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Table of Contents
Illustrations …………………………………………………………………………………… 2
Introduction ……..……………………………………………………………………………. 4
Scope of the Report ………………………………………………………………………. 5
Evaluation Criteria ………………………………………………………………………. 5
Evaluation of Scaffold Materials ……………………………………………………………. 8
Natural Scaffold Materials ……………………………………………………………….. 8
Synthetic Scaffold Materials ……………………………………………………………… 11
Evaluation of Scaffold Manufacturing Techniques ……………………………………….. 18
Electrospinning ………………………………………………………………………….. 19
Molding ………………………………………………………………………………….. 20
Particulate Leaching ……………………………………………………………………. 21
Phase Separation of Emulsions …………………………………………………………. 22
Recommendations …………………………………………………………………………… 23
References …………………………………………………………………………………… 26
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Illustrations
Figures
Figure 1. A. Two units of cellulose joined by a beta glycosidic linkage ……………………….. 9
Modified from http://www.mansfield.ohio-state.edu/~sabedon/071amylo.gif
B. Two units of alginate joined by a beta-glycosidic linkage.
http://www.scientificpsychic.com/fitness/alginate1.gif
Figure 2. Two units of hyaluronic acid joined by a beta-glycosidic linkage …………………… 9
http://upload.ecvv.com/upload/Product/200801/2007621142832477566_
Hyaluronic_Acid_Heparin_Sodium.jpg
Figure 3. Triple-helical structure of native collagen …………………………………………… 10
http://www.3dchem.com/imagesofmolecules/Collagen2.jpg
Figure 4. Fibrin, in turquoise, holds together platelets (purple) and red blood cells (red) …....... 11
http://www.bss.phy.cam.ac.uk/~jrb75/fibrin.jpg
Figure 5. A general ester bond …………………………………………………………………. 11
http://en.wikipedia.org/wiki/File:Ester-general.svg
Figure 6. PGA, PLA, and PLGA monomers …………………………………………………… 12
http://www.drugdeliverytech.com/Media/PublicationsArticle/001446.jpg
Figure 7. Cross-sectional view of compact and trabecular bone ………………………………. 13
Modified from http://www.onjcenter.com/images/bone.jpg
Figure 8. Crystalline hydroxyapatite …………………………………………………………… 14
http://content.answcdn.com/main/content/img/elsevier/vet/gr95.jpg
Figure 9. Powdered tricalcium phosphate ……………………………………………………… 14
http://www.runkai.net/en/cp/cp3-1.jpg
Figure 10. Water molecule showing electrical charges ……………………………………….. 15
http://www.amnh.org/learn/courses/images/W3E1_1.jpg
Figure 11. Structure of polyethylene glycol, one monomer shown ……………………………. 17
http://upload.wikimedia.org/wikipedia/commons/4/42/Polyethylene_glycol.png
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Figure 12. Structure of polyacrylamide, one monomer shown ………………………………… 17
http://www.tiptheplanet.com/images/c/c9/Poly.png
Figure 13. Diagram of general electrospinning apparatus ……………………………………... 19
Modified from http://www.ciam.unibo.it/polymer_science/images/
electrospinning.jpg
Figure 14. Linearly oriented fibers on rotating metal collector ………………………………... 20
http://www.people.vcu.edu/~glbowlin/images/electrospinning.jpg
Figure 15. Phase separation apparatus …………………………………………………………. 22
Modified from A.L. Wooten, “Agglomeration of cell-loaded polymeric
porous microbeads as a precursor to in vitro tissue generation.” PhD rotation paper,
Washington University, 2010.
Tables
Table 1. Sources and degradation times of linear aliphatic polyester polymer ………………… 13
Table 2. Majors classes of cross-linking initiators and representative chemicals ……………… 16
All images modified from http://en.wikipedia.org
Table 3. Effects of various process parameters on electrospun membranes …………………… 20
Table 4. Evaluation of Scaffold Materials ……………………………………………………... 24
Table 5. Evaluation of Scaffold Manufacturing Technique ……………………………………. 24
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Introduction
Every year, approximately 9000 Americans die while waiting for an organ transplant [1]. The
reason for this high mortality is that a patient who suffers serious organ damage because of
disease, accident, or congenital defect has very limited treatment options. Often, receiving a graft
or transplant from a human or animal donor are the patient’s only choices. These procedures
carry substantial risks: In addition to undergoing invasive and traumatizing surgery, patients who
survive a transplant operation must take immune-suppressing drugs for the remainder of their
lives to prevent organ rejections. These drugs increase patients’ susceptibility to other diseases
such as bacterial infections and do not always work. Even if the drugs successfully prevent
rejection, the transplanted organ itself still wears out within five to ten years [2], sending the
patient back to the operating room to undergo yet another dangerous surgical procedure.
A severe and prolonged shortage of donor organs exists in the United States and around the
world. Because of this shortage and the risks associated with transplant operations, the medical
industry needs to develop better alternatives to organ grafts and transplants, and the field of
tissue engineering has emerged in the past 20 years to fill this gap.
Tissue engineering seeks to create replacements for biological tissues that can repair or enhance
existing tissue. Tissue engineering often attempts to integrate living cells with manmade
materials to create structures that mimic the target tissue’s original biological functions. To
encourage research in this field, in 2009, the U.S. Congress has passed the “Organ Transplant
Shortage Act,” an $80 million bill funding tissue engineering research.
