Platelet Rich Plasma and Its Use in Androgenetic Alopecia and Alopecia Areata: A Systematic Review

Transcription

1 Platelet Rich Plasma and Its Use in Androgenetic Alopecia and Alopecia Areata: A Systematic Review MD. Scarlett Alvarado Carvajal March 2017 Barcelona, Spain

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3 INDEX Abstract 4 Introduction 6 Objectives 23 Materials and Methods 24 Results 28 Discussion 35 Conclusions 39 Thanks 40 Bibliography 41 3

4 Abstract Non cicatricial alopecias have an important negative effect on patients who suffer these diseases. Alopecia Areata is an autoinmune disorder while Androgenetic Alopecia is an androgen dependant condition. During the years, different treatments have emerged, however the high costs of some of theses treatments and low patient compliance make it difficult to get a complete remission. Lately, the trend in therapies and clinical studies seems now to focus with a new, more biologically oriented approach to male and female pattern hair loss: Platelet-Rich Plasma. The hypothesis is that growth factors released from platelets may act on stem cells in the bulge area of the follicles, stimulating the development of new follicles. The aim of this review is to study and analyze Platelet-Rich Plasma as an effective therapeutic method for Alopecia Areata and Androgenetic Alopecia. In order to do so, a research was realized in pages like PubMed, Medscape, Google, F1000 or Embase and a selection of 38 articles was made. The study found that growth factors act in the bulge area, where they interact with cells of the matrix, thus activating the proliferative phase of the hair. Some growth factors as the IGF-I have an effect on the rate of linear hair growth and extend the overall anagen phase. Therefore, it can be considered a useful tool for androgenetic alopecia treatment. On the other hand, PRP is also a potent anti-inflammatory agent, which can suppress cytokine release and thereby limit local tissue inflammation, making it helpful in alopecia areata. However, this study concludes that information on Platelet Rich Plasma on non-cicatricial alopecias is still needed in order to extrapolate results and safely recognize that PRP is effective in these two conditions. Resumen Las alopecias no cicatriciales tienen un importante efecto negativo en los pacientes que sufren estas enfermedades. La Alopecia Areata es un trastorno autoinmune mientras que la Alopecia Androgenética es una condición dependiente de andrógenos. Durante los años, diferentes tratamientos han surgido, sin embargo los altos costos de algunos de ellos y la baja adherencia al tratamiento por parte del paciente hacen que sea difícil obtener una remisión completa. Últimamente, la tendencia en terapias y estudios clínicos parece centrarse ahora en un enfoque nuevo, con una orientación mas "biológica" para la pérdida de cabello de patrón masculino y femenino: esto sería el 4

5 Plasma Rico en Plaquetas. La hipótesis es que los factores de crecimiento liberados de las plaquetas pueden actuar sobre las células madre en el área de bulbo folicular, estimulando el desarrollo de nuevos folículos. El objetivo de esta revisión es estudiar y analizar el Plasma Rico en Plaquetas como un método terapéutico eficaz para Alopecia Areata y Alopecia Androgenética. Para ello, se realizó una investigación en páginas como PubMed, Medscape, Google, F1000 o Embase y se realizó una selección de 38 artículos. El estudio encontró que los factores de crecimiento actúan en el área del bulbo, donde interactúan con las células de la matriz, activando así la fase proliferativa del cabello. Algunos factores de crecimiento como el IGF-I tienen un efecto sobre la tasa de crecimiento lineal del cabello y extiende la fase anágena total. Por lo tanto, puede considerarse una herramienta útil para el tratamiento de la alopecia androgenética. Por otro lado, el PRP es también un potente agente anti-inflamatorio, que puede suprimir la liberación de citoquinas y por lo tanto limitar la inflamación local del tejido, por lo que es útil en la alopecia areata. Sin embargo, este estudio concluye que la mas información sobre Plasma Rico en Plaquetas sobre las alopecias no cicatriciales sigue siendo necesaria para extrapolar los resultados y reconocer con seguridad que el PRP es eficaz en estas dos condiciones. 5

6 INTRODUCTION Background on Platelets A typical blood specimen comprises 93% red blood cells, 6% platelets, and 1% white blood cells. Platelets were first seen in the blood by French physician Alfred Donné in (1) Platelets are cytoplasmic fragments of megakaryocytes, formed in the marrow; they are small discoid cells (approximately 2μm in diameter) with a life span of about 7 to 10 days. (1,2) Platelets are responsible for hemostasis, construction of new connective tissue and revascularization and most of the research over the past century has been focused on this primary function. (1,3) Only in the past two decades have we learned that platelet activation in the body releases healing proteins called growth factors. (1) Wound regeneration commonly begins with clot formation and platelet degranulation, leading to the release of multifarious cytokines and coagulation factors, which modulate inflammatory response. (4,5) Platelets are activated by contact with collagen, exposed to the bloodstream after endothelial injury. (3) When they are activated, they release their stored intercellular mediators and cytokines from the cytoplasmic pool and release their granules containing growth factors that stimulate the inflammatory cascade and healing process. (1,2,3, 6) Three major storage compartments in platelets are alpha granules, dense granules, and lysosomes. (7) Within 10 minutes of blood coagulation, platelets release a burst of proteins from their α, δ, and λ granules. (2,4) The majority of the platelet substances are contained in alpha granules (7) More than 95% of the pre-synthesized growth factors are secreted within the first hour and platelets continue synthesizing more cytokines and growth factors from their mrna reserves for at least another 7 days. (2,3,4,7) More than 800 different proteins are secreted into the surrounding media, having a paracrine effect on different cell types: myocytes, tendon cells, mesenchymal stem cells from different origins, chondrocytes, osteoblasts, fibroblasts and endothelial, but cumulatively they may accelerate tissue and wound healing. (1,2,3,6) This further induces an internal signal-transduction pathway, unlocking the expression of a normal gene sequence of a cell like cellular proliferation, matrix formation, osteoid production, collagen synthesis, etc., thereby augmenting the natural wound-healing process. (3) 6

