There have recently been scientific articles about using stem cell therapy for “anti-aging” treatments. There has been buzz lately on the internet and in the press about the use of stem cell therapy for “anti-aging purposes”. This is something we have been involved with for some time now.
Even before the days of Ponce de Leon mankind has been intrigued the quest for true age reversal. Recently, there has been some buzz from the University of Miami. A study under the direction of Dr. Joshua Hare. Dr. Hare and his group recently published a study in the Journals of Gerontology. An editorial in the journal stated the following
“MSC transplantation is a promising and innovative approach for the treatment of frailty in older humans" and "stem cells might be the vehicles for youthful regeneration of aged tissues." What the Hare group did was to obtain bone marrow derived mesenchymal stem cells from young healthy volunteers. They then grew the cells in a lab increasing their numbers and gave the cells back to older individuals. So far, the studies have shown significant improvement in some aging perimeters. These cells that are transplanted are called allogenic cells meaning they are from a different patient. We have to commend Dr. Hare and his group for doing this study and advancing the world of stem cell science. Having been involved in the stem cell world for some time now I have seen similar findings.
Although I am not condoning it, mesenchymal stem cells have been used for years as an IV preparation. Typically, these were the patient’s own stem cells usually obtained from the breakdown of fat by an enzyme. Under current standards this practice is not allowed by the FDA. Patients would report improvement in a number of afflictions especially pulmonary problems. However, the evidence was mainly anecdotal. From a scientific point of view this may have occurred from the release of various growth factors. Taking it one step further, giving cells from a younger patient probably is beneficial in that the cells may have better vitality and more growth factors. But there is one big BUT here. We must realize that when we are using allogenic stem cells these are not immune privileged but they are immune evasive. Eventually the body will catch them and destroy them. Will this make a difference? No one can say for sure. I suspect a good arm of the study might be to use the patient’s own stem cells, expand them and give them back to the patient. Will we get the same results as a transfusion from a young patient? Another, intriguing aspect might be to use cells derived from a first or second degree relative. Also, do we want to use some of the plasma from the younger patient?
The next question to ask is if the mesenchymal stem cell is the best cell to reverse frailty? We know that our immune system is intimately related to the hematopoietic stem cell line, mesenchymal stem cells are also very important in immunity. Which cell would be the most important? We don’t know. However, expanding hematopoietic stem cells is a much more difficult task than expanding mesenchymal stem cells. Another question to ask if we have to expand stem cells? I suspect that antiaging treatments may be related to using an increased number of stem cells. One concern of mine is that we know when mesenchymal stem cell populations are expanded they seem to lose some of their immune modulatory aspects. Is this a problem? Perhaps it might be better to expand the number of stem cells while they are in the body. In another word, let the body increase the number of cells. We are aware that there does exist methods to expand cell numbers while they are still in the body. Furthermore, this does not run afoul of the FDA. There are some medications such as Neupogen that can do this. But Neupogen is expensive and can carry serious risks. Some safer methods include laser stimulation, certain supplements, hyperbaric oxygen and its offshoots. Will these techniques work better? Only studies will show. The other question to pose is if it would be better to use bone marrow aspirate intravenously. Bone marrow aspirate can be rich in a type of cell called a T-Reg cell. T Reg cells have a very important control in our immune system. They help to eliminate autoimmune diseases.
About a year or two ago there was some buzz about transfusing blood plasma from young rats to old rats. The results were very promising. The researchers found that the old mice had a reversal of many of the aging processes especially in the brain. The researchers found a protein in the blood of the young mice called GDF11. In this case we are not just slowing down the clock but we are actually turning it backward. There is controversy concerning the GDF11 protein. This is not universally accepted by the scientific community. Some studies showed it very beneficial while other studies showed no benefit or actually a negative benefit. I can confidently say the jury is still out on GDF11.
We have been involved in some antiaging/fragility studies in our office. These studies involve an “early” CD-133 stem cell. This cell is obtained from the patient after stimulation of the marrow with a few different supplements including intravenous CoQ-10. These cells are processed and activated in a propriety fashion and given back to the patient intravenously. We have seen improvements on a number of different levels. This stem cell is sometimes called a V cell or very small embryonic like stem cell. However, a “v cell” is not yet universally accepted in the scientific community. If we think of them as a primitive CD-133 than there is more acceptance.
These very small stem cells are thought to be capable of forming almost any type of tissue. For this reason, these cells are called pluripotent. As I have eluded to in other blogs these cells may be considered the body’s emergency stem cell supply. They also seem to be implicated in acupuncture. These cells are found in something called Bonghan Channels which are intimately related to acupuncture. The problem with the V cells is that they are normally difficult to activate. However, thinking out of the box researchers have devised ways of turning these cells back on. These are the patient’s own cells. The methods of turning them back on are proprietary. We have had the good fortune of working with these unique cells with some excellent results. The tantalizing question is if these cells can be harvested from a son or daughter or grandchild and given back to an older related adult. There is no reason why they cannot be used.
There are some other methods which will slow down aging. We know for sure that starvation will extend life but we are not sure about age reversal but it looks promising. It appears to turn on the surtuin 1 gene. Stimulating this gene may increase stem cell survival and ultimately our own survival. There are many supplements that we know may at least help slow down aging. Some of these include derivatives of beets, broccoli, and a host of other compounds. It appears that these compounds have a distinct effect on stem cells. These supplements can affect the stem cells in many different ways. They may affect the stem cell environment or as it is also called the stem cell niche. If the environment is hostile than the stem cell has a much less chance of success. Those supplements that effect the niche are many times considered antioxidants. The job of the antioxidants is to neutralize the effect of what is commonly called free radicals. These free radicals occur when the body utilizes either oxygen or nitrogen. We are beginning to realize that Nitric Oxide is extremely important to many body functions. Nitric oxide is now known to be a growth, immune, and neuromodulator, as well as a stimulator of stem cell proliferation.
