Autologous pure platelet‐rich plasma injections for facial skin rejuvenation: Biometric instrumental evaluations and patientreported outcomes to support antiaging effects

Peter A Everts PhD1  | Pedro Contreiras Pinto PhD2 | Leonor Girão MD3

1Gulf Coast Biologics, Research and Educational Center, Fort Myers, Florida

2PhD Trials Center, Lisboa, Portugal

3Clinica Dermatologica do Areeiro, Lisboa, Portugal

 

Correspondence: Peter A Everts, Gulf Coast Biologics, Fort Myers, FL. 

Email: peter@gulfcoastbiologics.com
Funding information EmCyte Corporation

 

 

KEYWORDS antiaging, anti‐wrinkle, biometric, pure platelet‐rich‐plasma, skin rejuvenation

Summary Background: During skin aging, a degeneration of connective tissue and decrease in hyaluronic acid polymers occur. Since platelet‐rich plasma (PRP) contains growth factors and various cytokines, it was hypothesized that it could play a role in fibroblast activation and type I collagen expression in human fibroblasts.

Objectives: This study was performed to assess the efficacy of autologous PRP injections for facial skin rejuvenation, measured by biometric instrumental evaluations and patient‐reported outcomes.

Patients and Methods: Patients signed an informed consent form. The EmCyte PurePRP® system technology was used to produce neutrophil‐poor PurePRP. The efficacy of the procedures was assessed by biometric parameters, and a patient outcome a self‐assessment questionnaire on each visit and at 6‐month follow‐up.

 

Results: Eleven volunteers were included in the study, receiving 3 PurePRP® treatments. A significant decrease in brown spot counts and area (P < 0.05) was seen after 3 months. Wrinkle count and volume were significantly reduced (P < 0.05 for total wrinkle appearance). Skin firmness parameters were significantly improved. Skin redness was significantly improved after 169 days post‐therapy for both the nasolabial and malar areas. A decrease in SLEB thickness was already noted at 2 months after the first injection, with an increase in SLEB density (P < 0.05 for both parameters), without affecting subcutaneous fat thickness. Self‐assessment at 6‐month follow‐up revealed an average satisfaction score of >90%.

Conclusions: A series of 3 PurePRP injections at 6‐month follow‐up resulted in significant skin rejuvenation as demonstrated by biometric parameters and confirmed by patient self‐assessment score.

INTRODUCTION
During skin aging, a degeneration of connective tissue and decrease in hyaluronic acid polymers occur due to intrinsic and extrinsic factors. There are cellular changes, alterations in dermal extracellular matrix (ECM) proteins, and dermal atrophy, with solidified dermal‐epidermal
junctions and fewer fibroblasts.1 Activation of dermal fibroblasts, remodeling of the ECM, and collagen synthesis is essential for aged skin rejuvenation. Since PRP contains several growth factors, cell adhesion molecules, and various cytokines, it was hypothesized that platelet‐rich plasma (PRP) could play a role in fibroblast activation and type I collagen expression in human fibroblasts.

Platelet‐rich plasma therapies and treatment protocols have evolved immensely over the past 20 years. Through laboratory, experimental, and clinical research, followed by meta‐analyses, physicians, medical practitioners, and scientists have gained a better understanding of the effects of PRP on cellular physiology, especially with regard to the functions of some of the specific biological components in the platelet proteome,2 affecting PRP treatment outcomes when used in regenerative medicine therapies. To optimize outcomes, based on patient‐specific underlying conditions and tissue type, the practitioner needs to have a clear understanding of when to use which specific PRP treatment protocols in a variety of medical indications, to obtain the desired regenerative and tissue repair effects. These various applications have given rise to considerable interest in the potential of PRP in facial rejuvenation and other esthetic applications. The functional design of existing PRP‐processing systems, with the subsequent final PRP production, varies tremendously. Different platelet concentrations and biological compositions are obtained by PRP device‐specific preparation protocols. Optimal blood separation is safeguarded by double‐spin PRP centrifuges with dedicated disposable concentration devices. These double‐spin PRP devices create a 3‐layer buffy coat stratum, based on different centrifugal forces and specific gravities of individual blood components (Figure 1). Single‐spin devices, or plasma PRP devices, prepare a product from the acellular plasma layer, excluding erythrocytes and leukocytes from the PRP preparation process, while collecting as many platelets as possible from the plasma layer.3 These differences in cellular compositions, and thus PRP characteristics, have recently been recognized in the literature.4,5 The use of a poor‐quality and inconsistent PRP product ultimately results in a lower treatment outcome. This was reported by Marques et al6 for many studies. In this study, we used a PurePRP®, double‐spin buffy coat product to obtain a specific concentration of platelets and other constituents, to achieve a clinically significant effect. We used several non‐invasive skin diagnostic techniques to objectively assess the PurePRP facial injection effects. The aim of this skin rejuvenation study was to assess the clinical effectiveness of 3 PRP injections in highly selected subjects, who also were asked to be compliant with the study constraints. Therapy efficacy was measured and calculated by specific, objective, biometric instrumental analysis protocols. Furthermore, we evaluated the patient‐reported outcomes of this treatment.