Within the field of tissue engineering, implantable tissue scaffolds are manmade structures that
assist the body in regenerating damaged or missing tissue. To be effective, a scaffold must be
physically and chemically compatible with a wound site, degradable by the human body’s
natural enzymatic processes, structurally stable for the time required to regenerate a damaged
tissue, and bioactive to encourage cellular growth and differentiation.
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Scope of the Report
At the request of the U.S. Senate’s Health, Education, and Labor Committee, the Center for
Scientific Review (a division of the National Institutes of Health), has prepared this
recommendation report evaluating the medical feasibility of new tissue scaffolds and presenting
funding recommendations. This report addresses the following topics:
•
Significant natural and synthetic scaffold materials scientists are currently studying.
•
Benefits and shortcomings of each material’s physical properties.
•
Important manufacturing techniques scientists are using to fabricate novel scaffolds.
•
Advantages, disadvantages, and potential applications of each technique.
Based on these areas, the report rates each scaffold material and manufacturing method
according to several key criteria and issues recommendations regarding which scaffolds show the
most promise for medical use and thus should receive the most funding.
Evaluation Criteria
In the context of clinical implantation in patients, the NIH will utilize the following six criteria in
this report, listed in order of importance, to evaluate tissue scaffolds and their manufacturing
techniques:
1. Compatibility – capability of two materials to coexist and function in the same environment.
In the context of tissue engineering, compatibility requires that an implanted scaffold be
both nontoxic and immune neutral. Nontoxic means that the scaffold material does not
harm or interfere with the human body’s internal functions. Immune neutral means that
the scaffold material does not trigger an inflammatory response from the patient’s
immune system. The inflammatory response is the immune system’s defense against
foreign biological invaders such as viruses or bacteria. Our immune system tends to
recognize and destroy any foreign organic material that enters the body, whether harmful
or not. This hostile response is the basis of organ transplant rejection [2], and our immune
system’s sensitivity makes compatibility a particular challenge when implementing
natural scaffold materials.
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2. Biodegradability – methods and time over which natural processes break down a material.
As patients participate in daily activities such as walking, sitting and bending over,
implanted materials undergo structural wear and tear that alter an implant’s shape and
location within the body. Damaged implants eventually cause pain and require surgical
removal. Therefore it is dangerous to leave a tissue scaffold implanted indefinitely in situ.
On the other hand, removing and replacing a scaffold poses its own dangers. After a
scaffold has been implanted and native tissues, organs, and blood vessels have grown
around the scaffold, it is impossible to remove the scaffold without causing severe trauma
to the implant site. Biodegradability requires that the human body’s own enzymatic
processes be able to digest an implanted scaffold material. Ideally, a particular scaffold
will degrade at the same rate its surrounding tissue grows [3]. Furthermore, the residues
of this breakdown process must be nontoxic, immune compatible, and excretable by the
liver and kidneys.
3. Structural Stability – a material’s ability to maintain its original form over time.
An implanted scaffold must be stable over its intended lifetime because a localized defect
in a scaffold, such as shrinkage, can become a defect in the regenerated tissue such as
lower bone density.
4. Bioactivity – ability to actively promote cellular growth, differentiation, and repair.
In the past, scientists pursued scaffolds that were as biologically inert as possible to avoid
the pitfalls of toxicity and immune incompatibility. Today, the field of tissue engineering
has undergone a reversal and is now seeking to develop implants that are not only fully
compatible with the human body, but that also dramatically improve the rate of tissue
regrowth. This quest for higher bioactivity is the driving force behind development of
new scaffold materials and manufacturing methods.
Bioactivity is also driving research toward smaller and smaller physical scales. Because
oxygen dissolved in blood can travel only about 200 µm [4], or the width of several
human hairs, cells growing in the center of an implanted scaffold are likely to become
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hypoxic, or oxygen-starved, and die within several days. To overcome this problem,
scientists are increasingly pursuing micro-sized scaffolds and experimenting with
different physical structures and chemical signaling compounds to encourage new blood
vessels to grow into and around a porous scaffold.
5. Applications – potential uses in various parts of the body for different types and sizes of
defects.
Different physical properties make each scaffold material suitable for different tissue
repair sites. For example, fibrous scaffolds are best for muscle repair because they are
structurally similar to muscle fibers, while minerals are best for bone repair because
bones contain high mineral content [3]. The most promising scaffold materials and
manufacturing techniques will be those that have either a broad range of potential
applications or demonstrated high applicability to a specific tissue type.
6. Cost – expenses incurred or saved during materials processing, manufacturing, and clinical
implementation.
Three key factors influence the cost of an implant. The first is customization. Medical
treatments are becoming increasingly personalized, and tissue engineering is no
exception to this trend. We anticipate that future implantable scaffolds will be tailormade for each individual patient in two ways: (1) custom molded to fit specific wound
sites and (2) seeded with a patient’s own stem cells, tissue grafts, or signaling molecules
to maximize bioactivity and minimize rejection. The more customized an implant is, the
higher its cost.
A second factor influencing cost is manufacturing. Mass production is the most effective
way of manufacturing any product, so scaffolds that are easy to mass-produce will be the
most cost effective in the long run. In issuing funding recommendations, we will discuss
potential challenges in scaling up current scaffold fabrication processes.
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The third factor that influences implant cost is clinical implementation. The more
invasive the implantation procedure, the higher the hospitalization, surgery, and recovery
costs. Scaffolds that require minimal surgery will be both cost effective and safer for
patients.