I am certain that stem cells will having an increasing importance in our quest to slow down aging and allow us to age gracefully without the trappings of aging. I think we need to remember that it will not be the stem cells alone but other aspects that will have a direct impact upon them. Hopefully one day we can make the hands of father time’s clock to start running backward or at least more slowly. Dr. P
Lubricin is naturally found in our body as part of the synovial fluid, the viscous substance that cushions and lubricates our joints. The above diagram is a quick synopsis of the effect of Lubricin on osteoarthritis. As we can see a lack of Lubricin will contribute to the formation of osteoarthritis. Lubricin is crucial to allow the two adjacent portions of a joint to glide on one another with minimal friction. It can be called the body’s Teflon system only it works much better than Teflon. Like many good things lubricin is lost over time due to the ravages of aging, injury and exercises. With the loss of lubricin the joint will become painful, stiff and cause the patient discomfort. When the patient reaches this stage, we try to use our tools that are available to us with Regenerative Medicine procedures. A heathier joint will hopefully have Lubricin production back in the mold. We must remember that Lubricin production not only decreases the painful symptoms of degenerative arthritis but also helps control the progression of the disease. Ultimately, this is what we try to accomplish with regenerative procedures. Now if we can combine Lubricin production with a regenerative stem cell procedure we may achieve better overall success.
Let us take a better look at the science of Lubricin. Lubricin is a surface-active glycoprotein secreted in a synovial joint. It plays an important role in cartilage integrity. In healthy joints, Lubricin molecules coat the cartilage surface providing boundary lubrication thus preventing cell and protein adhesion. Lubricin (also known as superficial zone proteoglycan SZP), is a protein expressed by superficial zone chondrocytes and is involved in lubrication of the articular cartilage surface, and also prevents deposits and degeneration of the cartilage and boundaries of the joint. It 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. These are the same factors produced by stem cells and other cells. Exposure of synoviocytes, chondrocytes and cartilage implants to pro-inflammatory cytokines such as interleukin-1(IL-1) and tumor necrosis factor-alpha (TNF), results in a marked reduction in the expression and/or abundance of secreted lubricin, with corresponding alterations in the amounts of cartilage-associated lubricin. Conversely, exposure to transforming growth factor-beta(TGF-Beta) significantly upregulates lubricin synthesis, secretion and cartilage boundary association. Of course, we must take these results with a grain of salt since they are studies performed in the lab rather than an actual real-life situation. In the field of Regenerative Medicine what happens in the lab does not always translate to real life situations. My suspicions are that these two situations will correlate with each other.
Lubricin plays an important role in joint physiology, and the loss of accumulation of lubricin may be a factor in the pathology of osteoarthritis. There have been some studies in mice where an arthritic condition was created and there were two treatment groups. The conclusion was as follows: supplemental intra-articular Lubricin reduced cartilage damage in the ACL transected rat knee 6 weeks after injury, while treatment with Hyaluronic acid did not. It was found that when the rats were treated with some growth factors which reduce inflammation and increase Lubricin formation than the damage was much lower to the joint in the long run. When people are thinking about joint lubrication they typically think of Hyaluronic acid. These two compounds are not the same. Hyaluronic acid is typically given as a shot to people who have osteoarthritis. It is approved by most insurance companies. It works fairly well in improving symptoms on a temporary basis.
In the case of Lubricin it is the load-bearing and non-load-bearing zones of the joint that give direction and regulation of certain growth factors (cytokines). The effect of certain compounds can have very interesting effects. For example, TGF1(Transforming Growth Factor-1) and heparin will bind and regulate the protein on the load- bearing zone in only 1 out of five times compared to simple saline. In most cases, TGF actually reduces binding of lubricin on the joint and inhibited SZP protein expression compared to saline or to the actual injection of lubricin. Same scenario for heparin. Let us take a better look at this. Heparin is used “to thin out” the blood and in Regenerative Medicine, it is utilized to prevent the coagulation of the platelets in PRP formulations and in Bone Marrow aspirations. So right off the bat if heparin is used than there may be some stimulation of Lubricin. We use heparin in all of our bone marrow aspirates. Now I realize that heparin may stimulate Lubricin production in the joint.
One of the most surprising things I have learned about Lubricin is the effect that saline has on its production. Saline is salt water
(sodium chloride) with the same concentration as our blood. We have found that saline with some other propriety components has a rapid and dramatic effect on the production of Lubricin in the joint. Using saline in a joint, especially a large volume, will stimulate the Superficial Zone Protein (also called Lubricin.) Adding certain cytokines to the mix will help neutralize inflammatory cytokines and further expand the production of lubricin. However, we have found the saline alone has a rather significant effect on the joint. This goes back to something I learned in doing thousands of arthroscopies. When we did arthroscopic surgery on an older person who had arthritis they initially felt better but the improvement did not last. What I realize what we were doing is that we were stimulating the formation of Lubricin. Using saline in a joint, especially a large volume as we do, will stimulate the Superficial Zone Protein (also called Lubricin.) This is a protein that is encoded with proteoglycan 4 (often mistakenly called PRG4) so basically your saline lubricates the joint. At one time, we considered saline a placebo but studies have now shown that saline is a therapeutic agent. Adding AIL-1 and IL-10 in specific ratios will neutralize the inflammatory cytokines and expand our lubricating and preparatory treatment. Joint motion increases the production of lubricin. That's why activity—specifically, exercises for joint mobility is so crucial to maintaining joint health and managing arthritic pain.
A research group from Cornell Univ (Bonassar et al) has discovered that Lubricin helps anchor Hyaluronic acid to the tissue surface. This in turn moves the cartilage in to a low friction regime. A quote from Dr. Bonasser “The implication of this finding is that the efficacy of HA treatment might depend on how much lubricin is in the joint at the time of injection, which could explain why clinical trials of HA have such variable outcomes and may also suggest new formulations of HA that might be even more effective in the clinic,”. Another intriguing twist is the possibility of using the arthrolavage with saline and the growth factors and then treat the patient with Hyaluronic acid. By first increasing Lubricin production we can increase the effectiveness of Hyaluronic acid injections into the joint. There are now studies to produce Lubricin in the lab. This process is called a recombinant DNA product. This means that many time the product is made by bio-engineered bacteria. Lubricin is just one of many new ideas that are arising in the field of Regenerative Medicine as it pertains to joint arthritis. It appears that it may be a good helper to stem cells.
