regenerative technologies. These methods, also

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known as tissue engineering, is the use of engineering practices in combination with cells and

biochemical factors to improve or replace biological tissues (What is Tissue Engineering?). As

new technology develops, stereolithography, a form of bioprinting, may be the most effective

way to produce bioscaffolds that have desirable mechanical properties, are biocompatible, and

biodegradable when compared to other methods, such as 3D bioprinting and traditional methods

of scaffold fabrication.

III. Literature Review

In order for bioscaffolds to be effective they must meet the host tissue characteristics as

closely as possible. There are six main characteristics that, if met, characterize a good

bioscaffold. The characteristics include biocompatibility, blood supply, mechanical properties,

pore size, bioresorbability, and printability. The first, biocompatibility, is the ability of the

scaffold to allow bone cells to attach, proliferate, and form their own matrix with interconnected

pores. The scaffold should not create any toxic conditions which inhibit the formation of new

cellular structures (Bose et al., 2012). Hydrogels are a type of bioink used in bioprinting that are

“composed of hydrophilic polymers cross linked either through covalent bonds or held together

via physical intramolecular attractions” (Bishop et al., 2017). These are used in 3D bioprinting

because they are degradable as well as offer the support needed for cells to attach without

interrupting growth or creating toxic conditions (Wang et al., 2015). The second important

characteristic is blood supply. For a scaffold to be successful, it relies on a functioning

microvascular system that can deliver oxygen and nutrients to developing cells while also

removing metabolic waste products (Bose et al., 2012). The lack of a properly functioning

microvascular system is currently found to negatively impact the effectiveness of scaffolds

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(Gerhardt et al., 2010). The third element is mechanical properties. These properties relate to the

physical forces and deformations (e.g., stresses and strains) that are exerted on the scaffold. The

bioscaffold should match the mechanical properties of the target tissue in terms of stiffness,

deformation, modulus, and ultimate strength (Wang et al., 2015). The next important bioscaffold

feature is pore size. The pores present in the scaffold allow for the proper distribution of

nutrients and oxygen to the growing cells. The minimum pore size is 100 μm but the most

effective is a pore size between 200 and 350 μm. However the more porous a scaffold becomes,

the more complex the fabrication process and the mechanical properties decrease in strength

(Bose et al., 2012). In addition, bioresorbability is a key feature in the efficiency of a scaffold.

The scaffold, once implanted, must dissolve at an ideally controllable rate once cells have started

growing and the scaffold frame is converted to the appropriate structure (Bose et al., 2012). The

final scaffold feature that applies only to 3D printing, is printability. When printing with

hydrogels, the hydrogel needs to be “suitable for printer deposition” with the proper density of

cells as well as the cells ability to survive the printing process.

An alternate method that has been used for treating major bone defects is the use of bone

grafts. A bone graft is when a piece of healthy bone is taken from a donor site and implanted into

the injured area. The healthy bone can come from a donor site on the same patient where it will

be transplanted or from a third party donor, usually a cadaver. This procedure is usually used in

cases where there are major injuries. However, this method of treatment, while rare, can cause

“infection, bleeding, blood clots[s], nerve damage, complications from anesthesia, [and]

infection from donated bone” (Bone Grafting). In additional, the procedure’s “use is severely

hampered by its short supply and the considerable donor site morbidity associated with the

harvest” (Stevens, 2008).

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 Fabrication issues for traditional bioscaffolds include the inability to “precisely [control]

pore size, pore geometry, spatial distribution of pores and construction of internal channels

within the scaffold” (Sachlos et al., 2003). In addition, cells can only survive close to the surface

of the scaffold due to ineffective delivery of nutrients and oxygen and removal of metabolic

waste products deeper within the scaffold (Sachlos et al., 2003).

Materials that have been used in the fabrication of traditional bioscaffolds include

“bioactive ceramics, bioactive glasses, biological or synthetic polymers, and composites of

these” (Stevens, 2008). However the “brittle nature of bioactive inorganic materials” results in a

lower fracture toughness that does not match that of host tissue and cannot be used on its own in

“load bearing applications” (Stevens, 2008). When a glass-ceramic composite is used the

“porosity is reduced and the solid structure gains mechanical strength”. However, a major

setback for this material is its “limited strength and low fracture toughness (i.e., ability to resist

fracture when a crack is present)” which prevents its use on load bearing injuries (Gerhardt et al.,

2010). Another material used in scaffolds is polymer. While polymer “favors cell attachment and

promotes” cell migration, there are concerns that some types of the material will cause an

unwanted immune system response resulting in “the potential risk of disease transmission,

sourcing and poor handling, and weak mechanical properties” (Stevens, 2008). However, these

risks are not true to all polymers. The risks mentioned above are more true to the use of synthetic

polymers such as PVA, PCL, PLA, etc. whereas there are also natural polymers such as gelatin,

collagen, chitosan, and cellulose. According to Kasyanov et al. an additional limitation not only

related to polymers but to solid scaffolds in general is, “the low level of precision in cell

placement, especially when engineering multicellular constructs, an intrinsic problem with

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vascularization of thick tissue constructs, and the extremely laborious, slow and costly non

automated tissue assembly process” (2008).

