Tissue Engineering Red Blood Cells

MATSE 403
Matthew Niesslein Jeffrey Zugates Haodi Wu

Red Blood Cell Function & Physiology

Red blood cell (RBC) function is essential to respiratory and circulatory operation within the human body. They are the most abundant type of blood cell and account for approximately one third of all cells in the human body. As they move through these two systems, red blood cells interact with endothelial cells and alveoli in order to fulfill their purpose. Primarily, the function of red blood cells or erythrocytes is to deliver oxygen and release carbon dioxide to and from the body, respectively. The heart pumps deoxygenated blood to the lungs and oxygenated blood to the entire body. The deoxygenated blood contains RBCs carrying carbon dioxide. These cells navigate through the capillary beds of the lungs. The red blood cells then exchange the carbon dioxide with oxygen, which the alveoli has acquired though gas exchange with the lungs. RBCs exiting the capillary beds now contain oxygen, and are pumped back to heart, where it can make its way throughout the rest of the body. This function does not come without its share of complexity. The cells change shape as they move from the arteries to the tiny capillary beds surrounding the cells. A RBC undergoes intense mechanical stresses as it circulates and navigates these channels. RBCs are subject to high pressure when pumped out of the heart and are forced into the blood vessel walls and collide with neighboring cells. There is no repair mechanism for red blood cells. Their lifespan is relatively short, and requires constant production of new cells.

Figure 1: Exchange of Carbon Dioxide across Red Blood Cell and Alveoli

The formation process of red blood cells is known as erythropoiesis which occurs only in the red bone marrow or myeloid tissue (1). Erythropoiesis continually replaces new cells: a rate of 3 million new cells entering the bloodstream per second (1). This process is regulated by the hormone erythropoietin. This cytokine is released by the kidneys when low oxygen levels are detected in the blood (6). Erythropoietin, often referred to as EPO, initiates the production and differentiation of new hemacytes. This hormone is often misused in athletics for its performance enhancing attributes (6). The red blood cell structure is among the most specialized cells in the body with a thin central region and a thicker outer margin. Figure 2 illustrates the membrane of a red blood cell. The biconcave disc structure affects the cell function in three significant ways. It provides each cell with a large surface area-to-volume ratio, enables the cells to form stacks for better flow properties, and confers flexibility in order to enhance transport through narrow capillaries (1). These characteristics of RBCs permit their function to go unhindered throughout the body. By allowing the membrane and structure of the cell to distort and become flexible in miniscule cellular beds, the cell then has the most surface area in contact with the endothelial cells.

Figure 2: Side and Top View of a Red Blood Cell The surface area-to-volume ratio is a crucial factor for oxygen transport and release to peripheral tissues. The greater the surface area per unit volume, the faster the exchange of oxygen from the red blood cell interior to the surrounding plasma. The protein responsible for the absorption and release of oxygen is hemoglobin. Hemoglobin accounts for more than 95% of a red blood cells intracellular proteins (1). A hemoglobin molecule contains four globular

protein subunits. Each subunit is comprised of a polypeptide chain. The hemoglobin molecule contains two alpha chains and two beta chains (Figure 2a). The hemoglobin chains each enclose a non-protein pigment complex called heme (Figure 2b). The heme unit has a bound iron ion in the ferrous state (Fe+2) that can bind reversibly with an oxygen molecule. The binding and dissociation of oxygen from the iron ion in heme is the mechanism for oxygen transport in the body.

Figure 3: (a) Hemoglobin Structure, (b) Heme Structure The iron-oxygen interaction is weak which allows for oxygen to easily dissociate from the heme unit without causing any damage to the oxygen or heme molecules. The amount of oxygen bound to hemoglobin depends on the concentration of oxygen in the plasma. Peripheral capillaries contain high levels of carbon dioxide, and this elevated level increases the concentration of carbon dioxide in the plasma. Therefore, oxygen levels are low and oxygen dissociates from hemoglobin. Carbon dioxide will bind to the alpha and beta chains of hemoglobin forming carbaminohemoglobin (1). The hemoglobin structure is in the taut conformation or T state and the heme iron atoms have a low binding affinity for oxygen (2). In the capillaries of the lung, the plasma oxygen content is high and carbon dioxide levels are low. When RBCs reach these capillaries, oxygen is absorbed, bound to hemoglobin, and carbon dioxide is released. Hemoglobin is in the relaxed configuration or R-state and the iron atoms have a high binding affinity for oxygen (2). Any structural defects of red blood cells will affect the outcome of oxygen transfer and can result in life threatening conditions. The types of problems associated with red blood cells include thalassemia, polycythemia, sick cell anemia, and malaria.