Above we can see what an actual cytokine “patchless patch” looks like. These patches are absolutely unique. They are the brainchild of Dr. Jo Serrentino. She has devoted a good portion of her life to the study of cytokines. I feel quite privileged to work with Dr. Jo Serrentino. She is my colleague, a mentor, and business partner. She and I have been working on a few cutting edge of the cutting-edge projects for a number of years. There is no doubt that her knowledge and inspiration have helped me design some very unique protocols. She is a fountain of knowledge. One day I may ask her how we can increase the effectiveness of stem cells injected into a joint. On another day, I might call her up to discuss some aspect of a V cell treatment that potentially increases the overall effectiveness of the treatment. She has been instrumental in helping us develop our protocol for the treatment of Osteoporosis with stem cells and different growth factors. Although our series is still small we are obtaining improvements of from 15-30% in bone density values in one year. We seem to be getting a bit off track from the Cyto-Amino Complexes.
We must realize that Cyto-Amino Complexes make use of cytokines. Cytokines are a broad and loose category of small proteins that are important in cell signaling. Their release has an effect on the behavior of cells around them. They are very involved with our immune system and help regulate it and balance it out. In Regenerative Medicine our immune system is of the upmost importance controlling the body’s reactions. Cytokines and the immune system regulate the maturation, growth, and responsiveness of particular cell populations. The environment that the stem cells are found in can cause them to have a profound effect downstream. In certain environments, the stem cells can cause the production of certain immune cells, such as T-Reg cells, that can decrease the effects of an autoimmune condition. Most every condition in medicine typically results from an imbalance in cytokines. If you are dealing with an osteoarthritis, tendinitis, or an autoimmune disease you can be sure that an imbalance of cytokines is at work. Some cytokines enhance or inhibit the action of other cytokines in complex ways. Cytokines are produced by stem cells and also found in platelets. Another source of cytokines is placenta and cord blood products. These are products which contain a multitude of growth factors. Realize that with current FDA regulations they do not contain live cells. If they do than this is in violation of FDA rules. If I physician is proposing to use these products as a stem cell procedure he is not being truthful with his patient. That is a discussion for another day.
I am most beholding to Dr. Serrentino for her guidance and teaching of cytokine therapy. Our journey with patches began with iontophoresis patches. These are patches that have a built-in battery. They look like a large Band-Aid. Like any battery they had a positive and negative end.
On one side, we would place a combination of growth factors and the other side would be saline. At the time, I thought these were quite revolutionary and at the time they were. We had a few different formulas that were utilized with this delivery technique. These patches had to stay on for about 12 hours. I was happy with the results but ultimately these were a stepping stone to what we call the patchless patch.
We have used cytokine therapy to enhance our procedures for a number of years now. I repeatedly get asked the question “am I too old for stem cell therapy?” The answer to this is a resounding no when we are also utilizing Cytokine therapy. Cytokine therapy is one of our secret weapons. A good clinical example of this is concerning a simple PRP injection. As we age the levels of Insulin Growth Factor-1(IGF-1) decrease significantly. IGF-1 is instrumental in the repair of cartilage tissue, tendons, etc. However, if you are starting with low levels because of age or advanced osteoarthritis the repair of that joint may not be forthcoming. This is the beauty of Cyto-Amino-therapy as Dr. Serrentino calls it. We can help correct these deficiencies and encourage repair. Age is no longer an insurmountable road block.
I asked Dr. Serrentino to write about cytokine therapy in her own words. She wrote the following summary for me: “Because initially cytokines were for research purposes only, they could not be used liberally in commercial products. The controversy over growth factors for example has been ongoing for decades; restrictions in using these valuable signaling molecules in medicine is still an ongoing issue, despite the research data proving their countless benefits and safety”. Signaling molecules or Cytokine Therapy, is indeed a boon to modern medicine. For example, the use of recombinant proteins such as IGF-1 to repair cartilage in osteoarthritis, or the use of recombinant Interleukin -10 as an anti-inflammatory can dramatically alter the results of a procedure. Many protocols have been established, but the availability of actual Cytokines in the right combination and dosage has been difficult to source and to regulate. Sources of real cytokines in commercially available products are usually from homeopathic sarcodes or nosodes, (a sarcode comes from an animal part) but these provide only the precursor components not the real thing. Recent advances in technology have enabled Dr. Serrentino to develop clones of signaling molecules that have shown better biological action than actual cytokines. Dr. Serrentino has coupled polyamino molecules with Nano technology to create Cyto-amino® Complexes that mimic the signals and the biological activity of many cytokines.
Dr. Serrentino further explains that: “Because these Cyto-amino complexes are engineered through Nano technology, they do not work like a supplement but rather go directly to the extracellular matrix where they signal cellular tasks. Just like cytokines they tell the cells where to go and what to do. Amino acids are the building blocks of proteins, and cytokines are small proteins, so we can synthesize a clone through certain biochemical techniques with readily available amino acids. But these new molecules are not supplements of amino acids, they do not work like supplemental amino acids. They have a unique structure that gives them the capacity to mimic the action of a bonafide cytokine with extreme precision. They are not dependent on serum levels or need to be broken down like supplements, they work exactly like cytokines at signaling pathways, thus we can use them to program repair, which is the basis of Cytokine Therapy. This Cyto-amino Complex ® goes straight to the extracellular matrix where it gives cells
directions in programming regenerative pathways. CYTO-AMINO COMPLEXES ® can provide some extremely effective practical products for medical therapy, especially regenerative medicine and stem cell therapies. Dr. Serrentino further states that “Exclusive formulations have been developed for Dr. Purita’s Stem Cell techniques and protocols and are solely available through his teaching venue.” This is indeed true we have utilized this science for several years. The true beauty of these complexes is that they are used as a topical with Tegraderm patches. Dr. Jo has combined these cytokines with penetrating molecules. These penetrating molecules deliver the growth factors much more efficiently than an injection. The combinations of growth factors can include FGF-6, FGF-12,
IL-1A, IL-3, IL-10 and a few others. These abbreviations stand for various types of growth factors that can be instrumental in achieving repair.
