Ganesh Sosale

Hydrogels for Organ and Cell Printing Overview

Introduction
Organ printing is very significant in regenerative medicine because it can provide a solution for
limited donor grafts for tissue and organ repair[11]. It involves layered deposition of cells and
gels to create a 3-Dimensional structure [11]. The concept of organ printing is shown in Figure
1. Hydrogels are polymer networks used as scaffolding materials for organ printing. They can
either be natural polymers or synthetic polymers that give the cells a supportive environment to
attach to and proliferate and differentiate in [11]. Hydrogels can either be stable chemically or
they may degrade [2]. Since there are many different types of hydrogels, each with differing
properties, a hydrogel with the most favorable properties for the required application will be
used.
Traditionally, creating 3D structures involved seeding cells on pre-shaped polymer scaffolds or

Figure 2 Shows how the different types

of stimuli can affect the swelling of a
smart hydrogel. [3]

Figure 1 - Overall Concept of Organ Printing. Shows how a 3D Structure is constructed. [12]

casting a cell-seeded hydrogel into a mold. This was

found to be inefficient and led to a heterogeneous cell
distribution. [11]

Research, recently, has gone toward creating cell-seeded implants that duplicate native tissues
in all aspects: anatomical geometry, cell placement, and microenvironment of the cells. For
these implants to be successful, the properties of the scaffold material, hydrogels, need to
ensure sufficient transport of nutrients, provide enough mechanical support and incorporate
multiple cell types. [11]

Types of Hydrogels
There are many different types of hydrogels, as in, hydrogels that are stimuli-responsive. They
are referred to as Smart hydrogels. Smart hydrogels can exhibit changes in their swelling
behavior based on the type of stimuli they are sensitive to. Figure 2 shows this concept. The
main types of hydrogels are: Thermosensitive, pH sensitive, Ionic Crosslinking, Photopolymerizable and Enzymatic. Thermosensitive hydrogels are sensitive to the temperature; as
the temperature decreases, the hydrogel hardens (forms a gel).Thermoresponsive hydrogels is
the most potential one for delivery of protein and peptides [1] Hydrogels can also be natural or
synthetic. Natural hydrogels are made from natural polymers and synthetic hydrogels are
synthesized from polymerization of vinyl monomers. Hydrogels must also have a certain
durability. A durable hydrogel is one that does not degrade. A degradable hydrogel breaks
down over time or due to a certain stimuli. The idea is to make it biodegradable so that the
body can absorb it without any complications.

Figure 3 Schematic of Hydrogel Classification. [3]

Properties of Hydrogels
A few examples of Thermosensitive hydrogels are: Agarose, Collagen, Gelatin and Matrigel.
Agarose are natural polymers. They are resistant to protein absorption, low cost, and have low
physical qualities. Collagen is also a natural polymer. It contains an adhesive extracellular

Figure 4 - The methods for formation of chemical hydrogel and

physical hydrogel. [5]

matrix component. Gelatin are natural polymers as well. They are weak at physiological
temperatures. Matrigels are synthetic polymers and are commercially available, but are very
expensive. Hydrogels can be natural, synthetic or a combination of both. There are two types
of hydrogel: Physical gel and Chemical gel (Figure 4). The different types of molecular
structures are: Linear polymers, Block polymers, Graft copolymers, Interpenetrating Networks,
or Polybeads. Some important properties of hydrogels are: Whether they are degradable or
non-degradable, Injectable or pre-fabricated, mechanical strength (how much force can they
withstand before breaking), ease of handling, shape and surface volume ratio, closed pores or
open pores, water content and character, chemical modification, whether bioactive components
are added, and sterilizability. [5]

Preparation of Hydrogels
There are many different types of methods to prepare hydrogels, depending on what type of
application the hydrogel will be used for and the desired structure. There are four main
methods for hydrogel preparation: Physical crosslinking, Chemical crosslinking, Free radical
polymerization, and Irradiation crosslinking.
Physical Crosslinking

