INSTITUTE OF REGENERATIVE AND MOLECULAR ORTHOPEDICS

DR. JOSEPH PURITA MD, FACS, FAAOS, FAAPM www.stemcellorthopedic.com and STEM CELL CENTERS OF AMERICA

Conditions that Promote Inflammatory Cytokines

1. Declining sex steroids -Testosterone, estrogen, androstenedione, also growth hormone (Anabolic)

2. Obesity3. Smoking4. Subclinical bacterial infections5. Increasing BMI6. Arteriosclerosis7. Diet8. Glucose9. Excess calories

Although adult stem cells have been considered an attractive source for cell therapy, their effectiveness and efficiency is hindered by a frequently low survival rate due to their exposure to a high cellular stress environment upon transplantation. This key limitation is observed when utilizing adult stem cells for regenerative purposes, as typical cell engraftment yields are extremely low (3%)

Inflammatory Markers and Mediators1. TNF-α 2. Nuclear Factor Kappa 3. Interleukins 4. C- reactive protein 5. Eicosanoids 6. Cyclooxygenase 7. Lipoxygenase

Multiple factors contribute to this low rate of cell survival, including the harsh environment of the recipient site (EXTRA CELLILAR MATRIX), harboring pro-apoptotic factors including hypoxia, malnutrition, pro-inflammatory cytokines and reactive oxygen and nitrogen species.

TNF-αAcute

Chronic

1. Cytokine released in response to cellular stress, damage andinfection2. Anti tumor compound produced by macrophages3. Activates bactericidal effects of neutrophils

1. Can lead to chronic inflammation Activates nuclear factor kappa-B which initiates inflammatory gene expression and pro-inflammatory enzymes including COX-2 andlipoxygenases2. Increases thrombosis3. Decreases cardiac contractility May be implicated in tumorinitiation and promotion (Kundu et al. 2008

C-reactive ProteinAcute ChronicAcute phase protein

synthesized by liver

during initialinflammatory response

“coats” damaged cells for

recognition by immune

cells

Strong association with

elevated risk of

cardiovascular disease and

stroke (Emerging Risk Factors Collaboration

et al. 2010)

Cyclooxgenases & Lipoxygenasesacute chronic

Catalyze first steps in synthesizing eicosanoids

converting omega-3 and omega-6 into necessary thromboxanes and

prostaglandins Lipoxygenases convert fatty acids into pro-inflammatory

leukotrienes necessary for local inflammatory mediation

Recruit WBC’s to site of inflammation

Chronic production of

pro-inflammatory

prostaglandins, leukotrienes,

thromboxanes, cytokines amplifying inflammatory response

IF WE ELIMINATE INFLAMMATION WE MAY TURN OFF CERTAIN DISEASES

HOW DO WE ELIMINATE INFLAMMATION? Certain forms of inflammation are necessary for the body to heal itself. Nsaids (Advil, Aleve etc.) and cortisone can relieve inflammation but have many serious side effects and actually stop the body from trying to heal itself. In the long term these are usually not effective in eliminating the disease process.

WHAT SEEMS TO BE THE ROOT OF INFLAMMATION? INFLAMMATION SEEMS TO BE RELATED TO THE FORMATION OF FREE RADICALS. AS WE USE OXYGEN WE FORM FREE RADICALS.

UNDERSTANDING FREE RADICALS Here we see the chemistry. The free electron causes the damage.

WHAT PART OF THE CELLS RECEIVE THE MOST DAMAGE?

THE CELL POWERHOUSES, NAMELY THE MITROCHONDRIA

WHERE DO FREE RADICALS COME FROM?1. THE ENVIRONMENT (air pollution,

drugs, certain foods)

2. INTERNAL PRODUCTION (the bodies own production).

3. STRESS FACTORS (aging, trauma, disease and infection).

4. CHAIN REACTION resulting in DNA damage

FREE RADICALS ARE CELLULAR KILLERS!!!1. THEY DAMAGE DNA.

2. THEY ALTER BIOCHEMICAL

COMPOUNDS AND REACTIONS.

3. THEY DESTROY CELL MEMBRANES. 4. THEY DESTROY CELLS OUTRIGHT.

THEY WILL DAMAGE CELLS CAUSING THEM TO LOSE SOME GENETIC MAKE-UP. THESE DAMAGED CELLS GO ON AND CAUSE DISEASES.

WHAT CAN PROTECT US FROM FREE RADICALS? We must look at the ORAC level of foods and medications.