Every day I continue to learn the intricacies of cytokine therapy. This therapy is truly unique in all stem cell therapy. As time goes on I am sure Cytokine Therapy will continue to make a difference not just in my practice but in that of a fortunate few others. In some circles, there is a notion that stem cells are but a waypoint in our treatment modalities. Eventually, cytokine therapy may replace harvesting stem cells. Every day we are seeing a push now in the use of exosomes. Remember, cytokine therapy mimics stem cell therapy in a much more efficient way. Perhaps I should say in a different manner: Stem cell therapy tries to mimic Cytokine therapy. We will certainly hear more of this as time goes on. Right now, combining the two therapies seems to be the best of both worlds. Thanks Dr. P
This weekend I spent a whirlwind trip to Morocco. I left Miami Thursday night and was back Sunday night. I was invited there to share information with a very interested medical community. Their eagerness reminded me of myself when I began the journey into regenerative medicine over twelve years ago. At that time, I was like a sponge and they seemed to be the same. The faculty included doctors and new friends from Europe, South America, Asia (including India), North America, and Africa. This trip also represented a milestone for me. I have now lectured on every continent with the exception of Antarctica. Something else to check off on my bucket list.
My lecture was designed as a surgery of the field of Regenerative Medicine as it pertained to Platelet Rich Plasma (PRP) and Stem cell therapy. I started out discussing the various aspects of PRP therapy. I explained to the medical community the good the bad and ugly concerning PRP therapy. To try to give them some semblance of order I discussed a paper which I recently published with some colleagues from Brazil. The paper is a classification of PRP preparations. It addresses PRP like no other paper has. I wanted to make sure that the doctors were not lead astray but some sales people telling them that certain components of a PRP product were not necessary. These components are red and white blood cells. We now realize that the full complement of blood is needed to order to properly get the best functioning PRP product. I told them about the “urban legends” of PRP products.
The next portion of my discussion concerned the different types and uses for stem cells. They were given the mechanics of stem cell therapy. I discussed with them why certain cells seem to be favored over other cells. The two most important cells discussed were mesenchymal and hematopoietic stem cells. Each has a very distinct place in the realms stem cell therapy. The mesenchymal stem cell can be likened to a Navy Seal. They are very specialized. A mesenchymal cell is what is called an immune- modulator. What it does is to try to quell the inflammatory response in the vicinity of the other regenerative cells. Thus, as one can see it is much like a Navy Seal. I gets introduced into a hostile are, it may not survive, but its job is to secure the area to allow other cells to accomplish repair. The cells which accomplish repair are the hematopoietic stem cells.
Another very important portion of my talk concerned the relationship between the immune system and stem cells. The two are intimately related. One is very much dependent upon the other. How the immune system responds can lead to success or failure in our treatments.
I ended the talk discussing the various adjuncts that we use to enhance our therapies. These included stimulation of Nitric Oxide (NO). We know that NO will help increase stem cell output from the bone marrow. I discussed various methods that increase NO output including hyperbaric oxygen, supplements and laser therapy. This is a very important aspect of my therapy protocols. We also gave some clues of some of our propriety methods of preparing PRP and bone marrow products. We have methods allow us to harvest increasing amounts of cells. I also discussed the use and reason for using extra corporal shock wave therapy. This is a therapy which utilizes sound waves. It does it in such a way that it resembles a jack hammer. This type of therapy will help stem cells migrate to the area, increases growth factors, and increases micro-circulation. These factors can all enhance the chance of success.
Some of the other lectures proved to be quite interesting. I met my old friend Dr. Bansal from India. He gave an excellent lecture on very small embryonic like stem cells. He and I both have a special interest in these cells. He has had very good success in utilizing these cells for a variety of auto immune diseases. He has also had some success in the treatment of spinal cord injuries. All in all, it was an excellent meeting with excellent speakers. It was worth the short trip. Thanks Dr. P
This is not a trick question. I can actually think of a few places but none fit the category better than a spinal disc does. Disc degeneration is about the cellular microenvironment more so than other areas. Like the moon the microenvironment inside the disc is very hostile on many different levels. The disc environment is essentially a vacuum with no oxygen. The pH of the disc is very acidic which lends itself to causing cell death. The high osmolality and low pH of disk are deleterious factors to viability of stem cells. The next problem that the regenerative cells need to deal with in the disc is the biochemical profile of the disc. By this I mean certain types of chemicals called cytokines and proteinases that attack the cells. The cytokines are signaling molecules which in this case cause inflammation and thus a hostile environment. The proteinases are enzymes that digest and damage proteins of cells which is certainly not a good thing for stem cells. Some feel that a compound called A2M may be of some help in the struggle with proteinases. This assumption may hold some truth but it will not turn the tide. Even if the proteinases could be neutralized, the cells will still be attacked by inflammatory cytokines. A good analogy is to look at these inflammatory cytokines as “potent cosmic rays”. Just like on the surface of the moon, if other portions of the environment don’t get you the cosmic rays (bad cytokines) will. These cytokines are the bad guys. They are called IL-1, IL-6, and TNF. Platelet Rich Plasma (PRP) might temporarily neutralize the cytokines but unfortunately this is just not a lasting effect. They are a good start. We must also remember that injecting a disc might actually cause further degeneration to the disc itself. So, we can see that the environment of a disc is somewhat like the environment of the moon very hostile and unforgiving. What can we possibly do to increase our chances of success?
We would have to start by improving the environment. Right off the bat I can conceive of a few things. We would have to see if we have a disc that may have a tear in its’ surrounding wall. The wall is called the annulus. If it is present than it needs to be sealed. This can be accomplished by using some agents found in the blood and platelets which are able to do this. Once the wall surrounding the disc is sealed than attention needs to be directed to the center of the disc called the nucleus. Dealing with the nucleus is much trickier. We need to deliver “good” cytokines to the cells to nullify the effects of the inflammatory cytokines. Getting the cytokines to the depths of the disc use to be a daunting task. The idea was to inject the disc either with a PRP and/or stem cells. So far, by and large, the results have not been very promising. We need to affect that environment of the nucleus. We have a propriety method of delivering the growth factors down to the disc without violating the disc itself. We have very high hopes for this technology but only time will tell.
So, we have a way that we can eliminate most of the cosmic rays (inflammatory cytokines). Another method might involve the use of lasers. Under the right circumstances, lasers can increase ATP production to increase energy, increases Nitric Oxide production, stimulate mitochondrial respiratory complex. So, we can see there are definitely benefits to the laser when it is used properly.