7 Background on Platelet-Rich Plasma Platelet-rich plasma (PRP) means "abundant platelets that are concentrated into a small volume of plasma". (8) Platelet-rich plasma (PRP) is defined as an autologous preparation from whole blood (WB), in which platelets are concentrated in a small fraction of plasma. (1,9,10,11,12) This broad definition is considered to be the consensus definition by the International Olympic Committee in sports medicine. (11) It is an emerging treatment in the modern health sector known as 'orthobiologics'. The goal of this discipline is to enhance the body's innate ability to repair and regenerate. (1) It has been investigated and used in numerous fields of medicine. Recently, PRP has received growing attention as a potential therapeutic tool for hair loss. (12) Platelet-derived products include platelet-rich plasma (PRP), which can be used with or without previous platelet activation. Such preparations have been used since the 1970s and they have been increasingly popular since the 1990s. (3) The ideology behind PRP treatment is the reversal of the red blood cell:platelet ratio by decreasing red blood cells to 5% (which are less useful in the healing process) and more importantly concentrating platelets containing a powerful concoction of growth factors to 94%. (1) Although the optimal PRP platelet concentration is unclear, the current methods by which PRP is pre- pared involve reported 300% to 700% enrichment, with platelet concentrations consequently increasing to more than 1,000,000 platelets/ll. (13) Marx proposed that platelet count of 10 lakh/ml in 5 ml of PRP, as a working definition of PRP, based on the scientific proof of bone and soft tissue healing enhancement. (2) A normal platelet count in a healthy individual is between 150,000 and 450,000 cells per microliter of blood. (1,3) There is a scarcity of studies stating the concentration at which optimal stimulation occurs. (2) Rughetti et al found that the stimulation for proliferation of endothelial cells peak at and angiogenesis at platelets/ml, respectively. (2) Further, a bell-shaped response curve indicating a dose dependant nature has been shown to be associated with PRP. (3) Platelet concentrations of less than 1, /ml were not reliable for enhancing wound healing, and most studies have suggested that tissue reparative efficacy with PRP can be expected with a minimum increase of five times the normal concentration of platelets (approximately 1 7

8 million platelets/μl) (1,2,3), whereas much higher concentrations did not show further enhancement of wound healing. The ideal concentration remains to be defined. (1) Its action mainly depends on the released growth factors from platelets. (12,14) Platelet concentrates contain many powerful mitogenic and chemotactic growth factors, which regulate key processes involved in tissue repair, including cell proliferation, chemotaxis, migration, cellular differentiation, and extracellular matrix synthesis. (5,14) The concentrations of these various growth factors have been shown to be increased 3 to 7 times in autologous PRP. (4,5) Growth factors contained in their alpha granules, are transforming growth factor-beta (TGF-β), fibroblast growth factor-2 (FGF- 2), platelet-derived growth factors (PDGF-AB), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF), which play an important role in tissue healing. 10) (1, 8, 9, Seven fundamental protein growth factors that are actively secreted by platelet initiate all wound healing process [Table 1]. (2) Table 1 Platelet growth factors and their specific functions (2) 8

9 Insulin-like growth factor-1 plays a role in the metabolism of numerous cell types. It is chemotactic for fibroblasts and enhances the synthesis of proteins. (4) PDGF was discovered in 1974 and is ubiquitous in the body. (7) Various types of cells, such as endothelial cells and keratinocytes, produce PDGF, which is fundamental for cell growth and proliferation. (7,14) The principal activities of PDGF include angiogenesis, macrophage activation, proliferative activity on fibroblasts, and chemotaxis for fibroblasts and collagen synthesis. (4,7) TGFβ is a bone morphogenetic protein. It is a key factor in the promotion of the proliferative activity of fibroblasts, as well as the synthesis of type-i collagen and fibronectin. (4) It is important to note that growth factors are not the only elements present in significant concentrations in PRP (4) The proteins that act to promote cell adhesion (fibrin, fibronectin, and vitronectin) are another essential component of PRP. (5,15) These provide the structural support necessary for cell migration and for proliferation and 3- dimensional growth of tissues over these structures. Therefore, PRP has effects not only directly related to the target cells for the various growth factors but also has a broader effect as an extracellular matrix for the stimulation and repair and/or regeneration of the tissue. (15) Platelets in PRP also play a role in host defense mechanism at the wound site by producing signaling proteins that attract macrophages; PRP also may contain a small number of leukocytes that synthesize interleukins as part of a non-specific immune response. (5) Classification The literature on PRP is considerable, but the published results are often contradictory. It is very difficult to sort and interpret the available data, due to a large number of preparation techniques, terminologies, forms of these materials, and the endless list of potential applications. (2) The development of a wide range of preparation protocols, devices and centrifuges for varying indications have led to a number of different platelet, leukocyte, and growth factor concentrations. (8,15) As a result, the nomenclature for PRP products 9