Red Blood Cell Pathology

There are several inherited disorders that cause an abnormal production of hemoglobin. Three of the most common disorders are thalassemia, sickle cell anemia, and polycythemia. Thalassemia results from an inability to produce the necessary amounts of alpha or beta polypeptide chains of hemoglobin (1). The genes that code for the production of alpha and beta chains are either deleted or mutated. This leads to a deficiency in the amount of subunits, and thus lower counts of hemoglobin (7). Without the hemoglobin, anemia develops from the lack of oxygen exchange. Also, the rate of red blood cell formation is much slower, and the mature red blood cells are fragile and short lived. The oxygen carrying capacity also diminishes which leads to developmental and growth related problems for tissues throughout the body. The disease is named alpha thalassemia or beta thalassemia, depending on which of the two proteins contain the defect (7). The altered chains lead to misshapen subunits, causing the inability of oxygen to bind with the heme group. Immune and apoptosis responses can lower the hemoglobin concentration, thus lowering the hemocrit. As of 2005, more than seven percent of the world population is carriers of thalassemia (9). This statistic is increasing, with 400,000 babies inheriting the disease each day (9). The Asian Indian community is susceptible to the disease. In fact, there is a 25 percent chance that the disease will be passed on to their children (9).

Figure 4: Depiction of Thalassemia Disease Inheritance Sickle cell anemia results from a mutation in the amino acid sequence that code for the beta polypeptide chains of the hemoglobin molecule. The mutated hemoglobin molecule is called Hemoglobin-S and this mutated form changes the RBC structure when oxygen levels are low. The

Hemoglobin-S molecule has a glutamic acid residue instead of valine which decreases hemoglobin solubility under low oxygen concentrations (3). The RBCs are deformed because of this defect in the hemoglobin. The hemoglobin can form long strands that attach the cell wall, pull on the membrane, and deform the cell. When Hemoglobin-S releases oxygen into the tissues, the abnormal hemoglobin sticks together and forms clumps. These clumps cause red blood cells to stiffen and distort the cellular structure forming a sickle or crescent shape (Figure 3). The fragile, sickle-shaped cells are unable to transport oxygen effectively as the normal disc-shaped cells. The crescent-shaped cells can get stuck in smaller blood vessels and can break into pieces interrupting healthy laminar blood flow (4). When this blocking occurs, the patient can experience intense episodes of pain ranging from hours to days. Each person diagnosed has these episodes, whether they occur on a yearly or monthly basis. These sickle blood cells do not last very long due to their defect, and result in lower RBC count than normal. Blood transfusions or altering the protein is required in order to control this disease. Sickle cell anemia afflicts many people. In the United States, 1000 babies are born with the disease every year. The rates are much higher in Africa because people of African and Arab descents are more susceptible due to genetic coding. Nigeria reports that 45,000-90,000 babies are born every year with the disease. Twenty five percent of Africas population has the disease and less than 1% of Americans have the disease. However, about 8% of Americas African American population possesses the sickle cell trait. There has been more research effort in sickle cell anemia since it is one of the most common blood disorders in the United States. However, no overall cure has been discovered.

Figure 5: Sickle Cell Anemia: Comparison of Normal and Abnormal Red Blood Cells Polycythemia is a blood disorder that causes the overproduction of red blood cells. This leads to a thicker, more viscous blood. There are two types of polycythemia; polycythemia vera (PV) and primary familial and congenital polycythemia (PFCP). PV is caused by an over expression of the JAK2 gene due to a mutation which is essential in erythropoietin. This type is also known as genetic polycythemia, and only presents in older adults. Every year, around 200,000 individuals are diagnosed

with this ailment. PFCP occurs when the EPOR gene is mutated, and produces too many RBCs. The thickened blood is risky, as it increases the chance of heart attacks, strokes, and several other effects. Thalassemia, sickle cell anemia and polycythemia are genetically derived conditions that affect red blood cell function. However, there are many viruses and parasites that infect red blood cells. A common parasitic disease transmitted through mosquitoes is malaria. The malaria parasite is known as Plasmodium falciparum and the parasite invades red blood cells (5). Once the parasite is within the cell, it begins to multiply until the cell bursts open and parasites are released into the blood stream. P. falciparum causes the RBC surface to become sticky due to the parasites own adhesion proteins. The altered surface will stick to neighboring cells which causes the blood to clot in small vessels (5). Figure 4 shows an SEM image of an infected red blood cell (orange) surrounded by healthy red blood cells adhering to its surface. Due to the necessity and important functions of the red blood cell, much research has gone into the subject.