This method involves the crosslinking of polymers through physical interactions. It is one of the
most common methods for hydrogel preparation. The hydrogels that are prepared using this
method are under mild conditions.
There are three types of physical crosslinking:
Polyelectrolyte complexation (Ionic interactions), Hydrogen bonding, and Hydrophobic
association. [3]
a. Polyelectrolyte Complexation (Ionic Interactions): This method involves the formation of
polyelectrolyte complexes. Along the polymer backbone, there are pairs of charged
sites. The formation of polyelectrolyte complexes are the links between the charged
sites. The stability of the links depend on the pH of the system. [3]
b. Hydrogen Bonding: Hydrogen bonds are formed when an electron deficient hydrogen
atom combines with a high electronegative functional group. An example of this is a
gelatin-based hydrogel. There are many factors that affect this method of hydrogel
formation: polymer concentration, solvent type and temperature. [3]
c. Hydrophobic Association: This method is when polymers and copolymers from
structures that are separated by hydrophobic domains. They act as cross-linked points
for the entire structure. The mechanical characteristics are poor for this method
because of the low interfacial adhesion. It is advantageous due to the low cost though.
[3]
Chemical Crosslinking
This method involves the addition of a bi-functional crosslinking agent to a hydrophilic polymer
dilute solution. This is a suitable method for the formation of synthetic and natural hydrophilic
polymers. Hydrogels that have a high water content using the crosslinking of polyethylene
glycol and a lysine-containing polypeptide have been formed using this method. [3]
Free Radical Polymerization
This is the preferred technique for preparing hydrogels that are based on monomers such as
acrylates, amides and vinyl lactams. This method involves free radical polymerization steps:
initiation, propagation, chain transfer and termination. Initiation involves using certain initiators
are used for radical generation. The radicals react with monomers and are converted to active
forms and results in propagation. The long chain radicals then go through termination. This
method can be formed in solution or bulk. If large quantity of hydrogel is desired, solution
polymerization is the method to use. Bulk polymerization is faster than solution polymerization
because solvent removal is not needed. [3]
Irradiation Crosslinking of Hydrogel Polymeric Precursors
This method generates many reactive sites along the polymer strands. That leads to a large
number of crosslinks. Hydrogels can be formed using this approach by the irradiation of the
polymers in bulk or in solution. Irradiation of polymers in solution is the more favored approach
because it requires less energy for the formation of microradicals. Also, the efficiency of
radicals is higher in solution due to a lower viscosity. [3]
This method is advantageous over the other methods because no catalysts or additives are
needed to initiate the reaction during the irradiation process. Also, the method is simple and
can be controlled easily by varying the irradiation dose. It has been used to prepare acrylic acid
hydrogels. This is not a recommended method for preparing hydrogels from polymers that
degrade under ionizing irradiation. [3]

Hydrogels in Tissue Engineering

Figure 5 - Shows the various methods for treating injured or diseased
organs and tissues. [5]

Hydrogels can be used as an option of treatment for when certain tissues or organs fail. Refer
to figure 5. There are different ways of treatment. It all depends on the tissue or organ that
failed. A synthetic or natural substitute can be used but there are limitations. Synthetic polymer
replacement requires surgical procedures. Implants have achieved good success but there is
great promise with tissue engineering. It enables regeneration of the failed tissue. Hydrogels
have become increasingly popular. They can be used as tissue engineering scaffolds that have
pores large enough to accommodate living tissue. They can also be designed to degrade over
time, causing the release of growth factors and such enabling living cells to penetrate and
proliferate (Hydrogels for Biomedical Applications). The degradation of hydrogels can be
determined via enzymatic, hydrolytic, or environmental pathways. Depending on the tissue and
the time of regeneration, polymers with different degradation properties will be chosen. [10]
Hydrogels also have their advantages and disadvantages. Some advantages are: Provide good
transport of nutrients to cells and products from cells, can be easily modified with cell adhesion
ligands, be injected in vivo as liquid that gels at body temperature, and is usually biocompatible.
Some disadvantages are: They can be tough to handle, mechanically weak, difficult to load
drugs and cells and are difficult to sterilize. [5]
Connective Tissue and Hydrogels

Connective tissue is one of the main biological tissues that connects, supports and separates
the different tissues and organs in the body. All connective tissue (except for blood and lymph)
consists of three components: extracellular fibers, an amorphous matrix called ground
substance, and stationary and migrating cells.
Osteoarthritis and congenital bone
malformations are connective tissue damages and diseases. They affect over two million
patients in the United States each year. Currently, there are medications and surgery available
to treat the patients but result in limited pain relief. The alternative such as grafts are usually
unavailable. Tissue engineering can be used as an alternate method. Ideally, appropriate cells
would be placed in a scaffold and delivered to the site of injury in hopes of facilitating the
regeneration of the damaged tissue. Mesenchymal stem cells (MSCs) that are taken from the
adult bone marrow have the ability to differentiate into bone, cartilage and adipose tissue cells.
Bacterial Cellulose (BC) has shown promise as being a scaffold for tissue engineering and is
synthesized extracellularly as a hydrogel. BC is highly pure, biocompatible, hydrophilic, highly
crystalline, moldable, cost-effective, nanofibrous and is mechanically strong and contains a high
water content (99.8% water and .2% pure cellulose). Hydrogen bonding allows high BC
strength as well. BC has been implanted into rats with no sign of a foreign body response or
inflammatory response and has been used as a scaffold for many other treatments. [4] Refer to
figure 6 for the characterization of the BC scaffold.

