Máster en

Medicina
Cosmética, Estética
y del
Envejecimiento
Fisiológico

PRP In Facial Rejuvenation: An Extensive Study

of the Aging Process and the Role of Platelet Dr. Shatha Rawashdeh
Dr. Vanessa Torrico
Rich Plasma in Facial Rejuvenation as an Anti- Sejas.
aging Treatment.
Index:

Number Subject Page

1 Summary 2
2 Introduction 3
3 Antecedents 4
4.1 Introduction into Skin Aging 5
4.2 The Aging Process 6-7
4.3 Intrinsic/Chronological Aging 8-9
4.4 Extrinsic Aging/Photoaging 10-15
4.5 Photoaging and the Importance of Sun Protection 16-17
4.6 Clinical and Histological Manifestation of Aged Skin 18-19
5.1 Platelet Rich Plasma (PRP):Platelets 20-23
5.2 Platelet Rich Plasma (PRP):Definition 24-27
5.3 Platelet Rich Plasma (PRP):Growth Factors 28-32
5.4 Platelet Rich Plasma (PRP):Mechanism of Action 33-37
5.5 Platelet Rich Plasma (PRP):Obtaining and Injection PRP 38-42
6 Objectives 43
7 Materials and Methods 43
8 Results 44
9 PRP in Facial Rejuvenation: Review and Evaluation of Clinical 45-51
Studies
10 PRP in Cell Therapy and Regenerative Medicine 51-52
11 Methods to Improve Patient Satisfaction 53
12 Complications of PRP 53-54
13 Contraindications to PRP 55-56
14 Advantages,limitations and Precautions 56
15 Discussion 57-58
16 Conclusion 59
References 60-67

1
1. Summary.
The human body has a remarkable capacity to heal itself. Regeneration of new tissue is
accomplished by stem cells and is fostered by growth factors and hormones. Platelet-
rich plasma (PRP) is an emerging treatment in a new health sector known as
orthobiologics, which refer to growth factors and proteins that are naturally found in
the human body.

In humans, PRP has been investigated and used as a clinical tool for several types of
medical treatments, including nerve injury, tendonitis, osteoarthritis, cardiac muscle
injury, bone repair and regeneration, plastic surgery, and oral surgery. It has received
attention in the popular media because of its use in treating sports injuries in
professional athletes.

Aging of the skin, dermal components, and cells means that the skin texture and
appearance deteriorate and have been damaged. Aging affects the hands and soft tissue
of the face, neck, and décolleté. This is characterized by sagging jowls, thinning of the
skin, puffiness, age spots, and wrinkling.

In dermatology and cosmetic medicine, PRP has been used to treat acne, scarring, and
alopecia. It is also effective for skin rejuvenation and tightening around the eyes (for
thin crepe-like skin and fine lines) and in the following areas: cheeks and midface,
thinning skin on the neck, jawline and submalar regions, back of hands, décolleté, and
others (eg, knees, elbows, and upper arms, as well as for postpregnancy skin laxity).

This review shows platelet-rich-plasma (PRP) as an enhancer of the histological

characteristics of the tissues with platelet-derived growth factor receptors. A review has
been made of the literature on the platelet-rich plasma published in PubMed. This article
describes the aging process, the cellular biology of PRP, the technique for obtaining it, its
clinical applications and studies regarding PRP in facial rejuvenation and its role as an
antiageing mechanism.

Keywords

Platelet rich plasma - skin aging - skin rejuvenation –skin wrinkles-biostimulation-

photoaging –intrinsic aging - growth factors - cytokines- transforming growth factor-β -
platelet-derived growth factor - vascular endothelial growth factor

2
2. Introduction.

Autologous Platelet-rich Plasma (PRP) has attracted attention for skin rejuvenation. PRP
is derived from fresh whole blood, which contains a high concentration of platelets
(1). Various GF, including platelet-derived growth factor (PDGF), transforming growth
factor (TGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor
(IGF), are secreted from the α-granules of concentrated platelets activated by
aggregation inducers(2). These factors are known to regulate processes including cell
migration, attachment, proliferation and differentiation, and promote extracellular
matrix (ECM) accumulation by binding to specific cell surface receptors (3), Although the
optimal PRP platelet concentration is unclear, the current methods by which PRP is
prepared is reported to involve 300~700% enrichment, with platelet concentrations
consequently increasing to greater than 1,000,000 platelets/µl. (4)

Due to the presence of high concentrations of these growth factors, PRP has been used
in a wide variety of surgical procedures and clinical treatments, including the treatment
of problematic wounds(5)and maxillofacial bone defects, cosmetic surgeries(6), and
gastrointestinal surgeries(7). Recently, PRP has attracted attention in the field of
dermatology, specifically in the aesthetic field for skin rejuvenation.

The application of PRP stimulates the rejuvenation of photoaged facial skin, improving
its clinical appearance and inducing new collagen synthesis Since PRP secretes various
.

growth factors with roles in skin regeneration, it may be hypothesized that PRP may
induce the synthesis of collagen and other matrix components by stimulating the
activation of fibroblasts, thus, rejuvenating the skin. Though PRP is widely used in
clinical dermatology, experimental studies confirming the effects of PRP on aged
fibroblasts are very limited (8).

3
3. Antecedents.

Plasma rich in growth factors (PRGF) is an endogenous therapeutic technology that is

gaining interest in regenerative medicine due to its potential to stimulate and accelerate
tissue healing and bone regeneration. This autologous biotechnology is designed for the
in situ delivery of multiple cellular modulators and the formation of a fibrin scaffold,
thereby providing different formulations that can be widely used in numerous medical
and scientific fields. It has been shown that the application of a Platelet Rich Plasma
(PRP) enhances early wound healing (9, 10), and improves healing in diabetic ulcers (11).
Since the 1990s, the application of platelet preparations for wound healing has
outpaced that of isolated, exogenous growth factors.

Autologous platelet rich plasma (PRP) injections were first used in 1987 in open heart
surgery (12). Apart from the wide use of PRP to accelerate wound healing, there is
substantial clinical evidence regarding its use in other medical fields. For example, cross-
linked PRP forms a gel which is widely used in orthopedics, sports medicine, as well as in
maxillofacial surgery (13, 14), cosmetic surgery (15) and tissue engineering among
others.

Following the successful use of Platelet Rich Plasma in the field of medical pathology, it
is now being used in the aesthetics area with many advantages such as: tissue
regeneration and rejuvenation, induction of cell differentiation, extracellular matrix
formation, recruitment of other cells to the site of injury, and an increase in collagen
production, which can increase skin thickness and overall skin health. In addition, PRP is
nonallergenic, is an autologous physiological product, eliminates donor transmissible
infections, and is biological glue for tissue adhesion, especially in skin flaps, bone grafts,
and trauma. (16)

4
4.1. Introduction into Skin Aging.

Skin – the largest organ of the body – protects all the other organs from the external
environment. The skin is a complex organ with multiple structures and cell types and
divided into three layers: epidermis, dermis, and the subcutaneous tissue. The
epidermis is mainly composed of keratinocytes, pigment-producing melanocytes, and
antigen-presenting Langerhans cells. A basement membrane separates the epidermis
from the dermis, which primarily contains extracellular proteins produced by the
fibroblasts below.

The vascular supply to the skin resides in the dermis. The subcutaneous tissue consists
of fat cells that underline the connective tissue network. Type I collagen is the most
abundant protein in the skin connective tissue. The other extracellular matrix proteins,
which are a part of the skin connective tissue, are collagens (III, V, and VII), elastin,
proteoglycans, fibronectin, etc. The newly synthesized type I procollagen is secreted into
the dermal extracellular space where it undergoes enzymatic processing to arrange
itself into a triple helix configuration. (17)

With age the skin’s natural rejuvenation process slows drastically and the Skinbecomes
thinner, drier, and less elastic. (18)

Skin aging is particularly important because of its social impact. It is visible and also
represents an ideal model organ for investigating the aging process (19). The “biological
clock” affects both the skin and the internal organs in a similar way, causing irreversible
degeneration (20, 21).

However, Nicholas Perricone, a prominent American dermatologist, begins his book

with the words “Wrinkled, sagging skin is not the inevitable result of getting older. It’s a
disease, and you can fight it” (22).

5
4.2. The Aging Process.

Aging represents a biologic attrition at the cellular level resulting in decreased reserve
capacity and ability to perform normal functions occurs throughout an organisms’ life
span increasing the likelihood of death. Aging is thus the result of a genetic program or a
clock that is implanted in the genetic make-up of each species. One must also remember
that cumulative damage to the genes and proteins derived thereof, result in
compromised function and homeostatic failure. This leads the organism towards
premature aging and death, which in turn shall depend on its repair systems.

