3. Platelet Poor Plasma
• The low gravity
product of centrifuging
• Traditionally discarded
• Benefits not recognized
by most manufactures
and clinicians
Piss Poor Performance
PPP Garbage
Bag
CellularHealing.net
4. Key take away points
• Whole Bone Marrow and Blood Contain Important Plasma Proteins
• Centrifugation concentrates some, but not all cells into distinct
layers.
• Joints and Soft Tissue Have a Injection Volume Limits
• Coagulation cascade activates and maintains growth factor and
MSC delivery over time.
CellularHealing.net
5. Disclosures
• Consultant for Celling Biosciences – ART BMC™ - Austin, Tx.
2015- Present
• Consultant, Principle Investigator and Stockholder- Augmenstem™
and Plenastem™ AVM Biotechnology- Seattle Wa.- 2016- Present
• Former Consultants for:
• Biomet Biologics – GPS system
• Lilly – Forteo
• Bionicare – PEMF Knee brace
CellularHealing.net
6. What are cell based therapies?
• Types -
• — somatic cell technologies
• — cell immortalization technologies
• — ex vivo gene modification of cells using viral vector
technologies
• — in vivo gene modification of cells using viral vector
technologies
• — genome editing technologies
• — cell plasticity technologies
• — three-dimensional technologies
• — combinations of the above
• Orthopedics will normally use Somatic Cell Technologies:
• This technology uses cells from the human body that are purified,
propagated and/or differentiated to a specific cell product that
subsequently is administered to a patient for a specific therapeutic
treatment without further technological input.
• Components for use in orthopedics
• Platelets
• Plasma proteins
• Stem cells
• Chondrocytes – ACI
CellularHealing.net
7. Three Types of Stem Cells in Bone Marrow
• Hematopoietic- Produce Blood
and Vasculature cell
components
• Progenitor Cells- aka
Circulating Angiogenic Cells
(CAC) or Colony Forming Unit-
Hill Cells (CFU-Hill) -
advanced differentiation cells
found in vascular system
• MSC- Multipotent Stromal Cells
capable of differentiating into
bone (osteoblasts), cartilage
(chondrocytes), muscle
(myoctes) and fat (adipocytes)
• 0.001 to 0.01% of nucleated cells
in BMA being MSCs.
• MSCs may be small, but very
important part of homeostasis.
CellularHealing.net
8. Bone Marrow is Not Just Stem Cells
• Plasma Proteins
• Growth Factors
• Platelet Rich Plasma
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9. Platelets
• Found in blood and bone marrow
• Natural coagulation of platelets – Platelet activation (
platelet aggregation) = alpha (∝) granules = release of
growth factors:
• PDGF
• TGF-B
• IGF
• VEGF
• EGF
CellularHealing.net
10. Plasma Proteins
• Found in both blood and bone
marrow
• Integral part of buffy coat, PPP and
RBC sections.
• Normally discarded with PPP in most
recovery/separation systems.
• 2 best known :
• Alpha 2 Macroglobulin
• IL-1ra- IRAP
• Different functions such as
scavengers and cytokine antagonist
CellularHealing.net
11. Bone Marrow is Not
Just Super PRP
• Cassano and colleagues have directly compared the
cellular content of whole blood to that of platelet rich
protein (PRP) and BMAC.16 The results found that
BMAC:
• 11.8x the number of white blood cells,
• 19.4x the number of neutrophils and
• 2.5x the number of platelets higher than that of
PRP.16
• 172.5x the concentration of vascular endothelial
growth factor (VEGF)
• 78x the concentration of interleukin-8 (IL-8),
• 4.6x the concentration of interleukin-1beta (IL-
1β),
• 3.4x the concentration of transforming growth
factor- β2 (TGF-β2) and
• 1.3x the concentration of platelet derived
growth factor (PDGF).16
CellularHealing.net
12. Concept of separating human blood or bone
marrow
• Based on weight of specific cell
groups
• Gravity separates cells based on
specific gravity. I.e. Sed rate
• High gravitational fields from the
centrifuge creates gradients and
specific gravity of the cell aligns
cells into distinct layers based on
formula angular velocity squared
times radial distance of centrifuge.
• High gravity = Red blood cells
• Low gravity= plasma
• Mid gravity = Buffy coat layer
CellularHealing.net
13. Mid gravity components
• All cells are not 100% compliant
with specific gravity principle
• Not all separation techniques
separate specific cell types
completely.