The big question is what type of cell can we use in the disc to try to regenerate the nucleus? The answer to this is we really do not know. We can look at a few of the candidates. One candidate is a mesenchymal stem cell (MSC). This is a cell found throughout the body. Once of its main purposes is immune modulation. It can change the environment and make it more conducive for cells to accomplish repair. The problem with the disc environment is that the conditions are so harsh that the MSC would have a very short survival time. A hematopoietic stem cell (HSC) can many times help drive tissue regeneration. However, it will run into the same problems as the MSC a very harsh environment. So, the question becomes what else might be out there that can withstand such harsh conditions or better yet even thrive in these conditions. One such group of cells is called a Muse cell. The word MUSE stands for multi-lineage stress enduring cell. I have previously written about muse cells and will update about them soon in another blog. Muse cells come from a variety of sources. One the easiest and best sources of these cells is adipose tissue. In many of the studies these cells were produced by the use of collagenase. Collagenase is an enzyme that is used to break down adipose tissue into its component parts. We do not use this since it appears to be in violation of FDA guidelines on the provision of minimal manipulation. We have found a propriety way of obtaining these cells from adipose tissue in a very simple and efficient way. There are some doctors that state that producing MUSE cells will kill other cells. While that might be true if they are processed in a specific way. What we are doing is to specifically selecting out these cells. These are the cells we wish to use. Those doctors that state that these cells cannot be used are probably correct when they are using their own techniques. This is not the case in the scientific community.
One more thing which may help the survival of the stem cells may be the use of a hydro-gel. One such compound that has had success in animal models is called Laponite. We are also high on one of our techniques. This technique is called “Joint Jelly”. This is made from platelet poor plasma that we process in a specific way. The joint jelly may be a good temporary medium for the cells while they are in the nucleus of the disc.
So, it seems like we may have all the components that may ultimately lead to success in treating disc problems with stem cells. Only time will tell how successful we will be. Thanks Dr. P
The above diagram shows the difference between a normal cell and a cell which is in a senescent state. A senescent cell is a cell that has stopped replicating. Although senescent cells can no longer replicate, they remain metabolically active and commonly adopt a pattern of secreting pro-inflammatory cytokines, the up-regulation of immune ligands, which means fooling the immune system to not attack them. In general, these cells are trouble makers!
Recently there was a significant announcement of a discovery by a consortium. This discovery is called UBX0101. UBX 0101 has some remarkable tributes. It is a drug that may delay, prevent, or even reverse the progression of osteoarthritis. It has a rather interesting mechanism of action. We now know that osteoarthritis is an inflammatory process that is age related. There is a medication UBX0101 that will eliminate a senescent cell. Again remember, healthy cells that experience stress can enter a state known as cellular senescence. In this state, cells stop dividing and profoundly alter their metabolism, appearance, and interactions with their environment. The subcellular damage itself does not directly cause the visible signs of aging, but rather, as the damage accumulates and reaches a critical mass, cells cease to proliferate and acquire the deleterious “senescence-associated secretory phenotype” (fancy way of saying the cells have changed character and altered the compounds they produce) which then leads to the consequences of tissue breakdown to create an aged cell. Thus, senescence is a precondition for tissue aging, and this explains why aging is a gradual process that remains largely invisible during most of its progression. The subcellular damage includes shortening of telomeres, damage to mitochondria, and DNA double-strand breaks triggered by various genetic, and environmental factors.
The senescent cells will accumulate over time. These cells are capable of doing damage on a molecular level. They will accumulate growth factors, proteases, and inflammatory factors that disrupt normal tissue function. Senescent cells comprise a small number of the total cells. Senescent cells have long been implicated in aging. Some rapidly dividing cells reach a point when they can no longer safely replicate; their telomeres become too short to protect the cell’s DNA. At this point, a cell will either destroy itself through a process called apoptosis, become dysfunctional (and potentially cancer-prone), or turn replication off but continues to survive. The cells that turn off are known as senescent. They can perform some functions, but they also release molecules that may increase risk for a variety of diseases, including cancer.
These cells are capable of producing those cytokines which are responsible for pain and inflammation in a joint or even worse cause systemic problems such as diabetes. Cytokines are molecules which act as signals for the cells telling them what to do. Some of these cytokines are “bad”. They are named Interleukin-1, Interleukin-6, and Tumor Necrosis Factor. They are called master inflammatory cytokines. One of the cornerstones of PRP and stem cell regenerative theories are that they can overpower these inflammatory cytokines. Many times, they will make certain anti-inflammatory cytokines such as Interleukin-1 antagonist, Interleukin -10, Insulin-Growth Factor-1. The can be considered the master anti-inflammatory cytokines. Unfortunately, the inflammation war may not always be won. The bad cytokines will over whelm the good ones and repair will come to a standstill. Perhaps this is where UXB0101 may come into play.
It has been discovered that these senescent cells accumulate in the joint and do their bad deeds. UXB0101 will selectively kill these cells. In cartilage grown from human knees with advanced OA, UBX0101 selectively eliminated senescent cells, increased proliferation of healthy chondrocytes, and induced new cartilage growth. This represents a new group of therapies called senolytic medicines to selectively eliminate senescent cells and thereby treat age-related afflictions such as osteoarthritis, atherosclerosis, eye diseases. When I did further investigation, I discovered that there are a number of compounds that have senolytic activities. Many of these are over the counter supplements. There is a good bit of interest in these compounds.
This brings us to a whole new field of possible therapeutic agents that may help with aging and Regenerative Medicine. These senolytic agents are generating a good bit of scientific interest. These agents may be a tremendous asset for anti-aging. There is now a race to study some drugs that can have senolytic properties. I did a good bit of investigating and found some supplements that have senolytic properties. Studies have shown the benefits of combining tocotrienols with quercetin, a flavonol. Flavonols are phytochemical compounds found in high concentrations in a variety of plant-based foods and beverages. They are common in many fruits and vegetables. Quercetin also has dual and complementary actions with regards to aging cells. Like tocotrienols, quercetin can induce senescence and promote cell death in numerous types of cancer cells. Like tocotrienols, quercetin has the opposite effect in healthy cells, delaying senescence in younger cells and rejuvenating older cells to rid them of their abnormal, age-promoting function. Tocotrienols are members of the vitamin E family of essential nutrients, naturally found in grains and nuts. However, newer information indicates that for other activities including antioxidant, cholesterol lowering, and anti-cancer effects that the tocotrienols (T3s), especially delta-tocotrienol (delta T3) exert more profound effects. I found some interesting information concerning the strengths of Vitamin E. So, while in terms of vitamin E activity the order of potency would be: alpha T > beta T > gamma T > alpha T3 > delta T > gamma T3 > delta T3.
