Platelet-rich Plasma as a Potential Treatment for Noncicatricial Alopecias.

Androgenetic alopecia (AGA) and alopecia areata (AA) are common hair loss disorders affecting both men and women. Despite available therapeutic options, search for new, more effective treatment is constant. Platelet-rich plasma (PRP) could be effective in promoting hair growth: (1) To present PRP and its mechanism of action in promoting hair growth and (2) to evaluate its preparation methods and its therapeutic potential in noncicatrial alopecias in a systematic review. An international bibliography search, through five databases, was conducted to find articles regarding PRP's action on hair loss. Growth factors in platelets' granules of PRP bind in the bulge area of hair follicle, promoting hair growth. In our systematic review, 14 articles matched our criteria, including 12 articles for AGA and two for AA. PRP is a potential useful therapeutic tool for alopecias, without major adverse effects. Nevertheless, due to the small number of conducted trials, further studies are required to investigate its efficacy.

INTRODUCTION

Since 1970s, platelet-rich plasma (PRP) has received significant attention as an application for tissue repair and hemostasis during the healing process of ulcers and undermined wound surfaces.[12] PRP is an autologous concentration of platelets in concentrated plasma.[3] It is considered to be a rich source of autologous growth factors (GFs),[4] which appear to enhance angiogenesis, extracellular matrix remodeling, and cellular effects as cell proliferation and differentiation.[5] Since then, it has been investigated and used in numerous fields of medicine, such as orthopedics, oral and maxillofacial surgery, plastic surgery and also dermatology.[6789] Recently, there have been little peer-reviewed studies investigating the clinical results of PRP applications as an alopecia treatment and no review articles regarding PRP's mechanism of action on hair follicle (HF) despite its possible promising results. Objectives of this article are to present PRP and its potent mechanism of action in promoting hair growth and to evaluate its preparation methods and its therapeutic potential in noncicatrial alopecias in a systematic review.

PLATELET-RICH PLASMA

Platelet-rich plasma is defined as a volume of the plasma fraction of autologous blood with an above baseline platelet concentration (usually more than 1,000,000 platelets/μL).[101112] PRP's regenerative potential depends on the levels of released GFs.[1314] Alpha granules of platelets contain GFs, which upon activation, are responsible for the initiation and maintenance of the healing response.[61516] PRP is known to carry more than 20 GFs[5131417] and other protein molecules, such as adhesion molecules, chemokines, which interact to promote inflammation, cell proliferation, differentiation, and regeneration.[181920]

Growth factors and hair

Main GFs stored in α-granules of platelets are platelet-derived growth factor (PDGF), transforming growth factor-b (TGF-b) and vascular endothelial growth factor with their isoforms.[5] They bind to their respective receptors, acting in tissue angiogenesis and stimulating the healing and growth of new organic structures.[212223242526272829] GFs act in the bulge area of the follicle, where primitive stem cells of ectodermal origin are found, giving origin to epidermal cells and sebaceous glands. In matrix, germinative cells of mesenchymal origin are found at the dermal papilla (DP). Interaction between these two kinds of cells as well as with binding GFs (PDGF, TGF-b, and VEGF) activate the proliferative phase of the hair, giving rise to the future follicular unit.[9]

Various types of cells, such as endothelial cells and keratinocytes, produce PDGF, which is fundamental for cell growth and proliferation. In vitro, cytokines, which are proven to be positive and negative regulators of HF growth activity, modify the expression of PDGF isoforms in HFs.[23] Furthermore, PDGF induces and maintains anagen phase in mouse hair cycling.[30] PDGF signals are involved in both epidermis-follicle interaction and the dermal mesechyme interaction required for hair canal formation and the growth of dermal mesechyme.[31]

Androgen-inducible TGF-b1 from balding DP cells is an inhibitory paracrine mediator in and rogenetic alopecia.[3233] Thus, it has been well established that the progression of androgenetic alopecia (AGA) is associated with androgen and TGF-b1 levels.[34]

Vascular endothelial growth factor appears to be a major mediator of HF growth and cycling providing direct evidence that the improved follicle revascularization promotes hair growth.[35]

Insulin-like growth factor 1 (IGF-I) also appears to play a critical role in promoting hair growth.[223637] IGF-I, produced by DP cells acts on keratinocytes' IGF-I receptor, promoting hair growth through stimulation of the proliferation of keratinocytes in HFs.[3037] In several reports, IGF-I and IGF-II prevented the HF from developing catagen like status[38] and Sheong et al. showed that IGF-I has a significant effect on the rate of linear hair growth and extends the overall anagen phase.[39]

