CPD: Understanding PRP Efficacy

Understanding PRP Efficacy

Dr Priya Shah explores the difficulty in determining the efficacy of platelet-rich plasma as a monotherapy for skin ageing

2022

Platelet-rich plasma (PRP) is an emerging skin rejuvenation treatment. Being readily available, cost-effective for both patient and practitioner, and having minimal downtime and side effects, PRP presents an attractive treatment option suitable for all skin types. Media-induced popularity of the ‘vampire facelift’ and PRP for self-healing, as well as clinic advertising campaigns claiming high patient satisfaction, are merely anecdotal in contributing to PRP hype and public misunderstanding of PRP efficacy.1,2

Where PRP fits in with antiageing PRP is a non-ablative treatment preserving epidermal integrity upon administration but it has the capability to elicit epidermal and dermal changes for skin rejuvenation, increasing dermal papillae and releasing growth factors (GF). It stimulates natural collagen with regenerative healing.3 Healthy skin architecture results from a fine balance in the dynamic equilibrium between extracellular matrix (ECM) deposition and matrix metalloproteinases (MMP) degradation.4 Most antiageing treatments target the dermis using injury to stimulate fibroblasts resulting in deposition of collagen, elastin, glycosaminoglycans (GAGs) and also fibrillin at the dermoepidermal junction (DEJ).5 Treatments using microinjury show fine, well-organised fibres with old dense abnormal elastic material moved downward towards the reticular dermis due to neocollagenesis.6 In a study of 24 participants, El-Domyati et al. reported significant objective quantitative increase in collagen types I, III, and VII after multiple microneedling sessions for atrophic acne scars, with a reduction in abnormal elastin level.7 Increased production of GAGs such as hyaluronic acid (HA) and chondroitin-4/6-sulfates has been stimulated in cultured human adult fibroblasts by recombinant human interleukin-6.8 Being just one of many chemokines capable of stimulating paracrine/autocrine ECM deposition by fibroblasts this is an example of the many pathways that could be positively influenced in skin rejuvenation by regenerative PRP treatment. As HA production reduces with age, skin treatments focus on HA delivery through topical means or direct intradermal injections avoiding the need to cross the epidermal barrier. Currently retinoic acid (RA) is regarded as a gold standard topical skin rejuvenating treatment having been widely researched.9,10 Topical all-trans RA can restore collagens I and VII, restore the fibrillin-rich microfibrillar network of the papillary dermis,11 reduce MMP-1 expression, normalise GAG production and reduce wrinkle appearance.12 Finding a treatment comparable to RA capable of positively targeting and inducing deposition of key structural skin components at multiple cellular and molecular levels which leads to true skin rejuvenation would be desirable of PRP treatment to demonstrate efficacy. With no single treatment offering mechanisms to restore the juvenile condition of skin, this topic remains of high interest to the scientific aesthetic industry. Currently, optimal rejuvenation is achieved using multi-modality treatment to satisfy patient needs, tolerance and goals. Some non-surgical treatments have side effects or fail to yield desired results. PRP has been studied both as a monotherapy for enhancing natural regenerative potential, or in combination with more established treatments such as hyaluronic acid fillers, microneedling and laser resurfacing for adjunctive or synergistic effects through acceleration of wound healing.2

PRP mechanism of action PRP is derived by centrifuging a whole blood portion to remove red blood cells and leave a concentrate of conditioned plasma rich in platelets. This high platelet concentration plays a critical role in wound healing, inducing clotting and initiating repair with increasing experimental and clinical evidence identifying platelets as modulators of inflammation and tissue regeneration.13 Exogenous or endogenous platelet activation induces release of bioactive peptides from their α-granules.13 These include GFs, cytokines and ECM modulators that mediate signalling pathways, stimulating cell regeneration and repair.14 Hence upon reinjection into the skin, PRP can mediate and enhance an individual’s own autologous regenerative potential (Figure 1).15 As illustrated in Figure 2, PRP potentially induces not only ECM remodelling by increased MMP expression to remove photodamaged ECM components and stimulates fibroblast proliferation and collagen synthesis, but also PRP affects blood vessels, adipose-derived stem cells and keratinocytes, thus influencing skin repair and rejuvenation at many levels by deposition of key structural components. 15 The plasma component of PRP also contains fibrin, fibronectin and vitronectin which bind cell adhesion molecules which in turn induce cell migration, attachment, proliferation, differentiation and ECM accumulation.16 Platelets are used in regenerative medicine for alopecia, dental, oral surgery, orthopaedic and dermatological