The somatic cells have telomeres at the terminal portion of the eukaryotic
chromosomes which consist of many hundreds of tandem short sequence repeats
(TTAGGG) predetermining the number of times the cell can divide before it senesces.
The enzyme DNA polymerase that replicates cellular chromosomes during mitosis
cannot replicate the final base pairs of each chromosome, resulting in progressive
telomere shortening with each cellular division. A critically short telomere will
compromise gene transcription and signal cellular senescence which is otherwise better
known as “apoptosis”. (23)

Human keratinocytes approach replicative senescence after 50–100 population

doublings in culture and remain permanently arrested in the G1 phase of the cell cycle.
The telomere is just one of the three molecules which were found to be crucial for
replicative senescence. In addition, keratinocytes have an increased resistance to
apoptosis, thus giving a time window for DNA and protein damage to accumulate. (24)

6
The skin, being the ultimate protective barrier between the internal organs and the
environment, is exposed to ultraviolet (UV) irradiation and to a lesser extent to other
DNA damaging agents such as cigarette smoke, automobile exhaust, and professional
exposure. UV irradiation causes formation of pyrimidine dimers and the benzo[a]pyrene
from cigarette smoke causes formation of guanine base pair adducts. All this moves
hand-in-hand with damage from endogenous agents such as reactive oxygen and
nitrogen species (ROS/RNS) generated by all aerobic cell species as part of their routine
metabolic processes. (25)

Amino acid racemization and interaction of amino acid groups with reducing sugars
(Maillard reaction) result in an altered or total loss of protein functions which does the
dermal collagen proteins.

Skin aging is influenced by several factors including genetics, environmental exposure

(UV radiation, xenobiotics, and mechanical stress), hormonal changes and metabolic
processes (generation of reactive chemical compounds such as activated oxygen
species, sugars and aldehydes). All factors together act on the alterations of skin
structure, function, and appearance. Yet solar UV radiation unquestionably is the single
major factor responsible for skin aging. (26)

7
4.3. Intrinsic /chronological Aging.

Intrinsic aging depends on time. The changes occur partially as the result of cumulative
endogenous damage due to the continuous formation of reactive oxygen species (ROS),
which are generated by oxidative cellular metabolism. Despite a strong antioxidant
defense system, damage generated by ROS affects cellular constituents such as
membranes, enzymes, and DNA. (27, 28)

It has a genetic background, but is also due to decreased sex hormone levels. The
telomere, a terminal portion of the eukaryotic chromosome, plays an important role.
With each cell division, the length of the human telomere shortens. Even in fibroblasts
of quiescent skin more than 30% of the telomere length is lost during adulthood. (29)
The enzyme telomerase is responsible for its maintenance. It seems that telomeres are
responsible for longevity. (30) The progressive erosion of the telomere sequence (50–
100 bp per mitosis) through successive cycles of replication eventually precludes
protection of the ends of the chromosomes, thus preventing end-to-end fusions, which
is incompatible with normal cell function.

The majority of cells have the capacity for about 60 to 70 postnatal doublings during
their lifecycles, and thereafter they reach senescence, remaining viable but incapable of
proliferation. This event facilitates end-to-end chromosomal fusions resulting in
karyotype disarray with subsequent apoptosis, thus serving as the “biological clock”.
(31)

8
Skin aging is affected by growth factor modifications and hormone activity that declines
with age. The best-known decline is that of sex steroids such estrogen, testosterone,
dehydroepiandrosterone (DHEA), and its sulfate ester (DHEAS). Other hormones such as
melatonin, insulin, cortisol, thyroxine, and growth hormone decline too. At the same
time, induced levels of certain signaling molecules such as cytokines and chemokine’s
decline as well, leading to the deterioration of several skin functions (32). Also, the
levels of their receptors decline as well (33).

At the same time, some signaling molecules increase with age. One of these is a
cytokine called transforming growth factorbeta1, which induces fibroblast senescence.
Cellular senescence is a result of molecular alterations in the cellular milieu as well as in
DNA and proteins within the cell. All of these changes gradually lead to aberrant cellular
response to environmental factors, which can decrease viability and lead to cell
death. (34)

9
4.4. Extrinsic Aging/Photoaging.

Extrinsic aging develops due to several factors: ionizing radiation, severe physical and
psychological stress, alcohol intake, poor nutrition, overeating, environmental pollution,
and exposure to UV radiation. Among all these environmental factors UV radiation
contributes up to 80%. It is the most important factor in skin aging, especially in
premature aging.

Both UVB (290–320 nm), and UVA (320–400 nm) are responsible, and the skin
Alterations caused by UV radiation depend upon the phenotype of photoexposed skin
(27, 35). UVB induces alterations mainly at the epidermal level, where the bulk of UVB is
absorbed. It damages the DNA in keratinocytes and melanocytes, and induces
production of the soluble epidermal factor (ESF) and proteolytic enzymes, which can be
found in the dermis after UV exposure. UVB is responsible for appearance of thymidine
dimers, which are also called “UV fingerprints.” That is, after UVB exposure, a strong
covalent bond between two thymidines occurs. With aging, this bond cannot be
dissolved quickly, and accumulation of mutations occurs. Affected cells appear as
sunburn cells 8 to 12 hours after exposure. Reduced production of DNA can be observed
during the next 12 hours. Actinic keratoses, lentigines, carcinomas, and melanomas
represent delayed effects. A mnemonic for UVB is B as in burn or bad.

UVA penetrates more deeply into the dermis and damages both the epidermis and
dermis. The amount of UVA in ambient light exceeds the UVB by 10 to 100
times, but UVB has biological effects 1,000 times stronger than UVA. It is accepted that
UVA radiation plays an important role in the pathogenesis of photoaging, so the
mnemonic for UVA is A as in aging (35).

10
The exact mechanism of how UV radiation causes skin aging is not clear. The dermal
extracellular matrix consists of type I and III collagens, elastin, proteoglycans, and
fibronectin, and collagen fibrils strengthen the skin. Photoaged skin is characterized by
alterations in dermal connective tissue. The amount and structure of this tissue seems
to be responsible for wrinkle formation. In photoaged skin, collagen fibrils are
disorganized and elastin-containing material accumulates (36). Levels of precursors as
well as cross-links between type I and III collagens are reduced, whereas elastin is
increased (37, 38).

UV radiation increases the production of collagen-degrading enzymes, matrix

metalloproteinases (MMPs), and the xerodermapigmentosum factor (XPF), which can
also be found in the epidermis. XPF induces epidermal-dermal invagination,
representing the beginning of wrinkle formation. At the base of wrinkles, less type IV
and VII collagen is found. This instability deepens the wrinkles.

Each MMP degrades a different dermal matrix protein; for example, MMP-1 cleaves
collagen types I, II, and III, and MMP-9 (gelatinase) degrades type IV and V and gelatin.
Under normal conditions, MMPs are part of a coordinated network and are regulated by
their endogenous inhibitors (TIMPs). The imbalance between activation and inhibition
can lead to proteolysis (39). The activation of MMPs can be triggered by UVA and UVB,
but molecular mechanisms differ depending upon the type of radiation. UVA radiation
can generate ROS that affect lipid peroxidation and generate DNA strand breaks (40).
On the other hand, within minutes after exposure UVB radiation causes MMP activity
and DNA damage. These effects can be observed after exposing human skin to one-
tenth of the minimal erythema dose.

The degree of skin damage following long-lasting UV irradiation also depends on the
skin phototype according to Fitzpatrick. In lighter complexes (types I and II) more serious
degenerative changes are elicited than in types III and IV, in which melanosomes in the
upper epidermal layer serve as relatively good UVA and UVB protection. (41)

11
Glogau developed a photoaging scale that is used to clinically classify the extent of
photodamage (Table 1) (27, 42).

Type Characteristics

Typical age 20s to 30s

1: No wrinkles Early photoaging
Mild pigmentary changes
No keratosis
No or minimal wrinkles
Typical ages late 30s to 40s
2: Wrinkles Early to moderate photoaging
in motion Early senile lentigines
Palpable but not visible keratoses
Parallel smile lines beginning to
appear laterally to mouth
Typical age 50 or older
3: Wrinkles at rest Advanced photoaging
Obvious dyschromias,
telangiectasias
Visible keratoses
Typical age 60 or older
4: Only wrinkles Severe photoaging
Yellow-gray skin
Precancerous lesions
No normal skin

Table 1:Glogau’s photoaging classification (27, 42).

12
Another environmental factor contributing to premature aging is smoking. “Smoker’s
face” or “cigarette skin” is characteristic; implying increased facial wrinkling and an
ashen and gray skin appearance (43, 44). A prematurely old appearance is a symptom of
long-term smokers. Yellow and irregularly thickened skin is result of elastic tissue
breakdown due to smoking (45) or to UV.