Mechanism of action and molecular signaling pathways

In spite of studies conducted so far, the precise mechanism by which PRP promotes hair growth has not been properly studied. Li et al. proved that activated PRP increased the proliferation of human DP cells through an increase in phosphorylation of extracellular signal-regulated kinases (ERK) and Akt.[7] Although ERK signaling contributes to the regulation of cell growth, Akt has anti-apoptotic effects in many cell types.[40] Activated PRP also increased levels of the anti-apoptotic protein Bcl-2, protecting cells from apoptosis.[741] Furthermore, activated PRP appeared to contribute to the formation of hair epithelium and the differentiation of stem cells into HF cells, through an upregulation of b-catenin, strongly expressed in the bulge region of the human anagen HF,[42] and to prolong anagen phase of hair cycle, through an increase in expression of fibroblast growth factor-7.[7] Apart from these, PRP increased proliferation of epidermal and HF bulge cells, revealed by an increase in Ki-67 (marker for cell proliferation) in AGA.[43] In alopecia areata (AA) too, an increase in Ki-67 was noted[44] and PRP appeared to act as a potent anti-inflammatory agent, suppressing release of inflammation cytokines.[45]

MATERIALS AND METHODS

The systematic review followed the CONsolidated Standards of Reporting Trials (CONSORT) 2010 Statement. Searching in international bibliography through PubMed/MEDLINE, ClinicalTrials.gov, Scopus, Cochrane Central Register of Controlled Trials and Google Scholar/Web of Science databases with publishing-time restriction till October 2014, we identified 57 articles. Keywords used were “PRP AND hair”, “PRP AND hair loss”, “PRP AND hair”, “PRP AND hair loss”, “PRP AND alopecia” and “PRP AND AGA”, “PRP fibrin AND hair”. No language restrictions were imposed. The study selection process and the exclusion criteria for trials are described in Figures 1 and 2. We included clinical trials with patients, males, and females, independently of alopecia type as well as animal model trials studying the effect of PRP on hair. We considered conference papers whenever available, but we excluded them since the information was not sufficiently accurate to evaluate the quality measurement. Finally, 14 articles were included in our study, from which one was a case-report. All included trials in the systematic review are presented in Table 1.

Figure 1

Flowchart for search and selection of publications for systematic review

Figure 2

Trials exclusion criteria in each database

Table 1

List of included trials according to CONSORT 2010 statement

Mode of preparation of platelet-rich plasma and treatment protocols

Platelet-rich plasma is produced using different methods for platelet concentration through centrifugation and cell separation.[53] There have been several commercially available systems.[36947484950] Differences are also observed in its activation means, with calcium chloride and calcium gluconate to be the most common.[54] As a result, different platelet and growth factor concentrations occur, hence leading to different outcomes in trials.[3111214]

In Table 2, mode of preparation, activators, time and g or rpm of centrifugation are presented in summary.

Table 2

Mode of preparation, activators, time and g or rpm of centrifugation of PRP

Furthermore, there is no consensus protocol regarding exact concentration, dosing parameters and depth of injections of PRP as well as frequency and number of required sessions [Table 3].

Table 3

Treatment protocols, evaluation methods and results

Systematic review-results

The main findings from the 14 selected articles are described in Table 3. For an overview of the most relevant characteristics of these studies, we reported in Table 1 the following information: First author and year, institutions' country and period of publication, study design, number of subjects enrolled, follow-up, level of evidence, according to Center for Evidence-Based Medicine, Oxford, and quality of studies, according to grading of recommendations assessments, development and evaluation (GRADE). In Table 3, apart from the outcomes, we also reported number and frequency of applications, description of the application and evaluation methods.

The first study was published in 2006, two in 2009 and all the other after 2011. Only four studies reported in detail the country and the period of the study setting.[44505254] The 14 articles included 12 clinical trials,[94344464748505152535455] one study with an in vivo and an in vitro model[7] and a case-report.[49] Four of the publicated studies recruited only males,[9434952] 50% recruited both males and females,[44474850515354] two of them do not mention the sex of patients[4655] while the animal study included only female mice.[7] Eleven papers report either the mean age or the age range of patients. The smallest number of patients was one patient as described in the case-report[49] and the largest was 64 subjects.[53]

Most of the controlled trials and the case report included a placebo group either of subjects or of the half-head scalp of subjects treated with normal saline. Kang et al. used as placebo placental extract, while both treatment and control groups were simultaneously treated with finasteride per os.[48] Three studies had both a positive and a negative control,[74447] while six trials did not have controls.[505152535455] In one trial, PRP was simultaneously applicated with scalp microneedling while the control group was treated only with microneedling.[46]

Seven authors referred to the extent of alopecia in patients with AGA using Norwood and Ludwig Scale for men and women respectively, and in patients with AA using SALT score.[43445051525354]

Platelet-rich plasma's clinical application modes included embedment of follicles in PRP before male baldness surgery,[9] injections (subcutaneous, intradermal, interfollicular),[74344474849505152535455] simultaneous application with microneedling[46] as well as simultaneous infusion with CD34+ cells.[48]

In most articles, the activation methods were not mentioned. Commonest activators were calcium gluconate, calcium chloride and calcium chloride with thrombin.[794344515254]

There is no standard protocol regarding the frequency of PRP applications. The duration of the studies and the follow-up also varied [Tables 1 and 3].

