Platelet-rich Plasma: Understanding Mechanisms and Parameters that Determine PRP Efficacy.

An informal review of the literature on Platelet-rich Plasma in Dermatologic and Cosmetic practices.

Background

Platelet-rich plasma (PRP) is platelet concentrate prepared through centrifugation of autologous whole blood and is used in many different clinical point-of-care and surgical applications (Table 1). There are numerous PRP preparation systems available on the market and each generates a unique composition of platelets, growth factors, erythrocytes (red blood cells, or RBCs), and leukocytes (white blood cells, or WBCs). The efficacy of PRP highly depends on the values of individual blood components present in the final PRP concentrate. Variability in PRP formulations and concentrating devices derives from each commercial product’s characteristic centrifugation parameters, manufacturing materials, initial blood volume, choice of anticoagulant and platelet activator, temperature and pH conditions, extraction and cycle technique used to generate the PRP.

Table 1. Dermatological and Specialty Applications of PRP

Mechanism of Action

Platelet α-granules, dense granules, lysosomes and surface adhesion molecules all contribute to the underlying cellular signaling mechanisms that render platelet regenerative function (Figure 1). Most of the PRP literature, however, has focused on the role of platelet α-granules, which contain cytokines, chemokines, growth factors and other low molecular weight proteins involved in cell proliferation and differentiation (Table 2) (Kang et al., 2014; Lee et al., 2012; Eppley et al., 2006; Everts et al., 2020). Once activated, platelets degranulate and release their contents, which results in the initiation of cellular signaling cascades that promote tissue regeneration and repair. Platelet growth factors bind to surface receptors on osteoblasts, endothelial cells, stem cells, fibroblasts and other tissue-specific cells to activate intracellular signaling cascades resulting in the expression of genes and proteins that effectuate angiogenesis, collagen synthesis, decreased apoptosis and synthesis of extracellular matrix components (Mazzocca et al., 2012; Browning et al., 2012; Freire et al., 2012; Cho et al., 2011; Alsousou et al., 2009). The proposed mechanisms of PRP in cosmetic, dermatologic and aesthetic applications vary depending on the tissue site and location of use. (Pierce et al., 1989; Alsousou et al., 2009; Garcia et al., 2005).

Table 2. Platelet Growth Factors involved in Rejuvenation and Repair

Abbreviations
PDGF: Platelet Derived Growth Factor; TGF: Transforming Growth Factor; VEGF: Vascular Endothelial Growth Factor; FGF: Fibroblast Growth Factor; EGF: Epidermal Growth Factor; IGF: Insulin-like Growth Factor; CTGF: Connective Tissue Growth Factor; PDG: stromal cell-derived factor 1 alpha; CXCL: chemokine (C-X-C motif) ligand; PF4: platelet factor 4
Adapted and modified from Everts et al. 2006, 2020.

Figure 1. Platelet Granule and Growth Factor Anatomy

Abbreviations
BMA: bone marrow aspirate; EPC: endothelial progenitor cell; EC: endothelial cells; 5-HT: serotonin; RANTES: Regulated upon Activation Normal T Cell Expressed and Presumably Secreted; JAM: junctional adhesion molecules type; CD40L: cluster of differentiation 40 ligand; SDF: stromal cell-derived factor 1 alpha; CXCL: chemokine (C-X-C motif) ligand; PF4: platelet factor 4
Adapted and modified from Everts et al. 2006, 2020.

Measures of PRP Efficacy

Platelet & Growth Factor Concentration

Platelet Viability and Morphology

Although the concentration of growth factors (GFs) increases linearly with PRP concentration, studies have shown that the efficacy of PRP treatment does not necessarily follow a linear relationship with concentration of PRP and GFs. Instead, PRP follows common pharmacodynamic and pharmacokinetic properties, thus, is characterized as having an optimum dose which may vary across specialties and treatment indications. Studies involving the effect of PRP GFs on endothelial cell angiogenesis suggest that the optimal dose is a concentration of 1,500,000 platelets/μL (Giusti et al., 2009). Endothelial cell angiogenesis provides the framework to generate and rejuvenate vessels and capillaries which provides the tissue with a means to obtain nutrients from the body and regenerate. Other studies show an inhibitory effect of high platelet concentrations on bone regeneration (Weibrich et al., 2004).