Premature facial wrinkling is not reduced in women on hormone replacement therapy

(46). Genetic predisposition may also influence the development of facial wrinkling (47).
It seems that cigarette smoking induces the activation of MMPs in the same mode as in
persons with significant sun exposure (48). Smoking also reduces facial stratum
corneum moisture as well as vitamin A level, which is important in reducing the extent
of collagen damage (27).

The photochemical activity of smog is due to the reduction of air pollutants such as
nitrogen oxides and volatile organic compounds created from fossil fuel combustion in
the presence of sunlight. Emission from factories and motor vehicle exhaust are primary
sources of these compounds. The major targets of ozone in the skin are the superficial
epidermal layers; this results in the depletion of antioxidants such as alpha-tocopherol
(vitamin E) and ascorbic acid (vitamin C) in the superficial epidermal layers (49).

As stochastic damage is explained, the damage is initiated by random cosmic radiation

and triggered by free radicals during cell metabolism, which damages cell lipid
compounds, especially membrane structures. The free radical theory is one of the most
widely accepted theories to explain the cause of skin aging. These compounds are
formed when oxygen molecules combine with other molecules, yielding an odd number
of electrons. That is, an oxygen molecule with paired electrons is stable, but one with an
unpaired electron is very reactive and it takes electrons from other vital components. As
result, cell death or mutation appears (27).

13
Table 2gives an overview of the various epidermal, dermal, and clinical signs with which
one can differentiate between chronological aging and photoaging.

Aging types Epidermis Dermis Clinical

Thinner than Elastin fibers Skin is smooth,

normal with lower appear irregular in unblemished, but shows
cell growth, minor their arrangement, saggy appearance
abnormalities in whereas collagen
Chronological keratinocyte fibers begin to
aging regularity lower in number
Normal stratum and thickness
corneum
There is loss of rete
pegs here as well

Thick skin, with Excessive Smooth, leathery,

acanthosis followed production of reddened appearance
by atrophy of the elastin fibers in an with initially light
cells improper wrinkles, which later
High basal orientation, deepen, thus showing
Photoaging keratinocyte collagen fibres loss of collagen fibers
irregularity Stratum appear to thicken
corneum appears and then wear out
compact soon
There is loss of rete Appearance of
pegs here as well grenzzone

Table 2: Comparison of chronological aging and photoaging (50)

14
The picture below (Picture 2) is for a 69-year-old man presented with a 25-year history
of gradual, asymptomatic thickening and wrinkling of the skin on the left side of his face.

The physical examination showed hyperkeratosis with accentuated ridging, multiple

open comedones, and areas of nodular elastosis.
Histopathological analysis showed an accumulation of elastolytic material in the dermis
and the formation of milia within the vellus hair follicles.

Findings were consistent with the Favre–Racouchot syndrome of photodamaged skin,

known as dermatoheliosis.
The patient reported that he had driven a delivery truck for 28 years. Ultraviolet A (UVA)
rays transmit through window glass, penetrating the epidermis and upper layers of
dermis. Chronic UVA exposure can result in thickening of the epidermis and stratum
corneum, as well as destruction of elastic fibers. This photoaging effect of UVA is
contrasted with photocarcinogenesis. Although exposure to ultraviolet B (UVB) rays is
linked to a higher rate of photocarcinogenesis, UVA has also been shown to induce
substantial DNA mutations and direct toxicity, leading to the formation of skin cancer.
The use of sun protection and topical retinoids and periodic monitoring for skin cancer
were recommended for the patient. (51)

Picture 2: A 69 year old

man presented with a 25-
year history of gradual,
asymptomatic thickening
and wrinkling of the skin on
the left side of his face. (51)

15
4.5. Photoaging and the Importance of Sun Protection.

This part was included in this review based on the fact that exposure to sun is
responsible of 80% of photodamaged skin. So it should be emphasized that sun
protection in daily life is a must to maintain a healthy youthful skin.

The skin is equipped with two photoprotective mechanisms: the melanin in the lower
layer of epidermis, and the urocanic acid barrier of the stratum corneum,
Which reflects and absorbs a significant amount of UVB radiation. The thickness of the
stratum corneum appears to be highly significant for photoprotection (53).

Antioxidants provide protection against UVB-induced oxidative stress, especially in

stratum corneum lipids. Even systemically applied antioxidants accumulate in the
stratum corneum and play an important role against UV-induced skin damage (54)

The body has developed further defense mechanisms that protect against UV radiation
and dangerous free radicals. Antioxidants naturally occurring in the skin are superoxide
dismutase, catalase, alpha-tocopherol, ascorbic acid, ubiquinone, and glutathione. Many
of them are inhibited by UV and visible light. The antioxidant program consists of a diet
containing large amounts of vitamins A, E, and C, grape-seed extracts, coenzyme Q10,
and alpha-lipoic acid (55). The most highly recommended foods include: avocados,
berries, dark green leafy vegetables, orange-colored vegetables and fruits,
pineapples,salmon, and tomatoes.

The mainstay in the prevention of skin aging is photoprotection. UV filters are now
present in cosmetic products for daily use, such as makeup, creams, lotions, and hair
sprays.

16
The general requirements are that modern sunscreens should protect against UVA and
UVB rays and be photo-stable and water resistant Chemical UV filters have the capacity
to absorb shortwavelength UV and transform photons into heat-emitting long-
wavelength (infrared) radiation. Most of them absorb a small wavelength range.

They can be divided into three groups. The first group consists of molecules
that primarily absorb the UVB spectrum (p-aminobenzoic acid derivatives and zincacid
esters), and second of molecules that primarily absorb the UVA spectrum (butyl-
methoxydibenzoylmethane). The third group consists of molecules that absorb UVA and
UVB photons (benzophenone). A combination of different filters in the same product
renders the whole filter system photo-unstable that means that UV exposure causes
photochemical reactions that generate ROS with subsequent phototoxic and
photoallergic reactions.

Great efforts have been made to stabilize molecules in UV filters, which have improved
the efficacy of photoprotection with chemical UV filters.

Today there is a growing need for standardization and evaluation of UVA

photoprotection, while for UVB there is already consensus on the international level
(56).

The use of physical filters is encouraged. The most frequently used of these are
microparticles of zinc oxide and titanium dioxide with diameters in the range of
10 to 100 nm. They are capable of reflecting a broad spectrum of UVA and UVB rays.
They do not penetrate into the skin and thus have low potential for developing toxic or
allergic effects.

Today they are increasingly being used in combination with chemical filters. One
disadvantage of the inorganic micropigments is that they reflect visible light, creating a
“ghost” effect. This is one reason such sunscreens are often rejected by consumers (57,
58)

17
4.6. Clinical and Histological Manifestations of Aged Skin.

Xerosis, laxity, wrinkles, slackness, and the occurrence of benign neoplasms such as
seborrheickeratoses and cherry angiomas. There are histological features that
accompany these changes. In the epidermis, there is no alteration in the stratum
corneum and epidermal thickness, keratinocyte shape, and their adhesion, but a
decreased number of melanocytes and Langerhans cells is evident (23).

The most obvious changes are at the epidermaldermal junction: flattening of the rete
ridges with reduced surface contact of the epidermis and dermis. This results in a
reduced exchange of nutrients and metabolites between these two parts.

In the dermis several fibroblasts may be seen, as well as a loss of dermal volume (23,
52). A decrease in blood supply due to a reduced number of blood vessels also occurs.
There is also a depressed sensory and autonomic innervation of epidermis and dermis.
Cutaneous appendages are affected as well. Terminal hair converts to vellus hair. As
melanocytes from the bulb are lost, hairs begin to gray. Further reasons for graying are
decreased tyrosinase activity, less efficient melanosomal transfer and migration, and
melanocyte proliferation (59).

Factors that contribute to wrinkling include changes in muscles, the loss of

subcutaneous fat tissue, gravitational forces, and the loss of substance of facial
bonesand cartilage. Expression lines appear as result of repeated tractions caused by
facial muscles that lead to formation of deep creases over the forehead and between
eyebrows, and in nasolabial folds and periorbital areas. Repeated folding of the skin
during sleeping in the same position on the side of the face contributes to appearance
of “sleeping lines.”

18
Histologically, thick connective tissue strands containing muscle cells are present
beneath the wrinkle (24). In the muscles and accumulation of lipofuscin (the “age
pigment”), a marker of cellular damage, appears. The deterioration of neuromuscular
control contributes to wrinkle formation (26). The constant gravitational force also acts
on the facial skin, resulting in an altered distribution of fat and sagging. Skin becomes lax
and soft tissue support is diminished.

Gravitational effects with advanced years play an important role and contribute to
advanced sagging. This factor is particularly prominent in the upper and lower eyelids,
on the cheeks, and in the neck region.

