Preparation

of
CGF -
for
BIOFILLE
Aesthetic Application
R
Dott.ssa
Pederzoli Paola

2017
Introduced by
Dott. Pederzoli Paola

Medicine and Surgery

Dentistry and Dental Prothesis
Specialist scientific research aesthetic and regenerative medicine
Professor at Baris University
Skilled in the administering high dose of cholecalciferol (25-OH-VITD)
for the autoimmune diseases
1. purpose
The purpose of this Standard Operating Procedure (SOP) is to describe the standard procedures to prepare
Concentrate Grown Factors (CGF) and gel from Pour Platelet Plasma (PPP) to be uses in Aesthetic Medicine
Applications.
2. introduction
Concentrated growth factor (CGF) is an autologous biomaterial termed second-generation platelet
concentrate. CGF is a Novel technologies (speed gradient, Silfradent) that make possible the extraction of
the so called CGF contains autologous platelet growth factors and also present in CGF: tgf-b1, VEGF and
CD34+ cells.
The application of CGF in aesthetic medicine resulted in an excellent healing of skin age symptoms, photo
age, aesthetic corrections and as a filling material (combined with PPP gel).

3. Scope
This SOP applies to technical preparation of bio-materials to be used in aesthetic treatment using Silfradent
technologies.

4. responsibilities
This SOP can be applied in clinic only by Medical Doctor (main responsible), that should be qualified by
education, training and experience to perform his or her respective task (s) and the qualifications are
documented in CVs or other training records. Biological material (CGF and PRP gel can be prepared by
training professionals (qualified in: biochemistry, biology, clinical pharmacy or nurses).

4.1 Training requirements

Training requirements should be in accordance with the responsibilities of each individual in his or her
respective task (s).
5. Preliminari operations
5.1 Supplies

Medical devices:
Digital Camera
Medifuge Silfradent (blood centrifugal machine)
A.P.A.G. system Silfradent (gelation machine)
Round-up Silfradent (Mixer device)

Disponsables
Gloves
Hair Clips
Towels
Gauze Swabs
Sharps Bin
Clinical Waste Bin
Vacuette
Safety blood collection set and holder
2 ml, 5 ml and 10 ml syringes with a luer-lock system
Needle or cannula needle 0.45 x 13 mm (26 G x 1/2 ).
Needle 18 G x 75 mm
Tree way stopcock
Drugs and Cosmetic
Anesthetic Cream, EMLA or similar
Make up Remover
Distilled Witch Hazel
Silfradent cosmetic kit (Juventute cream, mask and lotion)

Others
Trolley
Medical stretcher
Magnifying lens
Medical marker

5.2 Blood Collection

Withdraw 18 ml of blood by piercing a superficial venous blood vessel with a 21-gauge needle from the
patients. The blood was drawn in two sterile Vacuette tubes without additives (capacity 9 ml each).
6. PROCEDURE

1. CGF and PPP preparation

Centrifuge immediately the blood tubes in a special machine (Medifuge MF200, Silfradent srl, Forl, Italy)
using the program named CGF (prefixed duration time: 14 min). Take into consideration that tubes will be
paired and be contain the same blood volume. Tubes be places inside the device one in front another.
In the meantime, perform a light massage on the face skin or area to be treated, in order to improve the
microcirculation and to relax the patient.

2. Fractions (CGF and PPP) separation

At the end of the centrifugation process (about 14 min), three blood fractions are obtained: (1) the upper layer
representing the liquid phase of plasma named platelet poor plasma (PPP) about 2 ml - 2.5 ml depending on
the hematocrit values, (2) the middle layer representing the CGF including the interface between white and
red part representing the richest part of platelets and leucocytes; about 2 ml - 2.5 ml depending on the
hematocrit values; (3) the lower layer representing red blood cells (RBC) because of it mainly contains
erythrocytes.

