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University of Pittsburgh
ENGR 0012

Session C1 2245 EXTRACELLULAR MATRIX: A PROMISING BASIS OF TISSUE REGENERATION Katelynn Thomas ([email protected]), Oluyinka Olutoye ([email protected]) Abstract— Regenerative medicine, a medical practice In the human substances branch of regenerative engineering, focused on the regrowth of human tissue, is not widely human genes and proteins are engineered for use as drugs. studied or practiced but is one of the most spectacular “The proteins are generally made using recombinant DNA advancements in medical science [1]. One cutting edge technology, sometimes known as gene splicing. Genes are technique in the field is the use of extracellular matriceschemical instructions that enable a cell to make a specific (ECM) to regenerate tissue. ECM signals naturally present substance.” [3]. In this engineering, scientists extract human stem cells in a patient’s body to differentiate and multiplyenes and put them inside cells, giving the genes an once applied to a wounded area [2]. The makeup of the environment to replicate. While controlling the replication of ECM allows it to be a part of virtually every type of tissuehe cells containing the human gene, the scientists extract the healing and gives it the potential to affect every human desired protein that they need from the cells. This process is being in the near future. more desirable than obtaining the protein directly from a Not a lot is known about the use of ECM to regenerate human, because it limits the possibility of the transference of tissue since it is a fairly new concept. Scientists are workingases. Types of drugs made from processes like this are to find out why the ECM functions the way it does and how insulin and the human growth hormone. These substances they can manipulate it more toward the regrowth of full are used and are relative to many humans on a daily basis. limbs. While addressing the ethical concerns and the overall importance of the extracellular matrix, this paper will Embryonic Stem Cells examine the procedure of extracting and using the ECM powder to regenerate tissue, as it is a complicated and Embryonic Stem Cell research involves the extraction of sophisticated procedure involving many steps and much cells from embryos for use in regenerating tissue in other vigilance. This will be done by highlighting the benefits ofrganisms. A stem cell is a cell that has potential to the ECM that make it better than other controversial differentiate into any type of cell it is influenced into prospective treatments and presenting information gathered becoming. For example, if a stem cell is put in the midst of from both research and a direct interview with Dr. Badylak.muscle cells, it will become a muscle; if a stem cell is put in the midst of epithelial (skin) cells, it will become a skin cell. Although the use of stem cells for regeneration of tissue Key Words—amputees, bioengineering, extracellular matrix, is promising, it is an extremely controversial topic due to the regenerative medicine, scaffold fact that in some cases, embryos that have full potential to R EGENERATIVE M EDICINE become a human being would have to be aborted simply for their cells to be used in other organisms. Due to this Regenerative medicine is a relatively new field of researchcontroversy, stem cell research has not been able to thrive in the United States. “Yet the key discoveries that will enable it that focuses on the regrowth and engineering of cells in organisms. Scientists involved in regenerative medicine areto develop have already been made, so it will arrive in due breaking boundaries that seemed extremely distant or even course—if society permits” [3]. impossible not too many years ago. “Unlike most medicines Novel Materials today, regenerative medicines use human cells and substances to regrow tissue. Early forms are already in use. Some 30 drugs based on human proteins are approved for The novel materials engineering aspect of regenerative sale in the United States, as are several therapies that containeering involves the use of medical devices that do not include any types of human cells to perform various tasks in human cells. But today's protein and cell-based drugs are the body. These medical devices have been precisely merely the harbingers of what is to come.” [3]. In the individual types of bioengineering, scientists are making engineered to an atomic scale so that, when they are inserted strides to innovate and better the lives of humans. The into one’s body, they do not cause negative disruptions in the body, and the body’s cells do not detect them to be different types of regenerative engineering are: Human foreign bodies or attack them. Examples of novel materials Substances, Cells and Tissue, Embryonic Stem Cells, and Novel Materials [3]. that are being implanted into humans are steel and plastic hip and knee joints, synthetic heart valves, and blood