Autologous platelet concentrates for facial rejuvenation

Abstract Autologous platelet concentrates (APCs) are promising therapeutic agents in facial rejuvenation since they are a great source of cytokines, growth factors and other biologically active substances. Obtained from the patient’s blood, they have the advantages of reducing immunological reactions, making the procedure safer, well tolerated, with minimal adverse effects and lower cost. Currently, they are used for facial rejuvenation both in combination with microneedling and in mesotherapy techniques, as well as to treat facial acne scars, melasma and wounds after laser ablative treatments. This review summarizes current knowledge on the use of APCs, ranging from basic concepts related to their composition and mechanisms of action to up-to-date information on their clinical efficacy. Methodology MEDLINE (PubMed) was searched from inception through 2021 for English language publications on APCs for facial rejuvenation. Results A total of 100 files were found. Based on the available literature, APCs for skin rejuvenation are safe and well tolerated. The most studied product is the first-generation material, platelet-rich plasma (PRP). Conclusions The results are in general favorable, but the quality of the studies is low. The second and third generation products, platelet-rich fibrin (PRF) and injectable platelet-rich fibrin (i-PRF), respectively, are easier to be obtained and, at least in vitro , seem to induce greater collagen production than PRP, especially under lower relative centrifugation forces, but to date only a few clinical trials evaluating these products exist. More high-quality trials with appropriate follow-up are necessary to provide adequate evidence that may help to improve the treatment regimens with APCs. Many aspects should be considered when designing clinical trials to evaluate APCs, such as the patients’ characteristics that best predict a favorable response, the optimal number of sessions and the interval between them, the characteristics of the studies and the development of better instruments to evaluate skin aging.

rejuvenation, ranging from basic concepts related to their composition and mechanisms of action to upto-date information on their clinical efficacy.
Methodology MEDLINE (PubMed) was searched on August 25, 2021 for English language publications on autologous platelet concentrates for facial rejuvenation. Search terms were: [facial rejuvenation AND (platelet rich plasma OR platelet rich fibrin OR injectable platelet rich fibrin OR iPRF OR PRF OR PRP)]. A total of 100 files were found. Titles, abstracts and full-texts were independently screened by two reviewers (MB and FM). One file was excluded because it was an editorial. Other 28 files were articles unrelated to facial rejuvenation and were also excluded.

Results and discussion
Composition and mechanisms of action of autologous platelet concentrates To understand the mechanism of action of platelet concentrates in facial rejuvenation, it is necessary to know the platelets. These cells are cytoplasmic fragments of megakaryocytes, formed in the bone marrow, approximately 2 µm in diameter. Platelets contain, in their α granules, protein growth factors with a capital role in hemostasis and wound healing: CTGF (conjunctive tissue growing factor), EGF (epidermal growing factor), FGF-2 and -9 (fibroblast growing factor), IGF-1 (insulin growing factor), PDGF αα (platelet-derived growing factor), PDGF αβ, PDGF ββ, TGF α (transforming growing factor), TGF β1, TGF β2 and VEGF (vascular endothelial growing factor). After platelet exogenous or endogenous activation, these α granules fuse with the cell membrane, in a process called degranulation (Figure 2). These growing factors are then secreted, bind to transmembrane receptors on target cells (undifferentiated mesenchymal cells, osteoblasts, fibroblasts, endothelial cells and epidermal cells), activating an intracellular signaling protein that causes the expression of a protein, which, in turn, triggers effects such as cell proliferation, angiogenesis, synthesis of collagen and extracellular matrix components, and reduced apoptosis. 