Fat depletion and accumulation at unusual sites contributes to the altered appearance
of the face (26). It affects the forehead, periorbital, and buccal areas, the inner line of
nasolabial folds, and the temporal and perioral regions. At the same time it accumulates
submentally, around the jaws, at outer lines of nasolabial folds and at lateral malar
areas. In contrast to the young, in whom fat tissue is diffusely distributed, in aged skin
fat tends to accumulate in pockets, which droop and sag due to the force of gravity (26,
27).

The mass of facial bones and skeletal bones reduces with age. Resorption affects the
mandible, maxilla, and frontal bones. This loss of bone enhances facial sagging and
wrinkling with obliteration of the demarcation between the jaw and neck that is so
distinct in young persons (28).

19
5.1. Platelet Rich Plasma (PRP):Platelets.

Platelets are the first element to arrive at the site of tissue injury and are particularly
active in the early inflammatory phases of the healing process (60).

They play a role in aggregation, clot formation, homeostasis through cell membrane
adherence, and release of substances that promote tissue repair and that influence the
reactivity of blood vessels and blood cell types involved in angiogenesis, regeneration,
and inflammation (61).

Platelet secretory granules contain growth factors (GFs), signaling molecules, cytokines,
integrins, coagulation proteins, adhesion molecules, and some other molecules, which
are synthesized in megakaryocytes and packaged into the granules through vesicle
trafficking processes (62). Three major storage compartments in platelets are alpha
granules, dense granules, and lysosomes (62).

Platelets mediate these effects through degranulation, in which platelet-derived GF

(PDGF), insulin-like GF (IGF1), transforming GF-beta 1 (TGF-β1), vascular endothelial GF
(VEGF), basic fibroblastic GF (bFGF), and epidermal GF (EGF) are released from alpha
granules (63). In fact, the majority of the platelet substances are contained in alpha
granules (see Table 3) (63).

When platelets are activated, they exocytose the granules; this process is mediated by
molecular mechanisms homologous to other secretory cells, uniquely coupled to cell
activation by intracellular signaling events (64).

20
General activity Cytogenetic
Specific molecules Biologic activities
categories location

Regulates the tissue factor-(TF-)

Tissue factor pathway
2q32.1 dependent pathway of blood
inhibitor; TFPI
coagulation
Plays an important role in
Kininogen; KNG 3q27.3 assembly of the plasma
kallikrein
Growth arrest-specific
13q34 Stimulates cell proliferation
6; GAS6
Carrier protein for platelet
Multimerin; MMRN 4q22
factor V
Clotting factors
Is the most important inhibitor
and related Antithrombin; AT 1q25.1
of thrombin
proteins
Protein S; PROS1 3q11.1 Inhibits blood clotting
Acts as a cofactor for the
Coagulation factor V;
1q24.2 conversion of prothrombin to
F5
thrombin by factor Xa
It participates in blood
coagulation as a catalyst in the
Coagulation factor XI;
4q35.2 conversion of factor IX to factor
F11
IXa in the presence of calcium
ions

Induces plasmin production

Plasminogen; PLG 6q26
(leads to fibrinolysis)
Plasminogen activator Regulation of plasmin
7q22.1
inhibitor 1; PAI1 production
Alpha-2-plasmin
17p13.3 Inactivation of plasmin
inhibitor
Fibrinolytic Inhibits cell-cycle progression
factors and Osteonectin; ON 5q33.1 and influences the synthesis of
related proteins extracellular matrix (ECM)
Histidine-rich Interacts with heparin and
3q27.3
glycoprotein; HRG thrombospondin
Thrombin-activatable
fibrinolysis inhibitor; 13q14.13 Attenuates fibrinolysis
TAFI
Carrier of specific growth
Alpha-2-
12p13.31 factors and induces cell
Macroglobulin; A2M
signaling

21
 Inhibits matrix
Tissue inhibitor of metalloproteinases (MMPs), a
metalloproteinase 4; 3p25.2 group of peptidases involved in
TIMP4 degradation of the extracellular
matrix
Inhibits serine proteinases
Complement
including plasmin, kallikrein,
component 1 11q12.1
and coagulation factors XIa and
inhibitor; C1NH
XIIa
Alpha-1-antitrypsin Acute phase protein, inhibits a
Proteases and
(serpin peptidase 14q32.13 wide variety of proteases and
antiproteases
inhibitor) enzymes
Modulates intracellular
Nexin 2; SNX2 5q23.2 trafficking of proteins to
various organelles

Platelet factor 4; PF4 4q13.3 Inhibition of angiogenesis

β-thromboglobulin
Platelet activation, inhibition of
Basic proteins (Pro-platelet basic 4q13.3
angiogenesis
protein; PPBP)
Endostatin (Collagen,
Inhibitors of endothelial cell
type XVIII, Alpha-1; 21q22.3
migration and angiogenesis
COL18A1)

Blood clotting cascade (fibrin

Fibrinogen; FG 4q31.3
clot formation)
Binds to cell-surface integrins,
affecting cell adhesion, cell
Fibronectin; FN 2q35
growth, migration, and
differentiation
Adhesive Induces cell adhesion,
Vitronectin; VTN 17q11.2
proteins chemotaxis
Thrombospondin I;
15q14 Inhibition of angiogenesis
THBS1
18p11.31- Modulates cell contact
Laminin-8
p11.23 interactions

Table 3: Some bioactive peptides present in the alpha granules of platelets. (63)

22
Among bioactive molecules stored and released from platelets dense granules are
catecholamines, histamine, serotonin, ADP, ATP, calcium ions, and dopamine, which are
active in vasoconstriction, increased capillary permeability, attract and activate
macrophages, tissue modulation and regeneration. These non-GF molecules have
fundamental effects on the biologic aspects of wound healing (65).

For their numerous functions, platelets have developed a set of platelet receptors that
are the contact between platelets and their surroundings; they determine the reactivity
of platelets with a wide range of agonists and adhesive proteins. Some of these
receptors are expressed only on activated platelets (60). Certain biological mechanisms
present in the platelets are shared with other cells, and therefore they contain some
common cytoplasmic enzymes, signal transduction molecules, and cytoskeletal
components (62).

The platelet lifespan is approximately 7 to 9 days, which they spend circulating in the
blood in their resting form. When adhered to exposed endothelium or activated by
agonists, they change their shape and secrete the contents of the granules (including
ADP, fibrinogen, and serotonin), which is followed by platelet aggregation (66). Initiation
of the signaling event within the platelet leads to the reorganization of the platelet
cytoskeleton, which is visible as an extremely rapid shape change (67).

23
5.2. Platelet Rich Plasma (PRP): Definition.

PRP has been used clinically in humans since the 1970s for its healing properties
attributed of autologous GF and secretory proteins that may enhance the healing
process on a cellular level (68). Furthermore, PRP enhances the recruitment,
proliferation, and differentiation of cells involved in tissue regeneration (69). PRP-
related products, also known as platelet-rich concentrate, platelet gel, preparation rich
in growth factors (PRGF), and platelet releasate, have been studied with in vitro and in
vivo experiments in the fields of surgical sciences mainly (70).

Platelets are activated either by adhesion to the molecules that are exposed on an
injured endothelium, such as von Willebrand Factor (vWF), collagen, fibronectin, and
laminin, or by physiologic agonists such as thrombin, ADP, collagen, thromboxane A2,
epinephrine, and platelet-activating factors (71).

Depending on the device and technique used, PRP can contain variable amounts of
plasma, erythrocytes, white blood cells, and platelets. The platelet concentration should
be increased above baseline or whole blood concentration. It is generally agreed upon
that PRP should have a minimum of 5 times the number of platelets compared to
baseline values for whole blood to be considered “platelet rich” (72).

This conclusion is supported by in vitro work showing a positive dose-response

relationship between platelet concentration and proliferation of human mesenchymal
stem cells, proliferation of fibroblasts, and production of type I collagen (73). This
suggests that the application of autologous PRP can enhance wound healing, as has
been demonstrated in controlled animal studies for both soft and hard tissues (74, 75).

24
Autologous PRP represents an efficacious treatment for its use in wound healing like
chronic diabetic foot ulceration due to multiple growth factors, is safe for its autologous
nature, and is produced as needed from patient blood. Like we said, key for self-
regeneration (76).

Upon activation, platelets release their granular contents into the surrounding
environment. The platelet alpha granules are abundant and contain many of the GFs
responsible for the initiation and maintenance of the healing response (62). These GFs
have been shown to play an important role in all phases of healing. The active secretion
of these proteins by platelets begins within 10 minutes after clotting, with more than
95% of the presynthesized GFs secreted within 1 hour. After this initial burst, the
platelets synthesize and secrete additional proteins for the balance of their life (5–10
days) (77).

