Download III. Case Studies - Engineering Computing Facility

Henry H.Y. Lo
1
Using System Biology for Biomaterials: Protein
Interactions with Biomaterial Surfaces
Lo, Henry H.Y.

Abstract— There is a long history in using a systemic approach
in understanding human biology. The advancement of computers
and information technology allows the possibility to achieve this
goal. Currently, many successes in system biology evolve in the
genome area. However, I believe that system biology should
cover a wider scope of study. As the use of various biomedical
implants increases every year, it becomes very important to
develop an approach in understanding the interaction of the
implants with the human body.
interaction with other atoms and molecules. These interactions
can be classified as: Segregation, adsorption and absorption.
Index Terms—Biomaterials, System Biology
I. INTRODUCTION
S
YSTEM biology is a cross-disciplinary field of study that
aims to integrate different types of biological information
such as DNA, RNA, protein, protein interactions, cells, tissues,
and their relationships to one another [1]. This biological
information would be used to obtain a general model of the
system. Despite the fact that the exact goal for system biology
is very debatable, a very broad objective can be interpreted as
the development of a predictive, preventive and personalized
biological technology [1].
The improvements in computer technology allow the
possible integration of biological data. Currently, the success
in the development of the genome project has generated much
attention and interest in the system biology area. Should
system biology only involve interactions among living
systems? - No! This is especially true when many biomedical
implants are used to treat injured patients every year. The
interactions of biomaterials and body tissues are very complex
and have many unknowns. It is important to develop a system
approach in understanding these problems.
II. BIOMATERIAL SURFACES
A. Background
It is a physical nature of all crystalline materials that their
surfaces contain crystal imperfections [2]. Since material
surfaces are thermodynamically more active due to higher
atom and molecular mobility, they represent active zones for
Manuscript submitted on October 21, 2003. Henry Lo is a master student
at the Inistitute of Biomaterials and Biomedical Engineering in the University
of Toronto. 4 Taddle Creek Road, Room 407 Rosebrugh Building, University
of
Toronto,
Toronto,
Ontario
M5S
3G9
Canada
(e-mail:
[email protected]).
Fig. 1. Molecular interactions at implant surfaces. Different molecules will
behave differently on biomaterial surfaces [2].
Segregation occurs when impurities or other second phase
particles diffuse from the bulk of the material towards the
surface. The cause of segregation can be associated with
chemical gradients and the supersaturation of secondary phase
particles/atoms. Thermodynamically, it is favourable for these
particles to diffuse out of the bulk material [2]. This diffusion
process often leads to two end results: Build-up of particles on
the surface or the release of particles into the body. The buildup of secondary particles/atoms on the surface can cause
localized composition change [3]. A different surface material
will lead to different chemical and mechanical properties that
may affect the body reaction (i.e. inflammation) to the
biomaterial. The second scenario that involves the release of
particles/atoms can be either beneficial or disruptive. The
controlled drug release of a coating is often desired but the
release of metallic ions is not [4].
Adsorption takes place when another molecule or atom
attaches itself onto a surface. For biomaterials, protein
adsorption plays a fundamental rule in the biocompatibility of
the material. When a biomaterial enters the body, the material
surface will immediately attract proteins and other molecules
from the body fluids to adsorb onto the material surface [5].
Firstly, the surface properties and specific properties of
proteins would determine the organization of the adsorbed
protein layer. The adsorbed layer may contain a combination
of albumin, fibronectin, osteonectin, vitronectin, fibrinogen
laminin, collagen, and covalently bonded short-chain
oligosaccharides [2]. The adsorbed layer then determines the
tissue cellular response. Since the cellular response largely
determines the biocompatibility of a biomaterial, protein and
surface interactions have great importance to a biomedical
Henry H.Y. Lo
device. In later part of the paper, more details about protein
adsorption will be discussed.
Absorption occurs when molecules diffuse from the surface
towards the bulk of the biomaterial. For some polymeric
biomaterials, absorption of water can lead to material swelling
[3]. Swelling can lead to many undesired consequences. For
example, the mechanical strength of a swelled polymer is
usually less than the original material. The absorption of water
disrupts many secondary bonds (hydrogen / van der Waals /
crosslink bonding) that give the polymer mechanical strength.