The efficacy of PRP concentrate depends on the concentration of growth factors released by platelets, which is determined by platelet viability and morphology. If platelets are prematurely activated during the preparation, separation or extraction processes, the growth factors will be released into plasma and disposed of. Manual preparation systems (e.g., BD Vacutainer yellow-top tubes containing ACD-A or Serum Separating Tubes) may seem appealing to providers looking for a cost-effective way to offer PRP to their patients. However, these products contain preservatives, like silica, that are optimized for laboratory and diagnostic testing, and thus, do not preserve platelet integrity. If platelet coagulation cascades commence prior to administration, the PRP treatment is ineffective. 

There are many intricate factors involved in determining final platelet and growth factor viability and morphology. Although many of these delicate variables are outlined below, please note this is not a comprehensive list. Further, this review almost entirely focused on PRP in dermatology, cosmetic, plastic surgery, aesthetic medicine and rejuvenation.

Erythrocyte Concentration

When preparing PRP, the number of RBCs should be negligible. Erythrocytes contain reactive oxygen species (ROS) and free radicals that produce harmful inflammation, initiate edema and cause pain during and after injection (Magalon et al, 2014). Moreover, RBCs deposit hemosiderin, which together with the release of free radicals, contaminate PRP and lower solution pH, thus, reducing platelet viability (Lei et al, 2009). For hair restoration, these combined effects create a catabolic environment that hinders the role of growth factors and induces telogen effluvium.

RBC contamination may occur during collection, centrifugation or extraction of PRP. If the chemical properties of the tube and spin conditions are unsuitable, RBCs may lyse and release their harmful byproducts, and oxidative stress may cause other RBCs to undergo eryptosis and inflammation (Everts et al, 2019). The number of erythrocytes in the final PRP product varies depending on many factors including, the presence of a serum gel separator, centrifugation parameters, manufacturing materials, initial blood volume, and choice of anticoagulant (Kumar et al, 2005). A major drawback of using tubes without a gel separator is that remnants of RBCs spill over and contaminate the PRP concentrate. Thus, many dermatologists and aesthetic facilities may choose a plasma-based extraction method containing a gel separator to meet their patient needs.

Leukocyte Components

The most contentious topic among PRP investigators is in regard to the role of WBC components found in the final PRP concentrate. Many studies contend that leukocyte-rich PRP (LR-PRP), which contains an abundance of neutrophils and other granulocytes creates unfavorable catabolic conditions in tissue and prolongs the inflammatory process The quality of a PRP concentrating device is further revealed by its ability to achieve an appropriate ratio of platelets to WBCs, as well as individual concentrations of specific WBC components. 

Macrophages and monocytes play a fundamental role in repairing damaged cells and maintaining tissue integrity (Fernandez 2005). Thus, the presence of monocytes in PRP is beneficial to the cellular rejuvenation process. Other leukocytes—namely neutrophils, basophils and eosinophilsrelease proinflammatory metalloproteinases and other cytokines that prolong inflammation, neutralize growth factors, damage non-injured tissue, degrade extracellular matrices, cause fibrosis and scarring (Kim 2015, Fitzpatrick 2017, Magalon 2014, DeLong 2012). Thus, the presence of granulocytes antagonizes the healing properties of PRP and should be minimized. Although the ratio of platelets to WBC’s is important, the process and methods used to separate PRP provides equally significant insight about the quality of PRP systems in practice.

Centrifugation Parameters

The process of centrifugal separation of blood components is a major contributor to the quality of a PRP system’s final product. Currently, a standardized system for evaluating PRP effectiveness does not clinically exist. There’s significant variation of cellular and molecular components when measured in different PRP collecting devices but using the same centrifugation protocols. Moreover, differences in centrifugal force, acceleration and spin time produce major disparities in overall yields, purity, concentration, viability, morphology and activation status of platelets (Fadadu et al., 2019). Spin protocol, shock absorbance and dampening ability further contribute to the final components found in PRP concentrate and the most important parameters are investigated below:

Single vs. Double Spin

Studies conducted comparing different centrifugation methodologies and their effect on platelet, leukocyte, and growth factor concentrations have not provided definitive evidence for one protocol over the other. Several studies suggest that single-spin centrifugation is better suited for achieving optimal platelet and growth factor values for procedures of the skin (Carofino et al., 2012; Mazzocca et al., 2012; Pachito et al., 2020), wheras other studies have supported dual-spin methods (Nagata et al., 2010). 

Spin Duration

Since it is highly efficacious to rely on a high centrifugal force during preparation of the autologous blood PRP, caution must be taken when setting the duration of spin to avoid damaging the platelets’ morphology. Studies have revealed that a duration ranging from 5 to 12 minutes of spin combined with the parameters chosen above is effective for maintaining platelet integrity (Croisé et al., 2020).