ORAC= OXYGEN RADICAL ABSORBANCE CAPACITY

The higher the ORAC number the better the food or medication is in fighting free radicals.

REMEMBER ELIMINATING FREE RADICALS SLOWS DOWN DISEASES, AGING ETC.

ORAC VALUES OF CERTAIN FOODS

FREE RADICAL FIGHTERS Many of the antioxidant fighters are called phytonutrients. These are biologically active compounds that are concentrated in the skins of fruits and vegetables. They are responsible for the flavor, color, and smell of fruits and vegetables. REMEMBER TRY TO EAT FIVE COLORS A DAY!!!

BEST METHODS FOR FIGHTING FREE RADICALS

1. DIET 2. EXERCISE 3. SUPPLEMENTS

DIET Use a Mediterranean type of diet. Low in bad fats, low in bad carbohydrates, low in red meats. This type of diet does wonders in reducing inflammation in the body. The Scripps Institute calls this the anti-inflammatory diet!!

REMEMBER FIVE COLORS A DAY!

EXERCISE1. Have physical exam by physician.

2 If you are able , embark on a

program that in short bursts can elevate heart rate to about 85% of your maximum cardiac rate. This will naturally increase the body’s secretion of HUMAN GROWTH HORMONE (HGH)

For every decade of age increase resistive exercises.

SUPPLEMENTS1. Are supplements really necessary?

2. Do they have a place in stem cell therapy? 3. Can they slow down aging?

4. Can they help reverse some of the aging

processes?

5. Which are the most important for our purposes? (stem cell production)

THE BEST SUPPLEMENTS FOR OUR PURPOSES?

1. Omega-3 fatty acids (fish oil)

2. Resveratrol3. Various phytonutrients

4. Vitamin D-3 and Vitamin C

5. Carnosine

6. Stem X Cell supplement 7. Curcumin UltraCur 8. Living greens from both the land and the ocean

FUCODIN9. Nitric Oxide producers such as Neo-4010.Collagen stimulating compounds CH-ALPHA

OMEGA 3 FATTY ACIDS = FISH OIL

1. Only be concerned with the content of the EPA and DHA products of the fish oil.

2. The EPA and DHA need to equal 3000mg. You may need to take 6000mg of fish oil to obtain 3000mg of omega 3s.

3. Put fish oil in the freezer and take them frozen. Use care if taking with blood thinner

EFFECTS OF OMEGA 3 FISH OIL ON CELL MEMBRANEThe cell membrane is the eyes and ears of the cell. The cell membrane is basically the brain of the cell. Omega 3s make the cell membrane more receptive to the growth factors.

OMEGA 3 FISH OILInterestingly, researchers found an inverse relationship between levels of Omega-3 in the blood and telomere shortening: the more Omega-3 found in a patient’s blood, the less telomere shortening the patient had undergone over those years. The telomeres are the DNA ends.

OMEGA 3 FATTY ACIDSAssociation of marine omega-3 fatty acid levels with telomere aging in patients with coronary heart disease was published in the January 20, 2010 issue of the Journal of the American Medical Association. This study showed that over approximately a 5 year period telomere shortening was significantly reduced in the Omega 3 group.

OMEGA 3 FISH OIL It is possible that Omega-3 acids could increase the activity of telomerase in human cells and/or reduce the impact of oxidative stress on telomeres.

RESVERATROL

Derived from red wine, resveratrol has demonstrated significant health benefits ranging from cardiovascular protection to anti-cancer effects. It is believed that resveratrol works by mimicking the effects of calorie restriction, the best anti-aging strategy to date, through mechanisms such as reducing oxidative stress, boosting energy production, and regulating gene expression.

RESVERATROL1. Activates the SIRTUIN gene. The Sirtuin

gene is normally inactive after age 40. This is called gene silencing. When the gene is active it triggers a survival mechanism that appears to extend life.

2. Research performed at Harvard University.

3. Seems to mimic many of the effects seen in calorie restrictions and exercise.

4. There seems to be increase in stem cell production.

RESVERATROL DOSAGE1. No clear cut answer.

2. One class of RED wine has between

0.5-1 mg of resveratrol.

3. Probably need to take at least 500mg of trans Resveratrol per day.

4. If taking blood thinner check the blood thinning.

GENE SILENCING As we age certain genes become inactive or SILENCED. Many of these genes protect us against diseases (cancers) and the ravages of aging. If these silenced genes can be turned back on they will protect us from these problems!!!!!! Much in the same way a 20 age old is protected from these diseases.