6,[16][17][18][19] Active secretion of these growth factors by platelets begins 10 minutes after activation, with more than 95% of pre-synthesized growth factors being secreted within 1 hour. 20 With skin aging, fragmented collagen fibrils accumulate, which impairs the growth of new After platelets activation, their α granules fuse with the cell membrane, in a process called degranulation. Their growth factors are then secreted, bind to transmembrane receptors on target cells (mesenchymal stem cells, osteoblasts, fibroblasts, endothelial and epidermal cells), activating an intracellular signaling protein that causes the expression of a protein, which, in turn, triggers effects such as cell proliferation, angiogenesis, synthesis of collagen and extracellular matrix components, and reduced apoptosis. With skin aging, fragmented collagen fibrils accumulate, which impairs the growth of new collagen fibers and disrupts the extracellular matrix. Activated platelet aggregates increase the expression of matrix metalloproteases , stimulating the removal of fragments of collagen fibrils. In addition, they contain several growth factors that stimulate fibroblasts to synthesize new, more organized collagen fibers, besides increasing the synthesis of hyaluronic acid, which binds to water, increasing the skin volume and hydration J Appl Oral Sci. 2022;30:e20220020 4/13 collagen fibers and disrupts the extracellular matrix. 21 Activated platelet aggregates increase the expression of matrix metalloproteases (MMP-1 and -3), stimulating the removal of fragments of collagen fibrils. In addition, they contain several growth factors that stimulate fibroblasts to synthesize new, more organized collagen fibers, 22,23 besides increasing the synthesis of hyaluronic acid, which binds to water, increasing the skin volume and hydration 24 (Figure 2). First generation autologous platelet concentrates: PRP PRP is an autologous plasma preparation with high concentrations of platelets derived from whole blood, 16 containing more than 800 bioactive molecules. 25,26 The normal concentration of platelets in the blood ranges from 150,000 to 450,000/µL. PRP, by definition, should contain more than 1,000,000 platelets/µL to promote increased tissue healing. 20 PRP preparations generally have a 4-to 8-fold higher platelet concentration than peripheral blood. 27 A linear relationship between the concentrations of growth factors and platelets in PRP has been reported. 28 Although there is still no consensus on the most effective PRP preparation, platelet concentrations higher than 6-fold those of peripheral blood may inhibit healing. 29 At last instance, the regenerative effect of PRP depends not only on its platelet concentration, but also on the number/type of leukocytes entrapped in the fibrin matrix, and the release of bioactive molecules at the site of injury. 30 PRP contains leukocytes, with catabolic and proinflammatory activity, in combination with plasma and growth factors, with anabolic function. These constituents must be in balance so that there is adequate tissue healing and growth, for which the PRP preparation process is fundamental. The two main methods of preparation are the "PRP method" and the "buffy coat" method. 6 The latter typically produces PRP with higher platelet concentrations. 31 There are several commercial kits for preparation of PRP. The composition of the PRP obtained from the different commercial kits varies remarkably. The purpose of PRP preparation methods is to concentrate platelets and to reduce red blood cells. However, the leukocyte levels cannot be neglected. Typically, the kits that employ the "buffy coat "method produce a concentrate containing higher amounts of platelets and red blood cells, but the content of leukocytes is also increased. 31 Variation in the content of cells and growth factors also depends on the RCF and time of centrifugation employed. Longer and more forceful centrifugation cycles may push platelets down, discharge growth factors and disrupt cellular integrity. 32 Typically, the bottom layer of red blood cells (RBCs) is discarded, but variable proportions of plasma and buffy coat lead to distinct platelet preparations. These preparations were classified according to the inclusion of the buffy coat (presence of leucocytes) and the use of anticoagulants (formation of fibrin matrix) into 4 categories: 1pure platelet-rich plasma (P-PRP); 2 -leucocyte-rich platelet-rich plasma (L-PRP); 3 -pure platelet-rich fibrin (P-PRF); and 4-leucocyte-rich platelet-rich fibrin (L-PRF) . 33 The last two categories are activated fibrin-based matrices, not a liquid platelet suspension.
They are called "second generation" PRP and will be discussed later. Figure 3 shows the main findings of laboratorial studies evaluating different preparations of autologous platelet aggregates.