MATERIALS AND METHODS
2.1 | Patients
This single‐center open‐label study was conducted according to the principles of the Declaration of Helsinki, Good Clinical Practice Guidelines, and General Principles of Portuguese Law (46/2004). The study was reviewed and approved by the IRB. Eleven healthy female volunteers between 45 and 65 years old were enrolled in the study. All women signed an informed consent form before treatment with 3 facial PRP injections. All injections were given by the same physician (L.G.). Eleven patients were enrolled in the study. Inclusion criteria were as follows: female patients with voluntary participation, age between 40 and 60 years, phototype according to Fitzpatrick’s classification between II and IV, and all skin types showing signs of photoaging, chronologic aging, or smoking habits. Exclusion criteria are indicated in Table 1. Furthermore, the volunteers had to agree to several study constraints, as specified in Table 2. The subjects were requested to note every day any reaction observed, and sensation of discomfort felt on the individual observation sheet they were given at the beginning of the study. This examination was performed visually under standard “daylight” source, before, during, and then after treatment.


2.2 | Biometric instrumental assessments
The efficacy of the procedures was assessed by clinical and biometric instrumental parameters in all subjects according to the study schedule as shown in Table 3. The efficacy of the treatment was also evaluated by the volunteer’s assessment schedule. Biometric evaluations consisted of using different instrumental devices. The VISIA‐CR system (Canfield, Parsippany, NJ, USA) was used to quantify the development of the antiaging effects by in a full‐aligned facial image. To evaluate qualitatively and quantitatively skin profile changes, to assess wrinkle count, depth, and volume, the Optical In Vivo Primos 3D Skin Device (GFMesstechnik GmbH, Berlin, Germany) was applied to the periocular area (left or right). Skin biomechanical evaluations to measure elasticity of the upper skin layers using negative pressure were performed with a Cutometer dual MPA 580 (Courage & Khazaka, Cologne, Germany) using a 2‐mm probe. Skin color measurements for luminance were obtained in the malar area with the tri‐stimulus color analyzer, Minolta Chromameter CR‐400 (Minolta, Osaka, Japan). The Dermascan‐C ultrasound device with a modified 20‐MHz probe (Cortex Technology, Hadsund, Denmark) was used to measure thickness and density of the subepidermal low echogenic band (SLEB), with calculations of subcutaneous fat thickness. All biometric instrumental protocol evaluations were performed in a fully controlled room and after an initial acclimatization process of at least 30 minutes in a fully controlled and acclimatized room (controlled temperature: T = 21 ± 2°C; controlled relative humidity: RH = 55 ± 10%).To avoid circadian changes, 2 evaluation periods were defined (morning: 9 am‐1.30 pm; afternoon: 1.30 pm‐6 pm). All the evaluations were performed during the same period of the day chosen by the volunteers.

 2.3 | Patient self‐assessment
All subjects were requested to complete a self‐assessment questionnaire on each visit and at 6‐month follow‐up. The questions of the assessments are charted in a spider‐web graph to represent the multivariate data in the form of a two‐dimensional chart, including the 8 quantitative variables after each injection and at D169 (Table 4).
 

Individuals who performed an antiaging or esthetic treatment prior to the study: Botox or Botox‐like products, peelings, plastic surgery, resurfacing with Laser, IPL, threats, radiofrequency treatments, hyaluronic acid treatment, Plasma‐Rich Platelets treatment, or any other specific treatments prone to change the skin aspect during the last 18 mo


Cutaneous marks on the experimental area which could interfere with the assessment of skin reactions (pigmentation problems, scar elements, over‐developed pilosity, ephelides, and naevi in too great quantity, sunburn, beauty spots, freckles, etc.)


