Platelet-rich fibrin to incorporate bioactive graft materials

CHAPTER

Platelet-rich fibrin to incorporate bioactive graft materials

7

H. Almeida Varela*, M.A.P.P. Noronha Oliveira†, J. Pereira§, J.C.M. Souza‡, N. Pinto¶, M. Quirynen‖ Federal University of Rio Grande do Norte (UFRN), Natal, Brazil* Federal University of Santa Catarina (UFSC), Florianópolis, Brazil† University of Minho, Guimarães, Portugal‡ University Fernando Pessoa (UFP), Porto, Portugal§ De Los Andes University, Santiago, Chile¶ University Hospitals, KU Leuven, Leuven, Belgium‖

7.1 ­INTRODUCTION The remodeling of bone tissue (e.g., after tooth extraction) can result in a loss of bone volume that can negatively affect the cranio-maxillofacial and oral rehabilitation [1]. The material used to reestablish such bone loss can be divided, according to its origin, in autogenous, xenogenous, and synthetic or aloplastic. Among several biomaterials, autogenous bone is often selected as the first choice for bone regeneration although some disadvantages are reported in literature such as available volume, morbidity, and surgical complications [2]. Bone substitutes with a low resorption rate, such as xenografts or ceramics, are chosen if a higher volume stability is desired [3–5]. Recently, bioactive ceramics have been the focus of intensive research. Some of the advantages shown by ceramic biomaterials are its bioactivity, micro-/nanoporosity, osteoconductivity, and non-immunogenicity. Thus, bioactive ceramics are suitable candidates for the manufacturing of bone-like scaffolds. Scaffolds support osteoconduction in bone neoformation as they allow the recruitment and migration of osteogenic cells [6]. These scaffolds can be embedded within bioactive molecules such as growth factors or antimicrobial compounds [7]. Platelet-rich fibrin (PRF), an autologous leukocyte- and platelet-rich fibrin matrix, rich in growth factors, has shown to improve tissue healing in different applications [8,9]. PRF reveals a dense fibrin network with nanometric fibers that can act as scaffold for cell proliferation, migration, and differentiation and for delivery of growth factors, leading to an enhancement of neoangiogenesis [10,11]. The cross-linking between the fibrin fibers mechanically stabilizes the architecture of the fibrin-based scaffold. This intricate nanostructure of fibrin shows extraordinarily elastic mechanical and biological behavior [12,13]. This chapter addresses the morphological and biological aspects of PRF and the protocols for its preparation. Also, the existing evidence on the effect of PRF on Nanostructured Biomaterials for Cranio-Maxillofacial and Oral Applications. https://doi.org/10.1016/B978-0-12-814621-7.00007-X © 2018 Elsevier Inc. All rights reserved.

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tissue healing is described. A novel strategy involving a mixture of PRF and bioactive ceramics is also reported in the present chapter.

7.2 ­MORPHOLOGICAL AND BIOLOGICAL ASPECTS OF PRF PRF consists of an autologous leukocyte- and platelet-rich fibrin matrix [14] that involves a fibrin tetra molecular structure, embedding leukocytes, platelets, cytokines, and stem cells [15]. PRF acts as a biodegradable scaffold [16] that supports cell migration and the accomplishment of microvascularization [17,18]. The morphological aspect of the fibrin network is shown in Fig. 7.1. Moreover, PRF may operate as a delivery system of cells and growth factors leading to enhancement of wound healing during the two first weeks [19–21]. The PRF clot and membrane contains most of the platelets and half of the leukocytes present in the initial blood harvest. Platelets are mostly activated and act as a cement to reinforce the strongly polymerized fibrin matrix (Fig. 7.1) [22]. Leukocytes (a majority of lymphocytes) are trapped within the fibrin network. They are still alive and ready for the tissue-healing process [23]. In fact, the natural architecture combines a wide cell population, large quantities of mediators (particularly platelet growth factors) into a strong natural fibrin matrix [22]. The cell composition of PRF implies that this biological material is a blood-derived living tissue and must be handled carefully to keep its cellular content alive and stable [11]. The PRF clot is formed by a natural cross-linking polymerization process during centrifugation in the presence of a physiological amount of thrombin [14]. The reaction process results in a three-dimensional organization of a fibrin network, as seen in Fig. 7.1 [22]. The activated platelets release a high amount of transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and thrombospondin-1 growth factors that stimulate migration

5 µm

(A)

100 nm

(B)

FIG. 7.1 Morphological aspects of PRF: (A) scanning electron microscopy (SEM) image recorded for L-PRF obtained by secondary electron (SE) mode at ×6,000. Black arrows indicate red blood cells, while white arrows indicate activated platelet. Gray arrows indicate fibrin network which is shown in details on (B) the right SEM image at ×35,000 magnification.