The blood is placed in a centrifuge

Withdraw blood Whole blood Separate the platelets

Platelet poor plasma

Platelet rich plasma

Red blood cells

After centrifugation Extract platelet rich plasma Inject area with PRP

applications.17 Before treatment implementation, medical professionals require both evidence of PRP efficacy and understanding of the mechanism of action to justify use for treatment.18 Data for PRP usage in dermatology is dispersed widely throughout the literature. Some studies indicate PRP induces neocollagenesis and deposition of other matrix components by fibroblast stimulation.19-22 Due to lack of standardisation of methodologies, there are a limited number of studies definitively confirming PRP efficacy for induction of fibroblast ECM deposition. Currently dermatological PRP use outpaces the evidence justifying a firm scientific background and this gap needs to be bridged.

The functions of growth factors GFs are bioactive signalling molecules controlling growth, organisation and maintenance of cell activity in an autocrine, paracrine or endocrine manner by affecting cell proliferation, differentiation, apoptosis, morphogenesis, metabolism, wound healing and tissue homeostasis. They bind to specific receptors activating downstream signalling pathways to regulate gene transcription and ultimately stimulate a biological response in keratinocytes, fibroblasts, endothelial cells, adipose-derived stem cells and macrophages (Figure 2). This facilitates skin regeneration through inflammation, collagen and elastin synthesis, distribution and remodelling, tissue granulation and angiogenesis encouraging tissue restoration. 16 Table 1 illustrates GF diversity. We now assume there are more than 1,500 GFs and regulatory proteins responsible for PRP action. An exact analysis is yet to be made.23

The regulation surrounding PRP and its use PRP application is not approved by the Food and Drug Administration in the US or by the Medicine and Healthcare products Regulatory Agency in the UK/EU. In the US, PRP separating devices are FDA approved through the 510(k) clearance process where lower risk devices can be considered equivalent to a previously cleared device in terms of safety and performance and not necessitating supporting clinical data. Patients must be informed of off-licence use. As per my research, in the UK, it was found that no central database exists listing all certified PRP devices. MHRA contracts out approval processes to various Notified Bodies (NBs). Manufacturers apply to a NB once they have the necessary Conformité Européenne (CE) marks for certification and they are then allowed to commercially sell their device.25,26 It can be concluded that PRP devices are poorly regulated in both the US and UK.

Problems with current PRP preparation systems and a call for standardised classification In a 2019 systematic review by Maisel-Campbell et al. the authors stated that failure to report PRP concentration was a significant study limitation, with only two out of 24 identified studies detecting an effective supraphysiologic platelet concentration greater than 1,000,000/µL.27 Optimal platelet concentration is indicated to be 2.5 times higher than baseline in order to induce potent fibroblast stimulation to produce endogenous HA and endogenous procollagen type 1.28,29 In 2009, Giusti et al. identified optimal concentration for angiogenesis stimulation as 1.5 million/µL with lower or higher concentrations displaying a lower angiogenic potential.30 Hence, further investigation is required to define ideal platelet concentration. Consideration should be given to interpatient homeostasis variation which cannot be controlled nor fully standardised.31 With individual variation of baseline platelet counts, inconsistencies exist in quantifying measurements with an ongoing discussion regarding variable factors including description terminology, classifications (Table 2), PRP preparation systems (e.g. activators, number, speed and time of centrifugations, temperature, test tube materials), ideal volume,

