P-53 PROTEIN: A NATURAL SENTRY, STEM CELL PROTECTOR, TUMOR SUPRESSOR, and ANTI-AGING CHAMPION
Could an elephant hold some of the secrets of longevity? The elephant is an animal which has great longevity. Scientifically speaking, this bucks the trends of nature. Typically, the more cells an animal has the more likely that a cancer or some other serious medical problem will arise and thus shorten life span. Yet the opposite is true for elephants, certain species of whales, and a host of other animals that have extreme longevity. The common thread for these animals is improved DNA repair and cell cycle regulation mechanisms to prevent DNA damage. This damage accumulates during the life course of the animal. The secret may be that these animals have extra amounts of the P-53 protein. This protein controls repair which in turn promotes longevity and resistance to age-related diseases.
Certainly, there is not just one factor which will increase longevity in large animals but there are some common threads. However, one very important thread concerns Telomeres which are the ends of the DNA in all cells. Every time our cells reproduce the telomere gets shorter until a certain critical length is achieved. This certain critical length sets a number of reactions into place. This is very evident in the following diagram. This diagram speaks of the work of Dr. Ron DePinho. It is called the unified theory of aging. This diagram comes from an article about a good friend, Dr. William Andrews. Dr. Andrews has a quest to search for substances which seem to stimulate telomerase. If we can stimulate telomerase we might prevent short telomeres. Remember that telomeres are the ends of the DNA which shorten every time our cells undergo division. Short Telomeres will cause the cell to becomes a wayward cell involved in all types of mischief. These cells are called senescent cells.
There is protein called P-53 which is activated by cells with short telomeres. The short Telomeres cause the cells to enter a phase called cell senescence. The diagram below explains better what causes senescent cells to arise and subsequently what problems senescent cell cause in the body.
What few people understand is that as cells age, some of them degenerate into a population of poorly functioning senescent cells that accumulate throughout our body. There is no value to retaining these lingering senile cells. Their continued presence underlies a myriad of degenerative disorders. Senescent cells should be purged from the body. The buildup of these dysfunctional senile cells contributes to decay of individual organs and greater susceptibility to disorders related to chronic inflammation and thus disease of aging. I have written a blog already on senescent cells. In that blog I spoke about some supplements which seem to attack senescent cells. The body has a much better way of attacking these cells. This method is called the P-53 protein. This is one of the foundations of giving elephants and other large animals their advantage in anti-aging.
This blog will target a very intriguing aspect of senescent cells called the P-53 protein. In the first diagram above we see that the P-53 activation is awoken by critically short telomeres. When the p-53 is activated it is a very good guardian. We can see the P-53 helps in cellular repair.
One of the main purposes of P-53 is to have some control over the cell division. If the cell has significant damage especially to its DNA than that cell is flagged and the cell is programed for death. This programed death of a cell is called Apoptosis. When the damage to the cell’s DNA is not that great than the P-53 protein will direct the DNA to repair itself. The following diagram is a bit more technical. It shows us what happens when P-53 is working properly and when it is damaged.
What we can see in the above diagram is that an abnormal P-53 may lead to cancer. For this reason, the P-53 protein is also called the tumor suppressor gene.
P53 is a tumor suppressor involved in a wide variety of tumor types. At least 50% of known tumors contain mutations of P-53 in humans and animals. These mutations are found in different cell and tissue types. This has made P-53 the one of the targets in cancer research and therapy worldwide.
Main stream medicine targets this tumor suppressor in gene therapy and uses the protein extensively in cancer therapy. Just like recombinant cytokines growth factors, there is a recombinant P-53 protein. Recombinant DNA technology is when we join together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Typically, bacteria can be programed by the recombinant DNA technique to produce medicines proteins etc. Nowadays Insulin is produced in this fashion.
The vision of using P-53 comes from the work of Dr. Biava who isolated the P-53 protein from the zebra fish, creating a recombinant protein. This P-53 protein carries the signaling and the function of the P-53 tumor suppressor factor allowing the practitioner to program the microenvironment of the cancer patient or anti-aging patient.
P-53 is a transcription factor. Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA. Regulation of transcription is the most common form of gene control. The action of transcription factors allows for unique expression of each gene in different cell types and during development. P-53 modulates gene expression and induces Cell Cycle arrest, senescence or apoptosis (cell death), thereby preventing tumor formation. P-53 is killing those bad senescent cells. So, it seems that P-53 is a smart protein able to detect and regulate oncogene (cancer causing genes) activity. When there is no risk, P-53 is flagged for inactivity and degradation which suppresses its expression so that it does not interfere with normal healthy cell proliferation.
P-53 IS A BIG PLAYER IN THE CELL CYCLE
Abnormalities in the Cell Cycle lead to tumors. The normal Cell Cycle has four phases: Synthesis of DNA
Mitosis (cell division), 2 gap phases; one before each active phase; that is, before Synthesis and before Mitosis (cell division) respectively. But regulation of the cycle is also dependent on tumor suppressor genes such as P-53.
Getting back to the reason why elephants seem to have excellent longevity is due to the fact that elephants have evolved unusual P-53 genes. While we only have one copy of the gene, elephants have 20 copies. Research teams have observed that the elephant’s swarm of P-53 genes responds aggressively to DNA damage. Like many things in life it is a numbers game.
If we were to recap the many facets of P-53 we would find the following:
MAIN PROPERTIES OF p53
Tumor suppression which can dramatically reduce the incidence of cancer.
Maintains genomic stability in somatic cells. This means that the cells will not develop mutations that leads to all kinds of problems.
Regulates transcription, especially in stress responsiveness. Remember transcription involves the production of messenger RNA which turns on or off a variety of genes.