It is important to avoid contamination with erythrocytes when collecting PRP, as they contain reactive oxygen species, which produce unwanted inflammatory reactions at the site, probably resulting in pain and edema for the patient. 34 There has been some discussion in the literature about whether the efficacy of the PRP is affected by the inclusion of leucocytes. Despite they might act as antimicrobial agents, 35 they may also release catabolic cytokines, leading to inflammation and fibrosis, which is more pronounced in the case of neutrophils. 36 When PRP is employed in soft tissues, there is no need of exogenous activation (with CaCl 2 or thrombin), since collagen is a natural activator of PRP. When PRP is activated, fibrinogen is transformed in fibrin, creating a fibrin membrane or cloth. 37 Interestingly, the pH of the platelet concentrate influences its regenerative potential. Preincubation of lysed platelet concentrate at close to pH 5.0 increases its content of available PDGF and its capacity to stimulate fibroblast proliferation. On the other hand, incubation at pH 7.1 increases TGF-β production, which increases collagen production. 38 However, the applicability of this concept to facial rejuvenation has not been evaluated so far.
The clinical efficacy of PRP depends on the release of bioactive molecules. Therefore, the composition  PRP increased the expression of G1 cell cycle regulators, type I collagen and MMP-1, accelerating the wound healing process.
Cho, Kim and Lee 22 (2012) PRF Influence of the RCF on leukocytes, platelets and growth factor release within fluid PRF matrices.
Reducing RCF according with protocol-II (177 g) led to significantly higher platelets and leukocytes numbers compared to protocol I (710 g). Protocol-III (44 g) significantly increased platelets and leukocytes compared to II and I. Protocol II produced significantly higher levels of of TGF-β1 and VEGF compared to -I.

PRF matrices (PRF, A-PRF and A-PRF+)
Growth factor release was measured over 10 days using ELISA for PRF matrices prepared using different relative centrifugation forces (RCF) and centrifugation times.
There was a higher release of growth factors from A-PRF+ when compared with the other matrices. Platelets were more homogeneously distributed within the A-PRF and A-PRF+ matrices, while in PRF they were located mainly in the lower portion.
Platelet concentrates were nontoxic to dermal skin fibroblasts; i-PRF induced greater migration and proliferation than PRP, as well as significantly higher mRNA levels of TGF-β, type 1 collagen and fibronectin.
Wang, et al. 67 (2019) PRP Effects of PRP on extracellular matrix remodeling through evaluation of human skin fibroblasts proliferation and migration, expression of human procollagen I alpha 1, elastin, MMP-1 and MMP-2, phosphorylation of c-Jun N-terminal kinase (JNK) and JNK levels.   In all studies that evaluated PRP for treatment of nasolabial folds, significant improvement was reported 42,43,46,48 , both by self-assessment or evaluation by physicians, as well as by biometric evaluation. In one of the studies, PRP was injected only once; 46 however, treatment used to be performed in two or three sessions, with a one-month interval.
Patients were followed up to six months. 48 Regarding the use of PRP for the treatment of the cheeks and malar area, eight studies were found. 43,[45][46][47][49][50][51][52] In general, the beneficial results reported were less evident than those noticed for the nasolabial folds. Hersant, et al. 50 (2021) did not find any beneficial effect of PRP when used alone, but only when employed together with hyaluronic acid (HA). The association between PRP and HA, applied in two steps (mesotherapy and dermaroller) was also proven to be efficient in the study by Hersant et al. 51 (2017). Alam, et al. 49 (2018) reported that patients and dermatologists rated PRP nominally but not significantly better than saline. Lee,et al. 52 (2019) reported that WSRS scores improved in one patient,  Both modalities yielded a significant improvement of periorbital wrinkles after the 2 nd session, with significantly better results on the plasma gel injected side; however, the improvement was not maintained for the following 3 months. Objective assessment did not show any improvement in periorbital hyperpigmentation.
Both modalities showed a statistically significant improvement in dermis thickness and cervicomental angle (measured by OCT), GAIS and patient satisfaction score.  (1 mL) Patients received monthly intradermal injections of i-PRF over 3 months. Efficacy assessed by objective skin analysis (VISIA ® ) and FACE-Q at baseline and at 3 months .
Significant improvement in skin surface spots and pores was seen at 3 months. Skin texture, wrinkles, ultraviolet spots showed numerical improvement. FACE-Q scales that measure satisfaction with appearance revealed significant improvement from baseline.
