(The above diagram encompasses most of the external factors that affect stem cells in our body.) A stem-cell niche is an area of a tissue that provides a specific microenvironment, in which stem cells are present in an undifferentiated and self-renewable state. Cells of the stem-cell niche interact with the stem cells to maintain them or promote their differentiation. We can see that the niche, as we call it, is very complicated and multi-faceted. If we understand the environment of the stem cell we can learn how to make procedures work more efficiently. This is easier said than done. We must realize that when we study the niche it is done in the lab (in vitro) and the results in the lab may not match what happens in real life (in vivo). Let’s break down the Niche components one at a time. The first aspect we will discuss is hypoxia and metabolism. Hypoxia, or a diminished amount of oxygen, is one of the most significant environmental factors affecting cells in many different ways. Hypoxia plays an important role in different aspects of cell chemistry such as metabolism, migration, proliferation, differentiation and, apoptosis (cell death). Hypoxia works thru many different elements such as hypoxia-inducible factors (HIFs). These are various growth factors that are produced by hypoxic conditions. HIFs are master transcription factors. This means that they mediate various events in cells. In molecular biology, a transcription factor is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA. More than 1000 genes are targets of HIF, regulated directly or indirectly by it. For example, transcription factors, enzymes, receptors, and membrane proteins can be induced or suppressed by hypoxia. Mesenchymal (MSCs) and Hematopoietic (HSCs)stem cells are important components of the bone marrow niche. The bone marrow niche is a state of physiological hypoxia. In another word, it is low in oxygen. Typically, when we harvest bone marrow aspirate we like to keep in hypoxic conditions. This may increase the survivability of the cells when they are reinjected into the body. This may be achieved by putting the aspirate in a vacuum container. In regards to metabolism, some substances are broken down to yield energy for vital processes while other substances, necessary for life, are synthesized. In contrast to differentiated cells (which use oxygen in a respiration system like we are familiar with), many stem cells appear to rely to a greater extent on glycolysis than on oxidative phosphorylation to generate adenosine-5ʹ-triphosphate (ATP). Glycolysis is an oxygen-independent metabolic pathway. Unlike oxidative phosphorylation (the common form of respiration achieved by breathing air), glycolysis can proceed anaerobically (without oxygen). This raises the possibility that the dependency of a stem cell on glycolysis is an adaptation to the low oxygen levels that are present in the body during development and in an adult stem cell microenvironment or ‘niche’. What this really means is that the stem cells are able to survive on very low concentrations of oxygen mainly because they do not have to use oxygen. Realize that when we utilize oxygen for metabolism we will create a byproduct called a reactive oxygen species (ROS). A ROS is more commonly known as a free radical. ROSs can cause damage to the cells in a variety of ways. They cause damage on a variety of levels. The shift toward glycolysis might minimize the production of reactive oxidative species (ROS), which could indirectly affect stem cell function. As an interesting aside, another aspect of anaerobic glycolysis is that it is energy system of choice for high- intensity activities that last from 30 seconds to about three minutes. This occurs in athletes. This system has 11 chemical reactions that incompletely metabolize glucose to provide ATP and a byproduct of metabolism that causes acute muscle soreness, namely lactic acid. We can also see that what affects the “stemness” of a cell is the type of respiration it utilizes. The diagram below is very similar to the first diagram that I demonstrated. It is a bit different. We see that there are a variety of components to the niche. These components act as a symphony orchestra. They are all important cogs in the function of the niche. The cellular components are very important to stem cell function. These components can have a direct effect on the cell. They will produce certain biochemical compounds which are called cytokines. These cytokines are a method in which cells communicate with each other. There are a few different ways that the cells communicate with each other. I have included a slide from one of my talks that discusses cellular communication. In this case, the effect can be any of the “crines” of cellular communication. These “crines” are found in a number of different compounds such as hormones, different types of growth factors, and exosomes. They will have either a direct or indirect effect on the cells. Many of these effects are mediated by the