In biomaterial devices, the process of segregation,
adsorption, and absorption occur simultaneously [2]. The
amount of interactions that occur depends on the device and
the location of the implant. These interactions are the
fundamental mechanisms that govern the biocompatibility of a
biomaterial.
B. Protein Adsorption and the Vroman Effect
Protein adsorption has great importance in the study of
biomaterials because protein will attach to the biomaterial as
soon as the implant is put into the body. However, the type of
protein that first attach onto the biomaterial may not attach
onto the biomaterial forever. This phenomenon is known as
the Vroman effiect.
Fig. 2. Schematic diagram of the Vroman Effect [2]
Figure 2 is a graphical explanation of the Vroman effect. In
this example, there are three types of protein to adsorb onto
the biomaterial surface. Protein C has the most chemical
affinity towards the implant while protein A has the least
affinity. However, protein A has the most mobility and
concentration in the body while protein C has the least. The
mobility of a protein can be characterized by its molecular
weight and shape. When a biomaterial first implanted into the
body, protein A, because of its mobility and high
concentration, would attach to the surface faster than protein
C. However, overtime, it has been noted that protein C will
replace protein A on the biomaterial surface. This is known as
the Vroman effect [2]. The protein mobility and concentration
determines the kinetics of protein adsorption.
III. CASE STUDIES
Many biomaterials applications would be benefited from the
development of system biology. Currently, many issues with
in the study of implant biocompatibility cannot be explained.
2
Although there is knowledge in understanding “what” would
happen to certain implants, there are many questions regading
to “why” things would happen. The lack of understanding
prevents the use of many synthetic materials as biomaterials.
The following are three sample cases that system biology
can be used to develop better biomaterials.
Current
challenges for using the biomaterial will be discussed along
with the current systemic development in the understanding of
the implant-material interaction.
A. Blood Interaction with Vascular Graft and Stents
Vascular grafts are soft and stretchy biomaterial (i.e.
polymer) that is used to replace diseased section of blood
vessels [3]. This repair is usually required when a patient
experienced a coronary occlusion caused by atherosclerosis.
Atherosclerosis is a degenerative disease of the arteries
characterized by the development of fatty and fibrous deposits
in the arterial wall at the underlying endothelial cells called the
intima. The clot begins when a small injury in the blood
vessel exposes the intima. These endothelial cells are adhesive
to fatty substance and the clot grows. Eventually, the clot will
block the blood vessel or the clot will break off and block
another blood vessel further down the blood stream.
Using vascular stents is another method of treating
atherosclerosis. Instead of replacing the blocked section of an
artery with a graft, a catheter is used to expand the blood
vessel and hold it in the expanded position. Damage to the
vessel smooth muscles is inevitable despite the fact whether a
vascular graft or stent is used. The damage often leads to the
development of another clot and the blockage of the blood
vessel once again. Therefore, in order to understand the
formation of the second clot, it is essential to understand how
the body stops bleeding, or the process of hemostasis [6].
Hemostasis is a three steps process: Vascular spasm, platelet
plug formation, and thrombus formation. When a blood vessel
is damaged (i.e. implanting a device) the first intrinsic body
response is the constriction of blood vessel to reduce and slow
down blood loss. Then the platelet plug formation starts.
When a blood vessel is damaged, the exposed endothelial
cells, intima, stimulates a protein called von Willebrand factor
(vWf) to accumulate at the damage site. The vWf protein
binds to the platelets (called thrombocytes) and stimulates the
platelets to secrete two chemicals called ADP and TXA2.
These chemicals cause the morphological changes in the
platelets that cause them to adhere to one another to form into
an aggregate that stimulates further formation of ADP and
TXA2. This positive feedback loop increases the rate of plug
formation and temporarily stops the bleeding.
The third step of hemostasis is thrombus or fibrin clot
formation. The aggregated platelets from the previous steps
allow the fibrin clot formation to take place. Fibrin is a
protein that traps other cells and molecules (i.e. cholesterol,
cells) in the blood and forms the basis of a clot. Fibrin is
formed from the activation of fibrinogen by a coagulation
factor called thrombin. Thrombin, on the other hand, is an
active form of another protein called prothrombin that is
Henry H.Y. Lo
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activated by yet another coagulation factor. The nature of
these coagulation factors with biomaterials has not been fully
understood [5]. There are many steps in the clot formation
process and the series of steps are shown below.