Hassan, Quinlan and Ghanem 69 (2020) Placebo-controlled split-face trial (n=30) Mid-cheek and nasolabial fold PRF matrix was injected in the midcheek and nasolabial fold on one side of the face and saline on the contralateral side. Primary outcome measure was the difference between pre-and posttreatment total VISIA ® scores at 6 and 12 weeks.
PRF matrix can improve skin quality compared to placebo, but texture was the only skin parameter that was significantly improved, which is consistent with the role of PRF matrix as a filler agent. The results seem to persist for at least 6 weeks. 4 sessions of i-PRF with 2 to 3-week intervals (Cleopatra technique). Results were assessed by photographs (initialprior to 2 nd session as well as initial-after completion of treatment) examined by blinded ratters.
Mean percentages of true answers of the blinded ratters attested the success of the treatment.

Nacopoulus and Vesala 70 (2020)
Prospective clinical trial (n=25) Perioral wrinkles After a single session of fractional CO 2 laser skin resurfacing plus intradermal injection of PRP, 5 drops of PRP mixed with "medical device" were topically applied twice a day for 12 weeks vs. hyaluronic acid .
PRP significantly improves moisture, amount of collagen fibers and skin elasticity Araco 76 (2019) Split-face RCT (n=27) Cheek rhytids of Glogau class II or greater Each participant received 3 mL of intradermal injections of PRP to one cheek and saline to the contralateral one. Primary outcomes were photoaging scores rated by 2 masked dermatologists. Secondary outcomes included participant self-assessment scores of improvement and satisfaction.
Fine and coarse texture improved with single PRP application, as evaluated by the masked participants. Participants and dermatologists rated PRP nominally but not significantly better than saline. Alam,et al. 49 (2018) Prospective singlecenter open-label (n=11)

Nasolabial and malar areas
Volunteers received 3 sessions of PurePRP (EmCyte system) at 1-month intervals, with a follow-up period after 6 months. Efficacy was assessed by clinical and biometric instrumental evaluation (Visia CR, Optical In Vivo Primos 3D Skin Device, Cutometer dual MPA 580, Minolta Chromameter CR-400, Dermascan-D ultrasound) and by selfassessment (questionnaire) A series of 3 PurePRP injections resulted in significant skin rejuvenation (decrease in brown spot counts and area, wrinkle count and volume, improve in skin firmness and redness) at 6-month follow-up as demonstrated by biometric parameters and patient self-assessment score Everts, Pinto and Girao 48 (2019) Prospective Split-face (n=24) Face 24 volunteers with photoaging were randomly divided into 3 groups according to treatment (1 session every 2 weeks for 6 sessions) performed on each side of the face (microneedling by dermarolling alone or combined with PRP or TCA 15% peeling). Photography and punch biopsies were performed before and after 3 months of treatment for clinical, histometrical and histological evaluation.
Combined treatment showed significant improvement compared to dermaroller alone. Significant increase in epidermal thickness was observed, especially in the treatment with TCA. Organized collagen bundles with newly formed collagen were observed in all three groups. Improvement of dermal structures was more evident after combination of dermaroller with PRP, which apparently is more beneficial for facial rejuvenation.
El-Domyati, Abdel-Wahab and Hossam 56 (2018) Continued on the next page   (2019) RCT (n=103 test and n=128 control patients) Forehead, cheeks and chin 103 patients with facial aging skin underwent nanofat and intradermal PRF injection (treatment group) and 128 patients underwent hyaluronic acid (HA) injection (control). Outcomes were evaluated by assessing pictures taken before and after treatment, and after 1, 12 and 24 months using the VISIA Skin Image Analyzer Facial skin texture was improved to a greater extent after nanofat + PRF treatment compared with HA. The first also and a higher satisfaction rate. Neither treatment caused complications, such as anaphylaxis, paresthesia or infection during follow-up. Nanofat-PRF injection is safe, effective and long-lasting method for skin rejuvenation. Liang,et al. 72 (2018) RCT (n=62 test and n=77 control patients) Facial soft tissue depression areas 62 test patients with soft tissue depression and signs of aging underwent combined nanofat, PRF and autologous fat structural transplantation vs. 77 controls (autologous fat transplantation).
