Acknowledgments

To my parents, family, and friends who have all sacrificed far too often in my pursuit of a career in academic dentistry.

To my classmates, colleagues, and mentors who constantly raised the bar and strived for better.

To Quintessence Publishing for your thorough input in the editing, illustrations, and design of this textbook.

To my family at Lakewood Ranch Dental in Florida who have made clinical practice as enjoyable an experience as can be on a daily basis.

To all leaders and researchers alike who have contributed enormously to the field of PRP/PRF and laid the foundation for this textbook to be written.

To the faculty in the Department of Biomedical Sciences and Cell Biology at the University of Western Ontario (London, Canada; BMSc, MSc), the Dental School at the University of Laval (Quebec; DDS), the Department of Music at Berklee College (Boston; MMus), the Department of Periodontology at the University of Bern (PhD, Dr med dent), the Department of Oral Implantology at Wuhan University (China; postdoctoral research fellow), the Plastic Surgery Department at Queen Mary University (London; clinical masters in facial esthetics), and the Department of Periodontology at the University of Illinois at Chicago (clinical masters in periodontology). Your education and mentorship has provided endless opportunities.

And lastly, to the team at Advanced PRF Education (www.prfedu.com) for making excellence in teaching a top priority.

Contributors

Fabrice Baudot, dds, msc

Private Practice

Saint-Gély-du-Fesc, France

Luis Bessa, dds

Director, North Clinic

Porto, Portugal

Mark Bishara, dds

Private Practice

Bowmanville, Ontario, Canada

Thomas Boas, msc (econ)

CEO, Puremed

Roskilde, Denmark

Luigi Canullo, dds, phd

Independent Researcher

Rome, Italy

Marco Antonio Castro Pinto, dds, msc

Professor, Department of Reconstructive Dentistry

Montemorelos University School of Dentistry

Nuevo Léon, Mexico

Raluca Cosgarea, dds, msc, phd

Professor, Department of Prosthetic Dentistry

Iuliu Hat¸ieganu University

Cluj-Napoca, Romania

Catherine Davies, mbbch, mba

Private Practice Specializing in Facial Esthetics

Johannesburg, South Africa

Massimo Del Fabbro, md, phd

Professor, Department of Biomedical, Surgical, and Dental Sciences

University of Milan

Milan, Italy

Scott Delboccio, dmd

Private Practice

Naples, Florida

Anika Dham

Research Student, Nova Southeastern University

Fort Lauderdale, Florida

Jonathan Du Toit, dds, msc

Department of Periodontics and Oral Medicine

Faculty of Health Sciences

University of Pretoria

Pretoria, South Africa

Meizi Eliezer, dds, msc, phd

Research Associate, Department of Periodontology

School of Dental Medicine

University of Bern

Bern, Switzerland

Masako Fujioka-Kobayashi, dds, phd

Professor, Department of Oral and Maxillofacial Surgery

School of Life Dentistry at Tokyo

The Nippon Dental University

Tokyo, Japan

Maria Elisa Galarraga-Vinueza, dds, msc, phd

Professor, School of Dentistry

Universidad de las Américas (UDLA)

Quito, Ecuador

Arun K. Garg, dmd

Private Practice Limited to Implantology

Miami, Florida

Stefan Gerber, md, dds, msc, phd

Assistant Professor, Department of Cranio-Maxillofacial Surgery

University of Bern

Bern, Switzerland

Ezio Gheno, dds, phd

Post-Graduation Program in Dentistry

Fluminense Federal University

Niterói, Rio de Janeiro, Brazil

Alfonso Gil, dds, msc

Resident, Fixed and Removable Prostho- dontics and Dental Material Science

University of Zurich

Zurich, Switzerland

Howard Gluckman, bds, mchd (omp)