The fibrin matrix formed following platelet activation also has a stimulatory effect on
wound healing. The fibrin matrix forms by polymerization of plasma fibrinogen following
either external activation with calcium or thrombin or internal activation with
endogenous tissue thromboplastin(73). This matrix traps platelets allowing a slow
release of a natural combination of GF while providing a provisional matrix that provides
a physical framework for wound stem cells and fibroblast migration and presentation of
other biological mediators such as adhesive glycoproteins (78, 79)

PRP with a platelet concentration of at least 1 000 000 platelets/μL in 5 mL of plasma is

associated with the enhancement of healing (80). PRP can potentially enhance healing
by the delivery of various GF and cytokines from the alpha granules contained in
platelets and has an 8-fold increase in GF concentrations compared with that of whole
blood (81).

25
The use of PRP to enhance bone regeneration and soft tissue maturation has increased
dramatically in the fields of orthopedics, periodontics, maxillofacial surgery, urology,
and plastic surgery over the last years. However, controversies exist in the literature
regarding the added benefit of this procedure. While some authors have reported
significant increases in bone formation and maturation rates (70), others did not
observe any improvement (82).

The wound-healing process is a complex mechanism characterized by four distinct, but

overlapping, phases: hemostasis, inflammation, proliferation, and remodeling (83). The
proliferative phase includes blood vessel formation by endothelial cells and bone
synthesis by osteoblasts. All these events are coordinated by cell-cell interactions and by
soluble GF released by various cell types. Recent reviews have emphasized the need for
additional research aiming to characterize PRP in terms of GF content and their
physiological roles in wound healing (84-86)

Thrombin represents a strong inducer of platelet activation leading to GF release (87). It

is also known that particulate grafts, when combined with calcium and thrombin treated
PRP, possess better handling characteristics and higher GF content (82). Typically,
thrombin concentrations used in clinical applications vary between 100 and 200 units
per mL (70), while platelet aggregation is maximum in the range of 0.5 to 4 units per mL
(88).

The basic cytokines identified in platelets play important roles in cell proliferation,
chemotaxis, cell differentiation, regeneration, and angiogenesis (79).A particular value
of PRP is that these native cytokines are all present in “normal” biologic ratios. The
platelets in PRP are delivered in a clot, which contains several cell adhesion molecules
including fibronectin, fibrin, and vitronectin. These cell adhesion molecules play a role in
cell migration and thus also add to the potential biologic activity of PRP. The clot itself
can also play a role in wound healing by acting as conductive matrix or “scaffold” upon
which cells can adhere and begin the wound-healing process (79).

26
PRP can only be made from anticoagulated blood. Preparation of PRP begins by addition
of citrate to whole blood to bind the ionized calcium and inhibit the clotting cascade
(63). This is followed by one or two centrifugation steps. The first centrifugation step
separates the red and white blood cells from plasma and platelets. The second
centrifugation step further concentrates the platelets, producing the PRP separate from
platelet-poor plasma (68).

An important point is that clotting leads to platelet activation, resulting in release of the
GF from the alpha granules, otherwise known as degranulation. Approximately 70% of
the stored GFs are released within 10 minutes, and nearly 100% of the GFs are released
within 1 hour. Small amounts of GF may continue to be produced by the platelet during
the rest of its lifespan (1 week) (70).

A method to delay the release of GF is possible by addition of calcium chloride (CaCl2) to

initiate the formation of autogenous thrombin from prothrombin. The CaCl2 is added
during the second centrifugation step and results in formation of a dense fibrin matrix.
Intact platelets are subsequently trapped in the fibrin matrix and release GF slowly over
a 7-day period. The fibrin matrix itself may also contribute to healing by providing a
conductive scaffold for cell migration and new matrix formation (78).

27
5.3. Platelet Rich Plasma (PRP): Growth Factors.

Platelets are known to contain high concentrations of different GF and are extremely
important in regenerative process; activation of the platelet by endothelial injury
initiates the wound-healing process (89). When platelets are activated, their alpha
granules are released, resulting in an increased concentration of GF in the wound milieu
(90).

There is increasing evidence that the platelet cell membranes themselves also play a
crucial role in wound healing through their GF receptor sites (77). GFs are found in a
wide array of cells and in platelet alpha granules (68). Table 4 gives an overview of some
of the more extensively studied GFs and their involvement in wound healing. There are
many more, both discovered and undiscovered, GFs. The platelet is an extremely
important cell in wound healing because it initiates and plays a major role in the wound
regenerative process (69).

The first discovered GF was EGF in 1962 by Cohen (70). It was not until 1989 before
clinical trials with EGF were attempted to demonstrate enhanced wound healing.
Studies did demonstrate that EGF can accelerate epidermal regeneration and enhance
healing of chronic wounds (72).

28
PDGF was discovered in 1974 and is ubiquitous in the body. It is known to be released
by platelet alpha granules during wound healing and stimulate the proliferation of many
cells, including connective tissue cells. In fact, thus far, high-affinity cell-surface
receptors specific for PDGF have only been demonstrated on connective tissue cells.
When released, PDGF is chemotactic for monocytes, neutrophils, and fibroblasts. These
cells release their own PDGF, thus creating a positive autocrine feedback loop (73).
Other functions of PDGF include effects on cell growth, cellular migration, metabolic
effects, and modulation of cell membrane receptors (74).

PDGFs were first identified as products of platelets which stimulated the proliferation in
vitro of connective tissue cell types such as fibroblasts (75).

The PDGF system, comprising four isoforms (PDGF-A, -B, -C, and -D) and two receptor
chains (PDGFR-alpha and -beta), plays important roles in wound healing,
atherosclerosis, fibrosis, and malignancy. Components of the system are expressed
constitutively or inducibly in most renal cells (74). They regulate a multitude of
pathophysiologic events, ranging from cell proliferation and migration to extracellular
matrix accumulation, production of pro- and anti-inflammatory mediators, tissue
permeability, and regulation of hemodynamics (75).

Inactivation of PDGF-B and PDGF beta receptor (PDGFRb) genes by homologous

recombination in embryonic stem cells shows cardiovascular, hematological, and renal
defects. The latter is particularly interesting since it consists of a specific cellular defect:
the complete loss of kidney glomerular mesangial cells and the absence of urine
collection in the urinary bladder (75).

29
PDGF-C and PDGFR-alpha contribute to the formation of the renal cortical interstitium.
Almost all experimental and human renal diseases are characterized by altered
expression of components of the PDGF system. Infusion or systemic overexpression of
PDGF-B or -D induces prominent mesangioproliferative changes and renal fibrosis.
Intervention studies identified PDGF-C as a mediator of renal interstitial fibrosis and
PDGF-B and -D as key factors involved in mesangioproliferative disease and renal
interstitial fibrosis (75, 76).

Fréchette et al., demonstrated that the release of PDGF-B, TGF-beta1, bFGF, and VEGF is
significantly regulated by the amount of calcium and thrombin added to the PRP and
that PRP supernatants are more mitogenic for endothelial cells than whole-blood
supernatants (91). Other GFs such as epidermal growth factor (EGF), transforming
growth factor-alpha (TGF-alpha), insulin-like growth factor-1 (IGF-1), angiopoietin-2
(Ang-2), and interleukin-1beta (IL-1beta) are also known to play important roles in the
wound-healing process (77).

30
In 2008, Wahlström et al., demonstrated that growth factors released from platelets
had potent effects on fracture and wound healing. The acidic tide of wound healing,
that is, the pH within wounds and fractures, changes from acidic pH to neutral and
alkaline pH as the healing process progresses (76). They investigated the influence of pH
on lysed platelet concentrates regarding the release of growth factors. The platelet
concentrates free of leukocyte components were lysed and incubated in buffers with pH
between 4.3 and 8.6. Bone morphogenetic protein-2 (BMP-2), platelet-derived growth
factor (PDGF), transforming growth factor-beta (TGF-beta), and vascular endothelial
growth factor (VEGF) were measured by quantitative enzyme-linked immunosorbent
assays. BMP-2 was only detected in the most acidic preparation (pH 4.3), which is
interesting since BMP-2 has been reported to be an endogenous mediator of fracture
repair and to be responsible for the initiation of fracture healing. These findings indicate
that platelets release substantial amounts of BMP-2 only under conditions of low pH,
the milieu associated with the critical initial stage of fracture healing (76).

Recently, Bir et al., demonstrated stromal cell-derived factor 1-α (SDF-1α) PRP from
diabetic mice. The concentration (pg/mL) of different growth factors was significantly
higher in the PRP group than in the platelet-poor plasma (PPP) group. The
concentrations (pg/mL) of SDF-1α (10,790 ± 196 versus 810 ± 39), PDGF-BB (45,352 ±
2,698 versus 958 ± 251), VEGF (53 ± 6 versus 30 ± 2), bFGF (29 ± 5 versus 9 ± 5), and IGF-
1 (20,628 ± 1,180 versus 1,214 ± 36) were significantly higher in the PRP group than in
the PPP group, respectively (79).