Evaluation of Scaffold Materials
The NIH broadly classifies scaffold materials into two categories – natural materials, which
come from existing biological matter, and synthetic, or manmade materials. The following two
sections of this report describe the physical properties of the most significant natural and
synthetic scaffold materials scientists are currently studying and evaluate the benefits and
drawbacks of each material.
Natural Scaffold Materials
In general, the advantage of using natural scaffold materials in tissue scaffolds is that natural
materials tend to have high bioactivity. The disadvantage of natural materials is that they are
more likely than synthetic materials to exhibit immune incompatibility. Immune incompatibility
occurs because organic matter contains molecule-sized chemical markers known as antigens.
Antigens are typically small proteins or sugars attached to the surface of cells. The human
immune system recognizes these antigens as foreign invaders and attacks them [2], causing
implant rejection. Therefore, the most important area of natural scaffold research is to mitigate
immune incompatibility. Three of the most promising natural scaffolds are polysaccharides,
collagen, and clotting factors.
Polysaccharides
The prefix “poly-” means many, and “-saccharide” refers to sugars. Polysaccharides are multiunit sugars. The most common polysaccharides consist of carbon rings joined to one another by
chemical bonds, called glycosidic linkages. A single polysaccharide molecule may contain
thousands of rings, giving the molecule fibrous properties. Glucose, the main component of cane
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sugar used in everyday cooking, is an example of a polysaccharide. In tissue engineering, the
most popular polysaccharides are alginate and hyaluronic acid.
•
Alginate is a component of the cell walls of brown algae, a plant that lives in shallow
ocean areas [5]. Its structure is similar to that of cellulose, or common plant fiber. Like
cellulose, alginate’s main function is to provide structural stability. And like cellulose,
alginate’s physical strength comes from the bond connecting sugar units to one another
B
A
Figure 1. A. Two units of cellulose joined by a beta glycosidic linkage. B. Two units of alginate joined by a betaglycosidic linkage.
(compare Figure 1A and 1B). This bond is called a beta-glycosidic linkage, and it is
strong because it resists chemical breakdown. In fact, doctors recommend eating
vegetables while dieting because cellulose fibers are difficult for humans to digest and
thus do not provide as many calories as other foods. In the context of tissue engineering,
alginate’s structural stability makes it an excellent candidate for building scaffolds.
Because tissue repair and regeneration take months to years to complete, a scaffold must
be structurally stable for such an extended period of time. Furthermore, alginate promotes
wound healing. In fact, the medical industry already incorporates alginate into bandages
for wrapping burn wounds.
•
Hyaluronic acid is a component of human
connective tissue. Connective tissue includes
ligaments, joints, and tendons. The two roles of
connective tissue in the body are to hold bones,
muscles, and organs in place and to facilitate
movements such as walking and running [2].
Evaluation of Novel Tissue Scaffolds for Implantation
Figure 2. Two units of hyaluronic acid
joined by a beta-glycosidic linkage.
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Hyaluronic acid has a similar structure to alginate and cellulose, including betaglycosidic linkages that render it structurally stable (see Figure 2). Research has found
that hyaluronic acid contributes to cartilage growth and sunburn repair, making it an
excellent candidate for improving the rate of tissue regeneration [3]. The cosmetics
industry already incorporates small concentrations of dissolved hyaluronic acid into facial
lotions, toners, and masks as a wrinkle-reducer. Hyaluronic acid also demonstrates a
favorable biodegradability profile, meaning that scientists understand how the human
body breaks it down. For example, we know that the average person contains about 15
grams of hyaluronic acid, five of which break down and regenerate every day [6].
Collagen
Collagen, the second most promising natural scaffold material, is
the major component of the extra-cellular matrix, or ECM, found in
all animal cells including humans. The extra-cellular matrix is a
fluid-filled bath that surrounds animal tissues. Its purpose is to
provide structural support, assist cell-to-cell communication, and
direct cell growth and repair [2]. This last role, directing growth
and repair, makes collagen highly bioactive and therefore an
Figure 3. Triple-helical
structure of native collagen.
attractive candidate for tissue scaffold engineering. Collagen’s
structure is a fibrous coil consisting of three protein strands
wrapped around each other (see Figure 3). However, collagen is not useful in its native form and
must undergo processing before implementation as a scaffold. This processing denatures, or
unravels, the coil so that engineers can reshape it into a desired structure. Processing requires
dissolving powdered collagen in boiling water, pouring the liquid mixture into a mold, and
letting the mixture resolidify by cooling. The first step, boiling in water, unravels the triple coil,
thus reducing collagen’s structural stability and changing its biodegradability profile. The most
abundant source of denatured collagen – gelatin – usually comes from the bones and hooves of
pigs, and thus poses immune compatibility challenges [2]. Major directions in collagen research
include characterizing the biodegradability of gelatin and improving its structural stability by
hybridizing gelatin with stronger materials.
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Clotting factors
Clotting factors are a class of molecules involved in the first step
of wound healing, namely forming a clot, or scab, to prevent
further blood loss. Clotting factors include numerous substances,
but tissue scaffold engineers are primarily interested in two
factors: fibrinogen and thrombin [3]. When combined, these two
factors react to form a solid fibrous mesh called fibrin, shown in
turquoise in Figure 4. Fibrin’s role is to bind together blood
Figure 4. Fibrin, in turquoise, holds
together platelets (purple) and red
blood cells (red).
cells and other tissues [2]. Its binding abilities give it potential
as a scaffold material. However, fibrin biodegrades extremely rapidly; it begins to break down
within several days after forming, and completes its degradation process within several weeks
[3]. Fibrin’s short life span makes it more applicable for short-term or emergency tissue
regeneration treatments and less applicable for long-term therapy. An alternative direction of
clotting factor research is to combine fibrin with more durable substances such as
polysaccharides or various synthetic materials to slow down its biodegradability.