7.2 ­ Morphological and biological aspects of PRF

and maturation of mesenchymal and epithelial cells [24]. These growth factors also stimulate biological functions, such as chemotaxis, angiogenesis, cell proliferation, differentiation, and modulation [25,26]. The addition of platelet derivatives into bone defects result in an accelerated rate and degree of bone formation [27–30]. VEGF is the most powerful and abundant component among the vascular growth promoters [31]. It plays a direct role in the control of endothelial cell behavior, such as proliferation, migration, and specialization [32,33]. In fact, the simple presence of this cytokine will be enough to start angiogenesis, and therefore, the combination of different VEGF isoforms can refine the development pathway of the network growth. PDGF is the first growth factor present in a wound-healing process that initiates connective tissue healing followed by bone repairing [34–36]. The most important specific activities of PDGF include mitogenesis, angiogenesis, and macrophage activation [37]. In fact, the chemoattraction and proliferation of osteoblasts and fibroblasts are accelerated by PDGF [38,39]. Inflammatory cells are also stimulated by PDGF to secrete other growth factors that drive the wound-healing process [40]. TGF-β1 belongs to the group of growth and differentiating factors including the bone morphogenetic protein (BMP). When released by platelet degranulation or actively secreted by macrophages, TGF-β1 and BMP act as paracrine growth factors, affecting mainly fibroblasts, marrow stem cells, and the preosteoblasts. TGF-β1 stimulates the proliferation of fibroblasts and their production of collagen I and III [41,42]. TGF-β1 represents a mechanism for sustaining a long-term healing and bone regeneration module evolving to a bone remodeling factor over time [37]. TGF-β1 also stimulates osteoblast progenitor cells and induces apoptosis of osteoclasts [43]. Although most studies have focused primarily on the effect of growth factors, the three-dimensional fibrin matrix also plays key roles in tissue repair [44,45]. Fibrinogen and fibrin, in conjunction with growth factors, effectively support adhesion and proliferation of cells by increasing collagen production [46]. Fibrin acts as an scaffolding biological material for adherent cells to accumulate at the site of tissue regeneration [45,46]. Additionally, fibrin is a carrier of growth factors in a wellcontrolled release system that sustain a proper bioactivity over the healing period [47,48]. The intricate architecture of the PRF fibrin matrix offers a proper mechanical behavior due to design and elasticity provided by the cross-linked monomer units [49–51]. During the formation of PRF, fibrils undergo lateral associations and form branches that result in a complex fiber network. The high degree of equilateral fibril branching results in the membrane elasticity [12]. The fine nanostructure of fibrin, after the gel point, has been physicochemically characterized to show dynamic behavior and complex hierarchy at different scales [13]. The morphology and mechanical behavior of PRF depend on the proportion of fibrinogen and thrombin. For instance, a low concentration of thrombin results in clots with thick fibers, less branch structures, and larger voids. [43–45,48] Fibrin fiber diameter affects the surface area available for cell adhesion and interactions during platelet activation [52]. A descriptive analysis of the fibrin network formed when using standard protocols to produce PRF demonstrates a dense network of fibers of about 90 nm thickness. The microspaces found in the fibrin network are filled by cells and growth factors. Furthermore, cross-linking between fibrin fibers

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­ echanically stabilizes the architecture of fibrin networks and controls the fibrim nolytic activity of plasmin [53]. Fibrin not only acts as a scaffold into which cells infiltrate but also provides molecular signals to direct cell function, since it contains binding sites for integrins, growth factors, and other extracellular matrix components including fibronectin [54]. Overall, the quality and quantity of fibrin fibers, in addition to growth factors, affect the potency and efficacy of PRF in tissue healing [20].