10 makes reference to the different fractions that can be obtained according to the method used. (15) According to the classification proposed by Ehrenfest et al. (2009), four main families of preparations can be defined, depending on their cell content and fibrin architecture: (2,8) 1. Pure Platelet-Rich Plasma (P-PRP) or leucocyte-poor PRP products are preparations without leucocytes and with a low-density fibrin network after activation. (2) The P-PRP concentrate consists of an undetermined fraction of buffy coat, containing a large number of platelets, but most leucocytes are not collected. After the first slow spin centrifugation, only the superficial buffy coat layer is pipetted out and prepared for next centrifugation, as shown in [Figure 1]. (8) Figure 1 Flowchart illustrating preparation of activated PRP (8) 10

11 2. Leucocyte- and PRP (L-PRP) consists of most of the platelets, along with leucocytes and some residual RBCs, suspended in fibrin-rich plasma. It differs from P-PRP only on the means of buffy coat layer collection in which the Platelet Poor Plasma (PPP) along with the entire buffy coat layer and superficial 1-2 mm layer of RBCs are pipetted out. (8) It is in this family that the largest number of commercial or experimental systems exists. Particularly, many automated protocols have been developed in the last years, requiring the use of specific kits that allow minimum handling of the blood samples and maximum standardization of the preparations. (2) There is currently disagreement in the literature over whether the presence of WBC in PRP provides any benefit. Proponents of L-PRP believe that the presence of WBC provides natural protection against infections and allergic responses. (10) Cieslik-Bielecka and colleagues were the first to report the application of PLRP in an infected highenergy soft tissue injury and indicated that the volume and concentration of platelets and leukocytes were adequate to induce the healing process despite concurrent infection. (1) Other authors do not recommend the presence of high WBC concentration because the presence of neutrophils, which are 65% of WBC and more than 95% of granulocytes, may be harmful as they may destroy surrounding tissue, even if the tissue is not injured. (10) 3. Pure platelet-rich fibrin (P-PRF) or leucocyte-poor platelet-rich fibrin: The term PRF is used synonymously with platelet-rich fibrin matrix (PRFM). (8) These preparations are without leucocytes and with a high-density fibrin network. (2) When P-PRP is mixed with activator and allowed to incubate for some time, a stable PRFM clot can be collected which has useful applications. (8) These products only exist in a strongly activated gel form, and cannot be injected or used like traditional fibrin glues. (2) Very low amounts of leucocytes are collected owing to a specific separator gel used in the device. (8) 4. Leucocyte- and platelet-rich fibrin (L-PRF) or second-generation PRP products are preparations with leucocytes and with a high-density fibrin network. (2) Here, blood is collected without any anticoagulant and immediately centrifuged. A natural coagulation process then occurs and three layers are formed: the RBC base layer, 11

12 acellular plasma top layer and L-PRF clot in the middle, which harvests platelet and leucocyte growth factors into the fibrin matrix. When pressed between two gauzes, the PRF clot becomes a strong membrane, which also has potential applications. (8) This classification system was largely cited, advocated, and validated by a multidisciplinary consensus conference published in (2) The importance of having such a classification is to better understand the relative success or failure of different PRP preparations. Additionally, this can establish an objective approach for future development and research. (1) Preparation of PRP Plasma with a platelet concentration of approximately 1,407,640 μl, or 5 times greater than the platelet count in normal blood, is the suggested working concentration for PRP therapy. (4) The PRP can be prepared by aphaeresis or can be separated from fresh anti-coagulated blood by simple centrifugation, which concentrates platelets up to six times the baseline count in whole blood (5) Different ways of preparing PRP have emerged: from conventional blood centrifugation to commercial systems; activated by adding collagen, calcium and/or thrombin, by glass contact or by freezing cycles; and the methodology continues to broaden. (3) As it is a source of growth factors, it can be used, in both liquid and gel forms, to promote the regeneration of damaged tissues. (9) PRP quality and efficiency is highly dependent on the protocol used for its preparation. (11) The authors employ a specialized table top cold centrifuge device. (2) The platelets need to be sequestered in high concentrations, enough for achieving therapeutic benefit and in a viable state at the same time, so that they can actively secrete their GFs. (8) Variables in the process that may influence the platelet integrity along with the composition and effectiveness of the PRP include the number of spins, centrifugal acceleration, and time period of centrifugation. (10) In general, PRP preparation is a sequential three-step process that involves blood collection, centrifugation to separate and concentrate the platelets, and activation of the 12

13 platelets by adding a platelet agonist. (10,11) PRP can only be made from anticoagulated blood. (7) PRP is obtained from a sample of patients blood drawn at the time of treatment. A 30 cc venous blood draw will yield 3-5 cc of PRP depending on the baseline platelet count of an individual, the device used, and the technique employed. (2) Preparation of PRP begins by addition of citrate to whole blood to bind the ionized calcium and inhibit the clotting cascade. One or two centrifugation steps follow this. The first centrifugation step separates the red and white blood cells from plasma and platelets. The second centrifugation step further concentrates the platelets, producing the PRP separate from platelet-poor plasma. (7) The process must be carried out under strict aseptic conditions as well as optimum temperature regulations i.e., C. (8) An important point is that clotting leads to platelet activation, resulting in release of the GF from the alpha granules, otherwise known as degranulation. A method to delay the release of GF is possible by addition of calcium chloride (CaCl2) to initiate the formation of autogenous thrombin from prothrombin. (7) There is no consensus on whether or not platelets must be previously activated before their application and with which agonist. Some authors activate platelets with thrombin or calcium, whereas others apply platelets without being previously activated, arguing that better results are obtained. (2) Platelets are activated either by adhesion to the molecules that are exposed on an injured endothelium, such as von Willebrand Factor (vwf), collagen, fibronectin, and laminin, or by physiologic agonists such as thrombin, ADP, collagen, thromboxane A2, epinephrine, and platelet-activating factors (7) Conventionally, bovine thrombin and calcium have been used for platelet activation. Once activated, the resulting PRP mixture must be injected immediately, as the subsequent secretion of the granule contents occurs rapidly. Activation via type-i collagen produces a less rapid release of the granule contents, which enable a delayed administration of PRP. (4) There is also a lack of standardization in the use of PRP and, therefore, generation of readily reproducible scientific evidence is hindered. In this context, the Spanish Agency for Medicines and Health Products (AEMPS) issued a report in May 2013 with the aim of establishing a framework for the use of PRP in Spain, the obligations of the manufacturers, and the information that the patients treated with PRP 13