Facial soft depressions and skin texture were improved to a greater extent after nanofat+PRF transplants, which led to overall satisfaction rate above 90%. Transplants combining nanofat, PRF and autologous structural fat granules are safe, highly effective and a long-lasting method for remodeling the facial contours and promoting rejuvenation.
Wei, et al. 73 (2017) Open label prospective study (n=31) Cheek 2 mL of PRP was mixed with 2 mL hyaluronic acid (noncross-linked, 1550 kDa). Mesotheraphy was performed at 0,1 and 2 months by injecting 4 mL of the mixture per cheek, in 2 steps. 1 st step: deep intradermal injections of PRP-HA; 2 nd step: spreading 1 mL of the mixture per cheek followed by intradermal punctures with a 1-mm SkinRoller. Outcomes were assessed before injection and at 1, 3 and 6 months (Cutometer MPA 580 and FACE-Q).
FACE-Q scores and biophysical measurements revealed significant improvement at 6 months compared with baseline.
Hersant, et al. 51 (2017) Split-face prospective study (n=20) Face Patients were randomly assigned to treatment (6 sessions at 2-weeks interval) by readymade growth factors (area A) or PRP (area B). Evaluation was made by GAIS (skin turgor and vitality) and OCT (epidermal and dermal thickness) Patients receiving growth factors had significantly higher burning sensation and lower degree of satisfaction. Improvement was more sustained in patients receiving PRP. Punch biopsy was taken from the right infra-auricular area before treatment and PRP was injected to the upper site of this area (1.5-2.0 mm deep), while saline was injected to the left infra-auricular area. 28 days after treatment, punch biopsy was performed on the PRP and saline injected sites. Mean optical densities (MODs) of collagen were measured.
In "intense" periods, consecutive treatments were performed 3-4 weeks apart. In "maintenance" periods, patients received up to 5 sessions at 8-10-week intervals. The mean number of injections was 3.6±2.0; range 1-8). Patients rated satisfaction with skin pigmentation, texture, and sagging. Overall results were rated by 3 physicians.
Significant improvement was noticed regarding skin texture, firmness/sagging and general appearance by the physicians and patients. The degree of satisfaction and improvement was directly correlated with the number of PRP injections.
Clinical efficacy was assessed by satisfaction scores, dermatologists' evaluation and VISIA analysis system After 3 months, PRP increased subjective scores of facial wrinkles, skin elasticity and texture and decreased duration of edema, crusting and erythema when compared to control. PRP combined with CO² laser had a synergistic effect on facial rejuvenation and shortened the duration of side-effects.
Hui, et al. 45 (2017) Continued on the next page Fragments of skin were removed before and 3 months after injection of fat plus PRP, fat (stromal vascular fraction -SVF) or adipose-derived stem cells and analyzed by optical and electron microscopy Fat plus PRP led to more pronounced inflammatory infiltrate and greater vascular reactivity, increase in vascular permeability. The addition of PRP did not improve the regenerative effect. The use of PRP was not advantageous for skin rejuvenation over the use of SVF-enriched fat or expanded adipose-derived stem cells. Due to increased vascular reactivity, the combination of fat plus PRP might be useful in situations when an intense angiogenesis is desirable, such as tissular ischemia Rigotti,et al. 78 (2016) Prospective openlabel (n=10) Forehead, malar, jaw and crow´s feet PRP was applied thrice at 2-weekintervals by a dermaroller and injected into the wrinkles of crow´s feet. Participants graded general appearance, wrinkle state, skin firmness-sagging and degree of pigmentation of their face before and 3 months after the last PRP procedure. 3 dermatologists also evaluated.