Synthetic Scaffold Materials
Polysaccharides, collagen, and clotting factors demonstrate that the major weaknesses of using
natural implant materials are immune incompatibility and structural stability. To mitigate these
problems, scientists have increasingly turned to synthetic scaffold materials. This section
describes the properties of three of the most promising synthetic scaffolds: polyester polymers,
calcium phosphates, and hydrogels.
Polyester Polymers
Polyesters are chemical compounds that contain multiple ester bonds. An
ester bond joins two chemical groups and contains one carbon atom bonded to
two oxygen atoms (see Figure 5). R and R′ denote generic chemical groups,
Figure 5. A general
ester bond.
which usually include additional carbon atoms. Polyesters already have many
applications, particularly in the clothing and furniture industries.
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In the context of tissue engineering, the most popular synthetic scaffold materials are linear
aliphatic polyester polymers [2]. Polymers are large molecules made up of smaller repeating
units connected by chemical bonds, similar to polysaccharides. “Aliphatic” means fatty, which
describes polyesters composed primarily of the elements carbon and hydrogen. The primary
reason for the popularity of these polyester polymers is that the FDA has already approved three
polymers for limited clinical use in humans [3]:
•
Poly-glycolic acid (PGA)
•
Poly-lactic acid (PLA)
•
Poly-lactic-co-glycolic acid (PLGA)
Figure 6 shows the repeating unit, or monomer, of
each polyester. The n and m denote varying
numbers of monomers. Chemists synthesize these
Figure 6. PGA, PLA, and PLGA monomers.
compounds using byproducts from oil purification, and they can control the length of the
polymers (the values of n and m) by adjusting reaction conditions including temperature,
pressure, presence of a catalyst, and concentrations of starting materials.
The human body contains enzymes such as butyrylcholinesterase and acetylcholinesterase that
degrade esters by hydrolysis, a reaction that adds water to compounds containing ester bonds [2].
After implantation in the body, poly-glycolic acid (PGA) breaks down within several months via
this ester hydrolysis mechanism [4]. On the other hand, poly-lactic acid (PLA) takes several
years to break down [4]. The reason for their differing biodegradability is the differing strength
of the ester bonds: Each unit of poly-lactic acid contains an additional -CH3 group compared to
poly-glycolic acid. This -CH3 group repels water, slowing down the hydrolysis process.
Differing biodegradability of PLA and PGA allows chemists to carefully tune the degradation
time frame of a scaffold by using PLGA, poly-lactic-co-glycolic acid. PLGA contains both PLA
and PGA units. By varying the ratio of PLA to PGA, chemists can synthesize polymers that take
from several months to several years to degrade. This flexibility renders polyester polymers
attractive for a wide range of short term to long term tissue engineering applications.
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Polyester polymers also offer versatility in manufacturing. Newly-synthesized polymers come in
powder or granulated form, and they can be dissolved, molded, or even woven into many
different structures with different physical properties. Other non-FDA-approved polyester
polymers include poly-caprolactone, poly-hydroxyl butarate, and poly-propylene fumarate.
Properties of these polyesters are summarized in Table 1.
Table 1. Sources and degradation times of linear aliphatic polyester polymers. [2]
Compound
Poly-glycolic acid
Abbreviation Degradation Time Source
PGA
2-3 months
petroleum
Poly-lactic acid
PLA
1-2 years
petroleum
Poly-lactic-co-glycolic acid PLGA
2 months-2 years
petroleum
Poly-caprolactone
PCL
> 2 years
petroleum
Poly-hydroxyl butyrate
PHB
> 2 years
fermentation
Poly-propylene fumarate
PPF
unknown
petroleum
Calcium Phosphates
Calcium phosphates are a group of inorganic minerals that make up about half of the total bone
mass in humans [2]. While each bone has a unique structure, all bones generally consist of two
layers: a solid outer layer called compact bone and a porous, soft core called trabecular bone,
which includes the bone marrow (see Figure 7). Calcium phosphate forms porous networks that
provide structural rigidity in both layers, but is denser and more
abundant in the compact outer layer. Within the trabecular layer,
soft tissues including blood vessels, collagen, and growth factors
contribute to a spongy, flexible texture. The bone marrow is the
site of greatest cell growth and cell turnover. Although both layers
are porous, trabecular bone is much more so, with a porosity of
Figure 7. Cross-sectional view of
compact and trabecular bone.
over 30% [2]. The most popular calcium phosphates for tissue
engineering are calcium hydroxyapatite and tricalcium phosphate.
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•
Calcium hydroxyapatite is the natural form of calcium
phosphate found in human bone. Its molecular formula is
Ca10(PO4)6OH2, and in its pure form, appears as sharp,
solid crystals (see Figure 8). Commerical hydroxyapatite
comes from two sources: The first and most common
source is by direct synthesis, where chemists allow
calcium and phosphorous precursors calcium
carbonate CaCO3 and ammonium hydrogen phosphate
Figure 8. Crystalline
hydroxyapatite. (http://content.
answcdn.com/main/content/img/els
evier/vet/gr95.jpg)
(NH4)2HPO4 to react in solution, forming
hydroxyapatite as a solid precipitate [7]. The second method involves heating coral
skeletons, which contain calcium carbonate, to initiate their transformation into
hydroxyapatite [7]. The heating process burns away protein residues, preventing immune
incompatibility. However, hydroxyapatite derived from coral tends to be structurally
weaker and less uniform than hydroxyapatite synthesized in the laboratory.