14 should receive. This document recognizes PRP as a drug product for human use. (15) Principles of Preparation For the preparation of PRP, blood collection must be performed without trauma to the vessel wall to ensure the integrity of the platelets. Centrifugation is the first step in PRP preparation, which requires the recovery of a large number of intact platelets. (11) Centrifugation is one of the most widely used processes in liquid-liquid or solid-liquid separation. (10) It is a process known as differential centrifugation, in which acceleration force is adjusted to sediment certain cellular constituents based on different specific gravity. (8,10) The difference in size and density of the particles in the various phases is the driving force responsible for the separation. In the case of WB, the centrifugal force and time drive the packing of erythrocytes at the bottom layer, the volume of plasma at the upper layer, and the recovery efficiency of platelets. (10) Thus, both platelet activation and the final properties of the PRP preparation are influenced by the centrifugation step. (11) There are many ways of preparing PRP. It can be prepared by the PRP method or by the buffy-coat method. (2) Procedure (2,8,15) PRP Method: 1. Obtain WB by venipuncture in acid citrate dextrose (ACD) tubes 2. Do not chill the blood at any time before or during platelet separation. 3. Centrifuge the blood using a soft spin. 4. Transfer the supernatant plasma containing platelets into another sterile tube (without anticoagulant). 5. Centrifuge tube at a higher speed (a hard spin) to obtain a platelet concentrate. 6. The lower 1/3rd is PRP and upper 2/3rd is platelet-poor plasma (PPP). At the bottom 14

15 of the tube, platelet pellets are formed. 7. Remove PPP and suspend the platelet pellets in a minimum quantity of plasma (2-4 ml) by gently shaking the tube. Buffy coat method: 1. WB should be stored at 20 C to 24 C before centrifugation. 2. Centrifuge WB at a high speed. 3. Three layers are formed because of its density: The bottom layer consisting of RBCs, the middle layer consisting of platelets and WBCs and the top PPP layer. 4. Remove supernatant plasma from the top of the container. 5. Transfer the buffy-coat layer to another sterile tube. 6. Centrifuge at low speed to separate WBCs or use leucocyte filtration filter. The double spin method is preferred over the earlier prevalent single spin method, as the therapeutic concentration of platelets was not achieved by the latter. The active secretion of prepackaged GFs begins within 10 minutes of clot initiation and 95% of the secretion is completed within 1 hour. Hence, PRP must be used on the treated site within 10 minutes of activation. The concentrated platelets remain viable for up to 8 hours and sterile if placed on a sterile surgical table. (8) They are difficult to be used in therapeutic uses for their regenerative ability by outpatient doctors in different indications, which need repeated sessions. (9) With regard to the preparation conditions, the AEMPS has established some minimal quality requirements that have to be met by the prescribing physicians. Sterility test and product traceability are considered mandatory, and patient follow-up is necessary. (15) Commercially Available Kits There are many PRP systems commercially marketed, which facilitate the preparation of ready to apply platelet-rich suspensions in a reproducible manner. All operate on a small volume of drawn blood (20-60 ml) and on the principle of 15

16 centrifugation. These systems differ widely in their ability to collect and concentrate platelets depending on the method and time of its centrifugation. As a result, suspensions of different concentration of platelets and leucocytes are obtained. Differences in the concentrations in platelets and WBCs influence the diversity of growth factors concentration. (2) Different in vitro assays have been developed to establish the cell and molecular content of the different commercial systems. Of note among the results obtained are the substantial interindividual variability and the lack of proportionality between platelet concentration and quantity or growth factors obtained with different methods. The clinical repercussions of these differences have still not been determined. (15) PRP devices can be usually divided into lower (2.5-3 times baseline concentration) and higher (5-9 times baseline concentration) systems. The high-yielding devices include Biomet GPS II and III (platelet count 3-8 ); Harvest Smart Prep 2 APC+ (4-6 ); ArterioCyte-Medtronic Magellan (3-7 ). The lower concentration systems include Arthrex ACP (2-3 ), Cascade PPR therapy (1-1.5 ), and PRGF by Biotech Institute Vitoria, Spain (2-3 ).Regen PRP (Regen Laboratory, Mollens, Switzerland).. When a commercial kit is used, the manufacturer instructions should be followed and a prior request is not necessary. The kit should, however, have the CE conformity mark granted for the specified use. (15) Safety of PRP The myogenic effects of PRP are only limited to augmentation of the normal healing process and is theoretically not mutagenic, as the GFs released do not enter the cell or its nucleus, but only bind to the membrane receptors and induce signal transduction mechanisms (8). Growth factors, after binding to membrane receptors, activate intracellular signaling cascades that promote normal genetic expression regulated by different control mechanisms. To date no systemic effect has been demonstrated of growth factors released after local application of PRP. (15) Adverse effects are rare but, as with any injection, there is always a small risk of injection site 16