Eczematous reaction still visible, scar, or pigmentary sequelae of previous tests on the experimental area


Pregnancy and breastfeeding


Intention to pregnancy in the next 3 mo after the start of the study


Forecast of vaccination during the study period or last vaccination within 3 wk before the study


Systemic disorders: cardiovascular, pulmonary, digestive, neurologic, psychiatric, genital, urinary, endocrine


Hematological or hemorrhagic diseases


Thrombocytopenia moderate or severe (


Allergy to colophony or nickel


Allergy or reactivity to drugs, food or cosmetic products previously observed, including perfumes or cologne products


Skin hyper‐reactivity


Intensive sun exposure within the month before the study


Forecast of intensive sun or UVA exposure (UV lamps) during the test period


Intensive or regular practice of one or several sports whose temporary interruption creates difficulties


Treatment with Vitamin A acid or its derivatives within 3 mo before the beginning of the study


Treatment with topical corticoids on the experimental area within 16 d before the study


Treatment with antibiotics, anti‐allergic, anti‐inflammatory (systemic or topical corticosteroid therapy) treatment with patent medicines containing vitamin A acid or its derivatives during the study (if therapeutic requirement: exclusion foreseen)


Individuals with a history of any dermatological disease or condition, including but not limited to active atopic dermatitis, psoriasis, eczema, active seasonal allergies, collagen diseases, or skin cancer within the past 6 mo


Individuals who have undergone a bilateral mastectomy with lymph node removal, a unilateral mastectomy with lymph node removal within the last year, or a bilateral axillary lymph node removal


Individuals with a history of immune deficiency or auto‐immune disease treated for malignancy within 6 mo prior to enrollment or who are currently under treatment for asthma or diabetes


Treatment with salicylic acid or any anticoagulant drugs during the study


Treatment with PUVA or UVB within 1 mo before the study

No application of products on the experimental area (except the suggested ones), particular any antiaging cosmetic products


No change in hygiene habits


No application of any cosmetic moisturizing products on the face or any makeup on face and lips, on the day of biometric evaluations and PurePRP injections


No drugs that interfere with the study outcomes (anticoagulant, immunosuppression, and salicylic acid drugs)


No change in the way of life or in the physical activity


No dietary activities, or any treatment that significantly impacts body weight


No exfoliating treatment on the experimental areas


Description of any treatment undertaken during the study and all eventual deviations

2.4 | Pure platelet‐rich plasma injection procedure
Subject preparation, injection, and aftercare. A local anesthetic (EMLA®, Astra Zenica, Cambridge, UK) was applied in all facial areas for at least 30 minutes before the injection. Prior to the injections, excess local anesthetic was removed, and the skin was disinfected with chlorhexidine. Small aliquots of PurePRP in 1‐mL insulin syringes were administered intradermally and subcutaneously, using a 13‐mm‐long 27G or 4‐mm‐long 32G needle (depending on the thickness of the skin to be treated). Volunteers underwent 3 sessions of PurePRP treatment at 1‐month intervals, with a follow‐up period after of 6 months. At the end of each injection procedure, platelet‐poor plasma, a byproduct of the PurePRP preparation procedure, was applied to the skin at all injection sites, and the treated area was then covered with polypropylene film for 10 minutes to promote skin penetration. After film removal and skin cleaning, a moisturizing cosmetic product was used (Toleriane Ultra Creme, La RochePosay, La Roche‐Posay, France). No ice packs were used after the procedure. Daily applications of sunscreen protection were recommended.

2.5 | PurePRP preparation 

The EmCyte PurePRP® system technology (EmCyte Corporation, Fort Myers, FL, USA) was used at point of care in the clinic, just before injection. Fifty milliliters of whole blood were pre‐donated in a 60‐mL syringe containing sodium citrate. Processing was in accordance with the instructions for use from EmCyte Corporation. In all volunteers, EmCyte’s proprietary Protocol‐A was carried out to produce PurePRP using the GS‐60 platelet concentrating devices and the 544E Executive Eppendorf centrifuge. The PurePRP is characterized as a double‐spin buffy coat product, with a low erythrocyte concentration and significantly reduced, pro‐inflammatory, neutrophils. The final PurePRP volume for the facial injections was standardized to 7 mL of PurePRP in all subjects. To compensate for the anticoagulant effects of sodium citrate, 0.05 mL of 10% calcium chloride was mixed with 1 mL of PurePRP prior to facial injection.