•
Tricalcium phosphate, molecular formula Ca3(PO4)2, is
called “bone dust” because it forms when burning
calcium hydroxyapatite. In its pure state, tricalcium
phosphate appears as a fine white powder (see Figure 9).
It already has several medical applications as an antacid
and calcium dietary supplement. Tricalcium phosphate
exists in nature as a rock mixed with sandstone and
phosphorous oxides [8]. Chemists can also synthesize
Figure 9. Powdered tricalcium
phosphate. (http://www.runkai.net/
en/cp/cp3-1.jpg)
tricalcium phosphate directly by reacting calcium and
phosphoric acid.
The advantage of calcium phosphates is that both hydroxyapatite and tricalcium phosphate are
osteoconductive and osteoinductive. Osteoconductive refers to their ability to promote both bone
cell adhesion and differentiation, while osteoinductive refers to their ability to induce new bone
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cell growth. Studies have shown that osteoconductivity arises from the attachment of bone
morphogenetic proteins that direct the formation of new bone cells [3]. On the other hand, the
major challenge of working with calcium phosphates is mimicking the porous structure of
natural bone. Pure calcium phosphates are usually solid crystals with geometry dictated by
atomic size and spacing. These crystals are brittle, meaning they fracture easily and are thus
difficult to mold into alternate forms. Promising calcium phosphate research seeks to combine
calcium phosphates with more flexible materials such as collagen in order to improve elasticity.
Hydrogels
Hydrogels are cross-linked polymers that can absorb a great deal of water, up to 99%, without
dissolving into liquid form [3]. Cross-linking is a chemical process that transforms linear
polymer chains into an interconnected, three-dimensional network by forming bonds between
polymer chains. Cross-linking typically enhances the mechanical and thermal stability of
materials. This enhanced stability allows hydrogel polymers to hold water while still maintaining
solid form, making hydrogels capable of filling tissue defects of various sizes and shapes. The
degree of cross-linking within a hydrogel correlates directly with its mechanical strength and
inversely with the amount of water it can hold: the more extensively linked a gel, the stronger the
gel is but the less water it can absorb. Hydrogels are advantageous in scaffold engineering
because the human body contains about 90% water [2], allowing hydrogels to achieve high
compatibility and bioactivity by providing a favorable environment for regenerative agents
including stem cells, signalling molecules, and growth factors. Designing hydrogels requires a
tradeoff between bioactivity from water content and structural stability from mechanical
strength.
Most hydrogel polymers are anionic, meaning they contain negative electric
charge. These negative charges are key to hydrogels’ ability to absorb water:
Water molecules of molecular formula H2O are highly electrically polar, with
oxygen atoms carrying partial negative charges and hydrogen atoms
Figure 10. Water molecule
showing electrical charges.
carrying partial positive charges (see Figure 10). Because opposite
electrical charges attract, negatively charged polymers can bind to the
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positively charged hydrogen atoms of water. Since polymers contain many subunits and
therefore many sites of negative charge, a hydrogel can bind many water molecules, resulting in
a high water-to-polymer ratio. Chemists can control the water content of a hydrogel by adjusting
the concentration of cations, or positively charged species, in the hydrogel mixture. Metal cations
such as sodium, Na+, and calcium, Ca2+, carry stronger positive charge than the hydrogen atoms
of water. In competition with hydrogen, these cations bind preferentially to negatively charged
polymers, neutralizing some of the polymers’ negative charges and preventing those sites from
binding water. The more cations in a hydrogel mixture, the less water it can absorb.
Chemists also control the water content of hydrogels by increasing or decreasing the amount of
cross-linking present. Cross-linking requires the use of a chemical initiator, a highly reactive
compound that breaks previously stable bonds between polymer units, allowing the formation of
new bonds. Because these cross-linking agents are very reactive, a major safety concern about
hydrogels is the potential toxicity of cross-linking initiators. Table 2 shows the most common
initiators and briefly describes their toxicity concerns.
Table 2. Majors classes of cross-linking initiators and representative chemicals. [9]
Initiator Class
Acid anhydrides
Representative Chemical
Acetic
anhydride
Toxicity Concerns
Corrosive, flammable,
respiratory irritant
Glutaraldehyde
Aldehydes
Corrosive, respiratory irritant
Azobenzene
Azo-compounds
Mutagen (causes DNA
mutations)
Hydrogen
peroxide
Corrosive, explosive,
respiratory irritant
Photoinitiators
Nitrogen
dioxide +
UV radiation
Carcinogen (causes cancer)
X-ray radiation
N/A
Carcinogen (causes cancer)
Peroxides
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The two major classes of hydrogels used in tissue scaffolds are polyethylene glycol (PEG) and
polyacrylamide derivatives:
•
Polyethylene glycol (PEG) polymers are available in a wide range of polymer lengths,
from tens to several hundreds of subunits long. Solutions containing longer polymers
typically have higher viscosity, or fluid thickness,
than solutions containing shorter polymers. Figure
11 shows the chemical structure of one subunit of
polyethylene glycol. Note the presence of oxygen
atoms, which bind hydrogen in water. In addition
Figure 11. Structure of polyethylene
glycol, one monomer shown.
to toxicity of cross-linking agents, PEG has the disadvantage of being non-biodegradable.