VITAMIN D At least 2,000 genes, or nearly 10% of your genes, have been identified that are directly influenced by vitamin D, which in turn impact a wide variety of health issues, from preventing the common cold and flu to inhibiting at least 16 different types of cancer. There’s even evidence linking vitamin D to the process of brain detoxification of heavy metals such as mercury.

VITAMIN D One of the most important genes Vitamin D up-regulates is for cathelicidin, a naturally occurring broad-spectrum antibiotic. Vitamin D's final metabolic product is a potent, pleiotropic, repair and maintenance, seco-steroid hormone .

A growing body of evidence indicates that rickets in children and osteomalacia in adults (both a softening of bones due to defective bone mineralization) are just the tip of a vitamin D-deficiency iceberg. Tuberculosis and various autoimmune diseases, such as multiple sclerosis, lupus, and type I diabetes have a causal association with low vitamin D blood levels. Vitamin D deficiency plays a causal role in Hypertension, coronary artery disease, congestive heart failure, peripheral vascular disease, and stroke. It is also a risk factor for metabolic syndrome and type II diabetes, chronic fatigue, seasonal affective disorder, depression, cataracts, infertility, and osteoporosis. At the bottom of the vitamin D iceberg lies cancer. There is good evidence that vitamin D deficiency is a causal factor in some 15 different common cancers. (NEJM 2007;357:266-81.)

WHAT TYPE OF VITAMIN D TO TAKE?

PATIENTS SHOULD BE ADVISED TO TAKE VITAMIN D3. OTHER FORMS OF VITAMIN D DO NOT HAVE THE BENEFITS AND CAN BE TOXIC TO THE LIVER IN HIGH DOSES. THESE FORMS OF VITAMIN D INCLUDE COD LIVER OIL. IT IS BELIEVED THAT VITAMIN D3 IS LIPID AND WATER SOLUBLE.

VITAMIN C

1.Very important in the production of collagen and therefore cartilage

2.Also acts as a good anti-oxidant

STEM X CELL STEM CELLS AND DEVELOPMENT 15:118–123 (2006)

Original Research Report“Nutriceutical Synergistically Promote Proliferation of Human Stem Cells”

PAULA C. BICKFORD, JUN TAN, R. DOUGLAS SHYTLE, CYNDY D. SANBERG, NAGWA EL-BADRI, and PAUL R. SANBE

STEM X CELL CONTENTS1. VITAMIN D-3 2000 UNITS

2. CARNOSINE

3. BLUEBERRY EXTRACT

4. GREEN TEA EXTRACT

5. A VARIETY OF ENZYMATIC EXTRACTS

Melatonin-N-acetyl-5-methoxytryptamine 1. Secreted by pineal gland as well as

extra pineal production 2. • Produced in darkness, suppressed by

light 3. • Concentrated in nucleus and

mitochondria 4. • Levels decline with aging - 10-15% per

decade 5. • Manages circadian rhythm of inner clock 6. • Controls sleep wake cycle

Scavenges Free radicals • Most effective FR scavenger of hydroxyl radical known

• More than Glutathione or Vitamin E • Hydroxyl radical damages mitochondria

• Protects DNA from Injury • Especially in Pharmacologic concentrations • Protects against pro-oxidation effect of Fe

• Herrera J et al. Melatonin prevents oxidative stress resulting from iron and erythropoietin administration Am J K idney Dis 2001 Apr; 37(4) : 750- 7

Protects lipids, proteins, DNA • Stimulates glutathione • Protects mitochondria • Protects against ischemia- reperfusion injury • Protects against ionizing radiation

Reiter RJ et al. Pharmacological utility of melatonin in reducing oxidative cellular and molecular damage. Pol J Pharmacology. 2004 Mar-Apr;56(2):159-70

MELATONIN =The ultimate anti-oxidant

Melatonin Dose - 1/2 hr before sleep

Try small dose 0.5 mg at first • If no unpleasant reaction can increase dose in increments to 3-10 mg

• Time release for people who wake up in middle of night

• Sublingual lozenge or drops for people who have trouble falling asleep

• Some tolerance develops but usually levels off at 3-10 mg

• For some people, less in more and better sleep at low dose 0.3 mg

CHLORELLAChlorella stimulates the immune system by increasing the number and activity of macrophages and polymorph nuclear leukocytes. It also stimulates interleukin production. A polysaccharide from the cell wall of chlorella also induces production of interferon. The photosensitizing components in chlorella have been identified as pheophorbides.