There is also no standard method for evaluating results. Global photographs were mostly used.[434748495052535455] Phototrichogram and dermoscopic images were also used.[43444748495254] Other methods included histologic examination, hair diameter measurements with micrometer, hair density index, hair pull test or manual counting.

We also considered potential risk of bias in studies, following the CONSORT 2010 Statement, including the presence of control group, placebo, randomization, blinding, intrapatient design (half-head study), sample size calculation, description of statistical methods, computer validation, information on safety and dropout.

Among all the articles considered, eight had a control group[79434446474849] and six of them used only saline (normal or phosphate buffered) as placebo.[7943444749] Two studies were randomized.[4344] All the others did not report randomization. Two studies were double-blind.[4344] None of the trials reported details on sample size calculation or study power. Finally, seven studies were considered of moderate quality, four of low quality and three of very low quality, according to GRADE [Table 1]. Eight from the included papers mentioned the statistical methods and/or statistical software used.[79434447484950515354] Information on side effects and safety of the PRP applications was described in 71, 42% of papers.[43444748495051525354] Two authors reported dropout.[5154]

Describing in detail the studies of moderate quality, Uebel et al. in 2006 documented an increase in graft survival of 2.4 follicular units/cm2 in patients whose grafts were stored in PRP for 15 min before implantation than in control areas (P < 0.001) on the opposite side of their scalps.[9] Despite the facts that the treated area was not clearly demarcated from the surrounding nontreated area, the study was not blind and evaluation methods included just hair counting, it was the first good-quality trial to propose the possible positive therapeutic effect of PRP on hair.

In another study, patients who underwent PRP with dalteparin and protamine microparticles (D/P MPs) sessions, which induce neovascularization, presented significant stimulation in hair crosssection, but not in hair number, comparing to patients who underwent PRP only or placebo sessions. PRP and D/P MPs and PRP facilitated hair growth but D/P MPs provided additional hair growth.[47] This study is characterized by excellent detailed protocol methodology. PRP was not activated as in previous studies.

Kang et al. injected CD34+ cell-containing PRP preparation in the scalps of 13 patients with AGA since CD34+ cells have an angiogenic potential, vital in hair growth, while they also injected interfollicular placental extract in 13 control patients. Three months after the first treatment as well as at 6 months, the patients presented significant clinical improvement in the mean number of hairs and mean hair thickness.[48] Phototrichogram, as well as pictures, were used for evaluation. Kang et al. did not give details about centrifugation.

Sclafani, after a series of three intradermal platelet-rich fibrin matrix injections, observed a significant increase in hair density of 47.4% ± 22.7% (P = 0.0031) at 2 months, of 106.4% ± 56.9% (P = 0.0277) at 3 months and of 75.1% ± 46.82% (P = 0.0606) at 6 months after the initial treatment. Patients who achieved <25% increase in HDI by 2 months after the initial treatment were more likely to have <25% improvement at 6 months after the initial treatment (100 vs. 16.7%, P = 0.0476).[51] This is a well-designed study with relative objective evaluation methods, but no controls.

Gkini et al. injected PRP in 20 patients, males and females, with AGA. Three months after the first treatment a significant increase in hair density was noted (170.70 ± 37.81, P < 0.001). At 6 months and at 1-year, hair density was also significantly increased, 156.25 ± 37.75 (P < 0.001) and 153.70 ± 39.92 (P < 0.001) respectively, comparing to that of baseline. Nevertheless, it was lower than that of 3 months. Patients were satisfied with a mean result rating of 7.1 on a scale of 1–10 without noting any remarkable adverse effects.[54] This is also a well-designed study, with statistical analysis, with relative objective evaluation methods, but no controls.

Cervelli et al. conducted a randomized, controlled, double-blind, half head study evaluating the effect of PRP injections on hair loss. At the end of the three cycles of treatment, the patients presented clinical improvement in the mean number of hairs and total hair density (P < 0.05). Microscopic evaluation showed the increase of epidermis thickness and of the number of HFs (P < 0.05) as well as an increase in blood vessels. An increase of Ki67 (+) keratinocytes was also reported (P < 0.05).[43] This study is characterized by excellent study design and protocol and includes many evaluation methods, subjective and objective.