Figure 6: SEM Image of Healthy Red Blood Cells Adhering to Malaria-Infected Cell

Red Blood Cell Research

The first tissue engineering procedure was performed in 1970 by an orthopedic surgeon by infusing chondrocytes with a bone spicule. This is the part of a bone that is responsible for cell growth and mass cell division. Unfortunately the research was unsuccessful and did not take. ESC, or embryonic stem cells have an amazing characteristic, they are undifferentiated. This means that they do not have become a special cell: blood cell, osteocyte, neuron, epithelial cell, or myocyte. This is useful, as researchers can purchase these specialized cells and differentiate them to the desired cell type. In fact, research was conducted at the Mayo Clinic in Rochester, Minnesota using ESCs to make red blood cells (8). The lab investigated the possibility of creating blood type O cells from the ESCs. This is the universal donor blood type. And with only 10 % of the population having this blood type would enable blood shortages to be a thing of the past.

More research is being conducted on hemoglobin, the active metalloprotein in RBCs and the primary carrier of oxygen and carbon dioxide. Without this protein, oxygen would not be able to be picked up in the lungs and carried to the capillary beds, as carbon is transported from cells and carried away. This hemoglobin is extracted from RBCs and is processed by cross linking or encapsulation (8). This allows for the removal of hepatitis, HIV and other pathological defects in the protein by using different sterilization techniques; filtration, pasteurization and chemical processing (8). And with HIV plaguing the population of the world, cries for cures and deterrents have exploded in the research community. Once the hemoglobin is removed from the blood, cross linked, and collected, it is studied on the spot or stored for use in future trials. Over the years of research, it has shown that by minimizing the degree of linking and encapsulation of this now modified hemoglobin has shown to lead to the best results. If the modified hemoglobin has many connections to each other, stronger and more destructive reagents are needed for the hemoglobin to return to its natural shape. This has shown to be ineffective, and one of the limiting factors in the process. Also, there is a requirement for 2,3-diphosphoglycerate (2,3 DPG) to be present in the 2,3-DPG binding site of hemoglobin. The presence of 2,3-DPG decreases the binding affinity for oxygen and allows oxygen to dissociate from hemoglobin (10). If modified hemoglobin is infused into the body without 2,3-DPG, then oxygen will not be easily released into the tissues. Another problem with releasing free modified hemoglobin into the bloodstream is toxicity. Each hemoglobin molecule degrades into dimers which are highly toxic to the kidneys and could affect renal function (10). The challenge lies in altering the biomaterial, hemoglobin, before it can be utilized as an effective blood substitute. Being a thirty year old research topic, modified hemoglobin has made a lot of headroom, and is continuing on its path. Currently, a version of hemoglobin modification is practiced in South Africa, and is in Phase III trials within the United States. The research is leading towards use in a variety of surgeries, some being trauma, cancer, cardiopulmonary bypass, and cardioplegia (8). And the greatest, if not most exciting point of the research is the fact the hemoglobin is universal and can be stored at room temperature. This ability for hemoglobin to be accepted by any individual arises from the fact that hemoglobin is a protein structure, and not an entire cell. Cells contain agglutinins that trigger immune responses. This is ideal for the trauma situation. A surgeon would be able to go to the shelf, grab the mixture, add a salt solution, and deliver the cells to the patient as an intravenous solution. Unfortunately, a product must undergo years of thorough research and development and yield positive results in clinical trials before use in patients. Hopefully, investigators will develop a RBC product that will be as effective as hemoglobin and be available for routine use.

References
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Images Figure 1 Photograph. The Circulatory, Respiratory, and Digestive Systems. Fall. 2004. <http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect20.htm>. Figure 2 Photograph. Blood: RBCs. Web. 26 Nov. 2011. <http://legacy.owensboro.kctcs.edu/GCaplan/anat2/notes/APIINotes6%20Blood%20RBC.htm>. Figure 3 Photograph. BaileyBio.com. 19 Mar. 2008. Web. 26 Nov. 2011. <http://www.baileybio.com/plogger/?level=picture&id=490>. Figure 4 Photograph. Stjude.org. < http://www.stjude.org/Images/hosp-hem-alpha01-0902.jpg>. Figure 5 Photograph. United Health Directory. Eye Site Media. Web. 26 Nov. 2011. <http://www.unitedhealthdirectory.com/diseases-and-conditions/sickle-cell-anemia/>. Figure 6 Photograph. Science Photo Library. Web. 26 Nov. 2011. <http://www.sciencephoto.com/media/259781/enlarge>.