We can see that the T3 tocotrienol has the most potency and the probable best chance of being a senolytic agent. Let us take a better look at Vitamin E more specifically Vitamin E T3. Here are some helpful diagrams.
The different forms of Vitamin E are what causes controversy in studies. Some forms of Vitamin E have very different actions. The Tocotrienol form, especially T3 is a very potent anti-inflammatory agent in addition to being a senolytic agent.
When one looks at some the most recent studies there is evidence that tocotrienols and quercetin reduce levels in the blood of inflammatory cytokines. When these inflammatory cytokines are suppressed than systemic inflammation is reduced.
Another promising senolytic agent is fisetin. Fisetin is flavonoid (a plant pigment that gives plants color, smell etc.) derived from various fruits such as strawberries. Fisetin also has direct activity as a reducing agent, chemically reacting with reactive oxygen free radicals to neutralize them. Based on lab studies, it appears that fisetin lodges in cell membranes and prevents oxidative damage to lipids in the cell membrane.
Now comes the tricky part. We do not think it is best to continue on some of these supplements on an indefinite basis. I suspect it would be best to take them on an occasional basis. We feel the best method would be to combine a few of these supplements in a propriety manner. This would eliminate a good number of the senescent cells. This elimination would hopefully have a cascade effect. This effect would significantly help reduce inflammatory cytokines. Essentially what we are doing is allowing the regenerative cells to have a better chance of achieving success. We now feel we are ready to launch a program to achieve a reduction in the senescent cell population.
Will senolytic agents be a game changer? Only time will tell. I am not a betting man but if I were a betting man I would bet on them. They might take us one step closer to the promised land. Thanks Dr. P
BOSTON BIOLIFE MEETING
The above pictures may be confusing to those who are history buffs. I am mentioning the Boston Biolife meeting yet I am showing pictures of Constitution Hall in Philadelphia. The simple reason for this was that the Boston Biolife Meeting was held in Philadelphia.
This was the first time I gave a lecture in conjunction with Boston Biolife. I must say I believe they have their act together. They have an excellent array of speakers including both PHD’s such as Dr. Peter Everts and Dr. Dmitry Shvartsman and an array of physicians some of whom were new to me and some who were old friends. Unlike any other field in medicine, Regenerative Medicine is one where there is a strong bond between PHD’s and MDs. It is only if we work together can we bring this specialty to the forefront of medicine. The target audience were those physicians who want get in depth information about the Regenerative Medicine field. They seemed ready, willing, and able to learn. What I believed these physicians realized is what can be found in the Boston Biolife insignia. In Regenerative Medicine, we need to be a physician, a scientist, a technician, and finally a physician who can bridge all these fields. We need to start as a physician. Having had the training as a physician, we need to understand the science and why we do what we do (scientist). Once we master these steps we need to know how to do the procedures properly (technician). When we achieve these goals we than need to act as a translational physician. A translational physician needs to put it all together. Many people may be asking what is a “translational medicine (TM)”? The Wikipedia definition is as follows: “an interdisciplinary branch of the biomedical field supported by three main pillars: bench side, bedside and community. The goal of TM is to combine disciplines, resources, expertise, and techniques within these pillars to promote enhancements in prevention, diagnosis, and therapies. Accordingly, TM is a highly interdisciplinary field, the primary goal of which is to coalesce assets of various natures within the individual pillars in order to improve the global healthcare system significantly”. These are the pillars of the Boston Biolife program. They teach science, techniques, and decision making. If I could have had these concepts when I started out it would have made an easier path for me.
I heard a good number of talks about spinal problems including the treatments of disk problems. Some good science was mentioned. We still have a way to go concerning the discs of the spine. Discs represent one of the harshest environments in the body for stem cells. Most will perish very quickly in the environment of a disc. I feel that perhaps one way to treat this problem might involve MUSE cells. Muse cells are very forgiving and actually seem to thrive in a stressful environment. We have a propriety way of obtaining the MUSE cells and are looking forward to utilizing them in a disc. This will be the subject of another blog. I did present some new information concerning a comparison of centrifuged vs non-centrifuged bone marrow aspirate. The non-centrifuged aspirate seemed to perform better on a number of different levels. The apparent reasons for this are mentioned in one of my other blogs. I suspect the field will ultimately move in the direction of non-centrifuged or at least some hybrid form of bone marrow aspirate. One other talk that rang true to me was that of Dr. Peter Everts. Dr. Everts spoke about Platelet Rich Plasma (PRP). He echoed my thoughts that there is much more to PRP than just platelets. Actually, a portion of my lecture was devoted to “Scary Urban Legends” concerning PRP. I showed some scientific reasons why we need to consider all components of the blood when we are discussing PRP.
The other aspect of the course was the practical aspects of the course. The actual preforming procedures such as a bone marrow aspiration on a cadaver. I happened to put my two cents in on certain techniques. One such technique were methods of performing a bone marrow aspiration which seems to give both higher numbers of regenerative cells and better clinical results.
All in all, this was a very good meeting representing different viewpoints but having a consensus of what seems to be important for the fledgling Regenerative Medicine doctor to grasp and learn. I had a very positive experience at this meeting. I think the same could be said about the physicians in the audience. Finally, the course organizer, Joseph Krieger gave some advice to the audience that I have been preaching for years. I actually have a slide on this. The saying is “the more we know the more we don’t know”. As my knowledge in the field expands so does my lack of knowledge. Thanks Dr. P
ARE ALL PRPs THE SAME? WHAT ABOUT PRXP?
In the Regenerative Medicine field, there has for some time been controversy concerning certain aspects of Platelet Rich Plasma or PRP as it is called. There seems to be a misconception in some circles that some components of a PRP are bad. There is a belief amongst many Regenerative Medicine doctors that a leukocyte poor (LP) PRP is more effective than a PRP product that contains leukocytes. Leukocytes are the white blood cells found in blood. Moreover, there is a perception that the only important component of a PRP are the platelets which contain the growth factors. When one delves deeper into the science of PRP we understand that this is far from the truth. As a matter of fact, there is little scientific literature to support the idea that LP-PRP is preferred over the Leukocyte Rich (LR) PRP. However, there is a copious amount of literature to support the concept that all components of the blood are necessary to achieve an optimal result.