Chemokines/cytokines

IL-1B PBP PF4 CCL5 SDF-1a CCL2

Small molecules

Ca2+ ADP Serotonin Epinephrine Histamine PRP

Growth Factors

CTGF HGF PDGF VEGF EGF TGF-B Adhesive proteins

Vitamin D-binding protein Plasminogen PAI TSP Fibrinogen Fibronectin Vitronectin a1-microglobulin

Proteases/antiproteases

a-2-macrogiobulin ADAMTSs MMPs

Macrophage Fibroblast Endothelial Cells Adipose-derived stem cells Keratinocyte

Enhance matrix proteases gene expression and clear tissue debris Increased I and III collagen expression Endothelial cell proliferation and promotes nagiogenisis Promoting the proliferation and secretion of adiposederived stem cells

Produces keratin and promotes epidermal repair

Collagen production, improved skin barrier structure, increased facial tissue volume, and decreased bone resorption ultimately promote facial rejuvenation

Name Abbreviation Proposed function

Platelet derived growth factor

Transforming growth factor ß PDGF Enhances collagen synthesis, proliferation of bone cells, fibroblast chemotaxis and proliferative activity, macrophage activation

TGF- ß Enhances synthesis of type I collagen, promotes angiogenesis, stimulates chemotaxis of immune cells, inhibits osteoclast formation and bone resorption

Vascular endothelial growth factor VEGF Stimulates angiogenesis, migration and mitosis of endothelial cells, increases permeability of the vessels, stimulates chemotaxis of macrophages and neutrophils

Epithelial growth factor EGF Stimulates cellular proliferation, differentiation of epithelial cells, promotes cytokine secretion by mesenchymal and epithelial cells

Insulin-like growth factor IGF Promotes cell growth, differentiation, recruitment in bone, blood vessel, skin and other tissues, stimulates collagen synthesis together with PDGF

Fibroblast growth factor FGF Promotes proliferation of mesenchymal cells, chondrocytes and osteoblasts, stimulates the growth and differentiation of chondrocytes and osteoblasts

Table 1: Growth factors in PRP and their biological functions24

technique, site, and frequency of administration.3,32-37 Additional cells remaining in PRP post-centrifugation add extra signalling proteins to the mix creating an unquantifiable number of potential outcomes with potential to act in anabolic, catabolic, proinflammatory, anti-inflammatory pathways and also immune response. PRP should contain minimal erythrocytes and leucocytes to limit proinflammatory cytokines producing free radicals and protease release respectively.32-34 Without optimal protocols, lack of standardisation creates differing PRP regenerative potentials making predictable treatment outcomes difficult to achieve. Three PRP classification systems have been proposed (Table 2). The clinical relevance of these classifications is yet to be evaluated.35,36 A 2019 literature review analysing all commercially available PRP separation systems showed vast variation in blood components and GFs, concluding future research should focus on component concentrations as well as optimal platelets, leukocytes, and GF concentrations for different fields of application.38 To date, no ideal classification system exists proving PRP efficacy with certainty for antiageing purposes and there are no comparative head-to-head studies investigating effects of different PRP preparations in skin rejuvenation. Fundamentally, establishing a better understanding of the biological and molecular basis of PRP is needed. Mass spectrometric analysis of PRP could help establish links with outcomes to improve the validity of PRP clinical efficacy.39

