Updates and Advances in Platelet Rich
Plasma and Biocellular Prolotherapy
Donna Alderman, D.O.
Medical Director, Hemwall Center for Orthopedic Regenerative Medicine
Co-Director, Institute for Biocellular Regenerative Medicine
American Osteopathic Association of
Prolotherapy Regenerative Medicine,
Conference May 2013
Overview of Lecture
1. Brief Overview of PRP
2. Is PRP Prolotherapy a Placebo? Video
3. Not all PRP is the same.
Concentration? Activation? RBCs, WBCs?
4. Importance of clarity when describing PRP
5. Update on PRP studies and World Anti-Doping Agency
6. Overview of Biocellular Prolotherapy
What it is, when to use it
Nomenclature
7. Alderman-Alexander Protocol sequence: Video
8. Conclusion
9. Questions
PRP Prolotherapy, like Dextrose
Prolotherapy, stimulates
musculoskeletal healing but also
provides growth factors to the tissue
(like adding fertilizer!)
There is mounting evidence that PRP
may initiate, enhance, or accelerate
healing of connective tissue.
Mishra, et al. Sports Medicine Applications of
Platelet Rich Plasma, Current Pharmaceutical
Biotechnology, 2012, 13, 1185-1195.
History of PRP
First introduced 1987 for use after open heart surgery to avoid
donor transfusion.machines were big though!
1990s introduced oral and maxillofacial surgery.
2006 introduced into Sports Medicine and Prolotherapy world
when Mishra (Stanford) published paper on the successful use of
PRP for lateral epicondylitis; (machines smaller, office size now)
2009 Heines Ward (Pittsburgh Steelers) reported using PRP for an
injured knee just prior to the Steelers winning
the 2009 Superbowl.
IS PRP PROLOTHERAPY A
PLACEBO?
VIDEO CAN BE FOUND AT:
http://www.prolotherapy.com/Dog.php
WHEN TO USE PRP?
PRP is used when:
When Dextrose Prolotherapy stops
being effective or levels out;
If degeneration, discrete tear(s) or
tendonosis;
Patient preference.
How works in various ways
Platelets contain powerful growth factors which increase healing and
angiogenesis.
Platelets also contain serotonin. When seratonin is released it promotes
recruitment of neutrophils to the area which boosts healing and
immunity . (Duerschmied et al., Blood Journal, February 2013)
Evidence that PRP cytokines and growth factors signal and recruit local
repair (mesenchymal) stem cells to the wound area, then help induce
them to heal needed tissue.
adult mesenchymal
stem (repair) cells
activated platelets
Platelets adhering
to injured tissue
In vitro: Five fold increase in Mesenchymal Stem Cell Growth with
PRP
Mishra et al. Buffered Platelet-Rich Plasma Enhances Mesenchymal Stem
Cell Proliferation and Chrondrogenic Differentiation, Tissue Engineering, 15:
(3) 2009; pg 431-435
Other things to know about PRP
PRP promotes differentiation of tendon stem cells into active
tenocytes (Zhang et al. 2010)
Shown to inhibit inflammatory processes in osteoarthritis by
decreasing a major pathway in the pathogenesis of OA
(Nuclear factor kB ) (van Buul et al. Am J Sports Med,
August 2011)
Possible protective qualities on cells (Baboldashti et al.
2011) (cultured human tenocytes protected from
dexamethasone and ciprofloxin)
* Increases viability of cells, used for many years in fat
transfer surgery (Abuzeni and Alexander 2001)
Not all PRP is the same
CONTROVERSY EXISTS AS TO WHICH IS BEST..
Concentration (1.1 to 10x)?
RBC content?
WBC content amount and types? Double
spins? Better to use low or higher?
Activation? (CaCl, exogenous thrombin)
Use of anticoagulant citrate dextrose (ACD)
makes acidic impact of ph? Buffer?
Various classification systems
GENERAL DEFINITION of PRP
Platelet Rich Plasma is autologous blood with
concentrations of platelets above baseline
levels*, which contains at least seven growth
factors**.
This definition has become too
simple!
*Hall, M. et al. Journal of the American Academy of Orthopedic Surgeons. 2009;27:602-
608.
**Marx, et al. Platelet rich plasma (PRP): A Primer. Practical Pain management, March
2008.
Traditional description of PRP (single spin)
Normal Blood
Platelet Rich Plasma
Classic PRP: Red cells go down to 5% and White cells stay the same
Marx and Garg. Dental and Craniofascial applications of Platelet Rich Plasma.
