Autologous Platelet-rich Plasma

What is plasma?
Fluid component of a persons blood
Contains platelets, white blood cells, stem cells, electrolytes,
enzymes, hormones, nutrients, anti-bodies, glucose, proteins, lipids
& albumin (powerful anti-oxidant), etc.
Why autologous?
Autologous means persons own (self donated) and not donated
from another person or from animal origin
Growth factors must be in genetically pre-determined ratios!
No risk of rejection and lower allergenic potential
How can A-PRP be obtained easily?
Venous blood sample is obtained from patients fore-arm
Centrifugation separates plasma & platelets & stem cells from red
blood cells

What is Autologous Platelet-rich Plasma (a-PRP)?
A-Platelet rich plasma is a concentration of human
platelets in a small volume of plasma measured as
1,000,000 platelets per mm
3
or 2-6 times the native
concentration of whole blood at a pH of 6.5 - 6.7
(whole blood pH = 7.0 - 7.2)
Also referred to as autologous platelet gel, plasma-
rich growth factors (PRGFs) or autologous platelet
concentrate
PRP is also a concentration of the 7 fundamental
protein growth factors that have been proved to be
actively secreted by platelets to initiate all wound
healing
PRP includes 3 proteins in blood known to act as cell
adhesion molecules: fibrin, fibronectin & vitronectin

RESEARCH &
DEVELOPMENT




Cell separation




Autologous cell culture




Autologous stem cells
culture




Cell differential



Tissue regeneration




Healing remodelling
DENTAL MEDICINE




Dental extraction
Dental implantation
DERMATOLOGY
INTERNAL
MEDICINE
GERONTOLOGY




Cutaneous
reconstruction and
transplantation




Ulcer and chronic
wound therapy
(e.g. after radio
therapy)




Re-implantation of
Autologous cells,
extemporaneous or
cultivated in-vitro
SURGERY




Cardio-vascular
surgery




Abdominal surgery




Maxillo-facial surgery


Orthopaedic surgery




Plastic & cosmetic
surgery/dermatology




Treatment of severe
burns
Fields of Application of A-PRP
Growth Factors Acting on Healing Cascade
Factor Name Principal Source Effects
PDGF aa
PDGF bb
PDGF ab
Platelet derived
growth factors
Activated thrombocytes Mitogenes of mesenchymal stem
cells promote the synthesis of the
extracellular matrix
TGF- alpha
TGF- beta
Transforming
Growth Factors
Activated thrombocytes Stimulation of DNA synthesis,
proliferation of various types of
cells. Favours the synthesis of
collagen
IGF- I
IGF- II
Insulin-like
Growth Factors
Activated thrombocytes Stimulates the proliferation and
differentiation of osteoblasts
EGF Epidermal Growth
Factor
Activated thrombocytes Stimulates proliferation and
differentiation of epidermis cells,
co-stimulating angiogenesis
VEGF Vascular Endothelial
Growth Factor
Leucosytes &
Endothelial cells
Stimulate angiogenesis & chemo-
attraction of osteoblasts
In addition the activated thrombocytes have onto their surface a multitude of
signalisation molecules eg. CD9, CD-W17, CD31, CD41, CD42a-d, CD51, CD-
W60, CD61, CD62P, CD63
How are Platelets Activated?
Dermal collagen & exposed endothelial collagen
Arachidonic Acid (inflammation pathway)
Thromboxane A2 (inflammation pathway)
ADP
Thrombin (bovine has high allergenic potential)
Substrate bound ligands of Glycoprotein II a / III b
Vasopressin
Adrenaline (20% patients no receptor!)
CaCl2
Thermal: controlled heat (Radio-frequency)
Vibration (?) via Vortex device
Cryo-activation