7.3 ­PROTOCOLS FOR PRF PREPARATION Starting from the earliest reports on the efficacy of fibrin gel, almost 30 years passed by before platelet-rich plasma (PRP) was identified as a promising reservoir of growth factors [28,52,54,55]. Initially, the inherent liquid state of PRP was converted to a gel state prior to clinical use. This conversion was achieved by adding bovine thrombin to PRP preparations to minimize the rapid diffusion of growth factors at the site of application. This procedure is a conventional method for clotting as reported in several articles [56–58]. In 2001, a novel technique was proposed to remove xenofactors [14]. From that moment, PRP clotting was achieved by stimulating only the endogenous coagulation pathway. This has simplified the preparation protocol. This well-known “second-generation platelet concentrate,” designated as leukocyte- and platelet-rich fibrin (L-PRF), has been increasingly used as a “PRP” substitute in regenerative medicine. The preparation of L-PRF was emphasized in the Consensus guidelines on the use of L-PRF from the first European meeting on enhanced natural healing in dentistry at the Katholieke Universiteit Leuven (Leuven, Belgium) in 2016. L-PRF is one of the four main groups of platelet concentrates for surgical use [59] and is frequently used in oral and maxillofacial surgery to enhance wound healing [60]. The L-PRF technology is very accessible and inexpensive in comparison with the many kinds of PRP [61]. The original L-PRF was developed as an open-access protocol, but the material and method were tailored in order to reach optimal performance [22]. For L-PRF preparation, blood (9 mL) is collected from a patient in specific glass-coated plastic tubes without anticoagulant and immediately centrifuged using a high-quality table centrifuge at 2700 rpm during 12 min (Fig. 7.2A). At the end of the process, a large L-PRF clot can be collected in the middle of each tube (Fig. 7.2B,C) and used in bone defects as illustrated in Fig. 7.2 D–H. This clot can be used directly to fill a tissue defect [29,62] or compressed into a membrane [63] or a fibrin cylinder [64] using the adequate equipment (Xpress kit, Intra-Lock, the United States) (Fig. 7.3) to avoid any damage [65].

7.4 ­PRF EMBEDDING NANO-POROUS BIOACTIVE CERAMICS Bone graft healing is a sequential process involving inflammation, revascularization, osteogenesis, remodeling, and incorporation into the host skeleton. The bone defect should be filled with a matrix that facilitates attachment, migration, and ­differentiation

7.4 ­ PRF embedding nano-porous bioactive ceramics

FIG. 7.2 Process of preparing L-PRF clots and membranes. (A) Venepuncture and blood collection. (B) L-PRF clot; and (C) clear separation: red blood corpuscles (RBCs) at the bottom, platelet poor plasma (PPP) on the top, and L-PRF fibrin clot in the middle. (D) L-PRF clots before compression into membranes. (E) Schematics of an intrabony defect filled with chopped L-PRF membranes parts and covered with L-PRF membranes. (F,G) Application of chopped L-PRF membrane in the defect. (H) Flap suturing, preferably with primary closure of the interdental papilla in the absence of stress. Adapted from Pinto NR, Temmerman A, Teughels W, Castro A, Cortellini S, Quirynen M. Consensus guidelines on the use of L-PRF from the first European meeting on enhanced natural healing in dentistry. Technical report. Catholic University of Leuven, Belgium; 2016, available on https://www.researchgate.net/ publication/309534770_Consensus_Guidelines_on_the_Use_of_L-PRF_from_the_1st_European_Meeting_on_ Enhanced_Natural_Healing_in_Dentistry.

of osteoblastic progenitor cells. As previously mentioned, the strong and flexible fibrin network of PRF will support cytokines enmeshment and migration of cells that are involved in angiogenesis and responsible for remodeling of new tissue. This is clearly different when compared with other kinds of platelet concentrates [8,14,66]. As seen in Fig. 4, PRF can be used in association with autografts, allograft, and xenograft materials [67–69]. L-PRF can also be mixed with particulate bone substitutes to produce a composite bioactive block for bone augmentation (Fig. 7.4A–F) [60]. In the case of extensive defects, the handling of bone grafts for bone augmentation can sometimes be difficult. The use of liquid fibrinogen mixed with chopped PRF membranes and particulate graft biomaterial becomes a good strategy. The liquid fibrinogen is slowly transformed into fibrin that can act as an autologous fibrin binder (AFB), as shown in Fig.  7.4C–F. Classic fibrin binders and AFB are commonly used by clinicians to improve the biomechanical properties of particulate bone grafts and to enhance their biological potential [70]. Different protocols have been proposed to obtain AFB [70,71] although on its cellular composition and biological properties are still scarce. Liquid fibrinogen is