Quintessence Publishing Co., Inc. 2005
Double Spin
PPP
Less red
Less white
Buffy Coat (WBC
and Platelets)
RBC and
some heavier
platelets
HIGH-DENSITY PLATELET RICH PLASMA
High-density platelet rich plasma (HD-PRP) is defined
as autologous blood with concentrations of platelets
at equal or greater than four (4) times circulating
baseline levels and which increases the important
bioactive protein load (growth factors) in a direct
correlative fashion.
Marx, R., Kevy, S., Jacobson, M. Platelet rich plasma (PRP): A primer. Practical Pain
Management, March 2008.
Hall, M., Bank, P., Meislin, R., Jazrawi, L., Cardone, D. Platelet-rich plasma: Current concepts
and application in sports medicine. Journal of the American Academy of Orthopedic
Surgeons. 2009; 27:602-608.
Reason why high density thought to be important:
Migration of repair cells (mesenchymal stem cells) to
injury site increases with platelet concentration
800
700
Number of
Human-
Mesenchymal
Stem Cells
600
500
400
300
200
100
0
---- Increasing Platelet
Concentration ----
227% increase in
stem cell
population with a
5x increase in
platelet/protein
load
X
SF
P
X
2.
5
D
PP
O
1.
25
Haynesworth, SE, Bruder, SP, et al; Mitogenic Stimulation of Human Mesenchymal Stem Cells by PRP Suggests
a Mechanism for Enhancement of Bone Repair, Presented at 48th Orthopaedic Research Society Meeting,
Dallas, TX, 2002
B
LO
5
X
ITS ALL PRP BUT.
We need to be more precise in how we describe
it, need better classification system;
Different concentrations and content seem to be
better for different tissues but studies vary.
White cells and inflammation??
Inflammation is how Prolotherapy works.so
do we want to throw the baby out with the
bathwater?
Some cases may need more inflammatory PRP
(severe chronic tendonosis).
However some cases may benefit from less
inflammatory PRP, i.e. acute injuries, muscle
and intraarticular.
UNDERSTANDING THE PROCESS:
SORTING OUT THE CONFUSION
Most systems separate based on weight.
Decreasing RBCs
decreases number of
platelets so may have to
use larger blood
volumes when reducing
Hct to get same number
of platelets. Also
removes heavier
plateletsdoes this
matter??
Platelets
Some platelets mixed in with WBCs
(buffy coat)
RBCs (and some heavier
platelets)
2% HCT
7% HCT
Increasing HCT
SPORTS MEDICINE CLASSIFICATION
SYSTEM OF PLATELET RICH PLASMA
Mishra, et al. Sports Medicine Applications of Platelet Rich Plasma,
Current Pharmaceutical Biotechnology, 2012, 13, 1185-1195.
Review of what was used in classic
studies
1A: Increased white cells, no activation, 5X or
more: (more studies available)
Mishra study 2006, effective for chronic severe
elbow tendonosis
Pearblossoms 2 year study 2012; significant
improvement in pain and disability in double blind
randomized controlled study for elbow
tendinopathy.
Type 4: Low or No WBC
athletes undergoing Achilles tendon repair
recovered faster than control group (Sanchez,
2007)
- Intraarticular knee injections showed significant
improvement. (Kon et al., 2010)
- Theorized in acute muscle injury that low WBC
may be better because of the already active
supply of neutraphils there during injury.(Toumi
and Best, Br J Sports Med 2003)
In China:
L-PRP: Leukocyte-PRP contains high
concentrations of platelets and leukocytes.
Researchers in China show L-PRP helps with
bone healing, acute soft tissue wound healing,
and repair of acute muscle, tendon, ligament,
nerve and cartilage injury caused by trauma.
Yuan et al. Applications of leukocyte-and platelet-rich plasma
(L-PRP) in trauma surgery. Curr. Pharm Biotechnol. June 2012;
13(7):1173-84.
Other interesting studies:
2013: PRP proposed for use to prevent infection
in implant surgery
Achieved consistent antimicrobial findings and
strong in vitro antimicrobial properties against
bacteria like methicilin-sensitive and methicillin-
resistant Staphyloccus aurea, Group A Streptococcus
and Neisseria gonorrhoeae.
Li and Li. PRP as a New Approach to Prevent Infection:
Preparation and In Vitro Antimicrobial Properties of PRP. J Vis
Exp. April 2013; 9(74).
Activation needed?