One of the most prominent problems encountered with bioscaffolds is the issue of

vascularization. Without proper vascular networks there will be insufficient delivery of nutrients

and oxygen as well as removal of metabolic waste products from the developing cells. Without

steady vascularization throughout the entire scaffold, cells will not proliferate throughout the

entire scaffold, instead remaining only where the proper nutrients can be delivered (Stevens,

2008). Although technology is improving, there are still major issues in the process of

bioprinting in the ability to print a vascular network to allow tissues to be clinically relevant. The

vascular network needs to include “multiple scales ranging from arteries and veins down to

capillaries” (Ravnic et al., 2017). Zhang et al. (2018) claims that a strategy used in bioprinting to

achieve a fully vascularized scaffold can generally be achieved in three steps, “i) bioprinting of a

network of solid fibers embedded in a hydrogel matrix encapsulating stromal cells; ii) selective

removal of the fibers to form perfusable channels; and iii) seeding of endothelial cells in the

interiors of the channels to build functional vessels”.

A new emerging method of fabrication of bioscaffolds is 3D bioprinting. This method is

similar to regular 3D printing except it uses a person’s own cells. There are several types of

bioprinting including inkjet, extrusion, laser assisted, and stereolithography. Bioink is the

material used in 3D bioprinting processes. This ink is made up of cells which are “embedded in a

printable material which aids proliferation of cells by maintaining a supply of nutrients, oxygen

and growth factors (GFs). Bioinks can be in the form of hydrogels, viscous fluids or micro-

carriers” (Kalyani et al., 2017). The first method of fabrication, inkjet printing, uses small

amounts of material dispensed onto a platform, drop by drop, to fuse with other droplets to form

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several merged layers (Wang et al., 2015). The advantages of inkjet printing “in hard tissue and

organ engineering are fast, cheap, readily available, and high resolution. The deposition

resolution can be adjusted … and the printing accuracy can be tailored … There are several

disadvantages of the inkjet bioprinting technologies: (1) the starting materials need to be

dissolved into liquid states at low viscosities; (2) the heat, ultrasound, and mechanical stresses

(especially shear forces) generated during the inkjet bioprinting have adverse effects on cell

viability; (3) it is difficult to update the required hardware and software for multiple cell type

assembling; (4) limited biomaterials used for cell loading because of nozzle (or head) clogging;

(5) only low cell numbers can be printed; and (6) finite printing height” (Wang et al., 2016). The

second method is extrusion where “mechanical forces are used to extrude material” through a

nozzle in a continuous stream onto a platform (Wang et al., 2015). While this method of printing

can print with bioink with a higher viscosity, “the cell viability is significantly compromised

with the increase of extrusion pressure due to the higher shear stress applied to the cells” (Gao et

al., 2018). In addition, laser-assisted printing has a “donor layer that responds to laser

stimulation” as well as an “energy-absorbing layer” with a “layer of bioink solution suspended

on the bottom”. During the printing process, a laser pulse targets certain areas of the absorbing

layer which then vaporizes pieces of the donor layer allowing the bioink droplet to fall onto the

printing platform and link with other droplets (Mandrycky et al., 2015). The final method of

printing is stereolithography. This method uses “light to selectively solidify a bioink in a layer-

by-layer process that additively builds up objects. These printers use a digital light projector to

cure bioinks plane-by-plane” (Mandrycky et al., 2015). The stereolithography printing process

“offers greater advantages due to the precise control on deposition of biologicals and high

resolution” (Cui et al., 2017).

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(a) A flowchart of different 3D bioprinting techniques and (b) Illustrations of more commonly used 3D

bioprinting techniques (Cui et al., 2017)

However, there are some issues that apply to all bioprinting methods. One of these is the

“organ blueprint”. This means that each individually printed scaffold will likely have to be a

slightly different size and shape, therefore not allowing the computer-aided design (CAD) model

be made automatically based on patient scans. In addition when printing with cells they are

subject to change during and after the printing process due to “tissue fusion, tissue compaction,

and tissue maturation processes” (Kasyanov et al., 2008). Additionally, not all 3D printing

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methods can be used in 3D bioprinting as they are only suitable to be used with certain materials

and some printing methods do not support the use of bioinks and can only be used with more

traditional materials. However, “Currently available 3D printing technologies allow a wide range

of materials to be printed using diverse ink formulations” (Ji et al., 2017).

IV. Data Collection

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 The research design chosen for this project most closely qualifies as descriptive

qualitative research with some quantitative data. All of the sources mainly utilized words to

describe the current state of 3D bioprinting technology. Each of the four sources displayed their

findings in very similar ways, so that the data was easy to compare to the remaining sources.

Findings were presented in paragraphs organized by 3D bioprinting subtype that established a

basic understanding of the fabrication process itself, as well as statistics related to the print type

and process.