Human Substances vessels. Cell and Tissue University of Pittsburgh Swanson School of Engineering April 14, 2012 1 Katelynn Thomas Oluyinka Olutoye Cell and Tissue Engineering evolved from reconstructive Hyaluronic acid also plays an important role in the surgery, the rebuilding of damaged body parts. As we function of ECM. It absorbs water and gives cells the ability become comfortable using human substances as medicines, to resist compression by its swelling force. It is abundant in we are also starting to use human cells as medicine. In type 2 the ECM around cells that bear a lot of wait, for example, regenerative medicine, cells will be removed from the body, cartilage in the knees and ankles. Hyaluronic acid also plays grown in culture, then reintroduced into patients.” [3]. a role as an environmental trigger by interacting with a Tissue engineering takes two forms. One involves building receptor on cell membranes called CD44 that signals for an organ or tissue outside the body by combining human cells to migrate. cells with appropriate materials, often a scaffold- like Collagen, Elastin, Fibronectin, and Lamnin are the structure to provide support. The other involves growing different types of fiber found in extracellular matrix. suitable cells in laboratory flasks, then injecting them into aCollagen is the most abundant protein in the ECM and gives tissue needing repair. The cells can often find their own way structure to resident cells while elastin gives tissues the to the sites where they are needed. This type of regenerative elasticity they need to perform their physiological functions. engineering has been the most successful thus far, after the For example, lungs need to expands and skin needs to be human substance branch of tissue engineering. Examples of able to stretch. Fibronectin allows cells to move and draw how tissue engineering is used are in the use of artificial skicollagen to the cells surface while lamnin gives ECM the for burn victims and the use of scaffolds as a structure on glue- like aspect it needs to help cells adhere to each other. which one can grow cells. The extracellular matrix is a Dr. Badylak, a bioengineer at the McGowan institute for major component of these decellularized scaffolds that are tissue regeneration, has used extracellular matrices to used in the regeneration. regenerate tissue. To do this, he uses ECM’s ability to use stem cells from the organism’s own body. Dr. Badylak E XTRACELLULAR M ATRIX extracts extracellular matrices from organisms and turns The extracellular matrix is the substance necessary for cell them into a powder that he places on wounded sites where stem cells are needed to come and differentiate into the growth in an organism. Rich with a variety of components, tissue what was present prior to the wound. ECM has many niches such as serving as a support structure for cells, separating different types of tissue and facilitating cell to cell communication during wound healing. The Decellularization and Preparation matrix often acts as glue between clusters of cells. The Decellularization of an organ or tissue is the removal of all extracellular matrix is mostly made up of macromolecules that are produced by the cells that are located within the cells and cellular contents from the sample leaving behind extracellular matrices as a scaffold on which new cells can matrix. As we will discuss later on, these cells also help to be grown. “Preservation of the complex composition and organize the matrix. The orientation of the cytoskeleton three-dimensional ultrastructure of the ECM is highly inside the cell can control the orientation of the matrix produced outside. Around most connective tissues, fibroblast desirable but it is recognized that all methods of decellularization result in disruption of the architecture and cells secrete macromolecules present in the matrix, however potential loss of surface structure and composition. Physical in certain specialized types of connective tissues, such as methods and chemical and biologic agents are used in cartilage and bone they are secreted by chondroblasts and osteoblasts respectively [4]. combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is Proteoglycans are one of the many components of the dictated by factors such as tissue density and organization, extracellular matrix. Proteoglycans are a result of geometric and biologic properties desired for the end glycosaminoglycan carbohydrates that attach to extracellular matrix proteins. The negatively charged proteoglycans product, and the targeted clinical application. [5]” There are varieties of decellularization methods varying on the type of attract sodium ions that, in turn, attract water that has the tissue or organ sample being decellularized or the desired ability to trap cellular growth factors. Therefore, use of the decellularized sample. proteoglycans are the component of ECM that contains “The most effective agents for decellularization