ASTRAGALUSWhen administered there is some evidence to suggest astragalus extract might increase proliferation and differentiation of bone marrow stem cells and progenitor cells. Astragalus also seems to have broad-spectrum in vitro antibiotic activity. It may have an effect on telomeres. This effect may be related to the production of telomerase.

ASTRAGALUSAstragalus has antioxidant effects. It inhibits free radical production, increases superoxide dismutase, and decreases lipid peroxidation. There appears to be a multitude of research of the compound in the medical community.

SUPEROXIDE DISMUTASE (SOD)Superoxide dismutase (SOD) is an essential enzyme found in all living cells. Some cellular processes produce reactive oxygen species (ROS) such as superoxide radicals that can impair cell function. SOD catalyzes the conversion of superoxide to oxygen and hydrogen peroxide, reducing damage from ROS.SOD isoenzymes contain copper and zinc, and iron or manganese.

Antioxidant Glutathione, or GSH Whey protein has been shown to increase your body’s stores of the antioxidant glutathione, or GSH. Glutathione is known to increase the integrity of telomeres. Glutathione is not a compound you can ingest directly with great success . It is manufactured inside your cells from its precursor amino acids, glycine, glutamate and cysteine. The most efficient precursor to GSH is n-acetyl cysteine.

ARGININE In the body, the amino acid arginine changes into nitric oxide (NO). Nitric oxide is a powerful neurotransmitter that helps blood vessels relax and also improves circulation. The suspicion is that arginine can mimic hyperbaric oxygen and by producing NO formation enhance stem cell production. It is also felt that arginine has a direct effect on the pituitary gland and stimulates HGH production. NOT AS EFFECTIVE AFTER AGE FOURTY. AFTER AGE FOURTY USE NEO-40!!!

NITRIC OXIDE (NO)NITRIC OXIDE PRODUCTION MAY BE PART OF THE HOLY GRAIL OF STEM CELL THERAPY IN THAT IT HELPS PRODUCE LARGE NUMBERS OF HEMATOPOETIC CELLS FROM THE MARROW. IT IS A SIGNALING MOLECULE WITH FAR RANGING EFFECTS

Fucoidan1. Complex carbohydrate obtained from

special species of brown seaweed. 2. A highly purified fucoidan

increases the expression of CD34+ cells and V cells.

3. Fucoidan is the "the plant equivalent of mother's milk.”

4. Fucoidan is very important in the diet of Okinawans who have some of the greatest longevity in the world.

BOVINE COLOSTRUM a high quality bovine colostrum providing an abundant amount of vital growth factors (IGF-1, IGF-2, transforming GFs A and B, epidermal-GF, fibroblast-GF and platelet- derived GF plus many immune factors including immunoglobulins, peptides, cytokines)

HORMONES The importance over the proper hormone balance can not be over emphasized. Typically the older patient may be lacking testosterone and/or estrogen. If these deficiencies are corrected the chance of success increases.

HORMONESIN ADDITION TO TESTOSTERONE AND ESTROGEN, ONE MUST ALSO OPTIMIZE THE THYROID AND ENTIRE ENDOCRINE SYSTEM.

AND

LOW LEVEL LIGHT THERAPY

Therapeutic Strategies1. Antioxidant therapy neutralizes the

deleterious effects of ROS and RNS, increasing nitric oxide bioavailability

2. Low level laser therapy (LLLT) dissociates Nitric oxide (NO) from cytochrome oxidase, hemoglobin and myoglobin, allowing immediate influx of oxygen and resumption of respiration. ROS levels increase and trigger downstream effects

3. Released Nitric Oxide(NO) causes vasodilation and activates guanylate cyclase (GC), increasing cyclic guanine monophosphate (cGMP) levels, which stimulates stem cell proliferation/differentiation

Nitric Oxide Mechanism Of Actions1. Now known to be a growth, immune,

and neuromodulator, as well as a stimulator of stem cell proliferation and it has a critical roles in analgesia, vasodilation and angiogenesis through c-GMP pathway.

2. Also it has anti-cancer, anti-inflammatory and anti-microbial activities by reacting with ROS and generating cytotoxic Peroxynitrite .

3. Nitroglycerin , L-arginine and other NO donors are widely prescribed medications

NADP+

ARE MITOCHONDRIA THE BODY’S CHLOROPLASTS?

1. THERE IS NOW STRONG EVIDENCE THAT THE MITOCHONDRIA ARE SIMILAR TO CHLOROPLASTS AND FURTHERMORE THEY MAY ACTUALLY PRODUCE LIGHT.