CGF
Withdraw with a needle 18 G x 75 mm (without removing the cap from tubes) 2 ml 2.5 ml of fluid interface
of CGF from each tube.
Activate CGF with calcium gluconate 10% (0.8 ml per 5 ml di CGF) or calcium chloride 10%
(0.2 ml per 5 ml di CGF)
E.g. 5 ml CGF + 0.2 ml calcium chloride
Lidocaine 0.5 ml x 5 ml CGF, could be added
PPP
Withdraw with a needle 18 G x 75 mm (without removing the cap from tubes) 2 ml 2.5 ml of fluid
PPP and placed in 2 ml / 2.5 ml syringes with luer-lock cap in a thermal heater: 1) at 75 C (A.P.A.G.
system, Silfradent, albumin denaturation programme) for eight min to obtain PPP gel, to be used as
filler matrix or 2) at 45 C (A.P.A.G. system, Silfradent, collagen stimulation programme) for 20 min to
obtain PPP activated, to be used as regenerating agent. At the end, remove the syringes and leave to
cool at room temperature in the dark.

6.3 Injection CGF or PPP

Clean and disinfect the skin
Apply anesthetic cream with occlusion by 45 min
Apply CGF or PPP+CGF (see Treatment steps*)
Clean and disinfect the skin
Apply soothing cream
 Characteristics

Differentiated acceleration

Speed and fractional alternating RCF

(does not allow the platelet degranulation)

Anti-static and anti-magnetic rotor

Constant temperature maintained

by self ventilation

Self-decontamination
 Medifuge MF200 Blood Separator

Relative Centrifugal Force

= RCF
RCF = FC/FG
FG = Gravity Force
FC = Centrifugal Force
2700 rpm = 282,6
= Radians per Second 2400 rpm = 251,2
3000 rpm = 314

Average RCF 770

CGF - LPCGF
Time From 0 to 2700 rpm to 29/30
Minutes Rpm - RCF Volt Acceleration seconds
Fg 25
2 min 2700 - 735 11,02V about
4 min 2400 - 580 9,57V about Time From 3000 to 0 rpm, Self Bleaching and Sterilizing
4 min 2700 - 735 11,02V about Deceleration stop to 33 seconds Minutes Rpm Volt
3 min 3000 - 905 11,95V about Fg 87/88
5 min 1200 about 4,74V about
 .P.C.G.F
iquid Phase of Co cent ated Growth Factors
Auto ogous Mesotherapy == 1. PP P
2.PRP
We will use :
.
3. CD34- stem cell
- M edifuge blood phase separator
.

4.RBC
- Butterfly needles - 5ml syringes
- Long needles - Green cap t ubes
- 2mI syringes - 30G 4mm need les
- 3-way

Process 1
Collect 18 ml of bl! d from a peripheral vein. PCG
:....... oo
F
..
Process 2
Place the tubes in the Medifuge blood phase separator.

...... - Process 3
- - -. Extract PRP and co34+ ce Is fractions and mix them using
a 3-way.
Process 4
..,,.........,. Inject LPCGF where needed.
 Biostimulation
L.P.C.G.F.
2,0 ml PRP activated with 0,4 ml of Calcium Gluconate 10%
0,6 ml CD34+

or

2,0 ml PRP activated with 0,4 ml of Calcium Gluconate 10 %

0,5 ml CD34+
0,1 ml of Vit. D 100 000 U
0,3 ml Lidocaine 2%

1 2 3 4

Collect two tubes of blood Place the tubes Extract PRP Inject LPCGF
from a peripheral vein in the and CD34+ cells fractions where needed
Medifuge and mix them using 3-way
blood phase
separator
 APAG denaturation device
(Activated Plasma Albumin Gel)

(Induces Collagen Formation)

Built with anti-static

and anti-magnetic materials

Operation with heating pulse and thermal

equilibrium at 75 degrees
A.P.A.G.
Activated Plasma A bumin Gel
t,::;:::======$
Autologous FIiing Material ==
1.PPP 2. PRP
= = = t 3. ( 0 34 tern cells
We will
use :
- Medifuge blood phase se.parator APA
- APAG denaturatio machne
- Butterfly needles - 26G 12mm needles G
- Long needles - 3-way
- 2 m I syringes
- 5 ml syringes
- 1 ml syringes of high quality (resistance in 75C)
- Green or white cap tubes

. *,- .9H- .t;9 mh fi .Q,.9 1s!!!