secreted factors. Some of these secreted factors can affect the production of messenger RNA. The messenger RNA can turn on certain genes in the cell that was otherwise “turned off”. By turning on certain genes we are able to have cells possibly start doing repair work. I cannot stress the importance of these secreted factors. They can lead to success or failure. The problem we sometimes run into is that the amounts and the actual secreted factors may be less than needed as we age. This may explain why as we age it takes us longer to get over injuries and illnesses. Another important component in the niche is the inflammation and scarring component. This portion of the niche (first diagram) also involves certain cells. These are very specialized cells called macrophages and T cells. They are intimately involved in our immune system. Our immune system and stem cells go hand in hand. Macrophages can be broken down into two different types. There is the M-1 and M-2 macrophage. The M-1 macrophage will lead to inflammation while the M-2 can lead to a reduction of inflammation. The diagram below further explains this: This diagram has many different things going on at once. In the middle of the diagram, we see a stem cell. Depending on the cells, environment, and other growth factors in the niche we can see a variety different effects. Some of these effects will be the formation of various autoimmune diseases courtesy of the T cells (think Rheumatoid Arthritis). While the macrophages under the right circumstances can help the release of T-Reg cells which will help eliminate or prevent autoimmune diseases. These effects are not just esoteric findings but have practical implications for real-world medicine. Remember, the cells in our body ultimately achieve the goal of repair. Where and which cells are present and active can cause or prevent serious problems. The next component of the niche deals with the extracellular matrix. Most cells release materials into the extracellular space, creating a complex meshwork of proteins and carbohydrates called the extracellular matrix (ECM). A major component of the extracellular matrix is the protein collagen. Collagen proteins are modified with carbohydrates, and once they're released from the cell, they assemble into long fibers called collagen fibrils. In the extracellular matrix, collagen fibers are interwoven with a class of carbohydrate-bearing proteoglycans, which may be attached to a long polysaccharide backbone. The extracellular matrix is directly connected to the cells it surrounds. Some of the key connectors are proteins called integrins, which are embedded in the plasma membrane. Integrins anchor the cell to the extracellular matrix. In addition, they help it sense its environment. They can act as the eyes and ears of the cell. They can detect both chemical and mechanical cues from the extracellular matrix and trigger signaling pathways in response. The integrin concept is very scientific but the concept is very important. The last niche component to address concerns the physical factors and their effects on the niche. In addition to the influence that an artificial ECM may have on cell shape, there is significant evidence that other physical properties of the ECM may also contribute to stem cell fate or lineage commitment. Cells that attach to a substrate have been shown to exert contractile forces, resulting in tensile stresses in the cytoskeleton. Interestingly, the relationship between these forces and the mechanical stiffness, or elasticity, of the ECM can have a major influence on cell behaviors such as migration, apoptosis (cell death), and proliferation. This represents a bit of a tour of the stem cell niche. Although, I have presented this in simplistic terms the niche is a very complicated environment which is getting more and more scrutiny. Is this the most important aspect of stem cell science? Probably not but it is difficult to say what is the most important aspect. Thanks, Dr. P

Extracorporeal Shock Wave Therapy (ESWT) is something that has been around for a while but it seems to be having a renaissance. ESWT has been proven as an effective and safe non- invasive treatment option for tendon and other pathologies of the musculoskeletal system. I many times get the questions from patients as to what effect the “electric shock wave” will have on the body? We must realize that extra corporeal shock wave therapy has nothing to do with electricity. There is nothing mystical or cryptic about a shockwave therapy. It is nothing more than a sonic boom or sound wave. When a shockwave enters tissue, it may break up and reflect the absorption of kinetic energy by the precise body structures (bone, fat, tendon, ligaments), which are exposed to the shockwave. All techniques of shockwave production (electrohydraulic, electromagnetic and piezoelectric) depend on the conversion of electrical energy to mechanical energy. When a sound wave is transmitted into tissue, there are two levels of transmission: low energy and high energy. Low energy has an analgesic effect by either disrupting the cell membranes partially or completely. When high energy (any energy greater than 0.28mJ/mm2) comes in contact with the damaged tissue, there is a direct biological interaction. The following diagram gives us a better idea of what we speculate some of the mechanisms of action of ESWT might be.I will discuss these aspects later in the article.In recent years much research has been done in Europe and other portions of the world. ESWT continues to take on increasing importance in the stem cell field. The reason for this are many faceted. For instance, there are many different aspects of ESWT has on various stem cells, their environment, and their ultimate destination. As an orthopedic surgeon, I used this therapy a number of years ago for both tennis elbow and heel spur syndromes. Not only did I use it but I had good results with it. It fell out of favor due to the fact that it was not reimbursed by the insurance industry.A few years ago, I became aware of ESWT again for its use in sports medicine and musculoskeletal problems. I think we are now understanding better the true mechanisms of how this is working. When I was lecturing in Brazil some time ago I met one of the worlds experts on shock wave therapy, a professor from a university in Munich Germany. We had quite a good discussion concerning the use of shock wave therapy in stem cell and PRP therapy. At that meeting, I had an epiphany when I realized that years ago when I used this technique I was actually creating an environment very conducive to stem cells. I go back to one of the basic concepts in Regenerative Medicine: “Cells not Doctors Heal Patients”. There were some surprising facts concerning the of the shock wave machines. They create a momentarily pressure that would be equivalent to an ocean depth of approximately 2000 ft. Luckily this pressure only lasts a fleeting moment. Under most conditions this would cause immediate destruction of the tissue and probably death. The machine works on a principle of two metal objects hitting each other at a speed of 50 miles per hour. This is the inner workings of the machine. It works on an air gun principle making one piece of metal hitting into another at the speed of fifty miles per hour. This creates a shock wave that is transferred to the tissue. AS I have previously mentioned there are also other types of machines generate shock waves in different fashion. But the machine I just mentioned is the type I am most familiar with and the one I use.What the shock wave actually does is create a bit of trauma to the area. This trauma also releases growth factors. In some respects, this is similar to those physicians that are proponents of Prolotherapy or for that matter PRP therapy. In Prolotherapy, some type of irritant such as high concentrations of dextrose (essentially sugar water) is injected into a tendon area or a joint. The idea is that the irritating solution will cause an inflammation. The inflammation will elicit certain cells in our immune system to secrete certain growth factors which attract stem cells. We now have the cascade of healing.These growth factors are what seems to begin the cascade of healing. When ESWT is performed it releases among other factors Vascular Endothelial Growth Factor (VEGF). VEGF is responsible for creating a vascular supply where there typically the supply is deficient. The lack of a vascular supply is what helps to cause many problems such as tendinosis. Tendinosis is a chronic problem that the body has given up on healing. Tendinitis is more of an acute problem. It is also thought that the ESWT releases certain growth factors that enable stem cell homing to a certain area. Homing is the principle where stem cells have a beacon to injured areas. This has been demonstrated by some fairly nifty experiments. In these experiments, stem cells were tagged with a tracer and were then injected into the body via an intravenous route. It was found that the cells seemed to congregate to the areas where the shock wave therapy was performed. Another growth factor produced is (PCNA) Proliferating Cell Nuclear Antigen. PCNA can help the cell repair its DNA etc.Another important aspect of shock wave therapy is the formation of Nitric Oxide. Actually, shock wave therapy produces an enzyme which is responsible for the formation of Nitric Oxide. This enzyme is called eNOS or endothelial Nitric Oxide synthase. Among other things eNOS is the mechanism by which hyperbaric oxygen has its success. The eNOS stimulates the formation of Nitric Oxide in the bone marrow. This Nitric Oxide than stimulates the bone marrow to release large numbers of stem cells into the circulation. The eNOS will also stimulate the formation of blood vessels in tissues where there seems to be a lack of blood flow. This is very typical of an area of injury such as a chronic tendinosis. We are now starting to realize that Nitric Oxide (NO) is one of the holy