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??