Regulates genes that control Cell Cycle arrest and cell death
Regulates homeostasis in cellular metabolism and Cell Cycle
Regulates autophagy. Autophagy is the process where sub-cellular organelles are destroyed and new ones are rebuilt to replace it. Think of it as similar to replacing parts on a car. Instead of replacing the entire car you just replace the defective parts. In autophagy old cell membranes, organelles and other cellular debris can be removed. This is done by sending it to the lysosome which is a specialized organelle containing enzymes to degrade proteins.
Involved in metabolic maintenance
Involved in fertility
Involved in cellular reprogramming
We can see the P-53 protein has far reaching tentacles. The P-53 protein responds to cellular stress in general. Oxidative stress in aging or degenerative pathophysiology can trigger the sentry to duty, so a recombinant P-53 protein can ostensibly be used in anti-aging therapies, degenerative conditions and prevention of tumorigenesis. This is the seminal point to this entire blog.
Rather than depend entirely upon our own P-53 protein, which potentially may have some mutations and loss of effectiveness, we are able to supplement it with a recombinant form of the P-53 protein. Remember, just like the elephant, it is a numbers game with P-53. We will have available a recombinant form of P-53. This will be supplied in the form of a patch which contains the P-53 with penetrating molecules. The patch is kept on overnight and then discarded. We feel this will be another nail in the coffin of aging. We still have a way to go but it is certainly a step in the right direction.
- Dr. P
In the diagram, we see one aspect on how we can increase stem cell numbers in some of our procedures in a safe and efficient way. We are constantly looking for ways to increase the stem cell output from the bone marrow. We know all too well that most of the regenerative cells that we inject into the joint typically survive for only a short period of time. Their release of growth factors is what gives the various signals for more cells to visit the area or, just as importantly, for some of the neighboring cells to help accomplish repair. It becomes a numbers game. The more cells we have in circulation the better chance we have to achieve success. Now, if we are able to find a medication that can increase stem cell numbers and at the same time have an effect on the health of the cartilage than we have something special. Parathyroid hormone (PTH) may be just the ticket.
For a number of years now we have been using a clone of Parathyroid hormone (PTH). Another PTH clone is known as Forteo. Forteo is used in the treatment of Osteoporosis. We are actually using another analog clone of PTH which has given us considerable cost savings and at the same time may work better. We first started using PTH as a stimulator of Very Small Embryonic Like Stem Cells (you can read about these cells on a previous blog). There is still some controversy to these cells but that controversy is on the wane. There is a receptor on the V cell surface that seems to stimulate these cells when the PTH binds to the cell membrane. It appeared to activate these V cells.
When we investigated PTH we were very intrigued by a number of scientific articles that show PTH can have some interesting effects on articular cartilage. The Univ. of Rochester is studying PTH for its ability to inhibit chondrocyte hypertrophy and induce cartilage matrix production. Chondrocyte hypertrophy is the beginning of the death spiral for chondrocytes which produce and maintain cartilage. Chondrocyte hypertrophy-like changes play a role in early and late stage Osteoarthritis (OA). Since not all cells in an OA joint are synchronized in their stage of the cell cycle, inhibition of hypertrophy-like changes might be a therapeutic target to slow down further OA progression. There are a number of studies that show that PTH is chondro-regenerative. There were many preclinical findings providing proof-of-concept that (PTH) may be useful for decelerating cartilage degeneration and inducing matrix regeneration in osteoarthritis patients. In addition to bone as a target, signaling downstream of the type I parathyroid hormone receptor (PTHR1) regulates chondrocyte differentiation. Parathyroid hormone-related peptide (PTHrP) a clone, is a potent stimulator of proliferation and collagen synthesis and suppressor of maturation. One classic article on this effect of PTH comes from the work of Dr. Eric Sampson and his group from Univ. of Rochester. They were able to show the chondro-regenerative properties of PTH. So, we can see that PTH may have some powerful effects on improving the status of articular cartilage and its underlying bone.
Another aspect of Parathyroid hormone is that it increases Prg4 expression which has been reported to inhibit articular cartilage degeneration in arthropathic joints. Lubricin is present in synovial fluid and on the surface (superficial layer) of articular cartilage and therefore plays an important role in joint lubrication and synovial homeostasis. Lubricin is the most lubricating and anti-adhesive molecule in the human body. Lubricin biosynthesis and bio distribution are mostly regulated by cytokines and growth factors. When first isolated, cartilage lubricin was called "superficial zone protein" (SZP) Lubricin, MSF, and SZP are now collectively known as Proteoglycan 4. Lubricin was originally identified as a lubricating glycoprotein present in the synovial fluid, specifically synthesized and expressed by articular chondrocytes (cells that makes cartilage) of the superficial zone. It is recognized to have a major protective role in preventing cartilage wear, synovial cell adhesion and proliferation and reducing the coefficient of friction of the articular cartilage surface. The best way to think of lubricin is that it is a very high-powered hyaluronic acid. Hyaluronic acid has been used for years as a treatment for symptomatic osteoarthritis of the knees. We are looking into getting a bioengineered form of Lubricin and Hyaluronic acid. Both Lubricin and Hyaluronic acid are more than lubricating agents. Lubricin and Hyaluronic acid as a biologically active molecules that regulates tissue repair process on multiple levels. They are signaling agents affecting many different cell processes. Hyaluronic acid (HA), one of the main components of the extracellular matrix, is considered a key player in the tissue regeneration process. It has been proven to modulate, via specific HA receptors that are located on cell membranes, inflammation, stem cell migration, and angiogenesis (formation of blood vessels). These are the main phases of healing. We typically use Hyaluronic acid in our treatment protocols. It is a very specialized form we are using. Unfortunately, many offices performing stem cell treatments do not go to this length and utilize Hyaluronic acid.