Force, Acceleration and Speed

For products that utilize a gel separator, a greater force is required to overcome the gel barrier and all RBCs and some granulocytes to traverse the gel. It is essential to determine the optimal force at which the concentration of PRP is the highest without compromising its morphology. Studies have revealed that a high-speed centrifugation during a short duration is best for achieving high platelet and growth factor concentrations. This force may range from 1500 to 2000g when using a single centrifugation approach (Dhurat & Sukesh, 2014; Croisé et al., 2020).

Anticoagulant

As a major parameter affecting platelet yield, morphology and function, the type of anticoagulant used has been overlooked by researchers in more than 53% of all PRP-related studies (Frautschi et al, 2017), which represents a major shortcoming in the PRP literature. Studies that have highlighted the impact of choosing the appropriate anticoagulant have demonstrated that Acid Citrate Dextrose Solution A (ACD-A) is the PRP gold standard (Araki 2012, Fukaya 2014, Wahlstrom 2007). Other researchers demonstrated ACD-A’s effectiveness over alternative anticoagulants in maintaining platelet viability and morphology (Lei et al., 2009; Giraldo et al., 2015; Singh et al., 2018). Solutions treated with ACD-A were most successful in maintaining platelet and alpha granule structure, whereas other anticoagulants resulted in a greater number of lysed cells and PRP contaminants. Samples stained and viewed under a TEM microscope revealed functional and morphological features that ACD-A exhibited over other anticoagulants, as well as a lack of acellular debris and aggregates, which together thwart premature platelet activation (Singh, 2018).

These studies and others established ACD-A’s capacity to function as a physiological buffer, maintaining a pH near 7.2 and providing optimal conditions to maximize platelet and growth factor concentrations (Arora, 2017). The concentration and effectiveness of growth factors are impacted by tissue pH, so having PRP concentrate buffered close to a physiologic range provides ideal conditions. Moreover, dextrose and other ingredients support platelet metabolism and viability, whereas citrate binds calcium and prevents premature coagulation (Arora, 2017). Together, ACD-A maintains ideal chemical properties to keep PRP buffered and intact, and thus, is the anticoagulant of choice for InclusionMD’s c-PRP concentrating devices.

Manufacturing Materials

Tubes manufactured using crystal glass and polyethylene terephthalate (PET) allow for a more effective and uniform PRP collection, centrifugation and extraction process that maximize platelet and growth factor concentrations (Bowen et al, 2014). The inner crystal layer is more hydrophilic than plastic, contains more tightly packed molecules to minimize wall rigidity, keeps out moisture, and allows for the thixotropic separating gel to readily move throughout the tube. The outer PET layer provides firm support, minimizes evaporation of anticoagulant solution, extends shelf life and maintains a prolonged vacuum that aids in the blood collection process. Smaller diameter tubes also help minimize surface area to atmosphere ratio, which prevents diffusion of CO2 out of plasma and thereby help maintain the system’s physiological pH (Collins et al, 2021).

Corporate Compliance

The US Food and Drug Administration’s (FDA) Center for Biologics Evaluation and Research (CBER) regulates the market for biologics products, like PRP, which fall under the guidelines in 21 CFR 1271 of the CBER Code of Regulations. Under these provisions, blood products like PRP are exempt and do not follow the FDA’s conventional regulations that would otherwise require clinical research or animal studies conducted prior to introducing a new PRP system to the market (Beitzel K et al, 2014). However, because the autologous nature of PRP renders it a low-risk procedure, PRP concentrating devices are class II medical devices that require FDA 510(k) clearance. 

To maintain an FDA “cleared” as opposed to an FDA “approved” medical device status, the FDA requires that the PRP collection device be similar to an already approved device, or predicate, and clearance is limited to indications of the predicate. Since the only approved on-label use for PRP is mixture with autograft or allograft bone to enhance graft properties, any use of PRP outside this context is considered “off-label.” Nonetheless, clinicians are free to use their best clinical judgement when applying PRP for an off-label procedure as long as the use is based on scientific knowledge that’s grounded in sound medical evidence (Harm et al, 2015).