There was significant difference in general appearance, wrinkle state, skin firmness-sagging before and after 3 PRP applications according to the patients´ evaluations. However, according to the dermatologists´ assessment, the only significant difference perceive was in skin firmness-sagging. Yuksel,et al. 47 (2014) Retrospective study (n=82) Face Recovery time and aesthetic outcome after treatment with fat grafting only (GI), fat grafting and PRP (GII), minimal access cranial suspension (MACS)-lift and fat grafting plus PRP (GIV) (GIII) and MACS-lift, fat grafting. Assessment was made by 10 plastic surgeons by a questionnaire and photographs Addition of PRP to fat grafting led to a significant reduction in the number of days needed to recover before returning to work/restart social activities (GI took 18.9 days vs. GII took 13.2 days, p=0.019). The aesthetic outcome of GII/GIV was significantly better than GI/GIII. The addition of PRP to facial lipofilling reduces recovery time and improves the overall aesthetic outcome of a MACS-lift. Willemsen, et al. 60 (2014) Prospective, randomized, splitface trial (n=20)

Infraorbital wrinkles
and skin tone 10 patients received a PRP injection in one side of the face and the other was treated with PPP; the remaining 10 were treated with PRP vs. saline (3 sessions at 4-week intervals). Evaluations performed at baseline and 3 months after the final treatment, including self-assessment questionnaire, subjective satisfaction scale and clinical assessment by 3 dermatologists (photographs). Erythema and melanin indices were evaluated spectrophotometrically.
Infraorbital skin treated with PRP presented significant improvement of tone and wrinkles compared with saline or PPP treated skin. After PRP treatment, the erythema and melanin indices significantly decreased Kang,et al. 79 (2014) Prospective openlabel (n=23) Face and neck

monthly injections (4 mL) of PRP (Regen Lab Kit). Evaluation performed by patients and physicians through questionnaires, dermoscope and photographs
No serious and persistent side-effects were observed. Overall, the results were satisfactory.
Redaelli, Romano and Marciano 59 Prospective openlabel (n=15) Nasolabial fold PRF matrix (Selphyl system) was injected into the dermis and immediate subdermis below the nasolabial folds. Patients were photographed before and up to 12 weeks after single injection. Evaluation performed using GAIS and WAS All patients were treated to maximal correction, with mean reduction in WAS score of 2.1±0.6, that 0.7±0.6 after 1 week but rose to 1.1±0.7 at 12 weeks after treatment. Fibrosis, restricted movement, irregularity hardness or lumpiness was not noticed by any patient. PRF matrix provides significant long-term reduction of deep nasolabial fold.

Second and third generation autologous platelet concentrates: PRF and i-PRF
PRF is obtained after a single centrifugation, without the need of using anticoagulants.  When used alone, PRF matrix provided significant long-term reduction of deep nasolabial fold. 70,74 It has been reported, using i-PRF with low RCF (combination of 60 g for 3 min and 208 g for 5 min), good results for the rejuvenation of the lower third of the face (nasolabial fold and labial commissure) after an intradermal application. 71 In another study, the effect of three monthly intradermal injections of i-PRF (low RCF 60 g, 3 min) in three facial regions was evaluated: malar area, nasolabial fold, and region above the vermilion of the upper lip. An improvement in skin texture, pores, wrinkles, as well as patient satisfaction was observed after three months. 69 However, additional studies are needed to establish the centrifugation protocol that leads to the best clinical effects. In addition, more high-quality trials with appropriate follow-up are necessary to provide appropriate evidence that may help to improve the treatment regimens.

Conclusion
Autologous platelet aggregates for skin rejuvenation are safe and well tolerated. PRP, the first-generation product, is more studied in the literature, with several clinical trials and case series, whose results have been complied in a systematic review. 5 The results, in general, are favorable, but the quality of the studies is low and additional studies are required. The second and third generation products, PRF and i-PRF, respectively, are easier to be obtained and, at least in vitro, seem to induce greater collagen production than PRP, 67 especially under lower RCFs. 64 However, only a few clinical trials evaluating these products are available to date.
More high-quality trials with appropriate followup are necessary to provide appropriate evidence that may help to improve the treatment regimens with autologous platelet aggregates. Several aspects should be considered when future clinical trials evaluating PRP are to be designed, such as the patients' characteristics that best predict a favorable response, the optimal number of sessions and the interval between them, the characteristics of the studies and the development of better instruments to evaluate skin aging.