Figure 6 - BC Scaffold characterization. SEM of BC scaffold (A) and Critical Point Dried BC
Scaffold (B). [4]

Injectable Hydrogels
Injectable hydrogels have been widely investigated as a scaffold for regenerative medicine.
Figure 7 shows the different crosslinking mechanisms for injectable hydrogels and the
interaction between the cell intergrins/receptors and biomolecules to the ECM polymers. The
feasibility of using hydrogels for cartilage regeneration was proven. Chondrocytes were
encapsulated within the hydrogel and the cells survived and proliferated for three weeks with
collagen type II and glycosaminoglycan (GAG) accumulating in the hydrogels. GAGs are

Figure 7 - Schematic of the Crosslinking Mechanisms. Mechanisms used for forming injectable
hydrogels (left) and interactions between cell integrin/receptors and biomolecules. [15]

carbohydrate polymers, such as heparin sulfate, chondroitin sulfate and keratin sulfate, which
are attached to the extracellular matrix (ECM) proteins. [14]
Injectable hydrogels can be used for greater therapeutic efficacy for protein drugs than
conventional drug delivery (oral or intravenous). This is because most proteins are degraded in
the stomach due to the low pH [9]. The ideal injectable hydrogel can entrap protein easily in a
hydrogel matrix and deliver the protein at specific sites for predefined periods, ranging from
days to weeks. This reduces the administration of protein frequency. Injectable hydrogels like
gelatin, hyaluronate, cellulose, and fibrin have been developed for this purpose.
Types of Injectable Hydrogels

There are four main types of injectable hydrogels: Fillers, Embolization Agents, Materials for
biosensors, and delivery systems. Fillers are used for correction of scars, wrinkles and lips.
They are also used as bulking agents to treat lipoatrophy in patients with AIDS. Embolization
agents are hydrogels that purposely occlude blood vessels to stop solid tumors from growing
and developing (block the feeding artery). Materials for biosensors use hydrogels to modify
properties as a function of biological signals. They are mainly used to measure glucose levels
of the blood. Delivery systems use hydrogels as carriers for controlled delivery of drugs. The
hydrogels slowly degrade after injection while giving off the agents. [10]
GOLD Standard for Hydrogels
Currently, Alginate is the most versatile natural-derived hydrogel employed as injectable cell
carriers for tissue regeneration [10]. Refer to figure 8 for a list of hydrogels and the types of
tissue they are compatible with. An alginate/hyaluronic acid hydrogel was seeded with human
Mesenchymal stem cells was constructed by spray. It was found that after 3 days, about 52% of
viable cells were in the scaffold. From day 7 to the end, the viability increased and the
metabolic activity also increased indicating that the conditions were not harmful for the cells.
This study shows that Alginate hydrogel is effective.
When considering connective tissue, BC is also considered a Gold standard. As said before, it
is highly pure, biocompatible, hydrophilic, highly crystalline, moldable, cost-effective,
nanofibrous and is mechanically strong and contains a high water content (99.8% water and .
2% pure cellulose). Hydrogen bonding allows high BC strength as well. BC has been
implanted into rats with no sign of a foreign body response or inflammatory response and has
been used as a scaffold for many other treatments. [4]
Future Directions
Although there is some success with engineered tissues, there are still many challenges ahead,
especially with hydrogel scaffolds in mimicking natural ECMs. Some challenges are: poor cell
penetration and irregular cell seeding because of the lack of proper temporal and spatial control,
difficulties with engineering tissues with multiple cell types and unique ECM composition, poor
mechanical properties of hydrogels limiting their applications to soft and non-load bearing
tissues and the lack of microvasculature. Lack of microvasculature leads to a loss of viability
and the function of seeded cells which results in improper transportation of nutrients and
signaling molecules. [3]