Specialist in Periodontics, Implantology, and Oral Medicine

Director of Implant & Aesthetic Academy

Cape Town, South Africa

Reinhard Gruber, phd

Professor, Department of Oral Biology

Medical University of Vienna

Vienna, Austria

Thomas Lau Hansen, phd

Puremed

Roskilde, Denmark

Tommy Hardon, dvm

Head Veterinarian, Haslev Dyreklinik

Haslev, Denmark

David Lee Hill, dds, msc

Private Practice

Chapel Hill, North Carolina

Søren Jepsen, dds, msc, phd

Director of the Department of Periodontology

University of Bonn

Bonn, Germany

Valerie Kanter, dds, msc

Professor, Department of EndodonticsUniversity of California, Los Angeles

Los Angeles, California

Dwayne Karateew, dds, msc

Director of Program in Periodontics

University of Illinois at Chicago

Chicago, Illinois

Tomoyuki Kawase, dds, phd

Professor, Division of Oral Bioengineering

Institute of Medicine and Dentistry

Niigata University

Niigata, Japan

Johan Lenz, dvm

Veterinarian, Jonas Tornell Veterinär

Ängelholm, Sweden

Marius Leretter, dds, phd

University of Medicine and Pharmacy of Timişoara

Vice Dean of Dental School

Timişoara, Romania

Victoria Lima, dds, msc

Research Fellow, Division of Periodontics

Institute of Science and Technology

São Paulo State University (UNESP)

São Paulo, Brazil

Richard J. Martin, dds

Private Practice Limited to Oral and Facial Surgery

Lewisville, Texas

Yuriy May, dmd

Private Practice

Farmington, Connecticut

Brian Mealey, dds, ms

Professor and Graduate Program Director, Department of Periodontics

University of Texas Health Science Center at San Antonio

San Antonio, Texas

Jacob Coakley Meyer, dvm

Veterinarian, Charlottenlund Dyrehospital

Charlottenlund, Denmark

Richard J. Miron, dds, bmsc, msc, phd, dr med dent

Group Leader, The Miron Research Lab

Lead Educator, Advanced PRF Education

Venice, Florida

Department of Periodontology

University of Illinois at Chicago

Chicago, Illinois

Omid Moghaddas, dds, msc

Assistant Professor, Department of Periodontology

Dental Faculty, Tehran Medical Sciences

Islamic Azad University

Tehran, Iran

Naheed Mohamed, dmd, msd

Private Practice

Oakville, Ontario, Canada

Vittorio Moraschini, dds, msc, phd

Professor, Department of Oral Surgery

Dental School, Fluminense Federal University

Niterói, Rio de Janeiro, Brazil

Ana Paz, dds, ms

Private Practice

Lisbon, Portugal

Michael A. Pikos, dds, msc

Director, Pikos Institute

Tampa, Florida

Nikola Saulacic, dds, phd

Assistant Professor, Department of Cranio-Maxillofacial Surgery

University of Bern

Bern, Switzerland

Benoît Schaller, dr med, dr med dent

Professor, Department of Cranio- Maxillofacial Surgery

University of Bern

Bern, Switzerland

Anton Sculean, dmd, dr med dent, ms, phd

Professor and Executive Director and Chairman

Department of Periodontology

University of Bern

Bern, Switzerland

Senthil Selvan, dds

Director, Jeya Dental Clinic

Theni, India

Samer Shaltoni, dmd, ms

Private Practice Limited to Oral Surgery Buffalo Grove, Illinois

Yoshinori Shirakata, dds, phd

Associate Professor, Department of Periodontology

Kagoshima University Graduate School of Medical and Dental Sciences

Kagoshima, Japan

Miguel Stanley, dds

Private Practice

Lisbon, Portugal

Robert Talac, md, phd

Director, Renaxis Spine and Orthopedic Clinic

Houston, Texas

Mustafa Tunali, dds, phd

Professor, Department of Periodontology

Haydarpasa Training Hospital

Gulhane Military Medical Academy

Istanbul, Turkey

Delia Tuttle, dds, ms

Private Practice

Lake Elsinore, California

Hom-Lay Wang, dds, msd, phd

Professor and Director of Graduate Periodontics

Department of Periodontics and Oral Medicine

University of Michigan School of Dentistry

Ann Arbor, Michigan

Hudi Xu, dds, phd

Research Associate, Department of Dental Implantology

School of Stomatology

Wuhan University

Wuhan, China

Yufeng Zhang, md, dds, phd

Professor, Department of Dental Implantology

School of Stomatology

Wuhan University

Wuhan, China

Abbreviations

The abbreviations listed here are used throughout the book and are NOT always spelled out in the chapters for ease of reading.