This is not where the story about the benefits of PTH stops. One of the missions of the regenerative medicine doctor is to come up with ways in which we are able to increase stem cell output from the bone marrow. We are aware that certain supplements may help in this endeavor. On such supplement that comes to mind is StemXcell which is licensed by the Univ. of South Florida. In some lab studies StemXcell was found to have similar effects as granulocyte colony stimulating factor (GCSF). GCSF is sometimes called Neupogen. Neupogen is used in cancer treatment to increase the numbers of white blood cells after for instance chemotherapy. GCSF will increase the number of white blood cells released into the system. At the same time, it will increase the number of other blood components released from the bone marrow including stem cells into the circulation. The problem with these medications is that they are very expensive and at the same time do present some substantial risks. Another product which increases stem cell output is Procrit. Procrit increases the number of red blood cells released into the circulation. Procrit and GCSF are manufactured by recombinant DNA technology meaning it is produced by Escherichia coli (E coli) which are bioengineered. Many of our medicines are produced by bio-engineered E. coli. We have utilized some of these recombinant DNA products in our lab.
What initially got our attention with PTH was the effect it has upon bone marrow. PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This is what has wet our appetite. We now have a safe method of increasing the number of circulating stem cells in the circulation. This is similar to the effect of hyperbaric oxygen. Thru the work of Dr. Thom of the University of Pennsylvania we have discovered that hyperbaric oxygen will increase the number of circulating stem cells in the circulation. Dr. Thom discovered that hyperbaric oxygen works on a nitric oxide (NO) mechanism. The enzyme responsible for the production of NO in this case is endothelial nitric oxide synthase. The NO will stimulate the marrow to release large numbers of stem cells. We will give a NO supplement to increase the effects on bone marrow. This supplement is called Neo-40. We have used it for years to help increase the marrow stem cell output. However, PTH seems to have a more profound effect on marrow stem cell release. The PTH is given in intermittent doses. Stimulation with PTH showed a significant increase of all characterized subpopulations of bone marrow-derived progenitor cells (BMCs) in peripheral blood (1.5- to 9.8-fold) similar to GCSF. Luckily, PTH has a much better safety profile than GCSF. Also, our source is much more reasonably priced. This is a piece of our Advanced Cellular Repair Division.
On doing research we find one more important aspect of PTH. PTH seems to increase the amount of a chemokine called SDF-1 also known to many PHDs as CXCL-12. The major role of chemokines is to act as a chemoattractant to guide the migration of cells. The receptor on the cells that attracts the SDF-1 is called CXCR-4. The CXCR-4 is expressed on many circulating progenitor cells. The CXCL-12 and the CXCR-4 axis is one of themain axis of stem cell migration and homing. SDF-1 acts as a “tractor beam” on stem cells much in the spirit of Star Wars.
We continue to expand our uses of PTH. Luckily, we have an excellent source of a clone of PTH which is both effective and cost efficient. We are using the PTH on an intermittent basis. As time goes on we will find other medications and supplements to complement our use of PTH. We are ever pushing the envelope. Thanks DR. P
Above we can see an article that was published back in 2016 about Peripheral Blood Stem Cells. Peripheral blood stem cells are another name for Very Small Embryonic Like Stem Cells (V-cells). These cells continue to gain acceptance in scientific circles. We have suspected for some time now that these cells seem to correct many health problems behind the scenes. They can be considered an emergency stem cell supply in the body. They are typically quiescent but come activated under stressful conditions that the body may encounter. Clinically, many studies show that subjecting these cells to four degrees centigrade, hypoxia, or both will cause activation of these cells.
Anecdotally, we have seen a number of interesting clinical effects from these cells. They seem to deal very well with various auto-immune diseases. For instance, they seem to deal well with osteoporosis which in many circles is considered an autoimmune disease. What the above scientific article conveys is that there is a positive change in telomere length in patients after treatment with these cells. This last sentence can have profound implications in the antiaging world. Let us take a better look at Telomeres and their ramifications. I have been intrigued with Telomere science for many years. In my practice I strive for methods which will at least slow down telomere degradation in various regenerative cells. We know that certain supplements can achieve this goal and some may even reverse telomere damage a bit. The following is a diagram on telomeres which are the end caps of DNA.
The yellow represents the Telomere. Every time our cells divide they lose a snippet of DNA which is the telomere. With each division of the cells the telomere becomes shorter and shorter. When the telomere reaches a certain critical length the cell typically dies. Unfortunately, telomere degradation is a relentless process. Your telomeres were in better condition this morning than when you will go to bed tonight. This is one of the reasons why it is recommended that babies save their cord blood. The telomeres in the cord blood stem cells will never be as good as they were on the day the baby is born. Furthermore, it is even recommended that adults may want to consider storing their stem cells in a bank to have a supply of cells with a reasonable telomere length. In some ways this is like an insurance plan. Someday those same cells may be used to correct a disease process that has developed. They may be used to save your life. Typically, these cells would need to be increased in numbers by culturing them. After the culturing process they could potentially be utilized in disease treatment. This procedure would need to be done outside the US according to current regulations.
Short telomeres are associated with many different diseases ranging from cancer, osteoarthritis, diabetes, and many neurological conditions. Some of these neurological conditions may include Alzheimer’s Disease which is one of the scourges of modern society. The following diagram shows the difference in size between an embryonic cell and adult cell as far as the DNA and its telomere is concerned. Embryonic cells have much higher regenerative capacity. The fact that the embryonic cells have longer telomeres contributes to their high regenerative capacity. However, embryonic cells have many pitfalls which limits their regenerative potential. Furthermore, there are ethical issues associated with them.