1. Formation of tri-dimensional
mesh (fibrin strand)or matrix.
*Platelets & Megacaryocytes vol.2 Dr J.Gibbins, M. Mahaut-Smith
2. Release of growth factors
by the thrombocytes and
leukocytes.
3. Chemo-attraction or
migration of
macrophages and stem
cells
4. Stem cells
proliferation &
mitosis
5. Stem cells
differentiation
(In addition ECM like fibronection,
vitronecton, thrombospondin)
The 5 Major Steps In The Platelet Activation Process
Timelines in Wound Healing
Benefits of a-PRP reported in the Healing Cascade
Physiologic response: time
%

W
o
u
n
d

c
l
o
s
u
r
e

Haemostasis

Inflammation


Tissue regeneration
Physiologic response: time
%

w
o
u
n
d

c
l
o
s
u
r
e

Fibrin
Plts agrgg
vWF
Leukocytes
Plts G. Factors
Chemo tactics
& mitotic
G. Factors
Extra Cell. Matrix synth.
& Cell differentiation
G. Factors
By
concentrating
specific cells,
wound
healing time
can be
shortened
significantly
Tissue
regeneration
Inflammation
Haemostasis
Wound
healing with
PRP
Wound
healing
without PRP


Tissue remodeling


Tissue remodeling
physiological process (days)
%

o
f

w
o
u
n
d

c
l
o
s
i
n
g

0
100
Journal of Oral & Maxillofacial Surgery, 2000; 58:45 Marx, Monteleone, Ghurani, Dr. Robert
Marx, University of Miami

0 30
Healing with
PRP
Control sample
physiological process (days)
P
a
t
i
e
n
t

d
i
s
c
o
m
f
o
r
t

(
p
a
i
n
)

0
10
0 30
Pain with PRP
Control sample
PAIN REDUCTION WOUND HEALING
Visible effect in time of Healing and Discomfort
(randomized study USA)
Advantages of A-PRP

Tissue regeneration & rejuvenation: neo-collagenesis (TGF & ), neo-
vascularisation (EGF & VEGF), & extracellular matrix formation (PDGF &
& ) NB: growth factors in genetically pre-determined ratio!
Bio-glue (fibrin glue): haemostasis & tissue adhesion in skin flaps, bone
grafts, trauma intra-surgery and post-surgery
Safety: non-allergenic & free from concerns over transmissible diseases
e.g. HIV, Hepatitis B & C, CJD, etc.
Autologous: no risk of rejection reaction
Wound healing time: increased
Physiological anti-biotic : anti-bodies & WBCs & proteolytic enzymes
Plasma includes: hormones, bio-transformed vitamins & other nutrients
Tissue engineering: in-vitro autologous tissue culture-medium.
Ease of use: dermal & hypodermal injections
Convenience: harvesting performed in doctors rooms (no external
laboratory required)
Cost effectiveness: 1 Plasma kit (2 tubes) delivers 12+ ml A-PRP
Contra - Indications
Platelet Dysfunction Syndrome
Critical Thrombocytopenia
Hypofibrinogenaemia
Haemodynamic Instability
Auto-immune disease
Chronic oral steroid therapy
Chronic topical steroid therapy of treatment area
Malignancy
Chemo-therapy
Sepsis
Acute & Chronic Infections
Chronic Liver Pathology
Anti-coagulation therapy (warfarin, aspirin)
Pregnancy (for cosmetic indications)
Potential Complications
(applicable to all dermal fillers)
Intra-vascular injection (thrombus/embolus)
- Venous
- Arterial
Nerve trauma (needle)
Secondary infection
NB: Beware of the peri-ocular area (eyelids) - no
coagulant used eg. thrombin or CaCl2
A-PRP Indications
1. Skin rejuvenation:
injection (intradermis)
mesotherapy (intradermis)
topical plasma & Dermaroller micro-needling (intradermis)
2. Fine lines & wrinkles: ditto
3. Volumetric filling :
large volume injection of plasma intradermis and hypodermis of the tear
troughs, eyelids, naso-labial folds, marionette lines, peri-oral areas,
cheeks, forehead, glabella, neck & back of hands
A-PRP mixed with fillers such as hyaluronic acid (Juvederm, Restylane,
Teosyal, etc) and calciumhydroxyl-appatite (Radiesse) = bio-active
filler containing growth factors
4. Acne scarring:
subscision injections & topical plasma & Dermaroller
5. Cellulite?
6. Striae (stretch marks)?
SKIN TREATMENT ACTION LEVELS
MULTIDISCIPLINARY PROGRAM FOR THE REJUVENATION
OF THE FACE
FILLERS
Wrinkles &
volumes
correction
BIO
STIMULATION
MyCells
Architectural skin
reconstruction/
reorganization
DERMO
COSMETICIS
PEELS
Renewal of the
corneal layer
Germinative layer
Stimulation
Dermis Stimulation
Hyperpigmentation
removal
Pre-Treatment Patient Preparation
& Combination Therapy