1

Evolution of Platelet Concentrates

Contributors

Richard J. Miron

Chapter Highlights

• Evolution of PRF and the reasons for its discovery
• Discussion of PRP vs PRGF vs PRF vs L-PRF, A-PRF, etc
• Biologic background of key steps involved during wound healing

Platelet concentrates have been utilized in medicine for over two decades because of their ability to rapidly secrete autologous GFs and ultimately speed wound healing. They have gained tremendous momentum as a regenerative agent derived from autologous sources capable of stimulating tissue regeneration in a number of medical fields.^1,2 Many years ago, it was proposed that by concentrating platelets using a centrifugation device, GFs derived from blood could be collected from a platelet-rich plasma layer and later utilized in surgical sites to promote local wound healing.^1,2 Today, it has been well established that platelet concentrates act as a potent mitogen capable of the following (Fig 1-1):

• Speeding the revascularization of tissues (angiogenesis)
• Recruiting various cells including stem cells
• Inducing the prompt multiplication of various cell types found in the human body (proliferation)

Fig 1-1 The three main GFs that are released from PRF include VEGF, a known inducer of angiogenesis; PDGF, a known inducer of cell recruitment; and TGF-β1, a known stimulator of cell proliferation. MSC, mesenchymal stem cell.

Wound healing is a complex biologic process whereby many cell types interact with one another as well as their local extracellular matrix (ECM) in order to repair and regenerate damaged tissues.^3–6 While many regenerative agents currently exist on the market to help speed tissue regeneration, it is important to note that the majority are derived from other human sources (allografts) and animal byproducts. These naturally create a foreign body reaction when implanted into host tissues. While the majority of such biomaterials do certainly favor improved healing, it has generally been recognized and accepted that the gold standard for the majority of tissue-regenerative procedures in basically every field of medicine has been the use of autogenous tissues.

Specifically in dentistry, platelet concentrates were introduced over 20 years ago by Robert E. Marx and colleagues with the aim of concentrating blood proteins as a natural source of GFs that would stimulate vascularization (angiogenesis) and tissue ingrowth based on the fact that blood supply is pivotal for tissue regeneration of all tissues.⁷ Wound healing has been described as a four-step process that includes (1) hemostasis, (2) inflammation, (3) proliferation, and (4) maturation^8–10 (Fig 1-2). Each phase overlaps one another and encompasses various microenvironments, including different cell types that assist in wound healing. Noteworthy are the implications of immune cells during biomaterial integration. In a study titled “OsteoMacs: Key players around bone biomaterials,” osteal macrophages were discussed as being key and pivotal cells during the wound healing process.¹¹ Thus, as tissue biology has continued to evolve, platelet concentrates have also seen significant advancement with respect to their ability to favor healing by incorporating immune cells (leukocytes). Various systematic reviews from multiple fields of medicine have now demonstrated their ability to support tissue regeneration across many tissue types and cell types. This chapter reviews the evolution of platelet concentrates.

Fig 1-2 Four phases of wound healing: (1) hemostasis, (2) inflammation, (3) proliferation, and (4) maturation. Noteworthy are the overlaps between each of the phases and the population of cells found in each category. Whereas lymphocytes typically arise at 7 days, the ability of PRF to introduce a high number at day 0 acts to speed the regenerative phase during this process.