We can see that the telomere has a repeating DNA sequence. What the diagram on the left is showing is that an enzyme called telomerase is actually extending the length of the telomere. The telomerase is adding back portions of the DNA.
Below is another fascinating diagram. This diagram was in reference to an article written in Popular Science magazine about a friend of mine Dr. Bill Andrews. Dr. Andrews has a quest of finding substances which will stimulate telomerase production. He is considered one of the world’s experts in the field of telomerase activation. I suggest that you go to his website www.sierrasci.comand read more about his work.
The above slide is actually the work of Dr. Ron DiPinho of Harvard. Dr. DiPinho proposed that short telomeres ultimately affect the mitochondria causing them to lose effectiveness and cause the “death spiral of the cell”. What we are seeing is that the production of an enzyme called P-53 seems to have a detrimental effect on mitochondria. The P-53 enzyme suppresses certain critical proteins that are responsible for proper mitochondrial function. If the cell is allowed to die there is no problem. If the cell continues to survive than the mitochondria produce free radicals which wreak havoc on the body on many different levels.
The cell is programed that when its telomeres reach a certain critical level the P-53 gene becomes active and it leads to cell death. This is a double-edged sword. In a perfect world the P-53 gene will cause the cell death on an aging cell. It is a very good protection for the body. In many research circles P-53 is called the tumor suppressor gene. We can see from the above diagram if the P-53 gene does not eliminate the aged cell many bad things begin to happen. There is a great deal of research concerning methods on how to turn on the P-53 gene. Another method of helping the P-53 gene is to use senolytic agents in the body. Senolytic agents are compounds which essentially kill senescent cells. The senescent cells are cells that should have died but instead remain alive and secrete many inflammatory growth factors. These “bad” growth factors are responsible for many different medical problems. Senolytic agents may be a good helper to the P-53 gene. Too much P-53 is just as bad as too little. Over stimulation of P-53 may adversely affect our stem cells and cause their untimely death. The best thing that could happen is too slow down cell aging so that we do not get free radical formation. One very good way to slow down cell aging is to actually reverse it. When we extend the length of the telomere we are reversing aging. What we are finding is that if we are able to preserve or lengthen the telomere length there may not be a need for the P-53 tumor suppressor gene. We will not have cells that have critically short telomeres.
Getting back to the original article, the cells that were studied in regards in telomere length were the leukocytes. Leucocytes are white blood cells. They represent a very important aspect of our immune system and actually have a profound influence on stem cells and their function. Stem cells and their efficiency are intimately tied to our immune system. Most studies that look at telomeres typically assess the cells of the immune system. The younger our immune system typically the healthier we are. Failures of our immune system lead to numerous diseases. In the paper here is a quote from the authors “We can only speculate that t phenomenon of increased telomeres may have been due to activation of telomerase through series of signaling pathways triggered by stem cells itself”.
It appears that V cells produce telomerase. In some circles this is one of the Holy Grails of anti-aging. The cells seem to turn on telomerase in other cells enabling these cells to go back in age. At least this appears to be happening in the immune cells. The significance of these peripheral blood stem cells cannot be over emphasized. When there is a process or cell type that will increase telomere length of immune cells we all have to sit up and take notice. These V cells may help correct many medical conditions and probably make our stem cells more healthy and efficient by increasing their telomere length. One observation we have seen is that when we combine a Vcell procedure with a regular procedure utilizing let us say bone marrow aspirate our results are better. I am not sure of the reason for this but improving the telomeres of the stem cells may be one explanation.
The relationship between the length of telomere and aging is a very complex process. Regenerative capacity adopted by the body’s constant use of stem cells is strictly interdependent with endogenous and environmental exposures and other sources of oxidative stress that eventually induce internal epigenetic changes in the cells. Are the V cells going to stop aging? The answer is of course not. But they may help slow it down. Time will tell with no pun intended. Thanks Dr. P
HYBRID BONE MARROW ASPIRATE, THE BEST OF BOTH WORLDS!
There is a continuing debate between using bone marrow aspirate in a non-centrifuged manner and using it in a manner where it is concentrated by centrifugation. Until recently bone marrow aspirate was essentially always concentrated. This question has generated much discussion and some heated debates in the Regenerative Medicine circles. The question is which group is correct? My answer is that both groups have valid points why their technology is the correct path to take. For this reason, we have come to the conclusion that a hybrid utilizing both centrifuged and non-centrifuged marrow aspirate works best. Bone marrow aspiration is very much technique driven regardless of the final preparation method. The purpose of this blog is not to discuss techniques but rather discuss aspects of centrifuged, non-centrifuged, and a hybrid marrow aspirate.
For some time now, we have been performing what we call the Purbred-HybridTM technique. We are utilizing both centrifugation and non-centrifugation to prepare our marrow. We realized that both preparation methods have their benefits. It has seemed to make a difference in our results clinically. Some time ago I became aware that centrifuging bone marrow may also have some drawbacks. We started doing some research on this subject. One question we started to ask is if there are some cells that are being discarded by centrifugation?
We felt that one type of cell that was being discarded was a V-Cell. This is a very unique stem cell. It is actually called a Very Small Embryonic-Like Stem Cell. In characteristics, it is very similar to an early CD-133 stem cell. There is still some controversy concerning V-Cells. I have previously written about this cell and do not wish to belabor the issue in this blog. We feel that V cells definitely have a contribution. This may be one of the more controversial differences between the two bone marrow preparation methods since V cells are not universally accepted. However, I would like to move past V cells instead keying in on other significant differences.