grails of stem cell therapy. NO has many different tasks in the body. It depends somewhat on what “neighborhood” in the body we find the Nitric Oxide in. The neighborhood effects its actions. We now know that NO is an extremely important signaling molecule in the body. It is known to be a growth, immune, and neuromodulator as well as a stimulator of stem cell proliferation. It has critical roles in analgesia, vasodilation and ATP production. NO release in the body is caused by many different factors. When we start combining shock wave therapy with laser therapy than we may stimulate significant amounts of NO. Laser therapy is similar to shock wave therapy in that it seems to have profound effects on the cell environment. Laser therapy will be the subject of another article.By utilizing shock wave therapy with the some the regimens that we already have hopefully many good things will happen. Using shock wave therapy with Platelet Rich Plasma (PRP) and or Stem cells will dramatically improve results.I do not know if shock wave therapy itself will be total game changer but I do know it will be an increasingly important part of our ever-expanding armamentarium of treatments. I am sure there will be more to come concerning this modality. I believe this will become an increasingly important aspect in the utilization of PRP and Stem cell therapy in the treatment of hip, knee, spine and other musculoskeletal problems. Thanks, Dr. P.“TOLD YOU IT WASN’T WHAT YOU THOUGHT IT WOULD BE”- DR.P

Extracorporeal Shock Wave Therapy (ESWT) is something that has been around for a while but it seems to be having a renaissance. ESWT has been proven as an effective and safe non- invasive treatment option for tendon and other pathologies of the musculoskeletal system. I many times get the questions from patients as to what effect the “electric shock wave” will have on the body? We must realize that extra corporeal shock wave therapy has nothing to do with electricity. There is nothing mystical or cryptic about a shockwave therapy. It is nothing more than a sonic boom or sound wave. When a shockwave enters tissue, it may break up and reflect the absorption of kinetic energy by the precise body structures (bone, fat, tendon, ligaments), which are exposed to the shockwave. All techniques of shockwave production (electrohydraulic, electromagnetic and piezoelectric) depend on the conversion of electrical energy to mechanical energy. When a sound wave is transmitted into tissue, there are two levels of transmission: low energy and high energy. Low energy has an analgesic effect by either disrupting the cell membranes partially or completely. When high energy (any energy greater than 0.28mJ/mm2) comes in contact with the damaged tissue, there is a direct biological interaction. The following diagram gives us a better idea of what we speculate some of the mechanisms of action of ESWT might be. I will discuss these aspects later in the article. In recent years much research has been done in Europe and other portions of the world. ESWT continues to take on increasing importance in the stem cell field. The reason for this are many faceted. For instance, there are many different aspects of ESWT has on various stem cells, their environment, and their ultimate destination. As an orthopedic surgeon, I used this therapy a number of years ago for both tennis elbow and heel spur syndromes. Not only did I use it but I had good results with it. It fell out of favor due to the fact that it was not reimbursed by the insurance industry. A few years ago, I became aware of ESWT again for its use in sports medicine and musculoskeletal problems. I think we are now understanding better the true mechanisms of how this is working. When I was lecturing in Brazil some time ago I met one of the worlds experts on shock wave therapy, a professor from a university in Munich Germany. We had quite a good discussion concerning the use of shock wave therapy in stem cell and PRP therapy. At that meeting, I had an epiphany when I realized that years ago when I used this technique I was actually creating an environment very conducive to stem cells. I go back to one of the basic concepts in Regenerative Medicine: “Cells not Doctors Heal Patients”. There were some surprising facts concerning the of the shock wave machines. They create a momentarily pressure that would be equivalent to an ocean depth of approximately 2000 ft. Luckily this pressure only lasts a fleeting moment. Under most conditions this would cause immediate destruction of the tissue and probably death. The machine works on a principle of two metal objects hitting each other at a speed of 50 miles per hour. This is the inner workings of the machine. It works on an air gun principle making one piece of metal hitting into another at the speed of fifty miles per hour. This creates a shock wave that is transferred to the tissue. AS I have previously mentioned there are also other types of machines generate shock waves in different fashion. But the