2. THE MITOCHONDRIA SEEM TO HAVE A PHOTO SENSITIVE PROPERTY WHICH HELPS THEM MAKE ATP.

3. IN ESSENCE THIS IS THE BASIS FOR HOW LASERS WORK!!

LLLT-NO-Antioxidants Improves Current Therapies

Platelet-rich plasma efficacy increased by blue LLLT

In-vitro blue light Irradiation (450 nm) of the whole blood leads to

1.increase in the nitric oxide-deoxyhemoglobuline disassociation2.shift the blood redox to reduction state3.decrease PN levels4.increase the NO bioavailability of the transfused PRP

STEM CELL THERAPY

1.LLLT, PRP, NO and Antioxidants increases the efficacy of the stem cells therapy.

2.Nitric oxide and PDGF stimulates stem cell proliferation and differentiation.

Blue Low Level Light Therapy

1. NO dissociates from deoxyhemoglobin in erythrocytes

2. Free NO then reacts with local ROS forming peroxynitrite

3. Peroxyredoxin reduces peroxynitrite to nitrite

4. Nitrite in converted to NO and H2O by deoxyhemoglobin

5. End results:– ROS levels are reduced– NO Bioavailability increased– Redox state is shifted to reduction

Mechanisms

ROS

ROS

ROS

ROS

Blue LLLT Mechanism

OONN

OONN

OONN

OONN

PN

PN

PN

PN

NO2-NO2-

Peroxyredoxin 2

NO2- NO2-

OONN OONN

OONN OONN

OOH

H OOH

H

OOH

HOOH

H

Peroxynitrite

Nitrite

Erythrocyte

deoxyHb

ROSROS

ROS

ROS levels are reducedNO Bioavailability

increasedRedox state is shifted

to reduction systemically

Mechanisms

Infrared & Red LLLT in Healthy Tissue

1. Under normal conditions, a certain proportion of cytochrome oxidase (CO) is bound to NO, inactivating it

2. Red and infrared LLLT dissociates NO from cytochrome oxidase, leading to increased aerobic respiration & ATP levels

3. Increased NO bioavailability leads to rise in cGMP & stimulation of stem cell proliferation & differentiation

4. ROS levels increase shifting redox state to oxidative

Desired result:• Increase respiration and generation of ATP• Create physiological oxidative state to stimulate

downstream effects

Mechanisms

II

NAD+ NADHNADPH

ADP+Pi

ATP

H+H+ H+

IV

H+

OONN

H+

H+

H+

H+

H+I

ATP synthaseInner

membrane

Outer membrane I

II

Inter-membrane space

Redox state shifts slightly to

Oxidation

Phototrophy GSSG

GSH

Mechanisms

Downstream Effects of LLLTCellular redox potential is shifted towards a physiological oxidation state, leading to:

A.Transcription of protective & stimulatory genes

B.Anti-apoptosis

C.Enhanced stem cell proliferation & differentiation

LLLT Increases Growth Factors and Cytokines LLLT

IL‐1 IL‐8

VEGF TGF‐B FGF HGF

Initial inflammatory phase

Neovascularization collagensynthesis

FibroblastsMyofibroblasts

Fibroblastproliferation and migration

NITROPHOTONICS1. There is probably no pathological condition

where nitric oxide does not play an important role as NO is ubiquitous throughout the body

2. The unique approach of using nitric oxide donors combined with antioxidants along with specific Low Level Light Therapies will have a significant impact on a variety of hard to treat pathologies

3. Activated stem cells can heal the underlying pathology by repairing damage

4.Photobiomodulation can balance the immune system and maintain homeostasis by activating mitochondria and increasing bioavailability of nitric oxide and stimulate the stem cells.

1. Molecular and cellular mechanisms of LLLT are becoming understood

2.Mitochondria are principle photoreceptors3. ATP, cAMP, NO, ROS are primary mediators4. Transcription factors (NF-kB, AP-1, etc.) are activated5. Pro-survival, anti-apoptotic, anti-inflammatory, pro-

angiogenic, pro-proliferation6. Whatever cells are designed to do will be improved by LLLT7. Cells with lots of mitochondria respond well – neurons,

cardiomyocytes, muscle cells, hepatocytes, kidney cells8. Biphasic dose response9. Muscles respond well in both exertion and repair10.Wound healing is major application11.Pain relief is significant12.Inflammation is reduced – arthritis, tendinopathy,

fibromyalgia

Summary

IV Laser treatment with red, green and blue laser

The intraarticular laser therapy

THANK YOU