Platelet-rich plasma was also studied in 45 male and female patients with AAAA. Patients treated with PRP had significantly increased hair regrowth, achieved significantly higher complete remission at 1-year, reported less relapses (31%) at 1-year and regrowth of fully pigmented hair (96%) compared to positive and negative controls.[44] This pilot study suggests that PRP may serve as a safe and effective treatment option in AA. Its importance is based on the fact that it was a randomized, double-blind, placebo-and active-controlled, half-head, parallel-group study. Nevertheless, there are not many details regarding method and preparation of PRP.

In these seven trials of moderate quality according to GRADE, main common deficit was the small sample. Six trials referred to AGA and one to AA treatment. Apart from Uebel et al., all the other authors proposed the use of PRP injections. All, except Takikawa et al., used activated PRP. Two did not have an intrapatient design.[4854] Regarding the frequency of applications, in three trials out of seven (42, 85%), three treatment sessions with an interval of 1-month were proposed.[434451] Each author proposed a different protocol for PRP preparation. Nevertheless, all of them reported a statistical significant increase in the mean number of hairs as well as in hair density. Concluding, PRP may serve as a potential treatment for hair loss for AGA, with encouraging results. The sole trial for AA proposed PRP use, as an alternative treatment option to triamcinolone. Nevertheless, further investigation is needed in both types of alopecia.

Apart from these studies, Betsi et al. also studied the efficacy of PRP injections in and rogenetic alopecia. This study, despite the large sample size of patients compared to that of other studies, lacks controls and objective evaluation methods.[50]

Khatu et al. injected PRP in male patients with AGA. This study, despite the objective evaluation methods, lacks controls and statistical analysis of the results.[52]

Furthermore, Sciavone et al. injected leukocyte PRP (L-PRP) with the addition of concentrated plasmatic proteins in patients with AGA. Despite the large sample size, this study lacks controls and objective evaluation methods (interpretation of results using Jaeschke scale).[53]

Greco et al. proposed the efficacy of simultaneous application of PRP with micro needling for AGA treatment.[46] Finally, articles of very low quality, according to GRADE, described the use of PRP injections in AA[55] and the use of PRP injections in a patient with adverse effects from the treatment with minoxidil and finasteride.[49] The in vivo model of Li et al., despite the high quality of the in vitro model, lacked evaluation methods.[7]

DISCUSSION

Despite the growing interest in regenerative medicine, few trials investigating PRP's efficacy on hair growth have been published. Most of the reviewed studies had important methodological deficiencies. Main flaws included lack of approved scientific devices for PRP preparation, lack of a reference protocol regarding the frequency of applications as well as the injected amount of PRP, heterogeneity in application modes, lack of controls, small sample size, lack of detailed reports in patients’ characteristics and used statistical methods. Furthermore, few studied referred to the safety profile of PRP.

Randomized controlled trials are considered to be the golden standard for proving the efficacy of a treatment, avoiding potential bias in the efficacy assessment. The use of blind or double-blind experiments and placebo are other strategies that improve the quality of the trials. The use of intra-patient design is an efficient strategy to reduce the number of volunteers in a trial, since it is reasonable to assume that the intra-subject variation is limited. For these reasons, the adoption of high-quality trial design studies, that is, placebo-controlled double-blind studies with randomization or intra-patient design, is strictly encouraged for testing the efficacy of PRP on hair loss.

The most important obstacles in trials evaluating hair growth are the absence of standard, reliable and objective noninvasive methods to evaluate hair loss as well as the results after PRP treatment. Global photographs as well as phototrichogram are the commonest evaluation methods. Global photographs give an overall picture of the treatment. Phototrichogram, in order to be performed in a strictly standardized manner with reproducible and comparable results, should be performed on a shaven part of the patients’ scalp, which is not easily applicable in patients, especially women. Other evaluation methods included hair pull test, dermoscopic photographs and hair counting with a magnifying glass, which are not reproducible and objective as they depend on doctor's ability and precision. Measuring hair diameter with a micrometer is also a subjective method as it depends on the choice of hairs. Nevertheless, an adequate means of measuring hair growth in the clinic over time in a reproducible, economical and noninvasive manner is not available, and all the above methods give an enlightening assessment of the results after treatment.

Among the 14 studies included in the systematic review, there were not sufficient information to conduct a meta-analysis for the overall quantification of hair growth. Platelet-rich plasma could be a possible useful treatment for noncicatricial alopecias. Nevertheless, it is still a highly controversial form of therapy. Larger, randomized blind, controlled trials, with approved devices for PRP preparation, are urgently needed as well as evidence-based data regarding concentration, dosing parameters and depth of injection to study its clinical efficacy. The safety profile of PRP should be reported in all these trials with a description of adverse effects, even if they are negligible.

CONCLUSION

This article describes in detail PRP and its possible mechanism of action in promoting hair growth. Furthermore, it is the first systematic evaluation of PRP's efficacy in hair loss treatment, following a rigorous methodological approach proposed by the CONSORT 2010 statement.