Current studies seek to improve its degradability by combining PEG with degradable
polyesters or by adding degradable bonds to the carbon backbone of the polymer [3].
•
Polyacrylamide (Figure 12) and its derivatives currently
have applications as cosmetic wrinkle-fillers and contact
lens materials. Like polyethylene glycol, polyacrylamide is
not biodegradable in its pure form [3]. An additional
toxicity concern unique to polyacrylamide is toxicity
associated with the precursor monomer, acrylamide. In its
Figure 12. Structure of
polyacrylamide, one monomer
shown.
unpolymerized form, free acrylamide is a potent neurotoxin – a dangerous chemical that
damages nerve cells [10]. Once polymerized however, polyacrylamide is non-toxic and
chemically unreactive. While polymerization reactions typically go to completion, trace
amounts of unreacted acrylamide may remain, posing health risks when implanted.
Scientists also harbor concerns regarding the potential release of free acrylamide into the
body if further technological development succeeds in making polyacrylamide
biodegradable [3].
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Evaluation of Scaffold Manufacturing Techniques
In addition to containing compatible, degradable, stable, and bioactive materials, a tissue scaffold
must also be the correct size and shape for a particular wound site. The physical property all
scaffolds must have, regardless of their final implant location, is porosity. Porosity is important
for two reasons:
1.
A porous structure contains higher surface area than a solid structure of the same
volume, allowing more cells to attach, grow, and replicate. Providing sufficient space for
cell growth is critical because cells secrete signaling molecules that cause neighboring
cells to die if grown in overcrowded conditions [2].
2.
A porous structure allows the diffusion of nutrients, wastes, oxygen, and carbon dioxide
in and out of the scaffold. Diffusion is the movement of a chemical, usually a liquid or
gas, from an area of high concentration to an area of low concentration. In the human
body, diffusion is the principle mechanism by which substances move between the
bloodstream and the tissues. The diffusion limit, or range, of oxygen in blood is about
200 µm, or the width of several human hairs [4]. Therefore in order to be viable, a
segment of tissue must be in close proximity to a blood vessel. A porous scaffold
provides enough room for blood vessels to grow inward and transport substances to cells
growing at the center of a scaffold.
To achieve a porous structure, researchers have developed the following four general synthetic
strategies:
•
Electrospinning
•
Molding
•
Particulate leaching
•
Phase separation of emulsions
The following sections describe each synthetic protocol, which materials can undergo each
protocol, and what physical properties the final products exhibit.
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Electrospinning
Many tissues in the body including
nerve cells, muscle, and connective
tissue have fibrous or threadlike
structures. Electrospinning is a
synthetic technique that produces
networks of fibrous mesh with higher
surface area than conventional textile
manufacturing techniques. The key to
the higher surface area and higher
Figure 13. Diagram of general electrospinning apparatus.
bioactivity of electrospun membranes
is the small diameter of electrospun
threads. Conventional textile manufacturing, such as silk spinning, typically produces threads at
least several hundred micrometers wide. On the other hand, electrospinning achieves nano- or
micrometer thin fibers by using high voltage to transform liquid solutions of dissolved polymer
into threads. First, droplets of dissolved polymer solution exude slowly through a syringe and
needle. The needle connects to a high voltage source, which imparts electrical charge to the
polymer droplet. An oppositely charged metal collector, situated about five to ten inches away
from the needle, attracts the charged droplet, forcing the droplet to elongate into a thin thread and
accelerate toward the collector (see Figure 13). As the thread travels toward the collector, the
liquid solvent in the thread evaporates, leaving behind a solid polymer thread. Using a syringe
pump – a machine that ejects fluid through a syringe at a constant low rate – provides a steady
flow of polymer solution that becomes a long, continuous thread.
Electrospinning is highly sensitive to process conditions such as polymer solution viscosity,
voltage, collector-needle distance, syringe pump rate, temperature, and humidity. Adjusting these
process conditions produces membranes with different properties. Table 3 summarizes the
effects of these parameters on the final membrane.
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Table 3. Effects of various process parameters on electrospun membranes.
Parameter
Effect on Membrane
Polymer solution viscosity Higher viscosity = thicker threads
Voltage (5 to 30 kilovolts)
Higher voltage = thinner threads
Collector-needle distance
Shorter distance = denser mesh
Syringe pump rate
Faster pump rate = thicker threads and denser mesh
Temperature
Higher temperature = thinner threads
Humidity
Higher humidity = thicker threads
Both natural and synthetic polymers can undergo
electrospinning. Popular electrospinning candidates
include poly-glycolic acid, poly-lactic-co-glycolic
acid, poly-caprolactone, collagen, and fibrinogen
[3]. The structure of the membrane changes
depending on the shape of the collector. The
Figure 14. Linearly oriented fibers on rotating
metal collector.
stationary metal plate shown in Figure 13 collects
randomly oriented fibers. A rotating collector can
collect linearly oriented fibers, seen in Figure 14.
The major drawback of electrospinning is that thus far, it produces only flat two-dimensional
membranes and cannot yet produce three-dimensional scaffolds. Attempts to produce threedimensional scaffolds by layering multiple membranes on top of one another have yielded
mechanically weak structures [3].
Molding
Unlike electrospinning, molding can produce three-dimensional scaffolds. In general, molding
works by pouring a dissolved scaffold material into a mold, letting the material harden, and
removing the mold to leave behind a scaffold with a specific shape and internal pore structure.
