Original Contribution

Journal of Cosmetic Dermatology, 0, 1--8

Platelet-rich plasma and hyaluronic acid – an efficient

biostimulation method for face rejuvenation
Betul Gozel Ulusal, MD
Department of Plastic Reconstructive and Aesthetic Surgery, Balikesir University, Balikesir, Turkey

Summary Background Cosmetic applications of platelet-rich plasma (PRP) are new, and reports
are scarce and dispersed in the literature. There are a variety of commercially
available kits and injection techniques, and the number and intervals of injections
vary. New investigations should focus on developing a standardized procedure for
PRP preparation and application methods to augment its efficacy and potency.
Objectives In this report, we aim to provide data and commentary to assist and add to
current guidelines.
Methods A series of 94 female patients with varying degrees of facial aging signs were
treated with PRP and hyaluronic acid (HA). Mean age was 53.0  5.6. The mean
injection number was 3.6  2.0. Platelet-poor and platelet- rich plasma parts were
mixed with 0.5 cc %3.5 hyaluronic acid and 0.5 cc procaine and injected with a
30G, 13-mm needle into deep dermis and hypodermis. Patients were asked to rate
their personal satisfaction with their skin texture, pigmentation, and sagging. In
addition, the overall results were rated by three independent physicians and the
patients themselves. The outcomes were peer-reviewed, and correlations between the
degree of the aesthetic scores and the number of injections were explored.
Results There was a statistically significant difference in general appearance, skin
firmness-sagging and skin texture according to the patients’ before and after
applications of PRP. A statistically significant correlation was found between the
number of injections and overall satisfaction.
Conclusions Compared to the baseline, the PRP and HA injections provided clinically
visible and statistically significant improvement on facial skin. The improvements
were more remarkable as the injection numbers increased.
Keywords: platelet-rich plasma (PRP) injection number, hyaluronic acid, facial
rejuvation, injection, biostimulation, collagen

altering their natural expression. Therefore, less invasive,

Introduction
affordable and effective nonsurgical tools are preferred.
The skin is the most visible indicator of age, which is Aging is accompanied by a decline in the homeo-
often psychologically distressing. Many patients want to static and regenerative capacity of all tissues and
revitalize their skin to gain a more youthful look without organs.1,2 Biostimulation for new collagen, elastin,
extrafibrillar matrix, and vascular network formation
Correspondence: Betul Gozel Ulusal, Associate Professor, Department of has the potential to restructure the skin and reverse
ß agis
Plastic Reconstructive and Aesthetic Surgery, Balikesir University, C the signs of aging.
Yerleskesi, Balikesir, Turkey. E-mail: [email protected] Regenerative medicine refers to research and treat-
Accepted for publication August 8, 2016 ments that restore damaged tissues. It consists of