of each cellular growth factors, which are major factors in the cell tissue and organ will depend upon many factors, including nutrition that is necessary for regeneration. the tissue’s cellularity (e.g. liver vs. tendon), density (e.g. The different types of proteoglycans in ECM are Heparin Sulfates, Chondroitin Sulfates and Keratin Sulfates. The dermis vs. adipose tissue), lipid content (e.g. brain vs. urinary bladder), and thickness (e.g. dermis vs. Heparin Sulfates are involved in blood coagulation and pericardium)” [5]. Whenever samples are decellularized angiogenesis (growth of new blood vessels in place of ECM composition is altered and the structure disruption, so previous blood vessels), and Chondroitin Sulfates are the proteoglycans that supply tensile strength to cartilage, when decellularizing, one is to chose a method that ill minimize these negative effects. allowing ECM to be a major source of cell support. Tissue can be sterilized by use of chemicals, biological substances, or physical techniques. Some chemicals used to 2 Katelynn Thomas Oluyinka Olutoye decellularize samples are acids, bases, detergents and alcohols. Acids and bases break down nucleic acids while The way that the extracellular matrix works is not fully detergents dissociate DNA from proteins and alcohols are understood, but researchers are working towards finding the used to lyse (burst) cells. answers. It is known that the ECM recruits the stem cells of Nuclease, Collagenase and Trypsin are three types of the body of the patient, but it is not certain how it does this. enzymes used to decellularize biologically. Nucleases cleave A study has shown that the ECM releases bioactive peptides nucleic acids that are left over after cells have lysed. Trypswhen it is degraded by protease to recruit cells known as is sometimes used to decellularize, but is not used often for progenitor cells [6]. The structural support of the ECM is it is extremely disruptive to ECM structure, because it what aids in the growth of the cells once they are recruited. breaks down the ECM that is trying to be isolated as well as Also, the physical and chemical conditions of the ECM the unwanted substances. Collagenase is used only when one provide a preferred environment for tissue to grow [7]. This does not mind losing a lot of the ECM along with the effectively tricks the body into regenerating rather than unwanted substances, because like trypsin, it breaks down scarring [2]. more than we would like it to. C URRENT STUDIES Physical techniques used to decellularize cells are used more often with full organs such as urinary bladders, small Many of the scientific studies involving the extracellular intestines and skin. These techniques involve extreme temperatures (freezing) and scraping to remove cells. matrix are currently being performed so the information known is preliminary. A study by the McGowan Institute for “Freeze-thaw processing effectively lyses cells within Regenerative Medicine and the University of Pittsburgh tissues and organs, but the resulting membranous and Department of Surgery is focusing on the healing process of intracellular contents remain unless removed by subsequent processing. A single freeze-thaw cycle can reduce adverse two different strains of mice in order to determine how regeneration is likely to occur. In this study sixty mice were immune responses such as leukocyte infiltration in vascular divided into fifteen groups. Each mouse underwent surgery ECM scaffolds. Multiple freeze-thaw cycles may be used to amputate the third digit of their right rear paw at the during decellularization and do not significantly increase the loss of ECM proteins from tissue [5].” Scraping is also an middle of the second phalanx. The wounds were then allowed to heal naturally for fourteen days. Every day one effective technique for tissue decellularization when group of the mice was euthanized so that the paw could be accompanied with the use of enzymes, hypertonic saline, or chelating agents that all help remove cells from their removed and observed. To be prepared for observation, the paw was submerged in a buffer for forty-eight hours and membranes. The use of hydrostatic pressure is one of the then submerged in formic acid to be decalcified. After this most effective techniques of tissue decellularization for it process, the paw could be stained and observed [8]. does not take much time and is more successful than techniques involving enzymes and detergents. The only con The main goal of this stain was to determine the ratio of collagen type I and III. On the first day, it was observed in to the use of hydrostatic pressure is degradation of the ECM both strains of mice that a blood clot and inflammatory cell by ice crystals that form, but this degradation can be infiltrate had formed, but a new epithelial layer had not prevented by increasing the temperature of the fluid that is
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