• Specific gravity techniques are
based on an average guess of
specific cell types following basic
principles of gravity.
• Stem cells are found in all layers
but on average most will gravitate
to Buffy coat or mid gravity layer.
CellularHealing.net
14. Plasma Proteins
• Plasma Proteins- Isolated/ Concentrate
Products
• Alpha 2 Macroglobulin (A2M)
• Isolated from blood, bone or plasma
• Very large and folded protein (Kringle
domain) designed to sweep and clean
protease enzymes
• May be found in 2-6x normal concentration
in GFC, blood and bone marrow.
• Therapeutic levels may be around 4-6 x
normal blood levels.
CellularHealing.net
15. Plasma Protein a2M
A2M has been shown to reduce 3 types
of destructive enzymes:
• Cytokines ( IL-1 and TNF)
• Matrix Metalloproteinases (MMP’s)
• ADAMTs (a disintegrin and
melalloproteinase with
thrombospondin)
• Thus, A2M has a broader effect in
reducing destruction.
Weng S et al, Identification of a2-macroglobulin as a master inhibitor of
cartilage- degrading factors that attenuates the progression of post
traumatic osteoarthritis, Arthritis Rheumatol. 2014 Jul: 66(7) 1843-53
CellularHealing.net
16. A2M Process
• Cytonics A2M process-
• 100-120 cc of blood drawn
and centrifuged for 4 min.
• Filtered for 20 min
• Produces final product with
4-6 x normal amount of A2m
• Cost is $1100 to $1400 per
kit.
CellularHealing.net
17. Scuderi A2M Study with FAC Pre-
test
• A study of 24 patients with lower back pain and MRI positive
for DDD were treated with concentrated autologous A2M.
• 13 males and 11 females were first tested for a biomarker
associated with lumbar disc disease (fibronectin-aggrecan
complex or FAC).
• When A2M was injected in FAC positive patients, the VAS
improvement was 4.9 +/- 0.9, versus 1.5 +/-1.2 in patients
with negative FAC.
• ODI scores in patients treated with A2M improved an average
37 +/- 9.3 points in FAC positive patients versus 9.4 +/- 11.9
in FAC negative patients.
CellularHealing.net
18. IL-1B and OA progression
• There is a direct relationship of IL-1B and
OA.
• Expression of IL-1β has a significant
positive correlation with joint space
width.
• The levels were also negatively
correlated with joint alignment (femoro-
tibial angle).
• Local expression levels of IL-1β are
associated with the severity of disease
• Mabey T, Honsawek S. Cytokines as biochemical markers for knee
osteoarthritis. World Journal of Orthopedics. 2015;6(1):95-105.
doi:10.5312/wjo.v6.i1.95.
• Ning L, Ishijima M, Kaneko H, et al. Correlations between both
the expression levels of inflammatory mediators and growth
factor in medial perimeniscal synovial tissue and the severity of
medial knee osteoarthritis. International Orthopaedics.
2011;35(6):831-838. doi:10.1007/s00264-010-1045-1.
CellularHealing.net
19. Interleukin 1 B wide ranging effects
• Regulates and initiates inflammatory responses
• Induces cyclooxygenase-2 (PTGS2/COX2)
• Is a suppressor of type II collagen and aggrecan
synthesis which are key constituents of cartilage
• Capable of inducing chondrocytes and synovial cells
to synthesize MMPs. Turns Cartilage cells against
each other
• Inhibits transforming growth factor-ß (TGFB)
• Induces the production of IL-6 and IL-8
• Increase in VEGF (The El Chapo ) AKA ”The
Tunneler”
• Synergistic action with PDGF for cartilage
degradation
CellularHealing.net
20. Plasma Protein IRAP™
• IRAP™ stands for intrluleukin-1 Receptor
Antagonist Protein which binds to a
chondrocyte receptor for Interluekin-1 Beta.
• Thus blocking the harmful effects of
Interlukein-1 Beta ( IL-1 B)
• Reducing IL-1 B will help reduce destruction .
*
• IRAP™ is used by many race trainers to
improve performances.
• Number 6 in 2017 Kentucky Derby was
named IRAP
• Attur M, Plasma levels of interleukin-1 receptor antagonist (IL1Ra) predict
radiographic progression of symptomatic knee osteoarthritis. Osteoarthritis
Cartilage. 2015 Nov;23(11):1915-24
CellularHealing.net
21. IRAP
• Human recombinant IL-ra (rHuIL-
1ra anakinra; Kineret™) - shows
promising results in RA patients.