2.6 | Adverse event monitoring
Adverse effects including erythema, edema, bruising, and altered pigmentation were assessed by questioning the volunteers and observing skin responses at admission and during all follow‐up visits.

TABLE 4Self‐assessment questionnaire


Q1. Do you notice an improvement in your wrinkles aspect


Q2. Do you notice your facial complexion more even


Q3. Do you notice your skin firmer


Q4. Do you feel your skin more moisturized


Q5. Do you feel your skin has healthier aspect


Q6. Do you feel your skin softer


Q7. Do you notice your skin more radiant


Q8. Do you notice an improvement on your skin’s visible youth fullness


2.7 | Statistical analysis
All data were analyzed using IBM SPSS Statistics‐20 (Armonk, NY, USA). The biometric instrumental efficacy data are expressed as numbered data. All continuous data comparisons were submitted to the Student T test or the Wilcoxon signed‐rank test. A 5% level of significance was used. The subjective data of efficacy were submitted to binomial testing. P < 0.05 was considered significant.

3 | RESULTS
All 11 enrolled female volunteers completed the study, receiving 3 PurePRP injections. The average age was 51 years (range 47‐60 years old), and 82% of the women were classified as Fitzpatrick skin type III. The treatment procedures and PurePRP preparations were all consistently performed without any complications. No skin reactions were noted after each procedure. None of the volunteers experienced any discomfort during the study, or during the follow‐up period. A well‐tolerated burning sensation was reported after the injections. Minor ecchymosis, which resolved within 3 days, was noted with no signs of inflammatory or allergic reactions. None of the subjects reported adverse events during the entire study period. There was a significant decrease (P = 0.029) in brown spot counts after 6 months and a 26.3% reduction (P = 0.004) in total brown spot area compared to conditions on admission (Figure 2). No significant changes in red spot counts and red spot area were observed. Mean wrinkle count (P = 0.000) and wrinkle volume (P = 0.049) were significantly reduced, compared to the count and measurement before the first PurePRP injection, respectively, 66.2 and 2 (Figure 3). Evaluation of the true wrinkle count and volume % changes in all subjects after 169 days revealed a relative transformation in relation to D0 of −37.2% and −11.5%, respectively (P < 0.05) (Figure 4). Figure 5 displays a significant % increase in skin firmness parameters in the malar area, compared to baseline. Also, changes in the nasolabial area demonstrated a significant improvement after 56, 84, and 169 days (P = 0.000). Skin redness for both nasolabial and malar area was significantly reduced, 29% and 44.3%, respectively, but only after 169 days post‐PurePRP procedures (Table 5). On the other hand, the luminance of the malar area improved significantly (P = 0.016) after the first PRP injection and continued to improve following the PurePRP procedures, until 3 months after the last injection (P = 0.006). A decrease in SLEB thickness (P = 0.021) was already eminent at 2 months after the first injection and continued to decrease at D169 (P = 0.033), with a simultaneous increase in SLEB density (P = 0.042) at 6‐month follow‐up (Figure 6). The increase in density, pixels/mm2, did not affect subcutaneous fat thickness (P = 0.224). All subjects respected the study constraints and completed the self‐assessment outcome scores. At 6‐month follow‐up, the average satisfaction score was >90%, as shown in Figure 7.

Tab3

4 | DISCUSSION
It is well known that during aging, epidermal and dermal changes in the skin are naturally occurring phenomena, with degradation of the ECM.7 Also, the cessation of collagen fiber and elastin synthesis, with degradation of proteoglycans, results in loss of skin elasticity.8 Furthermore, skin aging is characterized by flattened dermal‐epidermal junctions, dermal atrophy, and the presence of less fibroblasts. The remodeling of ECM and the activation of dermal fibroblasts are essential for rejuvenation of aged skin. It has been reported that the activity of PRP in facial skin rejuvenation induces the synthesis of new collagen by dermal fibroblasts via different molecular mechanisms. Particularly, PRP increases mRNA expression of type I collagen and metalloprotease‐1. As a consequence, enhanced dermal elasticity stimulates the removal of photo‐damaged ECM components.9 Choi et al10 confirmed these data, where they also showed an increase in type I collagen expression in human fibroblasts treated with PRP.