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FIG. 7.3 (A,B) Preparation of L-PRF plugs with Xpress kit (Intra-Lock, Boca Raton, Florida, the United States). (C) Schematics of an extraction socket filled with L-PRF plugs. (D) Accurate removal of “all” inflammation and granulation tissue. (E) Placement, one by one, of the L-PRF plugs in the socket and vigorous compression. (F) Stress-free suturing with a modified internal mattress or external mattress technique, primary closure is not necessary at all. Adapted from Pinto NR, Temmerman A, Teughels W, Castro A, Cortellini S, Quirynen M. Consensus guidelines on the use of L-PRF from the first European meeting on enhanced natural healing in dentistry. Technical report. Catholic University of Leuven, Belgium; 2016, available on https://www.researchgate.net/ publication/309534770_Consensus_Guidelines_on_the_Use_of_L-PRF_from_the_1st_European_Meeting_on_ Enhanced_Natural_Healing_in_Dentistry.

obtained by modifying the spin centrifugation forces. The fibrin coagulation could be deaccelerated at early time points via lower centrifugation speeds and by using nonglass centrifugation tubes, in order to generate a fibrinogen binder [71]. For instance, blood is collected from patient in sterile specific glass-coated plastic vacutainer tubes (9 mL) without anticoagulant. Vacutainer tubes are then placed in a high-quality table centrifuge machine at 2700 rpm for 3 min. After this process, one can clinically notice a yellow color region in the tube (liquid fibrinogen) with the remaining blood components below. The tubes are opened carefully to avoid homogenization of the material. The liquid fibrinogen is then collected from the tubes using a 20 mL syringe with an 18G hypodermic needle [70]. Liquid fibrinogen contains an amount of stem

7.4 ­ PRF embedding nano-porous bioactive ceramics

FIG. 7.4 Clinical preparation of a PRF block using 0.5 g particulate bioactive material (bone substitute). (A) Collection of L-PRF clots and (B) mix chopped membranes with bone substitute in Ti dish, and stirring gently while shaping it to the desired geometry and state. (C) L-PRF block ready for use and (D,E) its placement over implant with buccal dehiscence. (F) Schematics of L-PRF block for horizontal bone augmentation. Adapted from Pinto NR, Temmerman A, Teughels W, Castro A, Cortellini S, Quirynen M. Consensus guidelines on the use of L-PRF from the first European meeting on enhanced natural healing in dentistry. Technical report. Catholic University of Leuven, Belgium; 2016, available on https://www.researchgate.net/ publication/309534770_Consensus_Guidelines_on_the_Use_of_L-PRF_from_the_1st_European_Meeting_on_ Enhanced_Natural_Healing_in_Dentistry.

cells from the blood stream. Fibrinogen binder can be sprayed over the bone graft, resulting in clotting after a few seconds and encapsulation of bioactive graft particles (Fig. 7.4B,C). That leads to a formation of a compact block (Fig. 7.4C) for bone healing (Fig. 7.4D–F) [72]. The fibrin matrix of PRF can thus work as a scaffold for other biomaterials (Figs. 7.4 and 7.5), favoring neoangiogenesis, while the bone graft material serves as space maintainer for the regenerative volume. Biomaterial-embedding PRF supports the nucleation and accumulation of newly formed bone matrix. This combination gives the biomechanical strength necessary during the first steps of healing [10,11]. Considering that PRF is completely resorbed, leaving no residual particles in the regenerated sites, attention should be taken when choosing the bone graft substitute for mixing with PRF, since clinical data on resorption of such mixture are lacking.

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FIG. 7.5 SEM images of the combination between a fibrin glue network and BCP granules. (A) The morphology of the mixture between a fibrin glue network and BCP granules. (B) Fibrinbased glue between the BCP granules.