17 morbidity, infection or injury to nerves or blood vessels. Scar tissue formation and calcification at the injection site have been reported. (1) Given the autologous nature of PRP, it is considered a safe product that by definition lacks the potential risk of disease transmission implicit in the use of blood products from donors. (1,8,15) Based on the pioneering and long-term clinical experience of the oral-maxillary field with PRP, and thousands of patients being treated so far, the use of PRP is considered to be safe. (1) However, safety concerns with bovine thrombin have been raised about the potential transmission of Cruetzfeld-Jacob disease (mad-cow disease). (1,8,15) These have been refuted by some stating that the prion vector has been found only in the neural tissues of cattle, whereas thrombin is solely isolated from the blood and is also further processed by heating. (8) However, the systems that use bovine thrombin as activator are being phased out to avoid the development of coagulation disorders or secondary hypersensitivity. (15) On the other hand, use of CaCl 2 as an activator, automatically eliminates the above risks. (8) Rarely, development of antibodies against clotting factors V and IX leading to life-threatening coagulopathies have been reported. (1,8) To date, there is no compelling evidence of any systemic effect of a local PRP injection. (1) 17

18 Hair Cycle The hair follicle is a complex structure in constant activity and possesses a unique ability to regenerate itself throughout the life of the organism. (16,17,18) The mature follicle undergoes successive transformation from anagen (active hair shaft production) to catagen (apoptosis-driven regression) to telogen (resting phase with the involution of hair follicle). (17,18,19,20) The hair cycle has well-orchestrated kinetics regulated by interactions between mesenchymal and epithelial cells, although the intracellular signals remain unclear. (19) The starting phase is the anagen (or growth) phase. (16) Anagen-associated angiogenesis has been suggested as one of the important factors in active hair growth, due to the secretion of vascular endothelial growth factor (VEGF) by the keratinocytes of the outer root sheath and fibroblasts of the dermal papilla. (21) Cell division in the matrix of the hair bulb produces keratinocytes, which form the various layers of the individual hair shaft. Melanocytes in the hair bulb provide the keratinocytes with pigment. (16) Many growth factors play a fundamental role in the life-long cyclic transformation of the hair follicle functioning as biologic switches that are turned on and off during the different phases, controlling the active phase and promoting apoptosis to induce catagen and telogen. (18) After the hair reaches a certain length, which varies depending on body region, it enters the catagen (or degradation) phase, when cell division and pigmentation cease. (16) It starts in the melanogenic area (late anagen), propagates to the array (premature catagen) and then to the internal and external root sheath. An imbalance between various growth factors and cytokines, which maintain the anagen phase and promote apoptosis, can determine the beginning of the catagen phase. The diffuse apoptosis of the follicular keratinocytes leads to the follicular regression observed in this phase. (17) During the telogen (or resting) phase, a new follicle appears below the original follicle, the hair shaft grows back into the original follicle, and the previous hair shaft is shed. (16,17) It is unclear whether the hair is lost simply as a result of the new hair shaft's upward trajectory or whether there is a separate active shedding (exogen) stage. (16) The biological cycle of the hair follicles is not synchronized among the adjacent units, assuming a mosaic pattern in the scalp. (17) Studies on human head hair growth 18

19 have shown that at any point in time, most (80% to 90%) of the hairs are in the anagen phase, with 1% to 2% in the catagen phase and the remaining 10% to 20% in the telogen phase. (16,17) Normally, the anagen phase lasts between 2 and 8 years; the catagen phase last between 2 and 3 weeks, and the telogen phases lasts about 3 months. (17) Alopecia Etymologically, the word "alopecia" comes from the Greek άλώπηξ (alōpēx), which means "fox" and it is an allusion to the constant hair loss suffered by these animals during life. (17) Hair loss (alopecia) is a complex phenomenon that is not fully understood, it can occur as the result of a congenital or genetic disorder, or it can develop during lifetime. (16) Hair follicles are unique structures with exceptional regenerative potential. (18,23) They are believed to be crucial for epidermal homeostasis and reepithelialization after damage to human skin. Like other, more active and quickly proliferating organ systems, hair follicles may be easily disturbed in their normal growth cycle by systemic and local influences, including specific skin diseases. (24) Dysfunctions during the telogen or anagen stages appear to result in hair loss. Some forms of hair loss are associated with excessive shedding of hair, a prolongation of the telogen phase commonly referred to as telogen effluvium. (16) There are different causes of alopecia; some are natural occurring changes to severe immune diseases. Some examples are as the seen below: (16) Naturally occurring hair loss. Seasonal variation. Aging. Nutritional factors. Hormonal imbalances or changes. Mutations of the hairless gene. Autoimmune hair disorders. 19