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It is well known that during aging, epidermal and dermal changes in the skin are naturally occurring phenomena, with degradation of the ECM.7 Also, the cessation of collagen fiber and elastin synthesis, with degradation of proteoglycans, results in loss of skin elasticity.8 Furthermore, skin aging is characterized by flattened dermal‐epidermal junctions, dermal atrophy, and the presence of less fibroblasts. The remodeling of ECM and the activation of dermal fibroblasts are essential for rejuvenation of aged skin. It has been reported that the activity of PRP in facial skin rejuvenation induces the synthesis of new collagen by dermal fibroblasts via different molecular mechanisms. Particularly, PRP increases mRNA expression of type I collagen and metalloprotease‐1. As a consequence, enhanced dermal elasticity stimulates the removal of photo‐damaged ECM components.9 Choi et al10 confirmed these data, where they also showed an increase in type I collagen expression in human fibroblasts treated with PRP.
In recent years, PRP has been used in many medical areas, including indications to support acute and chronic wound healing. Research in wound healing models has provided interesting information with regard to the pathophysiology of photoaging, indicating that there are several parallel mechanisms between pathways involved in wound healing and those necessary for skin rejuvenation. Biological and biochemical processes are involved in wound formation which are similar to the needed changes to reverse the effects of intrinsic and extrinsic skin aging.

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11 A diversity of platelet growth factors (PGFs) with their specific characteristics (Table 6) is located in the platelet alpha granules and play pivotal roles in regenerative processes. Following the mechanisms in wound healing, PRP acts on skin aging through collagen remodeling, stimulating a thickening of the superficial layer of the skin, and simultaneously improving cell regeneration.12 Tissue repair, as in skin rejuvenation, and surgical wound healing are well orchestrated, and there is a complex series of events involving cell‐cell and cell‐matrix interactions, in which PGFs serve as messengers to regulate various regenerative processes.13 The effectiveness of PRP injections and therefore bio‐cellular activity is determined by which type of PRP is being used, plasma PRP or buffy coat PRP. Specific PGFs in combination with available platelet proteins, cytokines, and chemokines regulate fundamental cellular activities, including mitogenesis, angiogenesis, chemotaxis, formation of the ECM, and ultimately control the activity of PGFs.14 These adjunctive and obligatory effects (Table 7) should be part of the total biological activity of PRP. Commercially available whole blood separation systems are designed for the preparation of PRP; however, they produce major differences in cellular composition.3 A buffy coat PRP product is capable of enhancing cell proliferation and differentiation, cell migration, and ECM buildup, producing a concentration of platelets meeting the definition outlined by Marx of >1 million cells/μL.12,15 “PRP‐like” products (single‐spin plasma PRP devices) have a low to no platelet concentration in the final product and consist mainly of plasma. These devices will demonstrate a less significant to no effect when compared to a buffy coat (double‐spin) PRP product, which is rich in platelets and leukocytes capable of tissue regeneration such as monocytes. Moreover, most PRP technology systems are often based on antiquated devices and preparation protocols, using outdated science, a lack of solid research to show proof of concept of a newly developed product, and a too simplified view on the true regenerative capacities of all the PRP constituents. The consequential effects are that some PRP treatments show little, or even no therapeutic effects, and even deleterious effects have been reported, albeit these systems have not been adapted to meet current research findings. Platelet‐rich plasma should therefore be characterized as a small volume of plasma, with a substantial concentration of platelets, specific leukocytes, and minimal red blood cell contamination, since skin rejuvenation results from cell proliferation, angiogenesis, and cell migration aimed at remodeling the ECM.16 Magalon et al17 recently categorized the many PRP devices in classifications based on their specific characteristics and biological composition, such as the purity of PRP with regard to platelet concentration, leukocyte composition, and erythrocyte concentration, to provide support in selecting a system that meets the specific needs for a given indication. In this study, we used a 4th generation PRP system, where the design characteristics are centered on optimizing the efficacy of patients’ treatment outcomes based on the capability of preparing various bio‐cellular PRP protocol formulations. The EmCyte PurePRP® system can be categorized as giving a buffy coat PRP product.18,19 Following a density gradient double‐spin centrifugation protocol of whole blood, a buffy coat PurePRP product is created. The PurePRP® can be best described as an anticoagulated volume of plasma containing concentrated platelets, white blood cells, a fraction of red blood cells, including fibrin and cell adhesion molecules.

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