© 2016 Wiley Periodicals, Inc. 1

PRP and hyaluronic acid for face rejuvenation . B G Ulusal

three factors: cells, chemical substances and scaffolds. The mean number of injections was 3.6  2.0
Platelet-rich plasma is a rich source of growth factors (median: 3.0, range 1–8).
and provides a scaffold to support true tissue regener- The same preparation technique was used, and all
ation. Therefore, PRP is included within the field of applications were performed by the same physician
regenerative medicine. Precursor cell migration, prolif- (BGU).
eration and differentiation are intended biological Written informed consent was obtained before the
effects theoretically related to the PRP clinical procedure. Anticoagulants were stopped 1 week earlier
response.3–6 to prevent the formation of excessive bruising. Treat-
Loss of viscoelasticity is one of the primary signs of ment was avoided in pregnant and breastfeeding moth-
skin aging, followed by the appearance of visible wrin- ers. Autoimmune diseases such as Hashimato’s
kles. A key molecule involved in maintaining skin thyroiditis and metabolic disorders such as diabetes
hydration is hyaluronic acid (HA).7 The HA concentra- mellitus were not contraindications.
tion in the skin is determined by the complex balance About 12.5 cc of patients’ own blood was collected
between the skin’s synthesis, deposition, association in a syringe with 1.5 cc of 10% Na citrate. No activa-
with7 cellular structures and degradation. tor was used. Then, whole blood was instilled to a PRP
Recent basic research supports the idea that HA and kit (Dr B PRPTM) and centrifuged at 1800 rpm for 20–
PRP treatments can be advantageously associated 50 min until all red blood cells separate from plasma
without altering the original relevant characteristics of and the buffy coat became clearly visible. If the inten-
both products. When both products are injected, their tion was to treat the whole facial and neck region, the
effects might be additive to enhance the anabolic func- plasma and the buffy coat (platelet-rich and platelet-poor
tion of dermal fibroblasts.8,9 plasma) were collected in 5 cc syringes and infiltrated
However, the clinical outcomes of PRP and HA along the wrinkles with a 30G and 0.3 9 13 mm nee-
co-injection on aging facial skin have not been eluci- dle. Platelet-rich plasma portion was separately collected
dated for human subjects. if a specific local region was intended to be treated
The treatment protocol, including the number and (such as dark under-eye circles). In all cases, PRP was
intervals of injection and application methods, varies. mixed with 0.5 cc %3.5 hyaluronic acid gel and
In most clinical reports, up to three injections were 0.5 cc procaine. To reduce pain, local anesthetic cream
performed for cosmetic purposes.10 The number of and ice were applied before the procedure. Then, small
patients in these reports was limited, and the available aliquots of plasma were infiltrated 5–6 mm deep into
guidelines are arbitrary. Growth factors are not muta- the dermis and hypodermis through hundreds of
genic and whether additional injections have the micro-needle holes with a 30 G, 13-mm needle
potential to provide superior cosmetic results is not (Fig. 1). At the end of the treatment, ice and antibiotic
known yet. cream were applied locally and patients were asked to
In this article, the objective was to propose new avoid any contact with their face and neck for 3–4 h.
guidelines for optimal doses, treatment intervals, num- Vitamin C (1000 mg/day) and sun protection were
ber of injections and application method for PRP. We recommended.
also aim to analyse the effect of PRP and HA therapy The treatment was staged as “intense” and “mainte-
for face rejuvenation. nance” periods. In the intense treatment period, con-
To the best of our knowledge, this article presents secutive three treatments were performed 3–4 weeks
the largest series of patients who underwent PRP appli- apart. Patients were advised to receive at least three
cation with the intention of rejuvenating the face. treatments for more visible results. In the maintenance
Using the same kit and preparation methods, all appli- period, patients received up to five sessions at eight- to
cations were performed by the same physician (BGU). ten-week intervals.
The study data have the potential to clarify conflicting All of the treated patients were reached by phone
data in the literature and lead to updates in the appli- and asked to rate their satisfaction on a scale ranging
cation methods. from 0 to 3 (0 = no improvement, 1 = slight improve-
ment, 2 = moderate improvement, and 3 = good
improvement) for overall improvement and individual
Methods
satisfaction with skin texture, skin sagging/firmness
Between December 2012 and November 2015, 94 and skin pigmentation. In addition using the same rat-
female patients were treated for facial rejuvenation ing scale, three independent physicians rated the over-
using PRP and HA. The mean age was 53.0  5.6. all aesthetic improvement by assessing before and after

2 © 2016 Wiley Periodicals, Inc.