Fig. 4. Race for surface adherence. Note that the conditioning film on the
biomaterial surface; it mostly consists of proteins molecules [2].
Biofilms are formed by colonies of different type of
bacteria that adhere onto an implant surface. These films
consist of a slime layer of extracellular polysaccharide that
bond well with the biomaterial and bacteria. The presence of
this polysaccharide slime is very problematic. Firstly, it may
lead to chronic infections [4]. Secondly, the presence of
implant breakdown products may be utilized as bacterial
nutrition [2]. More importantly, the slime layer offers great
resistance to body host defence and antibiotics.
Fig. 3. Intrinsic and Extrinsic Coagulation Pathways. Please note that the
Roman numeric symbols only serves as a representation of coagulation
factors. They do not represent the order of appearance in fibrin clot formation
[6].
There are two pathways in fibrin clot formation: Intrinsic
and extrinsic pathway. In the case of a biomedical implant, the
presence of the biomaterial in the blood stream is believed to
trigger the intrinsic pathway while the surgical cut is believed
to trigger the extrinsic pathway [2]. Since the implant is
always present inside the blood vessels, there are many
stimulants in causing a clot formation.
Current techniques to prevent the formation of unwanted
clots involve the addition of anticoagulation factors onto the
surface of the biomaterial or through medications. Heperin,
coumadin, diypyramidole, ticlopidine, clopidrogel, reopro,
epifabitide, and tirofiban are many examples of the different
type of drugs that are currently available for clot preventions
[2]. However, some of these drugs are very expensive and
they have side effects. Since patients are often required to take
these medications on a regular basis, there are great demands
in finding a solution for the blood-clotting problem [2]. A
systemic approach in understanding the many different
procoagulation and anticoagulation factors would be beneficial
in applying the knowledge to biomaterials.
B. Implant-Tissue interface and bacterial biofilm
Biomaterials do not only interact with body tissues [3].
Infections caused by implant often leads to re-operation and
implant removal. For some implants, such as intravenous and
urologic catheters, have almost 100% infection rate after a few
days [2]. The reason why implants are so susceptible to
infections is that there is race between host body cell and
bacterial cell for surface adherence. In the presence of an
implant, the minimum bacterial concentration for infection
greatly decreases. This phenomenon is caused by the
formation of biofilms.
Fig. 5. Biofilm. The polysaccharide slime provides great protection for
the bacterial against body defence [2].
The slime layer, due to interactions with the biomaterial and
other bacterial species present, allows higher bacterial
metabolic activities inside the colonies. In some cases, the
biofilm acts as a “friendly” site for the transformation of nonpathogens or opportunistic pathogens into virulent organisms
[2]. Greater metabolic activity leads to more difficulties for
the body defence and promotes the detachment of colonies
towards nearby body tissues. This results in an inflammatory
response from body tissues surrounding an implant.
Although the mechanism is still unknown, there are some
relationships between biomaterial, organism and host location.
For instance, infected sites on metallic implants often contain
the s.aureus bacteria [2]. In order to prevent or minimize the
impact of bacterial infections, the current techniques involve
sterilizing the implant or applying antibiotic coatings [4].
Fig. 6. Effects of antibiotic drug coating.
Henry H.Y. Lo
Although current techniques can prevent infections to a
certain extent, there are many disadvantages. For instance,
current sterilization methods that use chemical, heat, or
radiations can degrade the biomaterial [3]. The chemical and
mechanical properties of a biomaterial can change because of
the sterilization operation. Antibiotic coatings have their
disadvantages too. It is difficult to select an appropriate
antibiotic due to the many types of bacteria on biofilms [4].
Furthermore, patients may develop antibiotic sensitivity and
the antibiotic may not reach all regions of the infected tissues.
Therefore, in order to fully understand the competition
between body cells and bacterial cells for the biomaterial
surfaces, it is necessary to understand the complex nature of
protein interactions among body tissues, bacterial cells, and
the biomaterial. This study is too complex to study as a whole.