PRP (1990s)

The use of platelet concentrates has slowly and gradually gained popularity over time, with a dramatic increase being observed in the past 5 to 10 years. This parallels precisely the massive increase in research articles being published on the topic. Despite this, it is important to review and highlight the pioneering work conducted by Marx and colleagues over 20 years ago, without which none of this textbook would exist.^12–14

Platelet-rich plasma (PRP), as its name implies, was designed to accumulate platelets in supraphysiologic doses within the plasma layer following centrifugation. The main aim of PRP was to isolate and further concentrate the highest quantity of platelets and their associated GFs for regenerative purposes, thereafter reimplanting this specialized supraconcentrate at sites of local injury. This concept has been the basis of thousands of research articles, with their protocols being utilized to favor wound healing in millions of patients.

Initial protocols typically ranged in duration from 30 minutes to 1 hour based on the centrifugation/collection systems and protocols utilized. The original concept was pioneered by Harvest Technology, where it was shown that over 95% platelet concentration could be accumulated, having the potential to help favor the regenerative phase of many cell types including soft tissues, epithelial cells, periodontal ligament cells, and bone cells.^15,16 Because these initial protocols were lengthy, anticoagulants were added to the blood collection tubes. These typically were various forms of concentrated bovine thrombin or sodium citrate.

Despite its growing success and continued use after its discovery, several reported limitations existed with these initial formulations of PRP. The 30-minute or longer technique was generally considered lengthy for routine dental or medical practice, and more importantly, the use of anticoagulants was shown to limit wound healing from reaching its maximum potential. Simply put, when injury is created following an open wound, a blood clot is one of the first steps that occurs in order for healing to take place. Shortly thereafter, cells and GFs get trapped within this newly formed ECM, and the wound healing process/cascade begins. By limiting the body’s ability to form a stable clot, wound healing is limited. Several studies have now demonstrated the superior outcomes of platelet-rich fibrin (PRF) when compared to PRP simply by removing anticoagulants from their formulations.^17–21 Even the pioneering research team behind the plasma rich in growth factors (PRGF) concept (Anitua et al) have since demonstrated more physiologic healing ability with anticoagulant removal.¹⁷

Another drawback of PRP was the fact that it remained liquid by nature (due to the use of anticoagulants), so when it was combined with biomaterials, a much faster delivery of GFs was observed (Fig 1-3). While an initial burst of GFs is typical of PRP therapy, a slower release of GFs over an extended period of time has been shown to better stimulate cell growth and tissue regeneration.^22,23

Fig 1-3 (a and b) GF release from PRP and PRF at each time point of PDGF-AA over a 10-day period. Notice that while PRP has significantly higher GF release at early time points, over a 10-day period, significantly higher levels are most commonly found with A-PRF due to the slow and gradual release of GFs utilizing slower centrifugation speeds. (Adapted from Kobayashi et al.¹⁹)

Much advancement related to PRP therapy has been made over the past 20 years, and two excellent textbooks have been written by its pioneers—Dental and Craniofacial Applications of Platelet-Rich Plasma by Robert E. Marx and Arun K. Garg (Quintessence, 2005), and Autologous Blood Concentrates by Arun K. Garg (2018). Its breakthrough features include the novel ability to concentrate platelets to supraphysiologic doses and further stimulate tissue regeneration across virtually all tissue types. For these reasons, PRP has not surprisingly been utilized in practically every field of medicine.

L-PRF (2000–2010)

Because the anticoagulants utilized in PRP prevented clotting, pioneering work performed by Dr Joseph Choukroun and Dr David Dohan Ehrenfest led to the development of PRF.²⁴ The aim was to develop a second-generation platelet concentrate focused on anticoagulant removal. Because anticoagulants were removed, a much quicker working time was needed, and centrifugation had to begin shortly after blood draw (otherwise, the blood would naturally clot). Furthermore, high g-force centrifugation protocols were initially utilized in an attempt to separate blood layers prior to clotting. The final spin cycle (initial studies ranged from 2500–3000 rpm for 10–12 minutes = ~700g) resulted in a plasma layer composed of a fibrin clot with entrapment of platelets and leukocytes. The main advantage of this fibrin matrix was its ability to release GFs over an extended period of time while the fibrin clot was being degraded.²⁵ Over the years, PRF has been termed L-PRF (for leukocyte platelet-rich fibrin) due to the discoveries that several leukocytes remained incorporated in PRF and that white blood cells play a central and key role in the tissue healing process. The most commonly utilized protocol today is a spin cycle at 3000 rpm for 10 minutes or 2700 rpm for 12 minutes (RCF-max = ~700g, RCF-clot = ~400g).