A systematic review was the most appropriate data collection method for the study. The

data required to answer the research question would require access to advanced technology and

extensive knowledge about the creation and care of bioprinting materials that is not achievable in

the situation. Based on the time constraint, creating bioink to test fabrication processes and

allowing the scaffold to proliferate while being supplied with the proper care, would be an

unreasonable expectation for this study. The journals used in the systematic review were chosen

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based on reliability of the source, currency, and similarity in data analysis methods as well as

tested variables. Currency was one of the most important factors because as the technology

further developments the statistical aspects of the data will change and outcomes will improve

overall.

Based on the data collected from the sources, stereolithography was determined to be the

most effective method of 3D bioprinting. Stereolithography is a relatively low cost method of 3D

bioprinting while also having a fast print speed and high accuracy. Compared to inkjet, extrusion

and laser assisted printing, stereolithography has the highest rates of cell viability, greater than

90% on average. Each type of 3D bioprinting has its own set of pros and cons that were more or

less consistent throughout the four sources used. Inkjet printing, while being low cost with high

cell viability and fast print speed, had only medium accuracy and the printing method does not

perform well with inks that have a high cell density or high viscosity. Another concern is that

during the printing process the cells can settle in the ink container and cause the printhead to

clog. The extrusion printing method is able to print in uninterrupted cylindrical lines, can print a

wider range of materials with higher viscosity but has a moderate cost, slow print speed, medium

accuracy, and low to moderate cell viability (40%-80%). Both the inkjet printing and extrusion

methods force the ink or hydrogel solution through a printhead which causes mechanical stresses

upon the cells and can damage them in the printing process, thus decreasing cell viability. Laser-

assisted printing has a high cost due to complex systems but also prints at a medium speed with a

high cell viability rate (>90%). One problem that may potentially be encountered with the use of

stereolithography and laser-assisted printing is the dependency on photosensitive materials. The

potential damages caused to cells by the use of ultraviolet (UV) light is relatively unknown.

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 It was surprising to discover that stereolithography has such a good combination of

features. The process is relatively unheard of or potentially referred to in a general term such as

simply 3D printing or additive manufacturing.

A large contributing factor to the argument of what fabrication method is the most

effective is purely determined based on situational application. The material chosen for the

printing process is determined by the type of tissue that will be repaired. Material choice restricts

the fabrication processes available to create the product. Another limitation is that 3D bioprinting

has not quite advanced to the point here fully printed tissues are implanted in humans for clinical

applications. Most trials focus on the various materials that can be used with a specific type of

bioprinting as opposed to using the same material and testing the result on various fabrication

methods.

Overall, the 3D bioprinting methods using UV light as opposed to force seem to be more

effective, especially for increasing cell viability and printing speed. The results of the study will

prompt further research and development of successful 3D bioprinting materials and technology.

As the technology further advances and the printed products are successful in replacing damaged

tissue in the body, the process can be applied to printing fully functioning organs from a person’s

own cells, eliminating the need of a transplant list and saving countless lives.

V. Conclusion

While bioprinting using one’s own cells to print replacement body tissues for

implantation into the body has not yet come to clinical trial in humans, the continuous

advancement of technology brings that milestone closer. According to Ji and Guvendiren (2017),

“3D printing has a strong potential to become a common fabrication technique in medicine as it

enables fabrication of modular and patient-specific scaffolds and devices, and tissue models,

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with high structural complexity and design flexibility”. While there are still major problems that

need to be addressed in the field of 3D bioprinting, such as vascularization and “organ

blueprints” (Kasyanov et al., 2008), the long term impacts of the advancement of this technology

could eliminate the need for bone grafts as well as organ transplants. As the population ages and

serious medical issues increase, the number of people awaiting transplants is likely to increase

even more than the 113,000+ number of men, women and children on the national transplant

waiting list as of January 2019 (Organ Donation Statistics). Many from these transplant lists

never make it as “recent estimates suggest that 22 people die every day waiting for an organ

transplant” (Ravnic et al., 2017). The further development of regenerative medicine and 3D

bioprinting systems will lead to an increase of the availability of personalized medicine.

Bioprinting, the future of regenerative medicine.

VI. References

Azizeh-Mitra Yousefi, Paul F. James, Rosa Akbarzadeh, Aswati Subramanian, Conor Flavin,
and Hassane Oudadesse, "Prospect of Stem Cells in Bone Tissue Engineering: A
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Bishop, E. S., Mostafa, S., Pakvasa, M., Luu, H. H., Lee, M. J., Wolf, J. M., . . . Reid, R. R.
(2017). 3-D bioprinting technologies in tissue engineering and regenerative medicine:
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Bone Grafting. (n.d.). Retrieved from Johns Hopkins medicine website:

https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/bone-grafting

Bose, S., Roy, M., & Bandyopadhyay, A. (2012). Recent advances in bone tissue engineering
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Cui, H., Nowicki, M., Fisher, J. P., & Zhang, L. G. (2017). 3D Bioprinting for ORgan
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Gao, G., Huang, Y., Schilling, A. F., Hubbell, K., & Cui, X. (2018). Organ Bioprinting: Are We
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