Components have to be broken down and study separately in a
systemic matter. By integrating smaller systems into one
bigger system, it is then possible to develop better techniques
in protecting patients against implant infections.
C. Modeling of human bone mineral phase content
The following is a case of using modeling techniques in
predicting and stimulating bodily environment with
biomaterials. Hydroxyapatite, a calcium phosphate compound,
is being modeled for the development of bone substitutes.
Currently, there are many researches directed towards using
calcium phosphates as biomaterials [7]. Their chemical
composition, biocompatibility, and degradation characteristic
make calcium phosphates suitable for biomedical applications.
Bones mainly consist of protein (collagen Type I and II) and
apatite. Apatite is the mineral phase of bone and it mainly
consists of calcium, phosphate, and small amount of other
minerals. The chemical similarity of calcium phosphates to
the mineral component of bone is the main reason why calcium
phosphate compounds are very popular biomaterials.
Many previous material studies have used pure
hydroxyapatite for their research [7]. However, the chemical
and mechanical behaviour of pure hydroxyapatite turns out to
be different from that of the natural apatite. Recent studies
have suggested that the “impurities” in natural apatite (i.e.
carbonates, fluorides, other alkaline earth metal ions) play a
significant role in determining the properties of the material.
Because of this, a research group from Osaka University in
Japan is developing a computer model in understanding the
crystallographic effects of elemental substitution of
magnesium, strontium, and barium [8].
The computer and graphics program that the group used was
a modification of the “PROTGRA” and “MODRAST-E”
program. “PROTGRA” is a program used for protein
stereochemical structure indication. The modified program is
able to illustrate the crystallographic changes when other
alkaline earth metal ions were substituted inside the model.
For instance, the model can predict the lattice expansion at the
calcium site when strontium and barium were put in. The
results agree with the actual experimental results provided by
the group. Furthermore, when calcium sites were substituted
4
with magnesium ions, the program is also able to correctly
predict the absence of crystallographic contraction. The
absence of contraction is due to the interactive forces with the
neighbouring ions.
Based on the above research, it is clear that the above
program is still far from a complete model for the apatite
material in bone repair. However, there are other researches
aiming towards this goal. For instance, the three-dimensional
modeling of the bone structure can now be done using
computer aided design programs (CAD) [9]. As compared
with the past, images from computed tomography (CT) scans
could not be efficiently used for engineering applications such
as finite element modeling. The conversion from CT data to
CAD models was difficult and expensive [10]. A new
protocol, called TRI2SOLID, claimed to be a simple and
inexpensive way of utilizing the CT data. Although the
protocol has only been tested to work partially on the femur
bone, it is nevertheless an improvement in understanding the
nature of bones and a step towards the goal of bone modelling.
IV. CONCLUSION
The three cases mentioned above are just examples of the
vast number of research opportunities in the area of
biomaterials and system biology. The amount of expertise
required to solve any of these problems needs a crossdisciplinary approach.
A better understanding in the
biological response to biomaterials surfaces is required for the
development of better biomaterials and designs. The use of
system biology is the ultimate tool in achieving this goal.
REFERENCES
[1]
What is systems biology, Institute of Systems Biology, Seattle, WA.
[Online]. Available: www.systemsbilogy.org
[2] R. Pilliar, “MSE 452: Biomaterials and Biocompatibility Course
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1992
[4] J. Anderson, J. Langone, “Issues and perspectives on the
biocompatibility and immunotoxicity evaluation of implanted controlled
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295-304, 1996.
[6] W. Germann, C. Stanfield, Principles of Human Physiology, San
Francisco, CA: Benjamin Cummings, 2002, pp. 407-409.
[7] N. Porter, “Solid freeform fabrication of calcium polyphosphate:
material characterization and assessment of processing parameters,”
MASc. thesis, Dept. of Materials Sciences and Engineering, Univ. of
Toronto, Toronto, Canada, 1999.
[8] M. Okazaki, M. Sato, J. Takahashi, “Space-cutting model of
hydroxyapatite,” Biomaterial, 16, pp. 45-49, 1995.
[9] G. Marsh, “A hard case for modeling,” Materials Today, pp. 32-36, Oct
2002.
[10] M. Vicemonti, C. Zannoni, L. Pierotti, “TRI2SOLID: an application of
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211-220, 1998.