machine I just mentioned is the type I am most familiar with and the one I use. What the shock wave actually does is create a bit of trauma to the area. This trauma also releases growth factors. In some respects, this is similar to those physicians that are proponents of Prolotherapy or for that matter PRP therapy. In Prolotherapy, some type of irritant such as high concentrations of dextrose (essentially sugar water) is injected into a tendon area or a joint. The idea is that the irritating solution will cause an inflammation. The inflammation will elicit certain cells in our immune system to secrete certain growth factors which attract stem cells. We now have the cascade of healing. These growth factors are what seems to begin the cascade of healing. When ESWT is performed it releases among other factors Vascular Endothelial Growth Factor (VEGF). VEGF is responsible for creating a vascular supply where there typically the supply is deficient. The lack of a vascular supply is what helps to cause many problems such as tendinosis. Tendinosis is a chronic problem that the body has given up on healing. Tendinitis is more of an acute problem. It is also thought that the ESWT releases certain growth factors that enable stem cell homing to a certain area. Homing is the principle where stem cells have a beacon to injured areas. This has been demonstrated by some fairly nifty experiments. In these experiments, stem cells were tagged with a tracer and were then injected into the body via an intravenous route. It was found that the cells seemed to congregate to the areas where the shock wave therapy was performed. Another growth factor produced is (PCNA) Proliferating Cell Nuclear Antigen. PCNA can help the cell repair its DNA etc. Another important aspect of shock wave therapy is the formation of Nitric Oxide. Actually, shock wave therapy produces an enzyme which is responsible for the formation of Nitric Oxide. This enzyme is called eNOS or endothelial Nitric Oxide synthase. Among other things eNOS is the mechanism by which hyperbaric oxygen has its success. The eNOS stimulates the formation of Nitric Oxide in the bone marrow. This Nitric Oxide than stimulates the bone marrow to release large numbers of stem cells into the circulation. The eNOS will also stimulate the formation of blood vessels in tissues where there seems to be a lack of blood flow. This is very typical of an area of injury such as a chronic tendinosis. We are now starting to realize that Nitric Oxide (NO) is one of the holy grails of stem cell therapy. NO has many different tasks in the body. It depends somewhat on what “neighborhood” in the body we find the Nitric Oxide in. The neighborhood effects its actions. We now know that NO is an extremely important signaling molecule in the body. It is known to be a growth, immune, and neuromodulator as well as a stimulator of stem cell proliferation. It has critical roles in analgesia, vasodilation and ATP production. NO release in the body is caused by many different factors. When we start combining shock wave therapy with laser therapy than we may stimulate significant amounts of NO. Laser therapy is similar to shock wave therapy in that it seems to have profound effects on the cell environment. Laser therapy will be the subject of another article. By utilizing shock wave therapy with the some the regimens that we already have hopefully many good things will happen. Using shock wave therapy with Platelet Rich Plasma (PRP) and or Stem cells will dramatically improve results. I do not know if shock wave therapy itself will be total game changer but I do know it will be an increasingly important part of our ever-expanding armamentarium of treatments. I am sure there will be more to come concerning this modality. I believe this will become an increasingly important aspect in the utilization of PRP and Stem cell therapy in the treatment of hip, knee, spine and other musculoskeletal problems. Thanks, Dr. P. "TOLD YOU IT WASN'T WHAT YOU THOUGHT IT WOULD BE"- DR.P

A multiple personality describes someone or entity with many personalities, each distinct and sometimes totally opposite. One side of a split personality may be amicable and easygoing, while the other side can be withdrawn or even violent. So, in essence we are dealing with a split personality. In a way, this seems to describe Nitric Oxide. Nitric oxide (NO), is one of the smallest signaling molecule known in the body. It has profound effects on the body. I have dealt with Nitric Oxide (NO) for a number of years due to its beneficial effects in the stem cell arena. I am a physician who has been a long-standing proponent of NO therapy. It is as I call it “one of the holy grails of stem cell therapy”. NO can act as a stimulator of stem cell proliferation and it has a critical role in analgesia, vasodilation, neuromodulation, our immune system and angiogenesis. Thus, these are impressive roles for Nitric Oxide. Yet when I read certain articles they describe Nitric Oxide as a potent free