Works Cited

Figure 6 Natural derived hydrogels as injectable cell carriers. [11]

1. Alexander, Amit, et al. "Polyethylene Glycol (PEG)Poly(N-Isopropylacrylamide)

(Pnipaam)
Based
Thermosensitive
Injectable
Hydrogels
For
Biomedical
Applications." European Journal Of Pharmaceutics & Biopharmaceutics 88.3 (2014):
575-585. Academic Search Complete. Web. 10 Feb. 2015.
2. Boucard, Nadge, et al. "The Use Of Physical Hydrogels Of Chitosan For Skin
Regeneration Following Third-Degree Burns."Biomaterials 28.24 (2007): 34783488. Academic Search Complete. Web. 10 Feb. 2015.
3. El-Sherbiny, Ibrahim M., and Magdi H. Yacoub. "Hydrogel Scaffolds For Tissue
Engineering: Progress And Challenges." Global Cardiology Science & Practice 2013.3
(2013): 1-27. Academic Search Complete. Web. 11 Feb. 2015.
4. Favi, Pelagie M., et al. "Cell Proliferation, Viability, And In Vitro Differentiation Of Equine
Mesenchymal Stem Cells Seeded On Bacterial Cellulose Hydrogel Scaffolds." Materials
Science & Engineering: C 33.4 (2013): 1935-1944. Academic Search Complete. Web.
10 Feb. 2015.
5. Hoffman, Allan S. "Hydrogels For Biomedical Applications." Advanced Drug Delivery
Reviews 64.(2012): 18-23. Academic Search Complete. Web. 13 Feb. 2015.
6. Ilkhanizadeh, Shirin, Ana I. Teixeira, and Ola Hermanson. "Inkjet Printing Of
Macromolecules
On
Hydrogels
To
Steer
Neural
Stem
Cell
Differentiation." Biomaterials 28.27 (2007): 3936-3943. Academic Search Complete.
Web. 10 Feb. 2015.

7. Imani, Rana, et al. "Evaluation Of Novel "Biopaper" For Cell And Organ Printing
Application: An In Vitro Study." Iranian Journal Of Diabetes & Lipid Disorders 10.(2011):
1-13. Academic Search Complete. Web. 10 Feb. 2015.
8. Kobayashi, Jun, and Teruo Okano. "Thermoresponsive Thin Hydrogel-Grafted Surfaces
For Biomedical Applications." Reactive & Functional Polymers 73.7 (2013): 939944. Academic Search Complete. Web. 10 Feb. 2015.
9. Kwon, Jin Seon, et al. "Injectable Extracellular Matrix Hydrogel Developed Using Porcine
Articular Cartilage." International Journal Of Pharmaceutics 454.1 (2013): 183191. Academic Search Complete. Web. 12 Feb. 2015.
10. Mironov, Vladimir, et al. "Organ Printing: Computer-Aided Jet-Based 3D Tissue
Engineering." Trends In Biotechnology 21.4 (2003): 157. Academic Search Complete.
Web. 10 Feb. 2015.
11. Munarin, Fabiola, et al. "New Perspectives In Cell Delivery Systems For Tissue
Regeneration: Natural-Derived Injectable Hydrogels." Journal Of Applied Biomaterials &
Functional Materials 10.2 (2012): 67-81. Academic Search Complete. Web. 10 Feb.
2015.
12. Natalja E., Fedorovich, et al. "Hydrogels As Extracellular Matrices For Skeletal Tissue
Engineering: State-Of-The-Art And Novel Application In Organ Printing." Tissue
Engineering 13.8 (2007): 1905-1925. Academic Search Complete. Web. 10 Feb. 2015.
13. Tritz-Schiavi, J., et al. "Original Approach For Cartilage Tissue Engineering With
Mesenchymal Stem Cells." Bio-Medical Materials & Engineering 20.3/4 (2010): 167174. Academic Search Complete. Web. 13 Feb. 2015.
14. Ward, Mark A., and Theoni K. Georgiou. "Thermoresponsive Polymers For Biomedical
Applications." Polymers (20734360) 3.3 (2011): 1215-1242. Academic Search Complete.
Web. 10 Feb. 2015.
15. Yang, Jeong-A., et al. "In Situ-Forming Injectable Hydrogels For Regenerative
Medicine." Progress In Polymer Science 39.12 (2014): 1973-1986. Academic Search
Complete. Web. 12 Feb. 2015.