.
1 Plllf:..
How to Make BIOFILLER
How to Make BIOFILLER
1 Blood sampling
-sampling 9-10 ml for one tube of whole blood
2 Separation of plasma
- Centrifuge with Medifuge MF200 (about 14
3 min)
4 Extraction of plasma (2 ml of PPP for tube)
Place the extracted plasma at medical warming device
(BIOFILLER making machine - APAG).
5 Set the program for A.P.A.G. (75C pulsed for 10-15 min max)
6 Keep at room temperature for 10 min.
7 Mix PPP gel with CD34+ (3-way connector)
After finishing all steps, assemble the syringes with needle or
cannula firmly.
The process for making BIOFILLER is very simple.
There will be a demonstration for How to make BIOFILLER.
PROTOCOL
NUMBER OF SESSIONS

3 sessions spread over 2 months

4 session after 6 months

5- session after 1 year

mantenance session every year

 A.P.A.G.: Activated Plasma Albumin Gel

Autologous Filling Material

1 ml PPP mix 0,3 ml CD34+

A.P.A.G. + SODIUM HYALURONATE:

1,0 ml PPP Gel treated with APAG

0,3 ml CD34+
0,5 ml PRP activated with 0,06 cc Ca Gluconate 10%
0,5 ml SODIUM HYALURONATE
 If we have an important lost of
tissue we inject the product
every 21 days

A.P.A.G. + 25-OH-VitD:

2,0 ml PPP Gel treated with APAG

0,6 ml CD34+
1,0 ml PRP activated with 0,12 ml Ca Gluconate 10%
0,2 ml 25-OH-VIT D ( 100.000 U.I.)
0,5 ml Lidocaine
The product can be more fluid with the addition:
0,4 ml CD34+
0,5 ml PRP activated with 0,6 ml Ca Gluconate
10%
MERITS:
1 Easy procedure 6 Easy repetitive treatment

2 Easy handling 7 No allergic reaction

3 Easy making 8 No graft rejection

4 Cost effective 9 Biodegradable

Available to make huge Procedure usually requires

05 volume easily 10 only topical

except some anesthesia
cases alone
I.C.
F
nducton of Collagen Formaton
Autologous Mesotherapy .
PPP
We will use:
stem
- Medifuge blood phase separator cell
- APAG Heating machine
LPCG
- Butterfly needles - 5ml syringes
- Long needles - Green cap tubes F
- 2ml syringes - 30G 4mm needles
- 3-way - 1 ml syringes of high quality

Process 4
Process 1
Insert the syringe with
Collect 18 ml of blood
PRP and CD34+ eel Is into
from a peripheral vein.
the heating machine .
Select "Activ. Collagen".
Process 2
Centrifuge the blood in Process 5
the Medifuge blood Remove the syringe with
phase separator. ICF and keep it at room
temperature for 10min.

Process 3
Extract PRP and co34 + Process 6 Inject ICF in
cells fractions and mix the treatment area.
them using a 3-way.
 I.C.F.: Induction of Collagen Formation
Put the syringe with 1,0 ml of PRP in APAG device
and select program I.C.F. (17 min at 44 C pulsed)

When the product is cool:

mix 1,0 ml of PRP treated with APAG + 0,3 ml
CD34+ + melatonin 10 mm

We will use:
- Medifuge blood p hase separator
- APAG denaturati on machine
- Butterfly needles
- Long needles
- 2,0 ml syringes
- 3-way
- 5,0 ml syringes
- Green cap tubes
- 30 G x 4 mm nee dles
- 1,0 ml syringes of high quality
 This picture is showing the BIOFILLER
and injecting needle, cannula.