• Normal BMC marrow produces
246ng/ml
• GFC can produce 4 x normal
amounts of Ilb-ra if concentrated
through micron filtering. .
CellularHealing.net
22. How Does BMC Reduce IL-1B?
• It’s a Ratio Thing!
• Fortier et al has shown that the ratio of IL-
1B: IL-RA in BMC is at least 4 x greater than
the 100:1 ratio required to suppress IL-1B
• You only need 1 IRAP Horse to rescue 100
receptors
• Cassano and colleagues has demonstrated
an increase in the IL-1β but also IL-1RA in
BMAC, therefore neutralizing the effect of
the raised IL-1β.16
• Additional IL-1RA is found in PPP.
IRAP
CellularHealing.net
23. Importance of Fibrin for Platelets
Fibrin is the activated form of
fibrinogen.
Fibrinogen is naturally found in plasma
Fibrinogen is necessary for platelet
aggregation
Platelet aggregation is critical to
homeostasis
Fibrinogen is the final stable
substrate of the coagulation
process.
CellularHealing.net
24. Platelet Rich Fibrin (PRF)
Used by endodontics for :
• Peridontal regeneration
• Superior to debridement alone. *
• Improved ingrowth of allograft
bone**
*Shah, Monali (2015). "Comparative evaluation of platelet-rich fibrin with
demineralized freeze-dried bone allograft in periodontal infrabony defects: A
randomized controlled clinical study". J Indian Soc Periodontol. 19: 56–
60. doi:10.4103/0972-124X.145803. PMID 25810594.
**Li, Qi; Pan, Shuang; Dangaria, Smit J.; Gopinathan, Gokul; Kolokythas, Antonia; Chu,
Shunli; Geng, Yajun; Zhou, Yanmin; Luan, Xianghong (2013). "Platelet-Rich Fibrin
Promotes Periodontal Regeneration and Enhances Alveolar Bone Augmentation". BioMed
Research International. 2013: 1–13. doi:10.1155/2013/638043. ISSN 2314-6133
CellularHealing.net
25. PRF Procedure
• No anticoagulants
• Remove clotted middle buffy
coat layer with sterile
forceps
• Sewn into the oral defect.
CellularHealing.net
26. Release of growth factors vary with
preparation techniques
PRF spun at low speeds reduces
G force to avoid forcing out
growth factors from the clot.
PRP- Released the highest initial
amounts of growth factors
PRF at ultra low speed =
Advanced PRF (APRF) released
greater total amount of gowth
factors over a 10 day period.
More clot= greater
sustained release
Kobayashi E1,2, Flückiger L3, Fujioka-Kobayashi M1,4, Sawada K1,2, Sculean
A3, Schaller B1, Miron RJ5,6. Comparative release of growth factors from PRP,
PRF, and advanced-PRF. Clin Oral Investig. 2016 Dec;20(9):2353-2360. doi:
10.1007/s00784-016-1719-1. Epub 2016 Jan 25.
CellularHealing.net
27. What if you could add the benefit
of high concentration of growth
factors over longer period of time?
• Use PRP with Fibrin Clot.
• Two ways to form the clot
in heparinized blood or bone
marrow.
• Add in – Thrombin
• Add in natural Fibrin from
PPP.
CellularHealing.net
28. Thrombin
• Converts soluble fibrinogen to insoluble strands of
fibrin usually with the additional of calcium.
• Three Types on the Market:
• B-Thrombin- from bovine pro-throbin activated by
tissue thomboplastin with calcium chloride.
Filtered to remove Factor V
• Pd- Thrombin- Human plasma derived from FDA
compliant plasma centers.
• Rh- Thrombin- Recombinant from Chinese Hamster
Ovaries (HMO)
CellularHealing.net
29. Safety Concerns about Thrombin
Not all bovine factor V is removed
and 15.6% of patients exposed to b-
Thrombin have anti-bovine
thrombin antibodies for up to 3
years after exposure. *
Increasing number of thrombin
induced coagulopathies forced FDA
to issue a “Black Box Warning” on
all thrombin products.
Over 100 reported cases in the
literature of thrombin induced
factor V and thrombin factor IIa.
* Singla NK, Ballard JL, Moneta G, Randleman CD, Renkens KL, Alexander WA: A phase 3b, open-label, single-
group immunogenicity and safety study of topical recombinant thrombin in surgical hemostasis. J Am Coll Surg.