Recently I read an article by Dr. William R. Parrish. The title of his article was: "Platelet rich plasma in osteoarthritis: more than a growth factor therapy". This title certainly rang true to my beliefs. I have been stating for years that having a PRP product with a minimal amount of white blood cells (Leukocytes) was probably not a good thing. Furthermore, Dr. Parrish stated that there is a notion (not his own) that other blood products such as red blood cells also contribute to an increased inflammatory response in a PRP product. The one concept that was ignored was that these studies that lead to the concept that WBCs and RBCs were detrimental were studies performed in the lab (in vitro).
Studies in the lab many times produce far different results than those in real life (in vivo). When we are studying a lab situation this is far different than real life. The environment is far different and thus the results can be far different. A review of the literature shows little in the way of support for the use of low concentrations of WBCs. The pendulum of research now supports the idea that greater concentrations of leukocyte and RBCs yielded a greater therapeutic effect on the patient. This is something I have known for the last ten years or so. I will concede that there may a bit more discomfort from a LR-PRP but at most this may last a day or so. We also have some propriety methods to help reduce this inflammation.
What is potentially so special about these other blood components. One thing we know is that platelets require some form of activation. Typically, when platelets come into contact with tissue (collagen) they become activated. RBCs help in platelet activation. RBCs will help generate additional amounts of thrombin. Thrombin is a very potent stimulator of platelet activation. When some stem cell processes are given intravenously the platelets are mixed with thrombin so that they will release their growth factors. We would not want to use platelets intravenously since they could form a blood clot in a vein leading to serious consequences. The growth factors would not be a problem.
Other factors that lead to the importance of RBCs is their content. RBCs are considered a sink of Nitric Oxide. (NO) is a gas which has numerous effects on cells. NO is a multifaceted compound that can act as a growth, immune and neuro-stimulator as well as causing angiogenesis (formation of blood vessels). They also contain Glutathione. Glutathione is a very potent antioxidant that can alter cell functions in a very positive way. It can help disable some free radicals at the site of injury. Free radicals rob the cells of energy and alter cell function in a negative way.
As Dr. Parrish pointed out eventually, the RBCs will be attacked by WBCs. When this occurs, the WBCs will release a variety of anti-inflammatory growth factors which will quell the inflammatory response and allow healing to occur. Many regenerative medicine docs continue to have the idea that RBCs cause an inflammatory response. Perhaps under certain conditions this might be true. However, this is usually not the case. A good clinical example of RBCs not causing an inflammatory response in what is called a "blood patch". A blood patch is used when a patient had a spinal tap (typically by accident) and then develops a severe headache. The headache results from leaking spinal fluid. A blood patch occurs when whole blood is taken from a vein and injected into the area of the spinal leak. The blood plugs the leak and the headache goes away. Moreover, the blood which is in direct contact with the spinal cord covering (dura) does not cause any type of inflammation. A similar example occurs when a patient suffers a bleed inside the knee. This bleed might be traumatic in nature such as an ACL tear. The blood in the joint does not cause an inflammatory response.
The leukocytes (WBCs) have a very important aspect in the overall scheme of regenerative therapies. As was pointed out in Dr. Parrish’s article there is an extremely important relationship between platelets and neutrophils. It seems that these two cells will work in a type of symbiosis helping to limit the magnitude and duration of the inflammatory response. These two cells work together to form an entity called a lipoxin. The lipoxin is produced by the platelet. Lipoxins are very potent anti-inflammatory molecules. The limit the neutrophil activation. They have far reaching effects but the bottom line is that they foster an anti-inflammatory environment.
What else might be thrown away when producing a PRP? One discard might be a macrophage. Macrophages come in two forms. They are an M-1 and M-2 macrophage. These are very important in our immune system. The following slides are from one of my lectures
In the above we can see the macrophages at work in their normal environment. Interestingly enough the mesenchymal stem cell can have a direct effect on these cells depending upon the environment. However, if we remove these cells from the PRP product than we cannot cause these reactions to occur. The second slide explains these actions.
Another type of white blood cell which is very important is called a neutrophil. Neutrophils have been for years thought to be culprits to instigate inflammation. Under certain circumstances this is true. In the course of a PRP injection neutrophils can have an opposite effect. They can actually quell an inflammation and help release anti-inflammatory compounds. Once the platelets become activated they will “prime” the neutrophils. A primed neutrophil will help to release anti-inflammatory growth factors and limit the inflammatory response. This is very important for otherwise more neutrophils would be called to the area and cause more inflammation.
We must also realize that there are some stem cells in a PRP product. These cells are called Hematopoietic Stem cells (HSCs). We now think that the HSCs are probably the cells that may help direct tissue repair. When we are trying to make a PRP product which is devoid in leucocytes we are also “throwing away” these valuable cells. A tantalizing thought is to give some medications that are able to help mobilize more of these stem cells into the circulation. There are some cancer therapies which stimulate the bone marrow to release more stem cells to the circulation but they have significant problems such as cost and potential side effects. We have come across a supplement which has a similar effect with a very reasonable cost and essentially no side effects. Some food for thought about the numbers of stem cells in a typical PRP product. HSCs (represent 0.06% of circulating TNCs) translates into 3-7K HSCs per ml of blood processed. Is this number significant? No one can say for sure but I would rather have stem cells present than not.