20 This review will be centered in two of this causes, hormonal imbalances and autoimmune hair disorders. More specifically, we will be talking about Androgenetic Alopecia and Alopecia areata. Alopecia areata universalis is an autoimmune disease caused by a peri- and intrafollicular infiltration of T lymphocytes and macrophages, thereby leading to defective anagen-stage hair shafts and miniaturization of the follicles. Similar anagen dysfunction appears to underlie the best-known form of hair loss in humans, androgenetic alopecia (male pattern baldness). In this case, the effects of dihydrotestosterone on genetically susceptible hair follicles presumably cause a dysfunctional anagen stage and miniaturization of the follicles. (16) Alopecia Areata (AA) Alopecia areata is a common, clinically heterogeneous, immune-mediated, nonscarring hair loss disorder. (22) It has a lifetime prevalence of approximately 2%. (23) The typical pattern involves the development of only a few patches that disappear very slowly as a result of regrowth from the periphery. (16) The aethiopathogenesis of alopecia areata is unclear and many factors including autoimmunity, genetic predisposition, emotional and environmental stress are thought to play important roles in its development. (22,24) Diagnosis can often be made clinically, based on the characteristic nonscarring, circular areas of hair loss, with small hairs at the periphery in those with early stages of the condition. The diagnosis of more complex cases or unusual presentations can be facilitated by biopsy and histologic examination. (23) The exact cause of Alopecia areata is unknown but it is characterized by a dysfunctional immune response, in which the hair follicle is invaded by lymphocytes (CD8+ T cells) and macrophages that target specific antigens of the anagen hair follicle. (16,23,25) Although spontaneous resolution and regrowth of the patch might occur in up to 50% to 80% of individuals in certain subgroups, it is also possible for additional patches to form, resulting in multiple areas of hair loss coalescing into a larger lesion or eventually involving the entire scalp, which is known as alopecia totalis, or it may eventually involve the entire body (alopecia universalis). (22,23,24,25) Other patterns of AA include the acute diffuse type, in which there is sudden hair loss all over the scalp, or 20

21 the ophiasis pattern, in which hair is lost on the posterior and lateral aspects of the scalp. (23) This disease has very limited treatment possibilities, and no treatment is either curative or preventive. (26) Since it is an autoimmune disease, therapies are mostly immunosuppressive; however, it is important to find new therapies and improve the effectiveness of the existing conditions. (13) Androgenetic Alopecia (AGA) Androgenetic alopecia (AGA) is a hereditary, androgen-dependent dermatological disorder more common in men (occasionally women), with significant negative impact on their social and psychological well-being. (18,27) By the age of 30, 30% of white men have androgenetic alopecia; by the age of 50, 50% do. (27,28) White men are four times more likely to than black men develop premature balding. (28) AGA is a progressive thinning of the scalp hair characterized a distinctive and reproducible pattern from the scalp. (12,1827,29) Bitemporal recession affects 98.6% of men and 64.4% of women, whereas mid-frontal hair loss affects nearly two thirds of women over the age of 80 years, and three quarters of men over 80 years have midfrontal and vertex hair loss. (29) In some men the loss over the vertex occurs more rapidly than the frontal loss; in others the entire frontal hairline marches back before a bald patch on the vertex develops. Less commonly, men bald in a Ludwig-type pattern, with preservation of their frontal hairline. (28) Diffuse hair thinning and sometimes increased hair shedding precedes the clinical appearance of baldness by a number of years. This is because the process of follicular miniaturization, which occurs in AGA, does not simultaneously affect all follicles within a follicular unit (FU). (29) Its etiopathogenesis is mainly androgen-dependent and modulated via the testosterone metabolite dihydrotestosterone (DHT), the expression of hair folliclerelated androgen receptor (AR); and genetic factors also have been implicated. (18,27) DHT-dependent cell functions depend on the availability of weak androgens, their 21

22 conversion to more potent androgens via the action of five alpha-reductase, low enzymatic activity of androgen inactivating enzymes, and functionally active AR present in high numbers. The predisposed scalp exhibits high levels of DHT and increased expression of the AR. (27,29) Conversion of testosterone to DHT within the dermal papilla plays a central role, while androgen-regulated factors deriving from dermal papilla cells are believed to influence the growth of other components of the hair follicle. (27) AGA is associated with a significant amount of psychosocial distress both in men and women. The angiogenic role of PRP has recently caught the attention of dermatologists and plastic surgeons, to explore its usefulness as a hair growth modality. (2) 22

23 OBJECTIVES Primary Objective Review, study and analyze Platelet-Rich Plasma as an effective therapeutic method for Alopecia Areata and Androgenetic Alopecia. Secondary Objectives Describe the physiopathology in Alopecia Areata and Androgenetic Alopecia. Research and review trials to evaluate the efficacy of PRP as treatment in noncicatricial alopecias. 23

24 MATERIALS AND METHODS A search of articles related to the subject of the present work was made from January 10, 2015 to November 22, The research was realized in pages like PubMed, Medscape, Google, F1000 or Embase. The following keywords and mixtures were used to search for articles: Alopecia Platelet Rich Plasma Alopecia Areata Androgenetic Alopecia Platelet Rich Plasma and Alopecia Platelet Rich Plasma and Hair growth Alopecia and growth factors This review focused mainly on articles whose main theme were Alopecia Areata, Androgenetic Alopecia and/or Platelet Rich Plasma. The articles used have publication dates that go from 1998 to Articles that had as main theme other pathologies although mentioning PRP as a treatment were discarded as well as articles in which the type of alopecia talking about was not areata or androgenetic. The idea was to obtain articles whose main idea was to talk about these three main concepts in order to speed up the review. Due to the small amount of studies made on this matter, all articles with studies that met the previous criteria were included, regardless of whether they were performed on humans or any other mammal. 24