The biggest discussion points for those favoring centrifugation is that centrifugation will result in a concentration of stem cells from the bone marrow aspirate. Those people in favor of centrifugation feel that taking a larger amount of marrow aspirate and then centrifuging it will give more stem cells. Typically, the type of stem cell we are talking about is a mesenchymal stem cell (MSCs). There is little dispute to this fact. I agree with it one hundred percent. The real question to ask are the mesenchymal stem cells the most important regenerative cell to enable success? There is no clear-cut answer to this question. However, we are becoming increasingly aware that a symphony of cells is needed for success. We need to move beyond the juvenile thoughts that the MSCs are really the only important cells in marrow aspirate. MSC numbers may be an indication of good aspiration techniques but does this translate into good clinical success? Are we cutting our nose off to spite our face? In our quest to increase MSC numbers by centrifugation are we sacrificing critical components of the marrow aspirate? Let us take a look at both sides of the issue.
What are some of the other benefits of centrifugation? Centrifuging does have benefit by stressing the cells. It can turn on certain metabolic switches driving the cells to repair and may well cause the production of a greater volume of growth factors. When all is said and done growth factors are important contributors to success in regenerative procedures. The growth factors work in many different directions including reducing local inflammation and at the same time encouraging repair. Centrifugation also has a positive effect on MUSE BM stem cells. Muse cells are pluripotent and can endure stressful situations such as those found in the joint. The stress of centrifugation may actually help the Muse cell since they by and large thrive in stressful situations. But not all is good since centrifugation probably will eliminate some of the Muse cells from the final product. Another negative side of centrifugation, is the fact that it will change the shape of many cells and the number of cilia and thus the cell surface receptors. Therefore, I believe, it will inevitably change the performance of the stem cells and other regenerative cells. So, we can see there is no clear-cut advantages of either technique.
Let us look at other advantages we get with centrifugation and non-centrifugation. Right off the bat we need to look at megakaryocytes and platelets. These cell types are what gives differentiation issues in bone marrow aspirate versus bone marrow concentrate. The bone marrow aspirate will contain more platelets so it will cause a different profile of cytokines. The cytokines from the platelets will cause the differentiation of cells to take on a different path, mainly hematopoietic differentiation. We know hematopoietic stem cells are one of the main drivers of tissue regeneration (the mesenchymal stem cell is important for immune modulation). For years we have been adding an additional PRP with every bone marrow injection. This seems to be a good equalizer.
Platelets may be a small difference between centrifuged and non-centrifuged marrow aspirate. The platelets are easy to add thus the difference can be equalized. There are some other components that are not so easy to add and they are lost in some of the centrifugation processes. One of these components is exosomes. The following diagram gives us an idea about exosomes. Exosomes are much like the body’s Fed-Ex system. They deliver growth factors where they are needed. The exosomes contained in virgin aspirate may be more potent and more numerous
than those found in centrifuged marrow. Many times, when we wish to concentrate exosomes we will use ultra-centrifugation. We are not sure of the overall effect that centrifugation has on exosomes but chances are we will lose some exosomes to centrifugation and possibility change their secretory profile. We must also realize exosomes maybe a product of the cell environment. The various cells can sense what type of growth factors may be needed based on their environmental stimuli. Centrifugation may affect the stimuli.
The neutrophils also play a huge role here in the two versions of bone marrow. The neutrophils basically control the oxygen uptake or “respiratory burst” that is so important to the viability of stem cells, especially vascular stromal cells. The respiratory burst does both good and bad. A respiratory burst will release free radicals (ROS) which can be bad for the overall environment of the regenerative cells (however they are very good at killing pathogens). At the same time the ROSs can have a profound effect on intracellular signaling pathways and ultimately in modulating gene expression. This following diagram shows the paradoxical effect of levels high free radicals on cells:
The opposite effect occurs in low levels of ROS.
Recent data have revealed that the respiratory burst is closely related to inflammation resolution in many instances. What else does the respiratory burst accomplish? Neutrophil respiratory burst-induced hypoxia activates macrophage erythropoietin signaling to promote acute inflammation resolution. This signaling is activated following inflammation. Pharmacological or genetic inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signaling. An interesting experiment was performed. In this experiment, a Macrophage-specific erythropoietin receptor-deficient mouse having chronic granulomatous disease (CGD) was utilized. These mice lacked the capacity for a respiratory burst, displayed impaired inflammation resolution and had symptoms of GCD. When given exogenous erythropoietin it enhanced this resolution of the CGD.
How does centrifugation come into play here? Mechanical stress like centrifugation causes major changes through reduced oxygen uptake especially in neutrophils. Centrifugation can negatively affect the “respiratory burst”. The reduced uptake of oxygen by the neutrophils caused by the mechanical stress of centrifugation will have a profound effect on transduction and differentiation and thus on the success of the bone marrow graft. There will be observable differences in the number of different cell types as well as in the downregulated pathways such as Macrophage 1 and Macrophage 2 conversion.
Macrophages have developed a unique ability to radically change their metabolism from a heal/growth promoting function, now known as M-2, to an inhibit/kill function, now known as M-1. This conversion is many times dependent on the “neighborhood” the macrophage is found. This neighborhood is the extracellular matrix. The macrophages are the center of the immune “solar system”. Through these innate responses, macrophages are the central controlling element for tissue integrity and host defense while at the same time directing other cells of the immune system. The following diagram is a good example of this concept.