CellularHealing.net
30. Large clot formation with Thrombin
40 to 45 cc clot within a confined knee
which needs to bend.
Size of clot had effect on number
of platelets :
• Smaller clots had 92.6% of vs. larger
with 67.2% *
Exercise reduced the stability of
the clot:
• Exercise has fibrolytic activity on
clot stability**
**Patelis N, Karaolanis G, Kouvelos GN, Hart C, Metheiken S. The effect of exercise on coagulation and fibrinolysis factors in
patients with peripheral arterial disease. Experimental Biology and Medicine. 2016;241(15):1699-1707.
doi:10.1177/1535370216660215.
*Kitamura Y,
Watanabe T,
Nakamura M, et
al. Platelet
Counts in
Insoluble
Platelet-Rich
Fibrin Clots: A
Direct Method for
Accurate
Determination. F
rontiers in
Bioengineering
and
Biotechnology.
2018;6:4.
doi:10.3389/fbio
e.2018.00004.
CellularHealing.net
31. Volume Limitations of using
Thrombin
• Volume:
• 60 cc of blood or BMA normally
produces 30 cc of PPP
• Additional 5 cc of thrombin
added
• BMC or PRP – additional 5-10
cc
• Total = 40 to 45 cc.
• Number of adult joints
allowing 45 cc of volume
injection :
• Knee
CellularHealing.net
32. General Fluid
Concentrate (Growth
Factor Concentrate)
• Made directly from PPP through
microfiltration removing water and
salts;
• Concentrates from 30 cc to 5 cc
• Additional increased
concentration of:
• A2M – up to 6 x normal to
reduce 3 types of destructive
enzymes:
• IL-1B RA- up to 4 x normal
amount
CellularHealing.net
33. Stable clot formation with natural Fibrin
• PPP has large levels of
Fibrin
• Filtering PPP with
embedded fiber filter
removing water and salts
concentrates fibrin x 4
normal.
• Mechanical mixing of GFC
with BMA or PRP forms
stable, small clot
CellularHealing.net
34. GFC Produces Higher
Concentrations Than PPP
• 12 samples of peripheral blood and bone
marrow were taken.
• Blood and bone marrow PPP was
concentrated to 5 x normal and thrombin
was added.
• P<0.05 greater amounts of VEGF and
A2M in GFC vs. PPP
• 1.7 increase fold of MSC proliferation
in GFC clot vs. PPP clot.
CellularHealing.net
35. General Fluid Concentrate Volume Dosing
Approximate Synovial
volumes of:
• Shoulders- 5-10 cc
• Hips- 5-10cc
• Elbow 3-5cc
• Knee- 13-15 cc
• Ankles- 5-8 cc
• Wrist – 2-3 cc
• CMC – 1 cc
Suggested GFC + BMC or PRP
• Shoulder- 10 cc ( 5 cc BMC + 5 cc GFC)
• Hips- 10 cc ( 5 cc BMC + 5 cc GFC )
• Elbow- 5 cc ( 3 cc BMC + 2 cc GFC)
• Knee- 10-15 cc ( 7-8 BMC + 5 cc of GFC)
• Ankle- 8 cc ( 5 cc BMC+ 3 cc GFC)
• Wrist- 2 cc BMC + 2 cc GFC
• CMC- 1 cc BMC + 1 cc GFC
CellularHealing.net
36. GFC + BMC mixing technique
Start with BMC or PRP
Use connector in Celling kit
Use syringes about 25% larger than final volume
to allow for foam
• 10-15 cc for knees,hips and shoulders
• 10 cc for ankle
• 5 cc for eblow, wrist , CMC
Mix vigoursly for 2-5 minutes till it becomes
thick and difficult to push.
Inject with 18 g needle to avoid clotting in the
needle .
Have patient sit still for additional 5-6 minutes
(normal clotting time)
CellularHealing.net
37. Summary Take Home Points
• Whole Bone Marrow and Blood Contain Important Plasma Proteins
• Centrifugation concentrates some, but not all cells into distinct
layers.
• Joints and Soft Tissue Have a Injection Volume Limits
• Coagulation cascade activates and maintains growth factor and
MSC delivery over time.
• GFC can help add in more plasma proteins, slowly release growth
factors and growth of MSC.
CellularHealing.net
38. Thank You:
Dr. Philippe Hernigou
Dr. Helene Rouard
Esatblissement Francais Du Sang- Ile-De France
CellularHealing.net