31
Name Cytogeneticlocation Biologicactivities

Transforming growth Controls proliferation, differentiation, and

19q13.2
factor, beta-I; TGFB1 other functions in many cell types
Platelet-derived Potent mitogen for connective tissue cells and
growth factor, alpha 7p22.3 exerts its function by interacting with related
polypeptide; PDGFA receptor tyrosine kinases
Platelet-derived
Promotescellularproliferation and inhibits
growth factor, beta 22q13.1
apoptosis
polypeptide; PDGFB
Platelet-derived Increases motility in mesenchymal cells,
growth factor C; 4q32.1 fibroblasts, smooth muscle cells, capillary
PDGFC endothelial cells, and neurons
Platelet-derived Involved in developmental and physiologic
growth factor D; 11q22.3 processes, as well as in cancer, fibrotic
PDGFD diseases, and arteriosclerosis
Insulin-like growth Mediates many of the growth-promoting
12q23.2
factor I; IGF1 effects of growth hormone
Fibroblast growth Induces liver gene expression, angiogenesis
5q31.3
factor I; FGF1 and fibroblast proliferation
Induces differentiation of specific cells, is a
Epidermalgrowth potent mitogenic factor for a variety of
4q25
factor; EGF cultured cells of both ectodermal and
mesodermal origin
Vascular endothelial
Is a mitogen primarily for vascular endothelial
growth factor A; 6p21.1
cells, induces angiogenesis
VEGFA
Is a regulator of blood vessel physiology, with
Vascular endothelial
11q13.1 a role in endothelial targeting of lipids to
growth factor B; VEGFB
peripheral tissues
Angiogenesis and endothelial cell growth, and
Vascular endothelial
4q34.3 can also affect the permeability of blood
growth factor C; VEGFC
vessels

Table 4:Peptidic growth factors present in platelet-rich plasma (PRP). (69)

32
5.4. Platelet Rich Plasma (PRP): Mechanism of Action.

PRP can potentially enhance healing process of musculoskeletal tissue by the delivery of
various growth factors and cytokines from the α-granules contained in platelets figure
1(71).

The basic cytokines,Table 5, which identified platelets, play important roles in cell
proliferation, chemotaxis, cell differentiation, and angiogenesis. Bioactive factors are
also contained in the dense granules in platelets. The dense granules contain serotonin,
histamine, dopamine, calcium, and adenosine (68). These nongrowth factors have
fundamental effects on the biologic aspects of wound healing.

33
Figure 1: A summary of the most important growth factors and cytokines
released from platelets. (71)

34
 Mechanisms Growth factors and cytokines Function

Important role in the

Proinflammatory
IL1, IL6, and TNF-alpha early responses of
cytokines
tissue repair.

Platelet-derived growth factor (PDGF)

[54], transforming growth factor (TGF)-
beta, platelet-derived epidermal growth
Help the
factor (PDEGF), platelet-derived
regeneration of
Growth factors angiogenesis factor (PDAF) [55], insulin-
tissues with low
like growth factor (IGF-1), and platelet
healing potential.
factor 4 (PF-4) [56, 57], vascular
endothelial growth factors (VEGF), and
endothelial growth factors (EGF)
Vascular growth factor (VGF), VEGF,
Angiogenesis platelet derived membrane microparticles Promote
factors (PMP), and peripheral blood mononuclear angiogenesis.
cells (PBMNCs)

In the dense granules

in platelets and have
Factors in other
Serotonin, histamine, dopamine, calcium, fundamental effects
mechanisms of
and adenosine on the biologic
PRP
aspects of wound
healing

Table 5: Growth factors and cytokines in PRP in different mechanisms

(68)

35
Platelet concentration is a rich source of various cytokines and growth factors, which
are activated after its injection into the target tissue. Platelets are activated
endogenously by coagulation factors (in some methods of preparing PRP, the activated
PRP is injected to the tissue). Following their attachment to special receptors on the cell
surfaces, some intracellular processes are activated, that facilitate extracellular matrix
(ECM) accumulation and improve cell proliferation and differentiation. Tissue
regeneration is resulted from cell proliferation, angiogenesis and cell migration (92, 93).

Matrix metaloproteinas proteins (MMP) are involved in aging process by degradation of

collagen and other extracellular matrix (ECM) proteins (94), this characteristic can be
used to benefit rejuvenation. They can help regeneration of dermis through omission of
collagen fragments that are harmful to the dermal connective tissue, and so, provide an
appropriate foundation for new collagen deposition (94).

In some studies aPRP (activated PRP) increases the expression of MMP-1 and MMP-3
protein. Thus, aPRP may cause ECM remodeling through stimulating the removal of
photo-damaged ECM components and inducing the synthesis of new collagen by
fibroblasts, which are in turn proliferated by their stimulation (95).

Another study showed that high concentration of PRP increased type I collagen, MMP-1
and MMP-2 expression in human skin fibroblasts (96).

In addition to above mechanisms, improving aging skin through PRP which is a dose–
response relationship has been recognized between concentrated platelet and
mesenchymal stem cell proliferation (97).

36
Another mechanism of PRP for skin rejuvenation is through acceleration of hyaluronic
acid production. Hyaluronic acid absorbs water and makes hyaluronic acid matrix
swelled which increases skin volume and turgor. It also promotes cell proliferation,
extracellular matrix synthesis and helps to the adjustment of the collagen fibers
diameter. Overall, it could enhance skin elasticity (98). All these processes and some
other unknown ones contribute to tissue rejuvenation through PRP.

Increased proliferation of human dermal fibroblasts

Increased expression of MMP* - 1 and MMP – 3 à removal of

photodamaged ECM
Increased production of procollagen type I peptide and expression of
collagen type I, alpha-I à Synthesis of new collagen

Increases expression of G1 cell cycle regulators à accelerates wound

healing
*MMP: Matrix MetalloProteinase

Table 6: Mechanism of action of PRP

in skin rejuvenation (99,100)

37
5.4. Platelet Rich Plasma (PRP): Obtaining and Injection PRP.

BLOOD COLLECTION

As your own (autologous) blood is used, it is first collected

by venesection, the same procedure used in blood tests or
blood donations.

The site of the venesection is usually a large vein in your

elbow. Up to 20 ml of blood may be collected. (4 tubes of
5ml)

38
PROCEDURES:
· TUBES: RegenLab, Inex, Glo Pro, Plasmolifting, Tubex.
· TUBES AND KITS: RegenLab, Inex, Selphyl, My Cells, Mesopras, BTI, Traylife.
· OUTSOURCED PROCESSES: Regennia.

39
40
CENTRIFUGATION

This blood will then be spun down in a centrifuge to separate the components. The
process takes 15 minutes. The platelet rich plasma (PRP) is then harvested and there are
a variety of different ways this can be done. Depending on your injury, up to 2-8 mL of
PRP will then be extracted from the centrifuged blood.

Force (RCF): PRP- Centrif: 3400 rpm for 5 minutes.

41
In the centrifugation tube 4 levels are obtained:

I. Deeper, sediment area, rich in erythrocytes and leukocytes. Should be discarded.

II. Zone platelet rich plasma (PRP)
III. Zone normal platelet in plasma. Should be discarded.
IV. Zone platelet poor plasma rich in fibrin

THE INJECTION

1) The patient is placed in an appropriate and comfortable position that allows for
sterility and access to the site of injection.
2) All necessary materials for the injection (PRP, additives, 4X4s, needles, US gel) should
have been planned and placed on a sterile table adjacent and easily accessible to the
physician.
3) The patient’s skin is cleansed appropriately and towels or drapes may be used to
create an aseptic field.
4) If local anesthetic will be used, it is to be applied with aseptic technique for 30
minutes before the procedure.

5) Injection is done with nappage technique, same as in mesotherapy.

42
6. Objectives.

To understand the process of aging, its causes and to emphasis on the role of
photoaging/extrinsic aging in wrinkled aged looking skin.

Toinvestigate the evidence concerning the management and use of PRP as an antiaging
mechanism in facial rejuvenation.

To Research the benefits of using PRP in Aesthetic Medicine.

To establish therapeutic uses and effects of PRP in skin rejuvenation, skin aging and
decrease wrinkles, fine lines and over all skin appearance.

7. Materials and Methods.

Articles and publications have been selected and revised. These have been mainly
chosen from PubMed, Medical journals like the New England Journal of Medicine.

High-impact articles, literature, meta-analysis, medical reviews and randomized case-

control studies with standardized measures and statistically significant results that were
currently in place were included.

The revised articles mainly have been chosen from the year 2002 up to this current year.

Articles that were not in English or Spanish were excluded for the review of the uses of
PRP in skin rejuvenation.

43
8. Results.