25 The basic criteria used in this review is summarized as following: Inclusion Criteria Exclusion Criteria Alopecia Any other disease or medicine branch (traumatology, plastic surgery, etc.) Alopecia areata or androgenetic alopecia Any other type or disease that involves alopecia Platelet Rich Plasma Minoxidil, finasteride or any other treatment for alopecia All studies done on any mammal In vivo In vitro A total of 1,051 results were obtained, of which the following were discarded because they didn t meet the criteria required to be included in this review: 193 articles were about PRP on acne scarring or other dermatologic uses. 144 articles talked about alopecia having other treatments like finasteride. 102 articles had other diseases that involve other types of alopecia as their main topic. 269 articles talked about PRP on plastic surgery. 298 articles were about PRP used in traumatology. 8 articles were repeated. 21 articles were discarded as they were inconclusive, errors in the translation or gave a very detailed biochemical view of the PRP in vitro 25

26 Article Level of Scientific Evidence (CEBM) Dhillon, Robinder S et al. (1) 4 C Dhurat, Rachita & Sukesh, MS. (2) 4 C Amable P. R. et al. (3) 4 C Wroblewski, Andrew P. et al (4) 4 C Ostvar, Omid et al. (5) 3b B Enoch, Stuart & Price, Patricia. (6) 4 C Sánchez-González, D.J. et al. (7) 4 C Arshdeep, Kumaran MS. (8) 4 C Hosny, Nada et al. (9) 2b B Perez, Amanda G. M. et al. (10) 2b B Perez, Amanda G.M. et al. (11) 2b B Gkini, Mari-Angeliki et al. (12) 2b B Zhen Jun Li, MS et al. (13) 3b D Gkini, Maria-Angeliki et al. (14) 4 C Conde Montero, E. et al. (15) 4 D Novak, Melinda A. & Meyer, Jerrold 4 C S. (16) Ramos PM, Miot HA. (17) 4 C Chaudhari, Nitin D. et al. (18) 4 D Sano, Shigetoshi et al. (19) 3b C Zhao, Jianzhi et al. (20) 2b B Cervelli, V. et al. (21) 1b B Villasante Fricke, Alexandra C & 4 C Miteva, Mariya. (22) Spano, Frank & Donovan, Jeff C. (23) 4 C Prie, BE et al. (24) 4 D Harris, John E. (25) 4 C Trink, A et al. (26) 1b B Singhal, Parul et al. (27) 3b C Sinclair, Rodney. (28) 4 C Recommendation (CEBM) 26

27 Sinclair, Rodney et al. (29) 4 C Schiavone, G. et al. (30) 2c C Khatu, Swapna S. et al. (31) 2c C Lin, Wei-hong et al. (32) 2b C Braga Silva, Jefferson et al. (33) 2b B Singh, Sukhbir. (34) 2c B Takikawa, M. et al. (35) 1b B Yang HS, Shin J. et al. (36) 2b B Wiedemeyeer K et al. (37) 4 C Godse, Kiran (38) 5 D 27

28 SYSTEMATIC REVIEW RESULTS The trend in therapies and clinical studies seems now to focus with a new, more biologically oriented approach to male and female pattern hair loss since new findings pointing out the influence of hair follicle stem cells in the hair growth cycle have been reported. (30) Platelet-rich plasma (PRP) has emerged as a new treatment modality in dermatology, and preliminary evidence has suggested that it might have a beneficial role in hair growth. (26) Proponents of platelet-rich plasma (PRP) technology suggest that its benefits include an increase in hard and soft tissue wound healing. (21) We now know that progenitor stem cells in the bulge area play a key role in igniting the hair cycle. (30) It is hypothesized that growth factors released from platelets may act on stem cells in the bulge area of the follicles, stimulating the development of new follicles and promoting neovascularization. (31) Several growth factors can promote cell cycle and proliferation and have the potential to rescue hair loss and facilitate hair cell regeneration in vivo and in vitro. (32) The main growth factors involved in the establishment of hair follicle are vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin 1-like growth factor, and fibroblast growth factor (FGF). Platelets release large amounts of platelet-derived growth factor (PDGFaa, PDGFbb, and PDGFab), transforming growth factor beta (TGFβ1 and β2), EGF, and VEGF. (18) Growth factors act in the bulge area, where stem cells are found, and they interact with cells of the matrix, thus activating the proliferative phase of the hair. (33) Germinative cells of the matrix, which are found at the dermal papilla, are of mesenchymal origin. (14,33) Interaction between these two kinds of cells as well as with binding GFs (PDGF, TGF-b, and VEGF) activates the proliferative phase of the hair, giving rise to the future follicular unit. (12,14,27) Takakura et al. revealed that PDGF signals in cell interactions are required for hair canal formation and growth of dermal mesenchyme, thereby opening newer perspectives for PRP in hair restoration. (8,14) Platelet derived growth factor signals, in fact, seem to play a role in hair canal formation, whereas vascular endothelial growth factor (VEGF) mediated angiogenesis involvement was demonstrated in the control of hair growth and follicle size. (14,30) The fibroblast growth factor (FGF) family is composed of 22 members with a wide range of biological functions involved in 28