We must ask the question which of these cells are we discarding with centrifugation. We must remember innate immune cells such as basophils and mast cells and other adaptive cells produce IL-4 and IL-13 priming M2 macrophage. IL-4 induced M2 macrophages expressed high concentration of IL-10, IL-1R antagonist, chemokines CCL22 and CCL17, and intracellular enzyme arginase-1. All of these ensure the recruitment and activation of immune response and immune-suppressive function of M2 macrophages. In addition to immune response, IL-4 induced macrophages stimulate arginase activity by converting arginine to polyamines and collagen precursors that are crucial for tissue modeling and wound healing. Macrophages were shown to be responsible for regeneration by actively metabolizing arginine with arginase into ornithine and urea. This is of upmost importance since macrophage production of ornithine is required for many repair processes because it is a precursor of the polyamines required for cell proliferation and collagen synthesis for extracellular matrix construction.
The M-1/ M-2 conversion is of extreme importance. We now know that M-1/ M-2 macrophage conversion can have a significant bearing on the ultimate success of a bone marrow graft. The following diagrams show this concept:
These slides show the relationship between the stem cells and the different types of macrophages. The environment that the macrophages are found determines what pathway they ultimately go down. Macrophages in M1 inhibit mode direct T cells to pro- duce Th1-like cytokines (e.g. IFN-γ) that stimulate specific cytolytic T cells and activate more M1 macrophages. In contrast, macrophages in the M2/ heal mode stimulate T cells to produce Th2-like cytokines (e.g. IL-4 and TGF-β) that cause B cell proliferation and antibody production and further amplify M2 responses such as collagen precursors. Thus, macrophages actually use T cells as the ‘middle man’ to further amplify M1 or M2 dominant responses. In this case the various cells of the immune system may be adversely affected by centrifugation. This can interfere with the M-1/M-2 conversion axis pushing it more towards the M-1 (inflammation) and away from M-2 (repair). This can have a profound effect on success of a bone marrow aspirate.
We can see that a bone marrow aspirate is much more than just mesenchymal stem cells. It involves a symphony of cells from the immune system. We are now very much aware that MSCs alone are not the only immune modulator nor are they the only mediators cell repair. The cells of the immune system are very much involved in the success or failure of a regenerative medicine procedure. We need to realize that many different cells such as MSCs, HSCs, and the whole array of immune cells are important for regenerative success. Our techniques for marrow aspiration have improved and at the same time so has our final product. I have gone from full centrifugation to no centrifugation to now the hybrid combining both techniques.
To quote one of my favorite Beatles songs “It’s been a Long and Winding Road” to come to these conclusions. We can now see that both centrifugation and non-centrifugation both have their merits and their drawbacks. We became aware of this some time ago and have used a hybrid technique (centrifuged and non-centrifuged marrow) for some time now. Our results are clinically better. In our final hybrid product, the unspun marrow aspirate predominates in volume over the centrifuged marrow aspirate. However, more marrow aspirate is harvested for centrifugation than non-centrifugation. I am also fortunate that in some of my clinics I have the ability to utilize a variety of cytokines such as IL-4 which helps regeneration by influencing macrophage polarization. As time goes on I am sure more changes to the hybrid protocol will be made. One thing I am sure about is that this is an ever-changing field that always requires rethinking and reevaluation. Thanks, Dr. P
IS ALL FAT CREATED EQUAL?
Fat is a very popular treatment modality in Regenerative Medicine. There have been many articles written about fat some condemning it as being far inferior to bone marrow aspirate while others give it the highest praise. The real question is where does fat (adipose tissue) lie on the Regenerative Medicine spectrum? Like many aspects in an emerging field, the studies that are mentioned need to be scrutinized further. Our many of the studies that utilize adipose tissue flawed? One of the problems that we have is that the studies concerning the use of adipose need to be more definitive. By this I mean we need a classification concerning the source of the adipose tissue. We are not just talking about the age of the patient, the location of the tissue, but also the patient’s lean body mass. The question becomes is all fat created equal or is there a major difference between patients having a different body habitus? The following two diagrams very succinctly give us a good idea of the differences in the cell content of adipose tissue between a lean and an obese patient.
These two diagrams speak volumes concerning fat cells in patients. We have known for some time that one of the major differences between obese and lean people is that the fat cells differ in size. The lean person has more cells per CC. than the obese person. If one gains weight the cells do not grow in number but increase in size. The opposite is true when one loses weight. The number of fat cells in the body is relatively constant. If a liposuction is performed than those cells are lost forever.
Typically, there is no replacing them.
The differences between obese and lean people goes much more beyond the number of cells. We do know that adipose tissue is a source of many different cells including stem cells and other regenerative cells. Remember nature works as a symphony. It utilizes many different types of cells to achieve repair (not just stem cells). If adipose tissue is a source of stem cells than logic would say that the more obese a person is the healthier they should be since overall, they have more stem cells which are available. This is where the logic is flawed. Why is it that an obese person who has belly fat has a propensity for diabetes and other diseases including an increased incidence of cancer? The answer lies in the cell profiles.
The obese patient has many more inflammatory cells. The inflammation caused by these cells leads to many medical problems. We now know that the cell profiles determine what the health of the patient might be. We look at the cell profiles in the above diagrams and can observe a significant difference in cell types.
Let us break this down further. In the second diagram we see in the lean adipose tissue anti-inflammatory cells far exceed the pro-inflammatory cells. While the obese patient has the opposite scenario.
Looking at things one cell type at a time we see the first cell to discuss is the iNKT cell. An iNKT is a type of Natural Killer T Cell (NKT). Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. NKT cells are cells that seem to be essential for several aspects of immunity because their dysfunction or deficiency has been shown to lead to the development of autoimmune diseases (such as diabetes or atherosclerosis) and cancers. They help produce a number of cytokine growth factors which help to suppress inflammation. Thus, we can see they are intimately involved in our immune system. Our immune system and stem cells are intimately intertwined. Lean fat has larger numbers of these cells compared to obese fat.
The second big difference between lean and obese fat is the in the number of M-2 macrophages. Macrophages are one of the white blood cells of the immune system. Depending upon the cell neighborhood they can either cause inflammation or reduce it.