IMPORTANT
We must use luer lock 1 ml disposable glass syringes (based test L.A.L.)
For the safe use of BIOFILLER, the needle or cannula must be assembled firmly
until we feel counter- pressure.
*Treatment steps:

A) Biostimulation Treatment
It is a mesotherapy treatment: inject the CGF with a needle 30G x 1/6 (0.30 x 6 mm) using a micro-ponfi
technique. Can be used in Alopecia using a mesotherapy gum and needle 32 G x 0.4 mm

B) Volumizer or Filler Treatment

Through a 3 way connector (extension line), mix PPP gel with CGF.
For example: 1.5 ml of PPP gel + 1 ml of CGF, shake to homogenizer.

Remember: CGF alone must be injected in the critical points in addition to being mixed with the PPP.
Mix should be using luer-lock syringes.

Inject with a needle or cannula needle 0.45 x 13 mm (1.2 x 26 G).

After the injection, perform a light massage in order to distribute the mix PPP+CGF uniformly in the
tissue.
7.forms/Templates to be used
Forms should be personalized
Medical History Form
Informed Consent Form
Information Leaflet
Post-treatment Instructions

8. Internal AND EXternal

references
1.Internal References
Silfradent APAG Brochure 2015
Silfradent Medifuge Brochure 2015
Silfradent Round-up Brochure 2015
Silfradent Juventute Brochure 2015

2. External References
ICH Harmonised Tripartite Guideline
for Good Clinical Practice.
Research Governance Framework for
Health and Social Care, 2nd edition,
2005
Rodella, L.F., Favero, G., Boninsegna,
R., Buffoli, B., Labanca, M., Scari, G.,
Sacco, L., Batani,
T. & Rezzani, R. (2011). Growth factors, CD34 positive cells, and fibrin network analysis in concentrated
growth factors fraction. Microsc Res Tech 74, 772-7.
Antonino Albanese, Maria E Licata, Bianca Polizzi, Giuseppina Campisi. Platelet-rich plasma (PRP) in
dental and oral surgery: from the wound healing to bone regeneration. Immun Ageing. 2013; 10: 23.
 Wish Well Healthy Beautiful
Juventute-Green

The continuous innovation in science and technologies

in addition to many years of experience in the marketing

of products and services in the world of aesthetics,

is a characteristic of Silfradent Cosmetic.
 Juventute

What is new in Juventute-Green cosmetic line:

Exclusive cosmetic line with buffer excipients that

stabilized growth factors and CD34+ cells.

Can enhance the results of existing protocols that use

PRP, PRP gel and CGF.

Can be mixed with vitamins as Vit. D and Vit. B and

Melatonin (protocols by Silfradent)
 Juventute

Helps the immune system with sensitive skin with

less collagen fibers

Stresses skin subjected to sun exposure, smoking

frequent cleasing and hair removal

Helps to reduce the effect of the changing season

and temperatures ristoring the physiological pH
 Juventute
Juventute-Green:
INNOVATION
(Improve the stabilities of stem cells
and the stability of the growth
Mask Juventus-Green factors)
MCH pH optimum, maintain
physiological conditions
pH Buers, prevents
Cream Juventute-Green changes in pH
Controlled osmolarity
CRE
Lotion Juventute-Green - Balances the physiological
pH of the skin
LOZ - Nutrient / antioxidant
Aging of the Skin

FREE RADICALS
Aging caused by the genes,
intrinsic aging (internal).

The other type of aging is

known as extrinsic
(external) and is caused by
environmental factors, such
as exposure to sunlight.
 Skin
Aging
kin
UVC UVB UVA
S
200----290------320-------400nm Aging

Carcinogenic
Burns
Phototoxic
Tan
Photoalergic
Max.10am-
2pm
Permanent
O2

OH
-

Collagen Type III / I

Fibronectin
 Mask Juventute-Green
MCH

Special mask that combines innovative

autologous growth factors
with precious natural active ingredients
 Cream Juventute-Green
Personalized Cosmetic
CRE
Juventute (Silfradent)
Preparation

Face Mask Juventute - Green MCH

Preparation: Mix 8 gr of Juventute - Green MCH with
3 mlL of autologous PPP.
Mix with homogenizer Round Up
(Silfradent) for 14 sec.
Professional use: apply in the same
session after treatment with PRP.