Most of the bone grafts are osteoconductive in nature. Thus, the adjunct use of PRF may be useful in bone regeneration due to its osteoinductive properties. Each given bone substitute has an individual healing profile concerning osteogenic potential and substitution rate. This must be taken into account when selecting adjunctive grafting materials for bone regeneration procedures [4]. Autogenous bone, collected from the patient itself, intra- or extraorally, is globally considered as the first choice considering osteogenesis, osteoinduction, and osteoconduction. Autogenous bone has significantly enhanced the formation of new bone when compared with bone grafts of allogenic, xenogenic, and synthetic (alloplastic) origin [4]. Human bone possesses a porous structure ranging from 20 to 400 μm. Such porosity is necessary for penetration, adhesion, growth, and proliferation of osteogenic cells, thus allowing new bone formation [73]. Despite being considered the first choice, autogenous bone presents some disadvantages such as donor-site morbidity and limited sources. The osteogenic effect of an autogenous bone graft at the implant surface during simultaneous bone regeneration procedures may be regarded as important to ensure osseointegration. However, the high rate of resorption of autogenous bone graft may not sustain long-term volume stability [74]. The ideal bone substitute not only should stimulate new bone formation and promote neovascularization but also should have a low substitution rate to maintain volume stability during ongoing remodeling over time. Although potentially osteoconductive and usually providing a scaffold for osteogenesis, bone substitutes possess an inferior osteogenic potential as compared with autogenous bone [74,75]. Biomaterials with a low resorption rate such as xenogenous or ceramics can provide a higher volume stability when compared with autogenous bone grafts [3–5]. Allogenic bone grafts (allografts), derived from a cadaveric donor, can be used in a mineralized or demineralized form. A few previous studies reported the presence of prions in allogenic grafts that has caused a controversial issue for clinical applications. The size of allogenic particles may vary between 250 μm and 2 mm. Allografts present a more rapid resorption and replacement by new bone when

7.4 ­ PRF embedding nano-porous bioactive ceramics

compared with xenografts [77,78]. Graft materials of xenogenic origin (bovine, equine, and porcine) are currently used worldwide and with a high acceptance rate. The best documented xenogenic bone substitute is deproteinized bovine bone mineral (DBBM). DBBM is essentially considered a hydroxyapatite (HA) ceramic after the removal of its organic components. It is often used for bone augmentation procedures when volume stability is desired due to its slow resorption and delayed healing. This results in the detection of residual particles several years following bone augmentation procedures [79–82]. In a systematic review conducted in 2013, Kim et al. concluded that bovine graft biomaterials may represent a risk for the transmission of prions to patients [76]. Particle size may normally range from 250 μm to 2 mm [83]. Increased porosity of bovine hydroxyapatite (BHA) seemed to benefit the osteoconduction process in dogs [83]. Synthetic bioactive ceramic materials are another alternative for bone regeneration favorable because of their well-controlled porosity, biocompatibility, osteoconductivity, and non-immunogenicity, thus being suitable candidates to be used in the manufacturing of bone-like scaffolds. Bioceramic materials such as calcium phosphates, bioactive glasses, and glass ceramics may also be designed to deliver biologically active substances aimed at repairing, maintaining, restoring, or improving the function of tissues [73]. Bioactive ceramics can be produced as porous or compact microparticles or coatings. Also, the size of their pores can be produced at micro- or nano-scale depending on the application [84,85]. Synthetic HA is one of the most commonly used bone substitutes. A minimal particle size of 10 μm is necessary for direct bone contact between bone and HA granules [86]. Granules with a 100–300 μm size have shown superior osteoconductive activity when compared with 1–2 mm particles [87]. Granule porosity exists between 60% and 80% [88,89]. Its osteogenic potential is lower than that for autogenous bone. However, the use of HA may be favored when volume stability of augmented sites is desired [4]. Pure-phase beta-tricalcium phosphate (β-TCP) provides a quicker bone remodeling than that for most bioactive absorbable ceramics [90]. However, this bone substitute is not indicated when long-term volume stability is desired. Bioceramics may be associated to combine the advantages and properties of both materials. Biphasic combinations of HA and β-TCP provide an excellent balance to achieve long-term stability and new bone formation [91]. HA should provide an initial bone response, while the β-TCP provides bone remodeling [92]. One of the most used calcium phosphates in dentistry is the biphasic calcium phosphate (BCP) with an HA/β-TCP ratio of 60/40 (%wt) (Fig. 7.5), which appears to mimic human cancellous bone structure [93]. Such ratio enables long-term volume stability although slows down the overall resorption capabilities. Comparatively with different bone grafts, BCP has revealed substantially higher osteogenic capacity, and it seemed to be as efficient as autogenous bone grafts [94,95]. The microstructure of the combination between a fibrin glue network (whiter arrows) and BCP (darker arrows) is shown in Fig. 7.5. The calcium phosphate grains composing the BCP granules exhibited areas of dissolution and needlelike crystal precipitation in the presence of fibrin glue (Fig. 7.5B).