 PRP and hyaluronic acid for face rejuvenation . B G Ulusal

scores for improvement in skin texture, pigmentation,

and sagging given by the patients and the mean over-
all improvement scores given by patients and physi-
cians are shown in Table 1.
Significant improvement was noted regarding the
general appearance, skin sagging/firmness, and skin
texture according to the grading scale of the patients
and physicians before and after the applications of
PRP. There were no statistically significant differences
between the overall aesthetic improvement score given
by patients (median: 2.0) and physicians (median: 2.0)
(P < 0.05 Mann–Whitney U-test).
The amount of aesthetic improvement and satisfac-
tion was directly correlated with the number of injec-
tions of PRP (Figs 2–4). However, no direct correlation
was found between the number of applications and the
mean score for skin pigmentation improvement. Pig-
mentation responded more favorably and earlier in
fair-skinned individuals.
A statistically significant increase in skin moisture,
minimization of skin pores and shiny and glowing skin
was the initial findings (Fig. 5), and rapid active acne
regression was noticeable in some cases (Fig. 6).
After the second and third sessions, gradual improve-
Figure 1 Method of injection with a 30G and 13-mm needle. ment in fine wrinkles and skin tone, especially at the
The depth of the injection is 5–6 mm, into the deep dermis and periorbital skin, was observed. Lower eyelid-cheek con-
hypodermis. tour was improved. In addition, some regression of
under-eye bags and sags was observed (Fig. 7). At later
digital photographs. On the assessments, five-grade sessions, due to the thickening and hydration of the
wrinkle severity rating scale (WSRS) was used; 0 = no dermis, dry and parchment-like skin gradually recov-
visible wrinkling 1 = mild wrinkling, 2 = moderate ered, and skin telengiectasis became less visible. (Figs 7
wrinkling, 3 = severe wrinkling, and 4 = extreme and 8). After six or more injections, the wrinkles had
wrinkling. mostly dissappeared and facial fullness was restored
For statistical analysis, GraphPad Prism 5.0 software (Fig. 9). The sagging cheek skin creating the jowls was
(San Diego, CA, USA) was used with a P value of improved to an extent, and sagging neck skin was
<0.05 considered statistically significant. tightened up moderately (Figs 4 and 9). However, the
total improvement in the upper lip deep wrinkles, jowls
and deep nasolabial folds was less visible even in
Results
patients who underwent six or more sessions. These
No significant or persistent side effects were observed. patients required additional interventions such as face
Transient mild facial edema was common, and about lift, filler, dermabrasion, or focused ultrasound for these
8% of the patients reported ecchymosis that lasted areas.
more than 10 days. Bruising was less common in
patients who smoked. No patients were found to have
Discussion
any evidence of inflammation or allergic reaction.
The most common patient complaints before the Aged skin is characterized by epidermal and dermal
treatment included the development of wrinkles, der- changes. The first stage is the degradation of the
mal atrophy and sagging facial skin at the periorbital ECM.11 The loss of skin elasticity is induced by a
region, jowls, blotchy dyspigmentation, and uneven breakdown of fibers such as collagen, elastin, or retic-
skin texture. ulin, whereas the degradation of proteoglycans results
The number of PRP injections, the mean WSRS and in decreased turgor and skin hydration.11–14 PRP has
the number of the patients in each group, the mean been reported to augment dermal elasticity by

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PRP and hyaluronic acid for face rejuvenation . B G Ulusal

Table 1 The number of injections, number of patients, mean WSRS, and improvement scores given by patients and physicians

Number of Improvement in Improvement in

Number of patients WSRS Improvement in sagging = firmness skin pigmentation Overall improvement Overall improvement
PRP injections (n = 94) (Mean) skin texture (MS) (MS) (MS) (patients’) (MS) (physicians’) (MS)

1 (n = 11) 1.8 1.83 0.66 0.54 1.66 0.50

2 (n = 19) 2.1 1.31 0.89 1.00 1.26 2.00
3 (n = 30) 1.8 1.96 1.60 2.90 1.80 1.73
4 (n = 5) 1.6 2.20 2.00 1.60 2.00 1.60
5 (n = 12) 2.0 2.91 2.50 1.91 2.50 2.16
6 (n = 7) 2.0 2.71 2.28 2.42 2.28 2.28
7 (n = 2) 3.5 3.0 3.00 2.00 3.00 2.50
8 (n = 8) 2.2 3.0 2.87 2.20 3.00 2.75

MS, mean score.

Overall Improvement Score: 0 = no improvement, 1 = slight improvement, 2 = moderate improvement, and 3 = good improvement.
WSRS: 0 = no visible wrinkling 1 = mild wrinkling, 2 = moderate wrinkling, 3 = severe wrinkling, and 4 = extreme wrinkling.