How to use:
Do facial cleansing with cleansing
milk, then apply a thin layer of
mask.
Potent anti-aging, rich in stemAfter
cells10-15 min remove very well
and autologous growth factors.
with warm-cold water then toning
with lotion Juventute - Green
LOZ. Before applying any other
product, wait at least 20 min.

Benefits: Juventute - Green MCH mask carries a powerful

 Cream Juventute GREEN CRE

Preparation: Mix 30 gr of Juventute - Green CRE with 1 ml of autologous CGF (CD34+) and 1 ml of
autologous PRP. NB: Mix with homogenizer Round Up (Silfradent) for 14 sec.

How to use: Do facial cleansing with cleansing cream, then apply a thin layer of cream, twice daily. After
15-30 min toning with lotion Juventute - Green LOZ. Do not apply any other product simultaneously.
The customized cream should be stored between 4-8C and it is stable for one month. NB: To pick the
cream use disposable spatulas.

Benefits: Juventute - Green MCH cream plays a powerful anti-aging action, rich in stem cells and
autologous growth factors. Juventute - Green CRE is an innovative regenerative and nourishing cream
which combines autologous growth factors with precious natural active ingredients (Green Beneficial
Substances).
Lotion Juventute-Green LOZ

15-20 min
LOZ returns the normal.
Physiological pH of the skin.
Strong moisturizing effect.
 LOTION
JUVENTUTE
GREEN LOZ
Without addition of autologous or artificial preparations.
Vial 200 ml.

How to use:
Apply after the use of the mask Juventute - Green MCH or 15-30 minutes after
the cream Juventute - Green CRE.
The lotion should be stored between 4-8C and is stable for one month.

Benefits:
Lotion Juventute - Green LOZ returns the physiological pH of the skin to its
normal and it has a strong hydrating effect (Green Active Substances).
 Results
Juventute-Green

Aesthetic: micro-peeling effect, increased tone and brightness of the skin,

enhances the natural color of the skin.

Anti-Age: removal of toxic substances present in the skin, both external

(pollution, smoke, etc.) and internal.

Functionally: act as a "natural regeneration."

GENTLE SMOOTH COOLING
 GENTLE COOLER MATERIAL
AND FORM

- Totally made of medical steel (AISI 630 X15TN )

- Antimagnetic (does not disturb the wound current)
- Keps the temperature for a long period
- you can choose the temperature by holding
it in an refrigerator
- has no microporosities so it can be used on
the wound
- can be sterilised in Autoclave
- fits easily in the hand
- round form allows to reach every part of the
face and neck
 GENTLE SMOOTH COOLING
ACTION

- does not interfere with the action of CGF/LPCGF

- stops swelling of the micro bruises caused by the injection
- levells the micro bruises caused by the injection
- reduces the pain
- CGF/LPCGF stays longer in place because the microcirculation is
reduced by cooling
- using gentle pressure the CGF APAG gel can be distributed
in the tissue
- refreshens the skin
- can be used in cosmetics and in orthopedics too
GENTLE SMOOTH COOLING
Graduate in Medicine and Surgery at Modena University
with an experimental thesis on Influence on the spine of
the stomatognathic during Evolutive Age
Graduate in Dentistry and Dental Prosthesis at Modena
and Reggio Emilia University
Graduate in Aesthetic Medicine at a Post-Universitary
Center in Ambulatory Medicine in
Bologna
Professor at Baris University For Training Course

Skilled in the administering high dose of cholecalciferol pederzoli.paola@gmail.com

(25-OH-VITD) for the autoimmune diseases