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The addition of osteoinductive properties to ceramics seems to be of great importance in the regeneration of critical-sized orthotopic defects in a large animal model [96]. Bioactive glasses, most widely used in biomedical applications, consist of a silicate network incorporating sodium, calcium, and phosphorus in different relative proportions [97]. Silica-based ordered mesoporous materials have attracted the interest of researchers, exhibiting highly ordered nanostructured surfaces and porosity higher than that obtained for the conventional sol-gel bioglasses [98]. The larger specific surface area of the nanoparticles should lead also to increased interface effects and contribute to improved bioactivity, when compared with standard micro-scale particles [99,100]. These silica-based ordered mesoporous materials exhibit the fastest in vitro bioactivity reported up to date. Mesoporous bioactive ceramics can also incorporate growth factors, osteoclasts inhibitors, and antimicrobial and antitumoral drugs [100]. Also, the lack of strength and bioactivity of bioceramics can be solved by adding biopolymers [101,102]. In turn, the addition of a ceramic phase to a biodegradable polymer may be used to favorably alter the polymer degradation behavior [103]. Synthetic scaffolds are aimed to provide temporary mechanical stability until new bone becomes synthesized, organized, and consolidated into a stable structure. Scaffolds obtained with bioactive glasses must display interconnected pores over 100 μm to allow bone cell ingrowths and angiogenesis [100]. Within the human body, pore diameters below 1 μm play a role in bioactivity and protein interaction. Pore diameters between 1 and 20 μm would determine the cellular behavior and the type of attached cells. Pores ranging between 100 and 1000 μm are responsible for cellular growth, blood flow, and mechanical strength, whereas pore diameters larger than 1000 μm would determine the shape of the biomaterial and its functionality [73]. Thus, materials used in tissue engineering should have a pore diameter ranging from 2 nm to 1000 μm and a coexisting macro-, micro-, and nano-scale porosity [73]. A minimum space of 100 μm among graft particles is essential to allow for neovascularization and bone formation [104,105]. Data regarding morphological aspects and main outcomes of some of the existing bone substitutes are summarized in Table 7.1.

7.5 ­EVIDENCE OF TISSUE HEALING PRF plugs and membranes are substitutes for connective tissue grafts in periodontal plastic surgery, intrabony defects and tooth furcation defects [8]. The use of PRF plugs and membranes as sole material for alveolar socket filling was shown to be beneficial for preservation of the horizontal and vertical ridge dimension for 3 [106] and 4 months [30] after tooth extraction. These membranes act as a barrier, avoiding epithelial cell invagination into the postextraction socket, when gingival wound closure is impossible or difficult to suture. PRF membranes can also be used in a second-stage implant surgery, allowing the gain of keratinized tissue. In these cases, no free gingival graft or soft tissue substitute is needed, thus reducing patient's morbidity and costs. PRF membranes are also of great efficacy in a case of a Schneiderian membrane perforation. The fibrin matrix can aid in membrane closure, thus avoiding

Table 7.1  Summary of the Characteristics of Biomaterials. Authors (Year)

Particle Size (μm)

Pore Size

Main Outcomes

Carvalho et al. (2007)

(a) Bovine hydroxyapatite (small granules) (b) Bovine hydroxyapatite (large granules) (c) Synthetic hydroxyapatite (small granules) (d) Synthetic hydroxyapatite (large granules)

(a) 150–200 (b) 300–329 (c) 150 (d) 300

In all samples, the granules had micropores (<10 μm)

Coathup et al. (2013)

Silicate-substituted calcium phosphate (SiCaP) with four different granules sizes

NA

Berberi et al. (2014)

(a) Calcium phosphate silicate hydroxide (b) Calcium gadolinium oxide phosphate (c) Calcium phosphate (d) Sodium calcium iron phosphate (e) Calcium phosphate silicate hydroxide (f) Calcium phosphate silicate hydroxide (g) Calcium phosphate silicate hydroxide (h) Calcium phosphate silicate hydroxide (i) Calcium phosphate silicate hydroxide (autogenous bone) (a) Nanocrystalline HA embedded in a matrix of silica gel (HA-SiO) (b) Deproteinized bovine bone mineral (DBBM) (c) Synthetic BCP with a 60/40% HA/ β-TCP ratio (d) Particulated autogenous bone

1000–2000 (100 vol%) 250–500 (75 vol%) 90–125 (75 vol%) 90–125 (50 vol%) (a) 0.26–8.92 (b) 174.62–1337.48 (c) 22.79–517.2 (d) 3.90–15.18 (e) 8.82–1337.48 (f) 174.62–1167.72 (g) 152.45–2301.84 (h) 39.24–1754.62 (i) 90.5–465.15