A question one may ask is why not just use whole blood? The problem with that is we will not get enough of the platelets. We have to concentrate the platelets. This is the whole idea behind Platelet Rich Plasma. The bottom line is to try to make full use of the tools nature has given us in blood. This probably gives us the best chance of success when using PRP for a Regenerative Medicine treatment. Thanks Dr. P
The Boston Biolife Meeting
Occasionally I am asked by some docs where I will be doing my next speaking engagement. I have tried to cut down on my international travel schedule. It will be creeping up in the fall with a few trips to Asia. In the meantime, I will try to stay local in the good old USA. This month I am scheduled to give a talk in Philadelphia at the Boston Biolife meeting. I am very much looking forward to this meeting. In looking at the faculty and topics presented it appears to be a well balance meeting presenting many different viewpoints. Probably more than any field, Regenerative Medicine demands an open mind. I can see this thru my own transformation. There were topics that I once considered gospel than I began to question them and finally discovered that what I once considered true was actually not so. Every meeting I attend I usually take back a small nugget of information. I am looking forward to doing so at this meeting. The meeting does not seem to have an ax to grind. They are not pushing one type of technique over another. What they are pushing is science and techniques. They want to present many different opinions. Too often meetings get tilted in their approach to Regenerative Medicine problems. The Boston Biolife approach is to give a solid background in Regenerative Medicine so that the physician can build upon this and better treat his patients. They do this by giving many different points of view. After being given information, the physician can make an informed decision on how to best treat his patients.
I am asked to give a lecture in the field of Regenerative Medicine as it pertains to musculoskeletal skeletal problems. What I will do is call upon my experience of performing many thousands of cases in Regenerative Medicine. I will tell what seems to work for me. More importantly, why it probably works for me. When one understands the “why” than they possess tools to improve upon the situation. To do things as a mechanic means to not understand the “why”. Unless one understands the “why” than improvement is an elusive goal.
I will introduce some our our newer concepts in the field of Regenerative Medicine. One topic that I suspect will garner interest is a procedure called Purekine. Purekine is our method of producing a IRAP product. IRAP stands for anti-interleukin-1 antagonist. This is one on the most potent anti-inflammatory agents known. It is similar to the German process called Regenokine. However, it is derived from the bone marrow.
We will also present some of our clinical data that compares centrifuged vs non-centrifuged bone marrow. This data has never been presented before anywhere in the world.
All in all I look forward to this meeting to present new material and at the same time learn some valuable nuggets of information from the other faculty. We all must realize one thing. Regenerative Medicine is a field that unlike any other there is a good chance that someone at the meeting can make a significant difference and change the course of events in the field of Regenerative Medicine. Thanks Dr. P
STEM CELLS AND THE IMMUNE SYSTEM (PART 2)
In part one we have discussed various effects that the stem cells have upon the immune system. Various stem cells will help in the release of certain type of immune cells. Some of these cells will stimulate an immune response while other will diminish the immune response. We are still missing one very important effect that they have on what we call macrophages and other cells.
As was mentioned in part one of the most important stem cells in dealing with the immune system is the mesenchymal stem cells (MSCs). As Dr. A Caplan states they are “medicinal signaling cells”. Their main function is not to cause tissue regeneration but to help in immunomodulation. In another word, they help water down an immune response. Without these cells, most repair would never be accomplished the area and the damaged area would remain unrepaired. The following diagram from one of my lectures is an excellent description of these cells:
As you can see like Navy Seals they are very specialized. They will not win the war but they can secure an area so that other forces (stem cells) can come in an accomplish repair. MSCs possess broad immunomodulatory properties. After activation, MSCs can secrete a variety of soluble factors, such as NO, IDO, PGE2, TGF-β, HLA-G5, TSG-6, CCL2, IL-1Ra, and IL-10. These are various growth factors or cytokines that are produced. Their names are not that important to the lay person. What we really need to know is that production of these factors can suppress the differentiation, proliferation, activation of various immune cell subsets. As a result, the immune response will be inhibited and local inflammation is suppressed by MSCs.
One effect of the MSCs that has not been mentioned is the effect that they have on macrophages. The Wikipedia definition of macrophages “Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign able to be accomplished. The body would destroy all reparative cells substances, microbes, cancer cells, and anything else that does not have the types of proteins specific to healthy body cells on its surface in a process called phagocytosis”. However, there is more than meets the eye here. The following slide from another of my lectures shows the importance of macrophages:
What we are able to ascertain from this diagram is that there are two types of macrophages. The following slide is an explanation of these two types of macrophages.
The MSCs will affect the microenvironment which ultimately determines the fate of the development of the macrophage. Obviously when we are fighting an infection we want to produce the M-1 type of macrophage to obliterate an infection. However, when we are performing a regenerative medicine procedure we want to produce an M-2 macrophage which will help with immune modulation. Anything we can do to help the MSCs will be a boon to regeneration. You may ask how do we do this very simply. If you were to look at the diagram you will see some growth factors with the following designations IL-1a, IL-10, and TGF-b. These are powerful growth factors that will nip inflammation in the bud. These are part of our propriety cytokine formulas. We are helping the regeneration. We are encouraging the release of M-2 macrophages which are immunomodulatory and also release growth factors which promote tissue repair. This is one reason why we do not feel it is wise to eliminate white blood cells of which macrophages are a part of that family from a Platelet Rich Plasma (PRP) mixture. We are also able to produce more M-2 macrophages by a process of photo-modulation. This occurs when a PRP product treated with certain wavelengths of light increases M-2 macrophage production. Macrophages are also important for other purposes. They help with what is called stem cell homing.
One more effect that the stem cells have upon the immune system involves the production of LL-37. LL-37 is a very interesting compound. It is one of the most effective anti-microbial compounds known to man and actually the only antimicrobial produced by man. It gives broad antimicrobial protection against bacteria, fungi and viruses. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin LL-37. Another interesting fact is that the effect of Vitamin D-3 on the function of LL-37 and chemokine production as well as an important role in immunomodulation. These anti-inflammatory and anti-infective roles of Vitamin D are becoming increasingly important.
The actual mechanism of LL-37 production is thought to be turning on a gene which helps production of LL-37. The vitamin D turns on this gene. Is there any practical example of the relationship between stem cells, vitamin D and the immune system? Perhaps one of the best examples occurs when a woman has a period. During a menstrual period, there is a setup for an infection. We have bacteria present in the mist of blood products at a temperature conducive for bacterial growth. This is a recipe for infection. Yet infection almost never occurs. Why is this so? We must remember one thing. Menstrual blood is very rich in mesenchymal stem cells. These mesenchymal stem cells release compounds including LL-37 which will luckily squash any type of infection. I have just scratched the surface concerning stem cells and our immune system but I believe we see how these important stem cells may be used in the treatment of many different conditions. Thanks Dr. P