Several other advantages also existed during clinical use because it avoided the need for dual-spin protocols requiring pipetting or various specialized tube compartments, which made the overall procedure much more user-friendly, cheaper, and faster when compared to PRP. Original protocols were purposefully designed to spin at high centrifugation speeds with the main aim of phase separation to occur as quickly as possible in order to separate the red corpuscle base layer from the upper plasma layer prior to clotting. Following centrifugation, a platelet-rich fibrin mesh was formed, giving it the working name PRF^26–28 (Fig 1-4). PRF has since been highly researched, with over 1,000 publications dedicated to this topic alone.

Fig 1-4 Layers produced after centrifugation of whole blood. A PRF clot forms in the upper portion of tubes after centrifugation.

Additionally, research teams from around the world have demonstrated the impact of leukocytes on tissue healing.^29–34 While it was once thought that the additional benefit of leukocyte incorporation into PRF was its main properties in improved host defense to foreign pathogens,^29–34 it has since been shown in well-conducted basic research studies that leukocytes are pivotal to tissue regeneration and favor faster wound healing also.^11,35–37 In dentistry, where the oral cavity is filled with bacteria and microbes, the inclusion of leukocytes was initially thought to play a pivotal role in wound healing by participating in the phagocytosis of debris, microbes, and necrotic tissues, as well as directing the future regeneration of these tissues through the release of several cytokines and GFs and orchestrating cell-to-cell communication between many cell types.

Tissue engineering with PRF

Tissue engineering has been an emerging discipline over the past decade, with major breakthroughs routinely being made every year. At its simplest foundation, tissue engineering requires three parameters: (1) a scaffold responsible to support tissue ingrowth, (2) cells that may act to promote tissue regeneration, and (3) GFs that stimulate the overall wound healing events. Unlike the majority of biomaterials currently available on the market, PRF actually contains each of these three properties (Fig 1-5). For comparative purposes, routine bone allografts contain a scaffold (mineralized cortical/cancellous bone) and GFs embedded in its bone matrix (such as bone morphogenetic protein 2 [BMP-2]) but have no cells. Recombinant human GFs typically have a GF (for instance, rhBMP-2) and a carrier (collagen sponge) but also lack cells. Certain stem technologies typically contain cells and also a delivery system (for instance a nanocarrier delivery system) but lack GFs. The ability to actually contain each of the three tissue engineering properties within a single biomaterial is quite rare and, more importantly, usually extremely expensive (think recombinant GFs and/or stem cell technology).

Fig 1-5 Three main components of PRF all derived naturally from the human body. These include (1) cell types (platelets, leukocytes, and red blood cells); (2) a provisional ECM 3D scaffold fabricated from autologous fibrin (including fibronectin and vitronectin); and (3) a wide array of over 100 bioactive molecules, including most notably PDGF, TGF-β, VEGF, IGF, and EGF.

PRF, on the other hand, is a particularly simple and inexpensive way to utilize the three principles of tissue engineering by utilizing a 3D scaffold (fibrin) that incorporates both regenerative host cells (platelets and leukocytes) and various GFs. These include PDGF, TGF-β, and VEGF, each of which is crucial during the regeneration process. Furthermore, the concentrated leukocytes (as opposed to simply platelets) in PRF have been well implicated as key regulators of tissue healing and formation.^{26–28,31,38}

A-PRF and i-PRF (2014–2018)

While much of the research performed in the late 2000s and early 2010s was dedicated to the clinical uses and indications of L-PRF discussed later in this textbook, major discoveries were made several years later from basic research laboratories. Following extensive clinical use and research with the original L-PRF protocol, it was discovered in 2014 by Dr Shahram Ghanaati that centrifugation carried out at relatively high centrifugation speeds (~700g) led to the great majority of leukocytes being located either at the buffy coat zone (between the red blood cell layer and the upper plasma layer) or more commonly at the bottom of centrifugation tubes (Fig 1-6).³⁹ It was expressed that the longer the centrifugation time is carried out, the more likely it is that cells get pushed further down the centrifugation tube. Similarly, the faster the spin centrifugation speed (higher g-force), the greater the proportion of cells found in the lower levels of centrifugation tubes.