High Dose Oral Vitamin C (1,000mg+) daily for 7 days
pre-treatment & post-treatment
Enhances wound healing (fibroblast stimulation)
Oral Vitamin A daily for 7 days pre- & post-treatment
Radio-Frequency
Immediately after treatment = platelet activation
Collagen fibre contraction (immediate)
Fibroblast induced neo-collagenesis (delayed)
Dermaroller & Topical A-PRP
micro-surgical needling
Induces growth factor release
Topical Vitamin A
Topical Vitamin C
MyCells Important Factor
Presence of Progenitor CD34 Bone Marrow
Mesenchymal Stem Cells in MyCells

Neocell Laboratories in Cape Town 2008
10 ml Venous blood sample yield 1.6 x 10 CD34
stem cells
6 ml post centrifugation plasma yield 1.2 x 10 CD34
cells = 80% yield!

Upper eyelid
Subdermal injections 0.2ml each x 3.
Total 0.6ml.
Lower eyelid
Subdermal 0.2ml injections 1 cm
apart. Massage evenly. Total 1-2ml.
Cheeks
Subdermal & intradermal injections
Linear threading technique 0.2ml per
injection. Total 3-5ml per side.
Naso-labial folds
Subdermal & intradermal injections
Linear threading technique 0.2ml per
injection. Total 2-3 ml per side.
Lips
Vermillion border injections
Linear threading technique 0.2ml per
injection. Total 0.4ml per quadrant.
Chin
Linear threading technique 0.2ml per
injection. Total 2-3ml per side.
Forehead
Intradermal injections 0.05ml. Total
for forehead 3ml.
Side-effects
Minor oedema
Seldom bruising
Eyelids can remain puffy for 2-3days
No infection
No allergy
PRP preparation(1)
Collect blood from
central vein of elbow
10cc for each tube
PRP and PPP
Gel separator
Red blood cell
Centrifuge:2058G, 7min.
After centrifugation
Mycells tube
PRP preparation(2)
PPP aspiration
PRP
PRP just before injection
Aspiration
of PRP
Aspiration and
Blow of PRP
PRP after PPP aspiration
Sleeve insertion
KUBOTA JUNICHIRO CLINIC
Now we are injecting the PRP using mesotherapy
like technique over the entire area to be treated.
Inject small spot (0.05cc to 0.1cc) into the skin.
Injecting layer is dermis and subcutaneous tissue.
How do we inject PRP into the skin?
PRP injection
PRP just before injection
We usually inject PRP using linear
injection and mesotherapy
technique over the entire area to be
treated.
Inject as small spot(0.05cc to 0.1cc).
Injecting layers are intra-dermis and
subcutaneous tissue respectively.
30G needle 1cc syringe
KUBOTA JUNICHIRO CLINIC
Case
70 year old female
She was injected PRP around lower eyelid
and nasolabial fold.
Pre-injection of PRP 1 month after injection
Skin elasticity ,wrinkles and tension improved !!
She looks much younger than before.

Dec. 2006 March 2007
June 2008
6 time PRP injection