Autologous Platelet-rich Plasma

• What is plasma?
– Fluid component of a person’s 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 person’s 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 patient’s 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 mm3 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
 Fields of Application of A-PRP
RESEARCH & DERMATOLOGY SURGERY
DEVELOPMENT INTERNAL
DENTAL MEDICINE    
    MEDICINE
GERONTOLOGY
Cardio-vascular
Cell separation         surgery
       

Cutaneous
Autologous cell culture Abdominal surgery
reconstruction and
Dental extraction transplantation    
   
Dental implantation
   
Autologous stem cells Maxillo-facial surgery
culture
   
    Ulcer and chronic
wound therapy
(e.g. after radio Orthopaedic surgery
Cell differential
therapy)    
       
Plastic & cosmetic
Tissue regeneration surgery/dermatology

   
Re-implantation of    
Autologous cells,
extemporaneous or Treatment of severe
Healing remodelling cultivated in-vitro burns
 Growth Factors Acting on “Healing Cascade”
Factor Name Principal Source Effects
PDGF aa Platelet derived Activated Mitogenes of mesenchymal stem
PDGF bb growth factors thrombocytes cells promote the synthesis of the
extracellular matrix
PDGF ab
TGF- alpha Transforming Activated Stimulation of DNA synthesis,
TGF- beta Growth Factors thrombocytes proliferation of various types of
cells. Favours the synthesis of
collagen
IGF- I Insulin-like Activated Stimulates the proliferation and
IGF- II Growth Factors thrombocytes differentiation of osteoblasts

EGF Epidermal Growth Activated Stimulates proliferation and

Factor thrombocytes differentiation of epidermis cells,
co-stimulating angiogenesis
VEGF Vascular Endothelial Leucosytes & Stimulate angiogenesis & chemo-
Growth Factor Endothelial cells 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
 The 5 Major Steps In The Platelet Activation Process

1. Formation of tri-dimensional 4. Stem cells

mesh (fibrin strand)or matrix…. proliferation &
mitosis…
3. Chemo-attraction or
migration of
macrophages and stem
cells…

2. Release of growth factors

by the thrombocytes and 5. Stem cells
leukocytes…. differentiation…

(In addition ECM like fibronection,

vitronecton, thrombospondin…)

*Platelets & Megacaryocytes vol.2 Dr J.Gibbins, M. Mahaut-Smith

Timelines in Wound Healing
 Benefits of a-PRP reported in the “Healing Cascade”

Tissue remodeling
% Wound closure

Tissue regeneration

Wound healing
Inflammation
without PRP
Haemostasis

Physiologic response: time

Tissue remodeling
By
By
% wound closure

Extra Cell. Matrix synth.

Tissue Extra Cell. Matrix synth. concentratin
concentratin
& Cell differentiation gg specific
regeneration specific
G. Factors
Wound healing cells,
cells, wound
wound
Chemo
Chemo tactics
tactics
&
& mitotic
mitotic healing
healing time
time
with Inflammation can
G. Factors can be
be
PRP Haemostasis Leukocytes shortened
shortened
Plts G. significantly
Fibrin
Fibrin Factors
significantly
Plts
Factors
Plts agrgg
agrgg
vWF
vWF

Physiologic response: time

 Visible effect in time of Healing and Discomfort
(randomized study USA)

WOUND HEALING PAIN REDUCTION

100 10
Healing with
PRP

Patient discomfort (pain)

Control sample
% of wound closing

Control sample

Pain with PRP

0 0

0 physiological process (days) 30 0 physiological process (days) 30

Journal of Oral & Maxillofacial Surgery, 2000; 58:45 Marx, Monteleone, Ghurani, Dr.
Robert Marx, University of Miami
 Advantages of A-PRP

• Tissue regeneration & rejuvenation: neo-collagenesis (TGFα & β), neo-

vascularisation (EGF & VEGF), & extracellular matrix formation (PDGFαα &
ββ & αβ) – 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 & WBC’s & 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 doctor’s 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

DERMO
COSMETICIS FILLERS

PEELS
Wrinkles &
volumes
correction
Renewal of the
corneal layer

Germinative layer BIO

STIMULATION
Stimulation MyCells

Dermis Stimulation
Architectural skin
Hyperpigmentation reconstruction/
removal reorganization
 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 10⁹ CD34⁺
stem cells
– 6 ml post centrifugation plasma yield 1.2 x 10⁹
CD34⁺ cells = 80% yield!
Forehead
Intradermal injections 0.05ml. Total
for forehead 3ml.

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.
 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
Mycells tube Centrifuge:2058G,
2058G 7min. Red blood
cellcentrifugation
After
PRP preparation(2)
Aspiration and
Blow of PRP
PRP

Sleeve insertion

PPP aspiration

PRP after PPP aspiration

Aspiration
of PRP
PRP just before injection
 How do we inject PRP into the skin?

• 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.
KUBOTA JUNICHIRO CLINIC
PRP injection 30G needle 1cc syringe

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.
 70 year old female
Case 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. KUBOTA JUNICHIRO CLINIC
Dec. 2006 March June 2008
2007
6 time PRP injection