Sterilization/Depyrogenation

Different sterilization techniques are used to render PRP and other medical devices “sterile” which must not be confused with rendering them “pyrogen-free.” A product that is sterile is void of any viable microorganisms (Lechman et al. 1976, Ashok 1994), whereas a product that is pyrogen-free has undergone more rigorous cleansing and must be confirmed not to contain pyrogens using either a Rabbit Test or Limulus Amoebocyte Lysate (LAL) Test. Although conventional methods to render a product sterile may be effective at removing the bioburden, sterilization techniques are ineffective at eliminating pyrogens and endotoxins. Further, sterilization techniques may generate pyrogens in the process of eliminating bacteria, whereby bacteria may release endotoxins and other remnants as byproducts after they denature (Van Belleghem et al, 2016).

InclusionMD Biologics: c-PRP Concentrating Systems

InclusionMD’s c-PRP Concentrating devices are class II medical devices with 510(k) clearance, which are relabeled and distributed on behalf of our manufacturer (510(k) Number: BK170136); our device manufacturer maintains a quality management system that’s ISO 13485 certified as well. All c-PRP collection tubes are manufactured using Crystel-PET and contain a polymeric, thixotropic gel separator with an optimized specific gravity of 1.05g/cm3 that helps maximize platelet, monocyte and growth factor concentrations while eliminating RBCs and trapping WBCs in the gel separator. Our PRP devices undergo a triple sterilization method using Cobalt^60 gamma irradiation technology. After sterilizing twice, our collection devices undergo a depyrogenation process in a unidirectional dry heat tunnel, before undergoing their third gamma irradiation sterilization. The quality and safety of our devices exceed the industry standards of our competitors and uphold all regulatory standards and requirements.

The i7 Slow-Speed PRP Centrifuge provides ideal conditions for platelet-tube centrifugation. The fixed-angle, operating speed, centrifugal force, and delicate rotation all work cohesively to maintain platelet, α-granule, and growth factor integrity. The centrifuge is manufactured with unique shock isolators to dampen vibrations and provide an automatic balancing mechanism to keep the tubes level throughout the cycle. Further, it operates under 60 decibels, functions without emitting micro-carbon dust particles, and accelerates and decelerates delicately as not to add torque to the system. The i7 can spin up-to 4000 revolutions per minute (rpms), but we’ve seen the highest concentration of Platelets when tubes are spun between 3700-3800RPM for 8-10 min. This machine provides state-of-the-art mechanisms for exceptional and consistent platelet yields, is one of the most affordable centrifuges on the market, and optimized for our c-PRP tubes.

Although case studies are currently being conducted on our refined c-PRP systems, our previous PRP systems provided exceptional yields for platelets, growth factors, WBCs and RBCs. Our new systems have a more optimized concentration of ACD solution A, as well as a gel separator with a more optimal specific gravity. This allows RBCs to separate from the final platelet mixture without causing them to lyse. The thixotropic gel’s specific gravity, taken with the crystal glass tubes and centrifugation parameters, allow RBCs and granulocytes to move past the gel without initiating contact and potentially lysing. As the centrifuge spins, the crystal walls heat the gel and allow it to alter its dimensions. The hydrophilic walls of the tube provide a mechanism for adhesion of the gel, which minimizes its surface area in the horizontal plane and provides a seamless process for RBCs and other WBCs to separate to the bottom of the tube based on density, while granulocytes are forced into the gel separator and trapped away from the final PRP concentrateBy enhancing our manufacturing materials and conducting trials to determine our systems optimal centrifugal force, speed and spin time we’ve strengthened the quality of our products and services. The InclusionMD c-PRP Concentrating systems allow for the safe and rapid preparation of autologous platelet-rich plasma. Although our PRP systems are only indicated for mixture with autograft and/or allograft bone, as deemed necessary by the clinical use requirements, it is optimized for “off-label” dermatological and cosmetic point-of-care procedures based on the most current scientific literature. 

Our next article in this series will provide more detailed insight about mechanisms of action and specific growth factor and cellular components involved in PRP for hair restoration, facial rejuvenation, and pigmentation disorders. After understanding the mechanisms which facilitate rejuvenation and repair, we will continue to explore the other parameters that impact PRP efficacy across various indications and specialities, such as pH, Temperature and the use of Calcium Chloride and other PRP activators. Sign up at the bottom of this page for updates through our newsletter and stay tuned! As always, feel free to reach out with any questions. 

 

A special thank you to the students in the InclusionMD Internship Program for contributing to this review.  

InclusionMD, Inc. 2021 - All Rights Reserved

Contributors: Vincent Berry, Mariam Duhaini, Aiyat Ali, Lamya Alsaidi, Adel Rizk

For more information about our PRP concentrating devices and other products visit our PRP Products Page or check out the manual below:

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