Fig 1-6 Histologic observation of leukocytes following centrifugation. Resulting white blood cells have been shown to be contained basically in the layers between the plasma PRF layer and the red blood cell clot. This finding demonstrated quite clearly that the g-force was excessive, necessitating the development of newer protocols aimed to improve the retention of leukocytes within the PRF matrix. (Reprinted with permission from Ghanaati et al.³⁹)

Pioneering research within his laboratory led to the development of an advanced PRF (A-PRF) whereby lower centri-fugation speeds (~200g) led to a higher accumulation of platelets and leukocytes more evenly distributed throughout the upper PRF layers. These newer protocols more favorably led to a higher release and concentration of GFs over a 10-day period when compared to PRP or L-PRF.¹⁹ In 2015 to 2017, our research team further demonstrated that optimization of PRF could be achieved by reducing not only centrifugation speed but also the time involved. The A-PRF protocol was therefore modified from 14 minutes at 200g as originally described in 2014 down to an 8-minute protocol.¹⁹

Following an array of basic research studies on this topic, it was observed that by further reducing the g-force and also the time, it was possible to obtain a plasma layer that had not yet converted into fibrin (ie, scientifically liquid fibrinogen but often referred to as liquid-PRF for simplicity). In a study titled “Injectable platelet rich fibrin (i-PRF): Opportunities in regenerative dentistry?”,²⁰ it was demonstrated that at lower centrifugation speeds and times (~60g for 3 minutes), a liquid-PRF (termed injectable-PRF or i-PRF) could be obtained. While these protocols typically produced minimal volumes (~1.0–1.5 mL), it was shown that both platelets and leukocytes were even more highly concentrated when compared to L-PRF or A-PRF (Fig 1-7).⁴⁰ This liquid-PRF layer could be utilized clinically for approximately 15 to 20 minutes, during which time fibrinogen and thrombin had not yet converted to a fibrin matrix (ie, remained liquid). This has since been utilized for injection into various joints/spaces similar to PRP, however with the reported advantages of a longer GF release time. Furthermore, the concept of “sticky” bone was also developed. Importantly, a different type of tube (plastic) was needed to minimize clotting, as will be discussed in detail in chapter 5.

Fig 1-7 Newer centrifugation protocols allow production of a liquid formulation of PRF found in the top 1- to 2-mL layer of centrifugation tubes following a 3- to 5-minute protocol. This liquid can be collected in a syringe and reinjected into defect sites or mixed with biomaterials to improve their bioactive properties. (Reprinted with permission from Davies and Miron.⁴⁰)

H-PRF and C-PRF (2019–Present)

Very recently, our research group discovered through a series of basic laboratory experiments that horizontal centrifugation led to significantly greater concentrations of platelets and leukocytes when compared to currently available fixed-angle centrifugation devices most commonly utilized to produce L-PRF and A-PRF. Simply, horizontal centrifuges are routinely utilized in high-end research laboratories as well as in medical hospitals because of their greater ability to separate layers based on density (Fig 1-8; see also chapters 2 and 3). Unlike fixed-angle centrifugation systems whereby the tubes are actually inserted at a 45-degree angle, in horizontal centrifugation systems (often referred to as swing-out bucket centrifugation), the tubes have the ability to swing out to 90 degrees once they are in rotation (Video 1-2). Amazingly, the original PRP systems developed by Harvest and Marx utilized and still use this technology.