Macrophages are found in two different activated forms called M-1 and M-2 macrophages as can be seen below.
The M-1 macrophages are macrophages which have many different names. Some people call them killer macrophages. The above diagram refers to them as the “dark side”. This is not necessarily a true identity for the M-1 macrophage. Under the right circumstances, they can save our lives by destroying invading bacteria. They are excellent in killing bacteria etc. but can cause tissue injury. The destroying of tissue is the dark side is the M-1 macrophage. Unfortunately, the inflammation is not always discriminatory. For regenerative purposes, the M-1 macrophage is not our friend. The M-2 macrophage belongs to the “Jedi Order”. The M-2 macrophage is instrumental in causing tissue repair. The “Repair" designation broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, and they turn off damaging immune system activation by producing anti-inflammatory cytokines like IL-10 and TGF-beta. I liken IL-10 to the body’s cortisone with no baggage. It is one of the body’s master anti-inflammatory cytokines. The following slide is a good summation of M-1 and M-2 macrophages.
These Macrophages are also found in PRP and Bone marrow aspirate.
Another cell which is very important in the adipose profile is called a Treg cell. The regulatory T cells are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. T cells can cause an inflammatory response. They are responsible for many autoimmune diseases. Recent research has found that the cytokine TGF-beta is essential for Tregs to differentiate from naïve CD4+ cells and is important in maintaining Treg cells. The bottom line is that Treg cells help protect the body from various autoimmune diseases such as Rheumatoid Arthritis, Multiple Sclerosis, Diabetes, and a host of other diseases. Now we can see the reason why there is interest in studying adipose in the possible treatment of autoimmune diseases.
The Treg cells can also be instrumental in repair. A source of Treg cells along with M-2 macrophages can be a potential weapon in the treatment of autoimmune diseases. I will admit that bone marrow aspirate also contains large numbers of Treg cells.
The real question becomes is there a method that would allow us to change the profile of the fat cells? Can we make obese adipose tissue behave more like lean adipose tissue? Can we change the cell profile to have more cells which will help in repair process? Can we make these cells more effective for autoimmune diseases?
These are very intriguing questions. It appears that the answer to these questions is yes. This can be done by changing the environment of the cells. In another word, change the cytokines the cells are exposed to. The purpose of this blog is not to say that the adipose tissue from obese patients cannot have benefits but that a lean patient’s adipose tissue may have more benefits.
Changing the environment of the cells is easier said than done. Some of this can be achieved by the use of supplements (such as Nitric Oxide generators and Curcumin) which may help improve the environment of the cells. A bigger method of improving the cell profile may be accomplished by Cytokine Therapy.
Cytokines are signaling proteins that are produced by white blood cells and stem cells. They help mediate and regulate immune responses, inflammation, and repair. By the addition of certain growth factors, we can cause a preponderance of let us say M-2 macrophages and Treg cells while at the same time suppressing other types of cells which lead to inflammation. We are pointing the adipose tissue towards repair.
When looking at these cell profiles and adipose in general there are many advocates of using fat sources after they are broken down into more of their component parts. This is typically done by using an enzyme. The product produced is called SVF or stromal vascular fraction. This technique and resulting product (SVF) is currently not permitted in the United States. We do not use any SVF in our procedures given the FDA regulations. It is currently permitted in many other countries.
I am certain that the addition of the growth factors such as IL-1A and IL-10 to all types of adipose tissue may have a significant effect on the use of fat as a Regenerative modality.
Thanks, Dr. P
I recently returned from lecturing at the meeting of the American Academy of Anti-Aging Medicine. When I toured the exhibit hall I noticed there were a number of vendors selling various derivatives of Marijuana for medicinal uses. Unfortunately, most of the sales people did not seem to have a true grasp on how the cannaboids which are found in Marijuana actually work in the body. I have written two blogs in the past about this medical phenomenon. I would like to again give an update. The diagram below gives us an idea of the various pharmacological actions of non-psychotropic cannabinoids. We can see that the effects are wide ranging affecting many different functions in the body. What we can assume is that ultimately these effects deal with the immune system and stem cells. These two entities go hand in hand.
The above diagram shows the various pharmacological actions of the many derivatives of Cannabis. Cannabinoid receptors are involved in a series of processes inside the human body, including the regulation of mood, pain sensation, appetite and memory. These receptors can be activated by endocannabinoids (produced by the human body) as well as by plant cannabinoids (like those found in hemp or cannabis), and they’re grouped in two main categories: CB1 and CB2. The above diagram shows the various components of the Cannabis plant. These components can have very different effects on the body. Why is THC psychoactive and CBD is not? When we’re talking about cannabis and psychotropic activity, we’re dealing exclusively with CB1 receptors, which are concentrated in the brain and the central nervous system. The difference between CBD vs. THC comes down to a basic difference in how each one interacts with the cannabinoid 1 (CB1) receptor. THC binds well with CB1 cannabinoid receptors. CBD has low binding affinity for CB1 receptor. The following diagram shows this concept more vividly:
A THC molecule is perfectly shaped to connect with CB1 receptors. When that connection happens, THC activates, or stimulates, those CB1 receptors. Researchers call THC a CB1 receptor agonist, which means THC works to activate those CB1 receptors. THC partially mimics a naturally produced neurotransmitter known as anandamide, aka “the bliss molecule.” Anandamide is an endocannabinoid which activates CB1 receptors. The endocannabinoid system is so large and vital that almost every organ on the body expresses cannabinoid receptors. The system is a vital bridge between the body and brain, and endocannabinoids like anandamide are the messengers that keep the system running.
