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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 3 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 Notes”, Sept. 2003, unpublished. [3] J. Park, R. Lakes, Biomaterials: An Introduction, 2nd Ed., Plenum Press, 1992 [4] J. Anderson, J. Langone, “Issues and perspectives on the biocompatibility and immunotoxicity evaluation of implanted controlled release systems,” Journal of controlled release, 57, pp. 107-113, 1999. [5] M. Griffiths, J. Langone, M. Lightfoote, “Biomaterials and Granulomas,” Methods: A companion to methods in enzymology, 9, pp. 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 reverse engineering methods to the creation of CAD models of bone segments” Computer methods and programs in biomedicines, 56, pp. 211-220, 1998.