BHA’s material characteristic itself rather than granules size accounted for the distinctive biological behavior. Increased roughness of the BHA's surface seemed to benefit the osteoconduction process in dogs SiCaP granules of 250–500 μm in size may be a more suitable scaffold when used as an injectable bone filler

NA

Different in terms of calcium concentration, particle size, and crystallinity, which may affect their in vivo performance

(a) 600 (b) 250–1000 (c) 500–1000 (d) 1000–2000

HA-SiO, 10–20 nm Other groups, NA

Osteogenic potential of HA-SiO and BCP was inferior when compared with autogenous bone. When low substitution rate is desired to maintain volume stability, HA-SiO, and DBBM may be favored

Broggini et al. (2014)

Continued

7.5 ­ Evidence of tissue healing

Chemical Composition (%wt)

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130

Authors (Year)

Chemical Composition (%wt)

Particle Size (μm)

Pore Size

Main Outcomes

Desterro et al. (2014)

Three different DBBMs

NA

DBBM with 15 nm HA crystallites showed a greater surface area and calcium release rate than the others

Dahlin et al. (2015)

(a) Synthetic BCP with a 10/90% HA/ β-TCP ratio (b) Synthetic BCP with a 60/40% HA/ β-TCP ratio (c) Deproteinized bovine bone mineral Porous poly-l-lactic acid (PLLA) scaffold compounded with borosilicate bioactive glasses (BBGs)

(a) 500–1000 μm (b) 500–1000 μm (c) 250–1000 μm

15 nm 39 nm Indeterminable crystallite size NA

Fernandes et al. (2017)

NA, not applied.

NA

Randomly interconnected porous (58%–62% of interconnectivity and 53%–67% of porosity) with mean pore diameters higher than 100 μm

Synthetic BCP with a 10/90% HA/βTCP ratio showed significant higher amounts of newly formed bone despite a higher remaining graft volume compared with the other groups PLLA-BBGs are not cytotoxic to cells, while demonstrating their capacity to promote cell adhesion and proliferation. They present a tailored kinetics on the release of inorganic species and controlled biological response under conditions that mimic the bone physiological environment

CHAPTER 7  Platelet-rich fibrin to incorporate bioactive graft materials

Table 7.1  Summary of the Characteristics of Biomaterials—cont'd

7.5 ­ Evidence of tissue healing

further complications [107,108]. PRF can be used as a sole filling material in sinus lift elevation, only if immediate implant placement is possible, as implants force the sinus membrane to be maintained in an elevated position [29,62,108,109]. A bone substitute should be associated to PRF when immediate implant placement is not possible during sinus elevation [29,62]. PRF allows a better implant stability in healed sites over time with less marginal bone loss [9]. Good results have also been shown during its use in immediate implant placement, being either as a filling material or for implant coating [110,111]. The avoidance of epithelial cell invagination by PRF membranes may be useful for guided bone regeneration. The principle of guided bone regeneration is to maintain a surgically created volume, thus excluding rapidly proliferating epithelial cells and fibroblasts and stimulating the migration of osteogenic cells and angiogenesis [4,112]. The ingrowth, proliferation, and differentiation of osteoblasts occurs during the first days [113] and the first provisional matrix forms in 7–14 days; thus, it is important that VEGF and TGF are present at that time. PRF acts as an immune regulation node with inflammation control abilities, including a slow continuous release of growth factors over a period of 7–14 days [17]. He et al. [114] reported that PRF membrane releases maximum levels of TGF-β1 until the 14th day. Randomized controlled trials using an association of PRF with allografts are scarce. Nonetheless, the existing ones have shown significant results [112,115–120]. The use of xenografts can counteract the ridge resorption in vestibule-oral dimension. On the other hand, their use in sockets has also been questioned as they may possible interfere with the natural healing process [121,122]. Xenograft particles can still be present for a long period of time and not be completely resorbed [82]. These remnants may decrease the final bone-to-implant contact when associated with osseointegrated implants [123]. However, PRF is completely resorbed, which may increase the proportion of “vital” bone. Lekovic et al. reported that PRF can improve clinical parameters linked to human intrabony periodontal defects. The combination of PRF with xenografts has shown good results for reducing pocket depth and in sinus floor elevation [124,125], with a faster bone healing [126]. However, the application of PRF in combination with deproteinized bovine bone graft did not bring an advantage nor disadvantage in sinus augmentation after a healing period of 6 months [127]. Some animal studies reported no additional effect of PRF on bone regeneration. In a study performed in the rabbit calvaria, Knapen et al. [128] noticed that PRF did not seem to provide any additional effect on the kinetics, quality, and quantity of the bone. Faot et al. [129] also reported that PRF treatment did not enhance bone healing in noncritical size rabbit tibiae defects after 28 days. There is still a lack in randomized controlled studies evaluating the effectiveness of PRF to repair large bone defects. Ezirganli et al. [130] recommended that PRF should not be used as a sole material in guided bone healing but deproteinized bovine bone graft or BCP instead. Also, there is still some controversy as some authors defend that a collagen membrane is necessary to avoid epithelial cell inclusion in larger bone defects. An in vitro study found favorable results for PRF membrane as a scaffold for human periosteal cell proliferation when compared to collagen membranes [131]. In another study performed in humans, a similar outcome was reported for PRF membrane ­regarding