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One of the most promising molding techniques described by Ma [11] uses small spheres of
paraffin wax as a template. When poured into a mold such as a small beaker or funnel, paraffin
spheres tend to sink to the bottom of the mold and pack together in an orderly fashion. When
heated briefly, these aggregated spheres melt slightly and stick together, producing a continuous
spherical network. After removal of the paraffin template, the gaps in the scaffold left behind
form an orderly and interconnected porous network.
Changing the size of the template paraffin spheres produces different sizes of pores in the final
scaffold, while changing the scaffold material produces different mechanical properties. To
undergo paraffin molding, a scaffold material must be soluble in a liquid that does not dissolve
paraffin wax. Varying solubility makes it possible to remove the paraffin template after the
scaffold hardens without dissolving the scaffold itself. Paraffin wax dissolves in specific
hydrocarbon-based solvents such as paint thinner, making it incompatible with some organic
polymers. Finding alternatives to paraffin for use as templates is a promising direction of
scaffold engineering.
Particulate Leaching
Particulate leaching is similar to molding in that it uses a template material to produce a porous
structure. In particulate leaching, developed by Mikos and Temenoff [12], salt crystals occupy
spaces that become pores upon removal of the salt. Salt crystals have the distinct advantage of
being soluble in water. Since most scaffold materials are insoluble or only slightly soluble in
water (to remain structurally stable when implanted in the body), soaking a dried scaffold in
water successfully removes the salt particles without affecting the scaffold. Thus, particulate
leaching is applicable to a wide range of scaffold materials. Particulate leaching can produce a
range of pore sizes depending on the size of the salt crystals used and the ratio between salt and
scaffold material. A major drawback of particulate leaching is that current technology cannot yet
finely control pore shape and inter-pore connections.
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Phase Separation of Emulsions
Many scaffold materials form emulsions when mixed with aqueous solutions. An emulsion is a
mixture of two liquids that do not dissolve well in one another. In an emulsion, the less abundant
liquid typically forms spherical droplets within the more abundant liquid. Everyday examples of
emulsions include mayonnaise (oil droplets in lemon juice) and vinaigrette salad dressing (oil
droplets in vinegar). Forming an emulsion requires vigorous agitation such as shaking to force
two incompatible liquids to mix. A common example is shaking salad dressing containers to
resuspend the oily top layer with the heavier bottom layer containing spices.
Aqueous solutions are liquids that contain mostly water. Because scaffold materials are insoluble
in water, chemists usually dissolve scaffold materials in hydrocarbon-based organic solvents
such as dichloromethane or toluene. These solutions are immiscible, or insoluble, with water.
Emulsions of organic and aqueous solutions, like vinaigrette sauces, are unstable and settle out
over time into separate layers. The principle of unstable emulsions is useful for fabricating tissue
scaffolds because when the aqueous layer is less abundant than the organic layer, the aqueous
layer forms droplets that become pores upon removal. The following synthetic protocol from
Choi [13] uses poly-lactic-co-glycolic acid and dissolved gelatin as an illustrative example of the
phase separation technique:
1. Poly-lactic-co-glycolic acid dissolves in an
organic solution of dichloromethane, forming a
2% polymer solution which is insoluble in water.
2. Powdered gelatin (denatured collagen) dissolves
in water, forming a 15% aqueous gelatin
solution, the less abundant aqueous layer.
3. A handheld emulsifier mixes the two solutions
vigorously, forming a uniform mixture
containing spherical aqueous droplets suspended
Figure 15. Phase separation apparatus.
in a continuous organic solution.
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4. After pouring the emulsion into a syringe and letting it sit, the emulsion begins to
separate into two layers. The top layer grows richer in aqueous gelatin, while the bottom
layer grows poorer in gelatin.
5. A syringe pump exudes droplets from the top layer through a narrow needle into a
collection bath containing cold water (see Figure 15).
6. After stirring overnight in the cold water bath, the dichloromethane solvent evaporates,
allowing the droplets to harden into PLGA beads containing gelatin.
7. Soaking the beads in hot water dissolves the gelatin droplets, which gradually seep out of
the PLGA beads, leaving behind interconnected pores.
The overall size of these beads depends on the inner diameter of the needle through which the
precursor droplets pass. The principle behind PLGA bead synthesis is that each bead acts as a
miniature scaffold that can be injected through medical needles directly into the wound site of a
patient without the need for invasive surgery. Producing beads of the correct size merely requires
using the correct needle when collecting bead droplets. The standard needle size is 18 gauge,
which produces beads several hundred micrometers in diameter, about the width of several
human hairs [4]. In vivo (live animal or human) studies have shown that cells seeded into these
porous PLGA microbeads can grow and survive when implanted into the shoulders of mice [14].
The next step of microbead scaffold research is to study the behavior of stem cells, signaling
molecules, and growth factors in interaction with PLGA beads. A toxicity concern is potential
poisoning from residual solvent, dichloromethane, which is a corrosive carcinogen [15].
Recommendations
Based on the previous evaluations of scaffold materials and scaffold manufacturing techniques,
the following tables tally up the benefits and drawbacks of each scaffold using a numeric system.
Each scaffold earns of 1 to 5 for each of the six categories from Scope of the Report, with 1
indicating low potential for success and 5 indicating very high potential for success. Regarding
cost, 1 indicates high cost and 5 indicates low cost.