PRP can be activated exogenously by
Thrombin fast, may make unusable if too fast,
not generally done with PRP Prolotherapy
Calcium chloride slow, physiologic, reverses anti-
coagultant, frequently done
Mechanical trauma- natural byproduct of the
injection (collagen release from tissue)
Avoid activating too much ahead of time. Ninety
percent of the prefabricated growth factors will be
released in the first ten minutes
PRP removed from Prohibited List for 2011
after finding current studies on platelet-derived
preparations do not demonstrate a potential for
performance enhancement beyond a potential
therapeutic effect.
http://www.wada-ama.org/en/media-
center/archives/articles/wada-2011-prohibited-list-
now-published/
What is BIOCELLULAR
PROLOTHERAPY?
Injection of autologous adult stem/stromal
cells from adipose combined with PRP or
BMAC (bone marrow aspirate concentrate),
then injected into injured joints or connective
tissue to stimulate repair.
Remember How PRP works
PRP growth factors signal locally available adult
stem cells..but signal is more like bluetooth than
wi-fi and if no locally available stem cells available
then tissue repair will be limited.
adult repair
(stem) cells
activated platelets
Platelets adhering
to injured tissue
Important point to remember
Chronic inflammation uses up the
local repair (stem)cells.* This is
when we need to add repair cells
to that area.
*Kotaro Yoshimura, MD
International Federation of Adipose
Therapeutics (IFATS) Conference, 2011
MAJOR SITES OF ADULT
MESENCHYMAL STEM CELLS
ADIPOSE TISSUE
and
BONE MARROW
People dont mind giving up a little fat
Cells can be easily harvested from the
adipose tissue in significant numbers; easy
to process; low donor morbidity
WITHIN BOTH OF THESE SOURCES
is THE MESENCHYMAL STEM CELL
Ligament
Tendon
2012 Study shows MSCs secrete
factors that inhibit inflammatory
processes in osteoarthritic
synovium and cartilage cultures.
Van Buul et al. Osteoarthritis Cartilage. Oct
2012 20(10): 1186-96.
AD and BM MSCs fundamentally
different cell types but similar in
action.
Several reports verify there are
more mesenchymal stem cells per
unit volume in adipose tissue than
in bone marrow (up to 500 to 1000
times as many ) (Mizuno, 2009;
Fraser, 2006, Stern 2005).
AD may be better for muscle/connective
tissue (Adipose-derived stem cells form
functional myotubes via mechanical
stimulation whereas bone-derived stem cells do
not, Engler et al. University of California,
presented at 2012 TERMIS meeting, Houston,
TX).
AD and BM MSCs display same ability to
differentiate towards chondrocytes
/osteoblasts (Noel 2008) although some
studies show BM better (Pacini, et al. 2007).
When to use Biocellular Prolotherapy
When results from PRP Prolotherapy have not
achieved desired results or leveled out;
Moderate to severe degenerative changes or
discrete tear(s); or
Patient preference.
Biocellular Terminology
Adipose sample termed Adipose Derived-Tissue
Stromal Vascular Fraction (AD-tSVF)
tissue used to differentiate from references in
the literature (particularly from lab papers)
referencing Stromal Vascular Fraction or cellular
SVF = cSVF which demands digestion, etc..
Trying to keep audience clear we are talking
about non-manipulated tissues versus the
isolated/ concentrates component parts
Clarification more terminology:
Adipose separation below.
Supernatant
(lipid layer)
AD-tSVF
Infranatant
(liquid/blood
bottom layer)
Alderman-Alexander Protocol
Published Journal of Prolotherapy Aug 2011
Since then many treatments done with high success rate
Set up (sterile)
TULIP PATENTED MICROCANNULA SYSTEM
cell friendly. Disposables and reuseables.
Infiltrator
(multiport)
Used to infiltrate area
with local anesthetic
Harvestor
Used to harvest
adipose tissue.
Johnny Lock: allows syringe to be held
in a locked position so that suction
maintained for fat harvesting
Injection Protocol
STEP ONE: Draw PRP or Bone Marrow
STEP TWO: INFILTRATION OF LOCAL
ANESTHESIA (fluid goes in)
STEP THREE PRETUNNELING (to separate fat)
STEP FOUR: HARVEST FAT
STEP FOUR: PREPARE AD-tSVF/PRP or AD-
tSVF/BMAC
STEP FIVE: TREATMENT USING ULTRASOUND
GUIDANCE
Not shown on Video:
First: Draw PRP, OR: If doing bone
marrow do that. Either one can then
spin while doing fat harvest
OR:
BONE MARROW
CONCLUSION
PRP and Biocellular Prolotherapy are
increasing in use; research and protocols are
being refined and.
Not all PRP is the same. Different types
appropriate for different problems. More
studies are needed.
Regenerative Medicine is the wave of the
future.
Questions??
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