and PRP treatments may have additive effects. It is

possible that HA added to PRP provided an ideal vis-
cous and hydrated environment for growth factor
functions.
This study revealed that number of injections has a
direct effect on clinical outcome. Restoring the facial
contours and tightening up the sagging skin of the
jowls and neck is one of the most difficult issues with-
out surgery. However, we showed that it was possible
to an extent with six or more injections. PRP injections
Figure 2 The amount of aesthetic improvement and satisfaction
can be performed many times until the targeted cos-
was directly correlated with the number of injections of PRP.
metic results are obtained because growth factors are
not “mutagenic”; they act through normal gene regu-
stimulating the removal of photodamaged ECM compo- lation.19
nents and inducing the synthesis of new collagen by The injection intervals can be adjusted according to
dermal fibroblasts via various molecular mecha- the grade of the aging signs and patient desire. The
nisms.15,16 outcomes from this series showed after three monthly
Hyaluronic acid is one of the most hydrophilic mole- injections, eight- to ten-week maintenance intervals
cules in nature, and when not bound to other mole- worked well. The injection depth reaching the superfi-
cules, HA binds to water giving it a stiff viscous cial musculoaponeurotic system (SMAS) layer of the
quality. As a result of HA’s ability to form hydrated, face may have also contributed to these favorable
expanded matrices, HA has also been successfully used outcomes. An increase in collagen and elastin, as well
in cosmetic applications.17,18 Moreover, hyaluronic as hyaluronic acid, has the potential to enhance the
acid has been reported to be an appropriate choice for viscoelastic properties of the SMAS layer20 and this
a matrix to support dermal regeneration and augmen- may explain why the patients’ facial contours obtained
tation.18 some fullness and enhancement. However, further
Early in our practice, PRP-alone and HA-alone treat- quantitative analyses for collagen and elastin content
ments were performed for about 10 months and then changes are required to verify this assumption.21 There
we abandoned. Because the eventual cosmetic out- are various sample preparation kits and although opti-
comes were mostly unsatisfactory, variable results were mal platelet concentration is still not known, a platelet
attained. In addition, “injection pain” was a great con- concentration of more than 1 million/lL (approxi-
cern. Then combination treatments with HA and PRP mately 4–7 times the mean levels) is generally
were commenced, and local anesthetic was added. regarded as the therapeutically effective concentration
Even after the first treatment, this combination ther- of PRP22 For this study, we have analysed the plasma
apy consistently provided satisfactory results for facial we obtained from the separation kit. The white blood
rejuvenation, which supports the suggestion that HA cell (WBC) and red blood cell (RBC) counts were zero.

4 © 2016 Wiley Periodicals, Inc.

 PRP and hyaluronic acid for face rejuvenation . B G Ulusal

Figure 3 (FRONTAL VIEW) Facial rejuvenation was more obvious with additional (more than 3) PRP +HA injections. Appearance of a
53-year-old patient before the onset of treatment (a) Appearance of the same patient after three PRP+HA injections (b). The facial skin
texture seems healthier. The pigmentation and fine wrinkles were improved. Appearance of the same patient after eight PRP+HA injec-
tions (19 months after the onset of the treatment) (c). Sagging facial skin at the periorbital and cheek region was further improved. The
nasolabial fold was better although no tissue filler was used. Dermabrasion was performed on to the upper lip because the response to
the treatment was poor.

Figure 4 (LATERAL VIEW)The same patient after eight PRP+HA injections. Tightening of the sagging cheek and neck skin is more
prominent.

The platelet concentration was 5–6 times higher hemorrhages and activate wound healing mechanisms
(1389 000/lL) compared to the baseline levels without damaging the epidermis. Post-traumatic regen-
(244 000/lL) (Fig. 10) However, the centrifugation eration of the skin forms a thicker epidermis with the
times varied between 20 and 50 min, and how these formation of new subepidermal collagen and elastin
periods affect platelet cell integrity and concentrations network. Needling is known to have a distinct poten-
require further investigation. tial to induce collagen and elastin formation apart
We injected platelet-poor plasma part along with from growth factor instillation.23,24
PRP with intention to inject more cells, further hydrate After the injection, we advised the patient to take
the skin, and to induce percutaneous collagen induc- 1000 mg/day vitamin C. This vitamin is essential for
tion by needling. Fine pricks create intradermal the formation of stable collagen I and III fibrils and

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PRP and hyaluronic acid for face rejuvenation . B G Ulusal

Figure 5 Significant increase in skin moisture, improved hydration and shiny skin which became evident within the first few months
after the PRP+HA application.

Figure 6 Rapid active acne regression was noticeable in some cases.

Figure 7 Due to the thickening and hydration of the dermis, dry and parchment-like sagging skin gradually recovered, and telengiecta-
sis became less visible.

thus has a supportive function in building up a duration of the botox. To the best of our knowledge,
healthy collagen–elastin matrix.25 no other method produces this effect. However, we
One interesting note is that this patient series, PRP cannot suggest an explanation for this with existing
and HA injection after botox application shortened the data.

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 PRP and hyaluronic acid for face rejuvenation . B G Ulusal

Figure 8 A change in the quality and texture of the skin which became evident after five sessions of PRP +HA injections.

Figure 9 Facial fullness was restored in this patient, and improvement in sagging was significant after seven sessions of PRP+HA
injections.

Figure 10 Platelet concentration was 5–6 times higher (1389 000/lL) compared to the baseline levels (244 000/lL) in the platelet
separation kit used in this series of patients.

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PRP and hyaluronic acid for face rejuvenation . B G Ulusal

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14 Harvell JD, Maibach HI. Percutaneous absorption and
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15 Cho JW, Kim SA, Lee KS. Platelet-rich plasma induces
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