Literature critique of current studies investigating PRP monotherapy efficacy Motosko et al. conducted a literature review analysing PRP data published from 2006-2015.2 This included seven studies using PRP alone for facial rejuvenation. They reported improvements in skin appearance to include texture, colour homogeneity, firmness, elasticity, solar elastosis, wrinkles, volume, dermal thickness, nasolabial fold severity, acne scars, erythema, melanin and patient satisfaction. From the 19 PRP monotherapy studies included in the Maisel-Campbell et al. systematic review (data studied inception to March 2015), four individual random controlled trials (RCTs), two individual cohort and 13 case series studies were identified.27 Although limited PRP literature for skin rejuvenation exists, meaningful effects and faster healing times with ‘75-100% of patients demonstrating physician-rated global improvement with 67% showing up to 50% improvement’ are claimed.27 Whilst best practice uses RCTs for evidence-based medicine, firm conclusions are not possible due to lack of PRP device standardisation, varying study protocols and inconsistent reporting. This makes quantitative analysis challenging, confusing our understanding of PRP mechanisms and where PRP fits in for comparative or adjunctive use within the non-surgical facial aesthetics arena. A large variation in outcome parameters is noted from studies so far. Clinical parameters are predominantly assessed ranging from patient satisfaction scores, physician scales, feedback questionnaires and pre- and post-treatment photography. Positive findings based on subjective patient satisfaction scores or objective practitioner observations using non-standardised outcomes and varying imaging techniques make data speculative and meta-analysis unrealistic, especially when performed in absence of quantitative evidence such as histological improvement, cytokine or gene assays. Literature analysis using the Motosko et al. literature review and the Maisel-Campbell et al. systematic review, demonstrates

that approximately one third of the PRP monotherapy studies utilise objective instrumental analysis. Du and Lei used objective instrumental measures, VISIA Complexion Analysis System and skin computed tomography, linking them to an in vitro human organotypic skin model.40 Disadvantages of this model include that it recreates only part of normal skin organisation and function and is unable to mimic wound healing so cannot be reliably studied.41 The platelet concentration of PRP was confirmed as 1009.91±219.

Classification

4-category: Based on differing leukocyte and fibrin content of the preparation37

DEPA34 • Dose of injected platelets • Efficiency of production • Purity of PRP • Activation process was advocated in 2016 utilising biological parameters used in cell therapy to standardise systems with rankings from A-D

FIT PAAW3 • Centrifugation force • Iteration of centrifugation • Centrifugation time • Platelet concentration (baseline of patient’s whole blood and final

PRP product) • Anticoagulant use • Use, type and amount of activator • Composition of white blood cells has also been proposed

Categories

1. Pure PRP (P-PRP)/leucocyte-poor

PRP or leucocyte-reduced PRP (lrPRP) 2. Leucocyte-rich plasma and PRP (L-PRP): adds to inflammatory cascade especially the neutrophils 3. Pure platelet-rich fibrin (P-PRF) 4. Leucocyte and platelet-rich fibrin (L-PRF)

1. Dose of injected platelets 2. Efficiency of production 3. Purity of PRP 4. Activation process was advocated in 2016 utilising biological parameters used in cell therapy to standardise systems with rankings from A-D

1. Centrifugation force 2. Iteration of centrifugation 3. Centrifugation time 4. Platelet concentration (baseline of patient’s whole blood and final PRP product) 5. Anticoagulant use 6. Use, type and amount of activator 7. Composition of white blood cells has also been proposed

Table 2: PRP classification systems3,31,34

x109/L. Collectively, results ‘indicated’ PRP treatment improved skin photoageing through regulation of MMP-1, tyrosinase, fibrillin and tropoelastin expression.43 Cameli et al., used a more robust study design correlating clinical, instrumental and flow cytometry assessment.42 This study confirmed an effective platelet concentration of 1680 x 109/L. However, lacking a control and not confirming histological changes, they left a significant gap in true justification for PRP use. Histological analysis of structural changes within the skin layers and correlation to mechanical and clinical changes would better justify the level of efficacy of PRP. Histological analysis was conducted in five studies (four case series and one individual cohort study), however, none identified mean platelet concentration of whole blood or PRP.4,43-45 Histological changes noted were increases in epidermal and dermal thickness, the number of fibroblasts, dermal collagen bundles, elastic fibre number and thickness and blood vessels however these studies were poorly designed, lacking scientific quantitative validation and relying on histological changes alone or by correlation to patient/ assessor scales complicated by variation in PRP devices and study protocols. Only Diaz-Ley and El-Domyati used blinded evaluators to minimise bias.4,43 A lack of scientific qualitative and quantitative evidence demonstrating histological improvement and without correlation to cytokine/gene assays means true PRP efficacy cannot be assessed due to variation in cellular content of each individuals’ blood.