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Table 4. Evaluation of Scaffold Materials.
Criteria
Polysaccharides
3 (immune
incompatibility)
3 (immune
incompatibility)
Clotting
Factors
3 (immune
incompatibility)
Biodegradability
5
3
2
5
5
2
Structural
Stability
Bioactivity
4
3
2
5
2
3
5
5
5
4
5
4
Applications
4
4
2
5
3 (Bone only)
4
Cost
3
5
1
2
4
5
Sum
24
23
15
26
21
22
Compatibility
Collagen
Polyester
Polymers
Calcium
Phosphates
Hydrogels
5
5
4
Table 5. Evaluation of Scaffold Manufacturing Techniques.
Criteria
Electrospinning
Structural Stability
2 (membranes are
Molding
Particulate
Leaching
Phase Separation
of Emulsions
5
5
5
4
2 (pores not
5
typically
millimeters thin)
Bioactivity
4
connected)
Applications
2 (twodimensional only)
5
5
5
Cost
4
3 (mold
destroyed)
5
2 (difficult to scale
Sum
12
13
12
up)
17
Based on these tabulated values, the most promising scaffold materials are natural
polysaccharides and synthetic polyester polymers, while the most promising manufacturing
process is phase separation of emulsions. In terms of research funding, the NIH recommends
preferentially funding projects with the greatest potential of achieving rapid clinical
implementation. To this end, the polyester polymers are particularly attractive because several
polymers have already garnered FDA approval for human use, dramatically speeding up the
process of clinical implementation.
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Funding should also go to projects that develop protocols for efficiently scaling up existing
manufacturing processes, which thus far have been carried out almost exclusively in small-scale
laboratories. A major roadblock to implementing scaffolds is guaranteeing the viability of cells
within large scaffolds. Because the diffusion limit of oxygen in blood is so small, only
microscale scaffolds such as polymer beads are currently useful. Larger implants will need to
promote the growth of blood vessels in order to become useful for medical applications. The
NIH recommends funding fundamental research that studies the growth mechanism of blood
vessels in an effort to induce new capillary growth in scaffolds.
Finally, because every scaffold material and synthetic technique exhibit unique advantages and
disadvantages, the NIH concludes that the most promising scaffolds will be hybrid structures that
combine multiple materials, thus taking advantage of favorable properties while mitigating
unfavorable properties. The Senate should give special funding priority to daring research
proposals that seek to study hybrid scaffold technologies. While tissue engineering is still a long
way from replacing organ transplants entirely, scientists around the world are aggressively
pursuing new tissue scaffolds, and the NIH is confident that the field of tissue engineering will
greatly improve the quality of medical care in the future.
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References
[1] S. Satel. (2006, May 15). Death’s waiting list. New York Times [Online]. Available:
http://www.nytimes.com/2006/05/15/opinion/15satel.html
[2] K. McCance and S. Huether, Pathophysiology: The Biological Basis for Disease in
Adults and Children. St. Louis, Missouri: Mosby, Inc., 2002.
[3] P.X. Ma, “Scaffolds for tissue fabrication,” Materials Today, vol. 7, no. 5, pp. 30-40,
May 2004.
[4] A.L. Wooten, “Agglomeration of cell-loaded polymeric porous microbeads as a precursor
to in vitro tissue generation.” PhD rotation paper, Washington University, 2010.
[5] D.J. McHugh, “Production and utilization of products from commercial seaweeds,” Food
and Agricultural Organization of the United Nations, Rome, FAO 288, 1987.
[6] R. Stern, “Hyaluronan catabolism: a new metabolic pathway,” European Journal of Cell
Biology, vol. 83, no. 7, pp. 317-325, Aug. 2004.
[7] K. Gross, “Synthesis of hydroxyapatite powders,” Azomaterials, Warriewood, Australia,
July 2004.
[8] U.S. Department of Agriculture: Bureau of Plant Industry, “Occurrence of beta tricalcium
phosphate in northern Mexico.” Beltsville, Maryland, 1951.
[9] Sigma Aldrich Product Catalog. (2010). Sigma Aldrich [Online]. Available: http://
www.sigmaldrich.com
[10] “Materials safety data sheets: acrylamide,” J.T. Baker, Phillipsburg, New Jersey, MSDS no.
A1550, Feb. 2009.
[11] P.X. Ma and J.W. Choi, “Biodegradable polymer scaffolds with well-defined
interconnected spherical pore network,” Tissue Engineering, vol. 7, no. 1, pp. 23-33, 2001.
[12] A.G. Mikos and J.S. Temenoff. (2000). Formation of highly porous biodegradable scaffolds
for tissue engineering. Electronic Journal of Biotechnology [Online]. Available:
http://www.scielo.cl/scielo.php?pid=S0717-34582000000200003&script=sci_arttext&tlng
=en
[13] S.W. Choi, I.W. Cheong, J.H. Kim, and Y. Xia, “Preparation of uniform microspheres
using a simple fluidic device and their crystallization into close-packed lattices,” Small,
vol. 5, no. 4, pp. 454-459, 2009.
[14] S.W. Choi, Y.C. Yeh, Y. Zhang, H.W. Sung, and Y. Xia, “Uniform beads with controllable
pore sizes for biomedical applications,” Small, vol. 6, no. 14, pp. 1492-1498, 2010.
[15] “Materials safety data sheets: methylene chloride.” J.T. Baker, Phillipsburg, New Jersey,
MSDS no. M4420, Aug. 2008.
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