The use of PRP in dermatology has taken on new meaning over the past few years,
research on. All studies on this topic vary in their methodology in one way or another;
however, the similarity lies in the results. All show the beneficial effects of PRP on
wound repair, differentiation and proliferation of adipose precursor cells, and
angiogenesis

Applications of PRP such as acne scars, fat grafting, wound healing, wrinkling around the
eyes, facial rejuvenation and hair regrowth have plenty of publications to boost about.
Newer combinations therapies such as PRP with carbon dioxide fractional lasers for acne
scars or microneedling for skin tightening effect.

Despite these abundant studies in various reviewed journals, we await evidence-based

data regarding the exact concentration and dosing parameters. All the studies are
conducted with different methodology. Questions such as, when to inject PRP? Will PRP
work better if injected weekly, daily, or monthly? For how long post procedure? Still
boggle the mind. For now, all we have are arbitrary guidelines. Instead of being helpful
and encouraging, these studies can often be confusing and misguiding to a doctor
planning to inculcate PRP in his daily practice. These unanswered questions, plus the
time and cost of equipment setup, are obstacles to widespread implementation of PRP.

It is undoubtable that the use of platelet-based formulations in cutaneous medicine will

continue to evolve. Hence to use PRP to the fullest extent, the principles, procedure and
indications need to be understood, standardized and simplified; only then, we can trust
this procedure completely and give maximum benefit to our patients.

44
9. PRPin Facial Rejuvenation: Review and Evaluation of Clinical Studies.

EVIDENCE TO SUPPORT ITS USE

Monthly intradermal injections of PRP in 3 sessions have shown satisfactory results in

face and neck rejuvenation and scar attenuation. (101)Shin et al.,(102) showed that a
combination of fractional non-ablative (erbium glass) laser therapy with topical
application of PRP, resulted in objective improvement in skin elasticity, a lower
erythema index and an increase in collagen density as well. Histological examination
showed an increase in length of dermoepidermal junction, amount of collagen and
fibroblasts in the treated skin.

PRP in combination with fractional ablative lasers (carbon dioxide) for deep wrinkles and
severe photodamaged skin, has also been shown to reduce commonly encountered,
transient adverse effects and decrease the downtime.(103)In a split face blinded trial,
PRP injections given monthly for 3 months, have shown good results for infraorbital
rejuvenation as well, without any obvious side effects.(104)

As varied number of products are available nowadays for skin rejuvenation like
mesotherapy solutions, adipose derived stem cells etc., whether PRP can be used in
combination with them to boost the aesthetic results, needs further clinical trials. Also,
comparative studies are lacking to contrast PRP with other treatments like topical
cosmeceutical preparations of growth factors, to be used after fractional laser
resurfacing.

45
SCARS AND CONTOUR DEFECTS

PRP has become a promising modality among soft tissue augmentation techniques.
PRFM has been used as a filler to correct deep nasolabial folds without any adverse
effects. (105)

As an adjuvant, it has been studied with autologous fat transfer procedures. An in

vitro pilot study, revealed that fat grafts when mixed with PRP resulted in greater
vascularity, fewer cysts and vacuoles, less fibrosis and overall improved survival and
quality of fat grafts as compared to saline.(106)This novel regimen was found to
maintain fat graft survival in a patient with facial contour defect for upto 2
years.(107)Data suggests that fat grafts can be admixed with PRP in treating traumatic
scars, and further can be followed by fractional laser resurfacing to give best
results.(108)

PRP injections in combination with fractional carbon dioxide resurfacing have shown
good results in acne scar resurfacing also, apart from skin rejuvenation. (109)

46
Role of Growth factors in skin healing

1. IN VITRO STUDIES

Knowledge of GF and their function is far from complete. Many of the known functions
were learned through in vitro study. Although many GFs are associated with wound
healing, PDGF and TGF-β1 appear to be two of the more integral modulators (110).
PDGF has activity in early wound healing (during the acid tide). In vitro studies have
shown that at lower pH (5.0), platelet concentrate lysate has increased concentrations
of PDGF, with an increased capacity to stimulate fibroblast proliferation (73). TGF-β
increases the production of collagen from fibroblasts (110)its release in vitro is
enhanced by neutral or alkaline pH, which correspond to the later phases of healing
(77).

Through modulation of interleukin-1 production by macrophages, PRP may inhibit

excessive early inflammation that could lead to dense scar tissue formation (111).
Insulin-like GF-I (IGF-1) has also been extensively studied for its ability to induce
proliferation, differentiation, and hypertrophy of multiple cell lines. Separate analyses of
GF in PRP have shown significant increases in PDGF, VEGF, TGF-β1, and EGF, compared
with their concentrations in whole blood (63,112)

47
IGF-1 has two important functions: chemotaxis for vascular endothelial cells into the
wound which results in angiogenesis and promoting differentiation of several cell lines
including chondroblasts, myoblasts, osteoblasts, and hematopoietic cells .(113)

TGF-β is a member of the newest family of proteins discovered. Two major sources of
this protein are the platelet and macrophage. TGF-β causes chemotactic attraction and
activation of monocytes, macrophages, and fibroblasts. The activated fibroblasts
enhance the formation of extracellular matrix and collagen and also stimulate the cells
ability to contract the provisional wound matrix (114). Macrophages infiltration
promotes TGF-β that induces extracellular matrix such as collagen and fibronectin;
however alpha-mangostin prevents the increase in this molecule in rats with Cisplatin-
induced nephrotoxicity (115).

2. IN VIVO STUDIES

In vivo study is much more complex due to the inability to control the environment. A
further complexing matter is the fact that the same GF, depending on the presence or
absence of other peptides, may display either stimulatory or inhibitory activity within
the same cell. Also, a particular GF can alter the binding affinity of another GF receptor
(116).

48
Release of PDGF can have a chemotactic effect on monocytes, neutrophils, fibroblasts,
stem cells, and osteoblasts. This peptide is a potent mitogen for mesenchymal cells
including fibroblasts, smooth muscle cells and glial cells (117) and is involved in all three
phases of wound healing, including angiogenesis, formation of fibrous tissue, and
reepithelialization.

TGF beta released from platelet alpha granules is a mitogen for fibroblasts, smooth
muscle cells, and osteoblasts. In addition, it promotes angiogenesis and extracellular
matrix production [59]. VEGF promotes angiogenesis and can promote healing of
chronic wounds and aid in endochondral ossification. EGF, another platelet-contained
GF, is a mitogen for fibroblasts, endothelial cells, and keratinocytes and also is useful in
healing chronic wounds (118).

IGF, another platelet-contained GF regulates bone maintenance and is also an important

modulator of cell apoptosis, and, in combination with PDGF, can promote bone
regeneration (119).

However, there are conflicting results with regard to IGF-1, where the majority of
studies reported no increase in IGF-1 in PRP, compared with whole blood. There are also
conflicting results regarding the correlation between the GF content and platelet counts
in PRP (120).The basis of these contradictions is not fully understood and may be related
to variability in patient age, health status, or platelet count.

49
Alternatively, differences in GF content and platelet count may be due to the various
methods of processing, handling, and storing of samples, in addition to the type of assay
performed. The diversity of PRP products should be taken into account when
interpreting and comparing results and methods for generating PRP (77).

VEGF, discovered 25 years ago, was initially referred to as vascular permeability factor
(121). In mammals, there are at least four members of the VEGF family: VEGF-A, VEGF-
B, and the VEGF-C/VEGF-D pair, which has a common receptor, VEGF receptor 3 (VEGF-
R3) (122). VEGF-A is a proangiogenic cytokine during embryogenesis and contributes to
vascular integrity: selective knockout of VEGF-A in endothelial cells increases apoptosis,
which compromises the integrity of the junctions between endothelial cells (123,124)
VEGF-B, which can form heterodimers with VEGF-A, occurs predominantly in brown fat,
myocardium, and skeletal muscle (125).VEGF-C and VEGF-D seem to regulate
lymphangiogenesis.

The expression of VEGF-R3 in adults is restricted to the lymphatics and fenestrated

endothelium (126). Neuropilin 1 and neuropilin 2 are receptors that bind specific VEGF
family members and are important in neuronal development and embryonic
vasculogenesis (127).

50
Megakaryocytes and platelets contain the three major isoforms of VEGF-A; after
exposure to thrombin in vitro, they release VEGF-A (128-131) VEGF-A alters the
endothelial-cell phenotype by markedly increasing vascular permeability, upregulating
expression of urokinase, tissue plasminogen activator, connexin, osteopontin, and the
vascular-cell adhesion molecule (132).