29 angiogenesis, embryonic development, cell growth, and tissue repair. The early literature reported that acidic fibroblast growth factor (afgf or FGF-1) and basic fibroblast growth factor (bfgf or FGF-2) may affect the growth of hair follicles. (32) It is now known that activated autologous PRP has been reported to induce the proliferation of dermal papilla cells by up regulating fibroblast growth factor 7 (FGF-7) and b-catenin as well as extracellular signal-related kinase (ERK) and Akt signaling. (21) Insulin-like growth factor 1 (IGF-I) also appears to play a critical role in promoting hair growth. In several reports, IGF-I and IGF-II prevented the HF from developing catagen like status 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. (14) Therefore, it constitutes a potent useful tool for androgenetic alopecia treatment. (12) The in vitro findings were confirmed by in vivo results, which showed faster growth of hair in mice treated with PRP every 3 days for 3 weeks than that in the untreated controls. (21,30) In addition to its proliferation-inducing effects, PRP is also a potent anti-inflammatory agent, which can suppress cytokine release and thereby limit local tissue inflammation. As AA is characterized by an extensive inflammatory infiltrate, responsible for secretion of a variety of inflammatory cytokines, it is probable that the anti-inflammatory effects of PRP may be of great benefit in this condition. (13) PRP has also attracted attention in plastic surgery and dermatology because of its potential use during facial plastic surgery and aesthetic, skin-rejuvenating effects. (13) As seen previously on this review, several articles and studies were found on PRP, yet very few were on alopecia areata or androgenetic alopecia and even less articles containing studies on this matter. However, in the following chart, there is a summary of the results from the studies found. 29

30 PRP Authors Study Design Sample Size Preparation and Follow Up Results Concentration Zeng Jun In vivo study n=6 Manual Double 3 weeks Near-complete Li, MS et injections of PRP in Spin Method hair regrowth al. mice to compare with 2 : PRP was observed 2012 control mice 2: Positive Concentration= in mice injected (13) Control 8.8x with activated (phosphate- PRP for 3 buffered weeks. saline) 2: Control (Fetal Bovine Serum) Three months after the treatment, the Singhal, Parul et al (27) In vivo study injections of PRP in individuals with AGA to be compared with control individuals with AGA. n=20 10: PRP 10: control (8: male and 2: female in each group) Manual Double Spin Method Concentration not documented 3 months patients presented clinical improvement in the hair counts, hair thickness, hair root strength, and overall alopecia. Patients treated Trink, A et al (26) Randomized, doubleblind, placebo- and active-controlled, halfhead, parallel- group study. n=45 15: PRP 15: Triamcinolone Acetonide 15: Placebo Simple Centrifugation Concentration 3-5x 12 months with PRP had significantly increased hair regrowth compared with those treated with TrA; 27% of patients treated with 30

31 TrA achieved complete remission, compared with 60% of patients treated with PRP, which is significantly higher than TrA- and placebo-treated patients. A significant reduction in Khatu, Swapna S. et al 2014 (31) Prospective study in which safety and efficacy of PRP injections in treating androgenic alopecia were assessed. n=11 Manual Double Spin Method. Concentration not documented 3 months hair loss was observed after 12 weeks. There was an average mean gain of follicular units per cm2. An improvement Schiavone, G. et al 2014 (30) Prospective study in which leukocyte platelet rich plasma (L-PRP) with the addition of concentrated plasmatic proteins was injected in patients with androgenetic alopecia n=64 42: men 22: women Single spin method (baseline) Concentration: 3x Double spin method (3 months) Concentration: 4x 6 months was observed in 62/64 patients by Evaluator 1 and in all 64 patients by Evaluator 2. The overall proportion of patients reaching a clinically important 31

32 difference was 40.6% and 54.7%, according to the 2 evaluators, respectively. Of the 20 patients, one patient had minimum hair Singh, Sukhbir 2015 A prospective study to evaluate the efficacy of platelet-rich plasma (PRP) in the treatment n=20 Not documented 1 year regrowth and also had a relapse. None of the (34) of chronic alopecia patients had areata any side effects, and all of them tolerated the procedure well. Takikawa, M. et al 2012 (35) A prospective placebo controlled split-scalp study to evaluate dalteparim/protamine as a new carrier for PRP in hair growth n= 26 13: PRP + D/P vs. Saline 13: PRP vs. saline Manual Double Spin Concentration: 6x 12 weeks PRP+D/P and PRP facilitated hair growth but PRP+D/P provided additional hair growth. Gkini, Maria- Angeliki et al (12) A Prospective cohort study to evaluate the efficacy and safety of PRP injections in the scalp of patients with androgenetic alopecia. n=20 18: male 2: female Single spin method (Regenlab SA) Concentration: 5.8x 1 year Hair density significantly increased, comparing to baseline. Patients were satisfied with a 32

33 mean result rating of 7.1 on a scale of No remarkable adverse effects were noted. Hair density Cervelli, V et al 2014 (36) A prospective, evaluator blinded, placebo half-head group study to evaluate the effects of PRP on active hair growth n=10 Single Spin Method Concentration not documented 1 year improved significantly in the treatment area compared to baseline, while decreasingly in the control area of the scalp. Zen Jun et al made the only study on mice included in this review, in which he reported a complete hair regrowth 3 weeks after the injections of PRP. (13) As for androgenetic alopecia, the chart shows articles such as the case-control study Singhal et al published in 2015, where the patients showed an improvement in hair count, thickness, hair root strength and overall alopecia 3 months after the treatment with PRP. (27) Khatu et al made a prospective study where a gain in follicular units was shown as the hair loss was significantly reduced 3 months after the PRP treatment. (31) Schiavone et al in 2014 made a prospective study with 6 months follow up in which the overall proportion of patients reaching a clinically important difference was 40.6% and 54.7%, according to the 2 evaluators, respectively. (30) Gkini et al said hair density was significantly increased, comparing to baseline after 1 year of treatment with PRP in a prospective cohort study. (12) For alopecia areata the chart shows how Trink et al in 2013 had a randomized, double-blind, placebo- and active-controlled, half-head, parallel- group study. From their 45 patients they sorted 3 groups of 15, where one group had placebo, the other had 33