Other study limitations include:

• Varying age range of study populations and uneven gender proportions.47,48 • Unjustified and imprecise sample size estimates.42,47,49 • Lack of industry regulation causing studies to be influenced by commercial interest with funding provided by manufacturers of centrifuges/blood preparation kits. The majority of studies do not justify reasons for device selection, and it is impossible to verify whether all these PRP types will achieve the same therapeutic effect and hence, evaluation is not standardised. • Varying volumes of blood drawn and processed (3.6ml-50ml) and differing PRP volume yields (0.2ml-7ml).50,51 • Variations in centrifugation systems: force, timing, single-spin, double-spin, anticoagulation and exogenous activation.46,48 • Varying platelet concentrations between patients: 524x109/L to 1680x 109/L.42,52,21 • Variance in the accepted number of treatments for efficacy (range is one to six).48,53 • Differing standardised intervals between treatments (two to four weeks).53,54 • Lack of a control making objective analysis difficult4,42,47 and variations in presence of blinding (no blinding, single-blinded, double-blinded).49,53,54 • Variance in PRP application techniques and sites of application across all studies: (topical application vs. intradermal injections).4,45,55,46 • Varying follow-up assessment periods (two to 52 weeks).4,49

The future of PRP Despite appearing promising there is a paucity of critical scientific evidence justifying PRP monotherapy use in skin rejuvenation. As discussed, there are a great number of study limitations and variabilities amongst the literature. Despite wide acceptance of off-label PRP delivery using a range of diverse treatment strategies with no optimal treatment protocols, high quality clinical research is urgently required within the dermatology community to ensure scientific evidence-based justification and that patients receive evidence-based treatments. A controlled prospective clinical trial establishing a standardised baseline is urgently required as to date, no double-blind RCTs have evaluated clinical, instrumental, and histological findings together using contralateral control groups and with known quantification of platelet yield in both whole blood and PRP. Only by comparing studies with standardised research protocols and justified sample sizes taking patient diversity into account and incorporating objective assessment, can we determine the real efficacy of PRP and remove any controversy surrounding use of PRP with justified clinical evidence.

Dr Priya Shah graduated dentistry in 2002 from King’s College London and has undertaken an MSc in Skin Ageing and Aesthetic Medicine. She is the founder of Dr Priya Shah Facial Aesthetics, London and a trainer with Botulinum Toxin Club (BTC). She focuses on delivering natural enhancements alongside educating patients on improving skin health. Qual: BDS, MSc in Skin Ageing and Aesthetic Medicine(Dist)

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Test your knowledge! Complete the multiple-choice questions below and go online to receive your CPD certificate!

Questions

1. PRP skin rejuvenation treatment is also commonly known as a…

2. PRP studies to date…

3. PRP treatment protocols…

4. An effective supraphysiologic platelet concentration is classed as...

5. PRP devices...

Possible answers

a. Vampire facelift b. Red cell treatment c. Regenerative facial d. Kardashian treatment

a. Always show gross volumetric facial changes b. Use standardised treatment protocols c. Show epidermal and dermal changes for skin rejuvenation with increasing dermal papillae d. Show increased skin wrinkling

a. Can be used on all skin types b. Can be used on oily skin c. Are always optimal d. Are performed at night only

a. Below 1,500/µL b. Greater than 1,000,000/µL c. Between 10,000 – 20,000/µL d. Greater than 5,000,000/µL

a. Are always standardised using similar preparation and treatment protocols b. Are well regulated in the US and USA c. Always lead to improved clinical, instrumental and historiological changes d. Are not well regulated and there are many study variables making data comparison difficult

Answers: 1. A, 2. C, 3. A, 4. B, 5. D