radical which can contribute to inflammatory disease and a whole host of other problems. This initially caused me thought, consternation, and confusion. So, who or what is the real Nitric Oxide? I would like to give a bit more information on this subject to try to clarify things for the clinical physician. I think this is also of interest to the lay public. Hopefully I can clear up some of the confusion confronting both the physician and the lay public. Actually, books have been written on Nitric Oxide and that still may not do it justice. There are certainly people much more qualitied than myself to discuss the topic of Nitric Oxide and its nuances. One of who comes to mind is Dr. Nathan Bryant. Dr. Bryant is considered one of the experts in the world on NO. I am fortunate to personally know Dr. Bryant. I will do my best to present the subject as a clinician to other clinicians Nitric oxide has three main personalities depending upon which “neighborhood” it is found in the body. The personalities stem not from Nitric Oxide itself but from which isoenzyme produces it. Since we must remember one basic thought: Nitric oxide is nitric oxide. What is of importance here is which isoenzyme produces the Nitric Oxide. The significance is where and how the Nitric Oxide is produced. Ultimately, this is what has a major impact on its function (or personality) and effect on the body. NO is produced by three different isoenzymes. The first form is called NOS-1or neuronal (n NOS), the second form is called NOS-2 or inducible (i NOS), and the third form is called NOS-3 or endothelial (e NOS). These represent the different forms of Nitric Oxide synthase (NOS). There is one other form of Nitric Oxide synthase that is produced by bacteria. We will skip the discussion on this form. The human nNOS gene is located on chromosome 12, iNOS on chromosome 17, and eNOS on chromosome 7. NO production is dependent on a number of biochemical reactions. Many diseases are associated with increased amounts of Nitric Oxide while others are associated with its lack of production. Neuronal nitric oxide synthase (n NOS) is mainly responsible for synthesizing nitric oxide in the neural tissue and the skeletal muscle. We can see that its environment is the nerve and skeletal muscle tissue. In mammalians, the largest source of nNOS in terms of tissue mass is the skeletal muscle. In the periphery, many smooth muscle tissues are innervated by Nitrergic nerves, i.e. nerves that contain nNOS and generate and release NO. Nitric oxide produced by nNOS in Nitrergic nerves can be viewed as an unusual neurotransmitter. The NO produced decreases the tone of various types of smooth muscle including blood vessels. Here, it has the acute properties of neuronal firing and ion channel modulation in the central nervous system. It also has a long-term effect on memory in the brain. Because of the important functions of nitric oxide, it's concentration must be highly regulated. Its functions also include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral Nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. It appears that n NOS has to be present in order for Viagra and Cialis to work properly. n NOS produces NO which ultimately allows these medications to work thru vasodilation etc. As we can see there are significant functions for the n NOS. We are just scratching the surface here but it gives some insight. Second form of Nitric Oxide synthase (NOS-2) or i NOS is perhaps the one form that causes some concern. The Nitric Oxide produced from i NOS can be associated with inflammation. iNOS is not normally present in tissues but expression is induced in inflammatory conditions and immune responses and hence often implicated as guilty by association. It is the large quantities of nitric oxide produced by iNOS that have associated it with injury. iNOS has been shown to have multiple biological effects. It is essential for normal healing in the skin and intestinal mucosa, for killing of certain bacteria, is potentially important in regulating T cell proliferation and differentiation (Th1vs Th2 immune system), and for regulation of leucocyte recruitment. These are profound implications in our immune system. We must remember that stem cells and our immune system are totally entwined. Countering these effects is the fact that iNOS can produce toxic metabolites. Perhaps this may be one of the bad personalities of the NOS forms. There is a large amount of data concerning iNOS expression in a variety of human diseases. I still need to temper my words a bit in that perhaps iNOS may not be the culprit we think it is. We are still far from understanding the precise role of iNOS activity in most of these diseases. In certain circumstances, it can indeed increase inflammation but maybe this is just a temporary phase. It is now clear that NO cannot be catalogued as either an anti-inflammatory or a pro-inflammatory molecule, but it can be considered a true inflammatory mediator. Although all three NOS isoforms