131

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mean percentages of vital bone formation and residual xenograft when compared to collagen membrane to cover a lateral bone window after sinus elevation [132]. To date, the number of studies evaluating the success of PRF in association with bioactive ceramics in humans is also scarce. One of the most used bioactive ceramics is synthetic HA. Elgendy and Abo Shady [133] reported that a nanocrystalline hydroxyapatite (NcHA) bone graft in combination with PRF demonstrated clinical advantages beyond that achieved by NcHA alone in the treatment of intrabony defects. Other studies showed that HA increases the effects of PRF in the treatment of human three-wall intrabony defects [134,135]. In a study performed in the rabbit calvaria, Acar et al. [136] found that the combination of PRF and a synthetic ceramic graft composed of HA and β-TCP resulted in a further significant increase in new bone formation compared with the sole use of PRF or HA/β-TCP. In another rabbit study, the addition of HA/β-TCP to PRF significantly increased the formation of new bone, providing a better substrate for bone healing [137]. The association of PRF and bone substitutes can also be used in immediate implant placement. The use of PRF mixed with an equine xenograft in a 50/50 proportion during immediate implant placement showed promising results and could be a natural complement for screw-guided bone regeneration [10]. Andrade et al. [71] described the hematologic profile of four AFB obtained via different protocols. The authors observed that the leukocyte, platelet, lymphocyte, monocyte, and neutrophil count were significantly higher in the i-PRF protocol. However, future studies are needed to assess the biological potential of each AFB. An in vitro study showed than one of those AFB, obtained without the use of anticoagulants, demonstrated the ability to release higher concentrations of several growth factors and induced higher fibroblast migration and expression of PDGF, TGF-β, and collagen 1 when compared with PRP. However, the authors state that animal research is necessary to validate the use of AFB as a bioactive agent capable of stimulating tissue repair [138]. Besides advantages in tissue healing, a reduction in postoperative discomfort and pain and antimicrobial effects due to the presence of leukocytes in the fibrin clot has also been described [17,139,140]. Some studies used composites of PRF and/or nanoporous bioactive ceramics as scaffolds for mesenchymal stem cells and showed good results [112,141].

7.6 ­CONCLUDING REMARKS PRF has shown clinical benefits on bone healing and osseointegration, and therefore, current in vivo studies are carried out on the combination of PRF and bioactive graft materials. PRF can be produced by a simple technique, and its association with other biomaterials may reduce treatment costs as less bone graft is needed to fill the bone defect. Despite the many applications of PRF, there is still no well-defined standard protocol for most surgical procedures [8]. The correct handling of PRF and the use of enough clots/membranes and bioactive graft materials for surgical site might be crucial to obtain clinical benefits from this technique [8].

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­FURTHER READING [1] Ryan EA, Mockros LF, Weisel JW, Lorand L. Structural origins of fibrin clot rheology. Biophys J 1999;77(5):2813–26. https://doi.org/10.1016/S0006-3495(99)77113-4. [2] Menezes  DJB, Shibli  JA, Gehrke  SA, Beder  AM, Sendyk  WR. Effect of plateletrich plasma in alveolar distraction osteogenesis: a controlled clinical trial. Br J Oral Maxillofac Surg 2016;54(1):83–7. https://doi.org/10.1016/j.bjoms.2015.09.027.