Fig 1-8 (a) Clinical photograph of a Bio-PRF centrifuge. (b) Photograph demonstrating the horizontal centrifugation concept. The tubes are inserted vertically (up and down), but once the device begins to rotate, the tubes swing out completely horizontally. This favors better blood cell layer separation with higher platelet and GF concentrations.

In 2019, an article on the topic demonstrated clearly that horizontal centrifugation could lead to up to a four-times greater cell content when compared to fixed-angle centrifugation.⁴¹ This represented a marked ability to greatly concentrate cells found within PRF, which were primarily being accumulated on the back distal surfaces of PRF tubes (Fig 1-9). The major disadvantage of fixed-angle centrifugation is that during the spin cycle, cells are typically driven along the back wall of the centrifugation tubes at high g-forces (Fig 1-10). This also exposes cells to higher compressive forces against the back wall, and cells must then separate by traveling either up or down the inclined centrifugation slope based on their respective cell density differences. Because red blood cells are larger and heavier than platelets and leukocytes, they travel downward, whereas lighter platelets travel toward the top of the tube where PRF is collected. This makes it relatively difficult for the small cell types such as platelets and leukocytes to reach the upper layer, especially granted that red blood cells outnumber in particular white blood cells typically by 1,000-fold (see chapter 2). Therefore, it is not possible to reach optimal accumulation of platelets or leukocytes using a fixed-angle centrifuge.

Fig 1-9 Illustrations comparing fixed-angle and horizontal centrifuges. With horizontal centrifugation, a greater separation of blood layers based on density is achieved owing to the greater difference in RCF-min and RCF-max. Following centrifugation on fixed-angle centrifuges, blood layers do not separate evenly, and as a result, an angled blood separation is observed. In contrast, horizontal centrifugation produces even separation. Owing to the large RCF values (~200g–700g), the cells are pushed toward the outside and downward. On a fixed-angle centrifuge, cells are pushed toward the back of centrifugation tubes and then downward/upward based on cell density. These g-forces produce additional shear stress on cells as they separate based on density along the back walls of centrifugation tubes. In contrast, horizontal centrifugation allows for the free movement of cells to separate into their appropriate layers based on density, allowing for better cell separation as well as less trauma/shear stress on cells. (Modified from Miron et al.⁴¹)

Fig 1-10 Visual representation of layer separation following either L-PRF or H-PRF protocols. L-PRF clots are prepared with a sloped shape, and multiple red dots are often observed on the distal surface of PRF tubes, while H-PRF results in horizontal layer separation between the upper plasma and lower red corpuscle layer.

Furthermore, by utilizing a novel method to quantify cell types found in PRF, it was possible to substantially improve standard i-PRF protocols that favored only a 1.5- to 3-fold increase in platelets and leukocytes. Noteworthy is that several research groups began to show that the final concentration of platelets was only marginally improved in i-PRF when compared to standard baseline values of whole blood.^41,42 In addition, significant modifications to PRF centrifugation protocols have further been developed, demonstrating the ability to improve standard i-PRF protocols toward liquid formulations that are significantly more concentrated (C-PRF) with over 10- to 15-times greater concentrations of platelets and leukocytes when compared to i-PRF (see chapters 2 and 3). Today, C-PRF has been established as the most highly concentrated PRF protocol described in the literature.

Conclusion

Platelet concentrates have seen a wide and steady increase in popularity since they were launched more than two decades ago. While initial concepts launched in the 1990s led to the working name platelet-rich plasma, subsequent years and discoveries have focused more specifically on their anticoagulant removal (ie, PRF). Several recent improvements in centrifugation protocols, including the low-speed centrifugation concept and horizontal centri-fugation, have led to increased concentrations of GFs and better healing potential. Both solid-PRF as well as liquid-based formulations now exist, with an array of clinical possibilities created based on the ability to accumulate supraphysiologic doses of platelets and blood-derived GFs. Future strategies to further improve PRF formulations and protocols are continuously being investigated to additionally improve clinical practice utilizing this technology.

References

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