are involved to a greater or lesser extent in the course of inflammation, the role of iNOS appears to be dominant. It is intimately related to the immune system and its workings. Perhaps I should also say that NO is one of the holy grails of our immune system. It appears the macrophages are involved in the production of NO. We must remember that there are two types of macrophages. There are M1 and M2 macrophages. M1 is pro-inflammatory and is implied in the production of iNOS. M-1 can help produce T cells which seem to be involved in autoimmune diseases. M-2 macrophages are anti-inflammatory and are involved in the production of T-Reg cells which appear to counteract autoimmune diseases. So, we can see that i NOS produces a high-level NO production which mediates a number of inflammatory and infectious diseases by acting both as a direct effector and as a regulator of other effector pathways. The different roles of NO in inflammation, often referred to as the NO paradox, is based mainly on the conflicting data showing the effects of NOS inhibitors of varying selectivity in different animal models. We still have a way to go to get a better handle on iNOS. The third form of Nitric Oxide synthase is NOS-3 or e NOS. This is the form that may have the most pertinence to the stem cell field. Data indicates that NO in the bone marrow acts primarily in a paracrine manner, as it is generated mainly by vascular cells of the bone marrow stroma. The stromal cell population also contains endothelial cells. Ongoing experimental and clinical wound healing studies have established NO as a critical mediator of normal tissue repair. Angiogenesis, granulation tissue formation, epidermal migration, collagen deposition, and microvascular homeostasis are significant processes critical to normal wound repair that are regulated by NO production and bioactivity. Some of the important work on Nitric Oxide generated by e NOS come from Dr. Thom’s classic article on NO and stem cell mobilization. Here is an exert from Dr. Thom’s article “HBO (hyperbaric oxygen) increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs (COLONY FORMING UNITS). Bone marrow NO concentration increased by 1,008 ± 255 nM in association with HBO2. Stem cell mobilization did not occur in knockout mice lacking genes for endothelial NO synthase. Moreover, pretreatment of wild-type mice with a NO synthase inhibitor prevented the HBO2-induced elevation in stem cell factor and circulating stem cells. We conclude that HBO2 mobilizes stem/progenitor cells by stimulating NO synthesis”. As we can see from the classic Thom article hyperbaric oxygen has a direct effect on e NOS production which has a direct effect on stem cell production via a NO mechanism. This is a truly significant aspect of e NOS. This is a very significant finding by DR. Thom. The actual mechanism of hyperbaric oxygen is by the effect of NO generated by e NOS on the bone marrow. The NO stimulates the release of stem cells into the circulation from the marrow. We must remember one of the most basic concepts of Regenerative Medicine: CELLS NOT DOCTORS HEAL PATIENTS! If we have more stem cells in the circulation than we increase our chances for success. Endothelial Nitric Oxide Synthase also has many effects on the cardiovascular system via its production of NO. For instance, it can help in angiogenesis of ischemic tissue. However, it is probably best that I leave the cardiovascular system for someone more qualified than myself to discuss. We can see Nitric Oxide encompasses cardiovascular, neuronal and immune systems. NO is found in many different neighborhoods in the body. The neighborhood influences the action. NO is involved in stem cell differentiation, epigenetic regulation and immune suppression. Stem cells trigger cellular responses to external signals on the basis of both NO specific pathways and concerted action with endogenous compounds including stem cell cytokines. As potency and interaction of NO with stem cells generally depend on the concentrations of NO and the presence of the cofactors at the active site, the suitable carriers for NO delivery is integral for exerting maximal efficacy of stem cells. So, it seems that we must develop strategies that address iNOS and its production of NO but at the same time increase available Nitric Oxide in the body which we know become deficient as we age. We need to be cognizant of the fact that as we age our production of NO diminishes. Furthermore, our ability to utilize certain supplements such as L-arginine also diminishes. We need to use strategies that target the cytotoxic and damaging actions of NO/RNOS without interfering with essential administration of NO donors. Luckily, we have some excellent NO donors such as Neo-40 which will help us turn the tide. Hopefully this serves a brief introduction to the many personalities of Nitric Oxide. This is a subject that will continue to grow in importance with time since it encompasses some of the basics of medicine. Thanks Dr. P