10. PRP in Cell Therapy and Regenerative Medicine.

PRP can be combined with cell-based therapies such as adipose-derived stem cells,
regenerative cell therapy, and transfer factors therapy (133).While this is a relatively
new concept, the strategy is appealing as the regenerative matrix graft delivers a potent
trilogy of regenerative cells, fibrin matrix, and GF (63) The applications are similar to
those for PRP alone with the added benefit of regenerative cell enrichment.

Verrier et al., demonstrated that cultures of human mesenchymal stem cell (MSC)
supplemented with platelet-released supernatant (PRS) had differentiation towards an
osteoblastic phenotype in vitro possibly mediated by bone morphogenetic protein-2
(BMP-2). PRS showed an osteoinductive effect on MSC, as shown by an increased
expression of typical osteoblastic marker genes such as collagen I, bone sialoprotein II,
BMP-2, and matrix metalloproteinase-13 (MMP-13), as well as by increased Ca++
incorporation (134)

51
Furthermore, the role of platelets in hemostasis may be influenced by alteration of the
platelet redox state, the presence of endogenous or exogenous antioxidants, and the
formation of reactive oxygen and nitrogen species (135). As discussed by Sobotková et
al., (135), trolox and resveratrol inhibit aggregation of washed platelets and PRP
activated by ADP, collagen, and thrombin receptor-activating peptide.

Antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit

platelet activation also by specific interaction with target proteins. In this context,
powerful natural antioxidants, like nordihydroguaiaretic acid (NDGA) extracted from
Larreatridentata (136), S-allylcysteine (SAC), the most abundant organosulfur compound
in aged garlic extract (AG) (137), sulforaphane (SFN), an isothiocyanate produced by the
enzymatic action of myrosinase on glucoraphanin, a glucosinolate contained in
cruciferous vegetables (138), and acetonic and methanolic extracts of
Heterothecainuloides, can be administered with ample safety margin in patients treated
with PRP (139).

52
11. Methods to Improve Patient Satisfaction.

The evaluation is essentially subjective. 95% of patients are satisfied with the
improvements in the appearance, texture, softness, touch, tone, brightness, etc. The
results can be objectified to measure and compare the parameters, such as
corneometry, hygrometry or sebometria.

PRP can be combined with other skin rejuvenating procedures such as: mesotherapy,
microneedling, fillers, botulinum toxin injections, various peeling, fractionated laser
therapy and radiofrequency biostimulation. (140)

12. Complications of PRP.

After the procedure there will be some pain, mild swelling or redness, of the skin
following injection of the platelet rich plasma - PRP. Bruising is also a possibility as the
needle is used to place the PRP into the skin. This may take a week or so to resolve.
There can be side effects. Here are the most common: (141)

Pain in the Injured Area: Some people who’ve undergone PRP therapy complain about
an acute ache or soreness in the spot of the injection. Sometimes this pain is even felt
deep inside the area, whether in the muscle or bone.

53
Infection: As with any injection-based treatment, infection is a slight possibility
regardless of thorough sterilization procedures.While a tremendous amount of
precaution is taken when injecting a patient with a PRP serum–intense sterilization
procedures are, in fact, followed closely for each treatment–sometimes an infection can
break out in the injured area.

No Improvement in Injured Area: While this is not necessarily a side-effect, we still

need to mention that not all athletes respond to a PRP injection. (Of course, this
particular type of sports medicine is undergoing more studies so we can understand
exactly why. Sometimes the original pain and soreness of the injury remains (it may
even get worse), even after an extended rest period after the PRP therapy.

Allergic Reaction: Some patients’ body will reject their own serum and react negatively
to the treatment. This is rare, but it does happen. Again, more studies need to be done
to understand why.

Blood Clot: Normally, a blood clot forms when there is damage to the lining of a blood
vessel, like with a cut. Because a PRP injection uses a needle (guided by a sonogram)
there is a chance that a artery or vein could be damaged. If that happens a blood clot
occurs and is treated like any normal clot.

Skin Discoloration: Sometimes the color around the skin of a PRP injection will appear
bruised. This could be normal, based upon your history of bruising.

54
13. CONTRAINDICATIONS TO PRP

Treatment with autologous PRP is generally considered safe in appropriately selected

patients. Potential candidates for treatment with PRP should undergo a pre-treatment
hematologic evaluation to rule out potential coagulopathies and disorders of platelet
function. (142)

Patients who are anemic and those with thrombocytopenia may be unsuitable
candidates for treatment whit PRP.

Other potential contraindications include hemodynamic instability, severe hypovolemia,

unstable angina, sepsis, and anticoagulant or fibrinolytic drug therapy. (143)

Absolute Contraindications:

• Platelet dysfunction syndrome

• Critical thrombocytopenia
• Hemodynamic instability
• Septicemia
• Local infection at the site of the procedure
• Patient unwilling to accept risks

55
Relative Contraindications:

• Consistent use of NSAIDs within 48 hours of procedure

• Corticosteroid injection at treatment site within 1 month
• Systemic use of coriticosteroids within 2 weeks
• Tobacco use
• Recent fever or illness
• Cancer- especially hematopoetic or of bone
• HGB < 10 g/dl
• Platelet count < 105/ul

14. Advantages, Limitations and Precautions.

PRP as autologous procedure eliminates secondary effects and unnecessary risks for
chemically processed strange molecules, is a natural reserve of various growth factors
that can be collected autologously, and is cost effective (144,145)

Thus for clinical use, no special considerations concerning antibody formation and
infection risk are needed. The key of our health and our own regeneration resides in our
own body. Nevertheless this treatment is not the panacea; it is only the beginning in this
new age of the regenerative medicine. Some clinical devices automatically prepare PRPs
are available at present.

56
15. Discussion.

The problem of skin ageing has captured public attention and has an important social
impact. Different therapeutic approaches have been developed to treat cutaneous
ageing and to diminish or prevent the negative effects of UVR.

The primary factor that causes human skin aging is its direct contact with the
environment; therefore, skin aging can be considered as a consequence of
environmental damage. The most important environmental factor involved in human
skin aging is ultraviolet (UV) irradiation from the sun. This sun-induced skin aging
(photo-aging) depends primarily on the degree of sun exposure and skin pigment.
Individuals who have outdoor lifestyles and those who are lightly pigmented will
experience the greatest degree of photo-aging (127).

The deleterious effects of solar radiation on the skin impact primarily on the dermal
connective tissue with accelerated breakdown and synthesis of collagen, and enhanced
inflammatory processes. These processes are directly responsible for the clinical
appearance of photo-aged skin: wrinkling, telangiectasia, laxity, pigment changes,
coarseness, dehydration, and loss of tensile strength (128)

Intradermal injection of growth factors produced remarkable clinical changes on aging

skin: restores skin vitality, increases the thickness of the skin, restores elastic consistency,
improves vascular flow, stimulates secretions and improves the smoothness and
appearance of the skin .The Growth factors regulate the remodeling of the epidermis and
dermis, and have a great influence on the appearance and texture of the skin.

57
The PRP treatments can be used on all skin types and tones. Minimal swelling, bruising,
and redness for the initial 12 to 24 hours are expected. A bruise at the needlestick site
may be visible for 2 to 3 days up to a week. Swelling from the fluid is what
the patient will notice first. During several weeks, the platelets stimulate growth factors,
which assist in more collagen stimulation. Treatment results vary but last up to 18
months in most patients. Biannual touch-up treatments will maintain the results.
As an initial treatment strategy, up to 4 sessions may be given. These are usually
performed 2 to 4 weeks apart.

PRP is effective particularly for fine wrinkles under the eyes that are difficult to treat by
conventional rejuvenation therapy. It is also effective for treating periorbital dark
circles, wrinkles on the face and neck, acne scars, saggy skin, eye bags, and nasolabial
folds.

58
16. CONCLUSION

Compared with other skin rejuvenation therapies, the clinical experience using Platelet
Rich Plasma (PRP) has demonstrated it to be a useful primary or adjunctive therapy for
the tissue rejuvenation.

Both superficial and deep dermal applications can result in skin rejuvenation and global
facial volumisation. PRP is a form of biostimulation that is safe and creates an
immediate, long lasting volumetric effect with natural looking results. The technique is
easy to perform and has virtually no side-effects.

Platelet rich plasma (prp) skin rejuvenation therapy utilizes the potential of your own
body to improve the overall texture and tone of your skin, naturally. It reduces wrinkles
and blemishes such as acne scars and may be used as part of your health and beauty
regimes to improve the overall condition of the skin, improving tone and texture.

Platelet Rich Plasma Skin Rejuvenation Therapy is a natural approach to improving the
condition of your skin, and you are using your own unique tissue growth factors and
healing properties to revitalize the skin by improving blood supply and stimulating
collagen formation, the results of which improves and increases over time with evidence
suggesting that the results of PRP Skin Rejuvenation Therapy lasts longer than chemical
treatments. PRP Skin Rejuvenation Therapy is specifically designed for you, by you!

59
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