Animal studies have taught us that anandamide can increase appetite and enhance pleasure associated with food consumption, and it’s likely responsible for some of the rewarding effects of exercise (e.g. the “runner’s high”). Anandamide plays important roles in memory, motivation, movement, pain, appetite, fertility, even potentially inhibiting cancer cell proliferation. THC is a “key” that so closely resembles anandamide that it activates CB1 receptors, allowing it to produce some of those same blissful feelings.
CBD, by contrast, is not a good fit with CB1 receptors. It’s categorized as an antagonist of CB1 agonists. This means that it doesn’t act directly to activate or suppress CB1 receptors—rather, it acts to suppress the CB1-activating qualities of a cannabinoid like THC. In other words, when you ingest THC and CBD, the THC directly stimulates those CB1 receptors, while the CBD acts as a kind of modulating influence on the THC. CBD does not stimulate these two receptors; instead, it activates other receptors, like the vanilloid, adenosine and serotonin receptors. By activating the TRPV-1 receptor for example, cannabidiol (CBD) plays a role in the mediation of body temperature, pain perception and inflammation. There are two forms of Cannabis, namely Marijuana and the Hemp plant. For cannabis to be legally considered hemp, it must contain no more than 0.3% tetrahydrocannabinol (THC) per dry weight. THC is the active compound in marijuana that causes its euphoric effect. The level of THC in hemp is 33 times less than the least potent marijuana strains, so it’s impossible for hemp to get a user “high.”
Unlike marijuana, hemp is naturally higher in cannabidiol (CBD), one of over 85 cannabinoids that have so far been identified in the cannabis plant. CBD is non-psychoactive and therefore won’t cause a high, making it safe for use by anyone, regardless of age.
Some time ago I a wrote a blog about the use of certain components of the marijuana and Hemp plants. It was a fairly short blog which I will include here. More and more states are proposing the legalization of Marijuana. There are numerous health claims about hemp oil which is. There may be merit to some of these claims by the action of Cannabis on stem cells and the immune system. There is some negative press. Recently, the FDA stepped in when a few companies were making claims about the use of Cannabis derivatives for the treatment of cancer. So far there is not sufficient evidence of Cannabis as an anti-cancer compound. Thus, these claims need to be retracted. Possibly with more studies and research this may be proven to be correct. However, there is a good bit of scientific evidence out there that shows some interesting aspects of the Cannabis plant. For those people looking for healthy benefits of Cannabis we must realize that the compounds for the health benefits typically do not have any aspect of hallucinogenic properties. Below is the blog I wrote some time ago and I will expand more on it:
"We use to think that marijuana was bad for one's health. Now we are not so sure about it. We need to clarify things a bit.
Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries for a variety of uses. The use of Cannabis or Marijuana (scientific name is Cannabis sativa) came before we were able to discover the active portion or substrate. This substrate is called endocannabinoid system. The endocannabinoid system has a number of components. The system consists of lipids, the receptors for the lipids and certain metabolic enzymes. The Cannabinoid signaling regulates cell proliferation, differentiation and it reduces cell aptosis or death. These receptors are found in the very early stages of life. The results of the Cannabinoid receptors depend upon molecular targets and cellular context involved. There are two main receptors which are called CB1 and CB2 receptors. These receptors seem to be involved in neural degeneration. They seem to be involved in all three germ layer formations. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. It is thought that the CB2 receptors may be most important. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes osteoblasts and osteoclasts, and epithelial cells. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease. Bone marrow and stem cells make endocannabinoids, these endocannabinoids interact with the cannabinoid receptors (Cannabinoid receptors have been found in nearly every cell in the human body). If cannabinoids can enhance stem cell migration and proliferation, this could be a powerful therapy. For instance, if you can increase the numbers and movement of stem cells to an injured tissue, you could vastly enhance the healing process. Lastly, the synthetic cannabinoid HU-210 is about 100-1000x times more potent than THC from Cannabis and this synthetic agent has been found to be neurogenic. Meaning that HU-210 can cause new neurons (brain cells) in the brain to form. However, this study was done in rats...and humans are different from rats. Will I prescribe medical marijuana for my stem cell patients? At present, I do not think I have enough information to make an intelligent decision about this. I suspect if someday I do prescribe this it will be some derivative of Cannabis. There are certainly some intriguing aspects of Cannabis but I feel the jury is still out. I suspect we will certainly hear more about this. Thanks Dr. P"
That was the blog I wrote some time ago. At this juncture, I do believe that Cannabis derivatives are here to stay and will eventually be incorporated into main stream medicine. I have further looked at the literature and there seems to be some very good science on the effect of Cannabis on stem cell workings. One of the intriguing aspects of the CB2 receptor is that it is found mostly in the immune system. At the University of South Carolina, a team discovered that THC could reduce the inflammation associated with autoimmune diseases by suppressing the activity of certain genes involved in the immune response. Its presence there interests scientists because the immune system triggers inflammation, and studies show marijuana can have an anti-inflammatory effect. When we start talking about the immune system we have a host of implications. We are aware that many diseases of aging may have some basis as an auto-immune disease. One of these the interests me is Osteoporosis. There may be both receptors at work. CB-2 works on the immune system while CB-1 is induced during osteogenic differentiation. Changes in endocannabinoid levels and/or CB2 receptor expressions have been reported in almost all diseases affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, autoimmune, lung disorders, pain and cancer. The prevalence of this trend suggests that modulating CB2 receptor activity by either selective CB2 receptor agonists or inverse agonists/antagonists depending on the disease and its progression holds unique therapeutic potential for these pathologies. I am still not at the point that I will prescribe the CBDs to patients. However, I will continue to watch and see how the science progresses.
More to come I am sure. Dr. P.
(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