Introduction

Different types of bone substitutes can be used to enhance bone healing such as autogenous, xenogeneic, allogeneic, and or alloplastic graft materials [1, 2]. Some of those bone substitutes reveal morphological aspects, physical properties, and chemical composition that lead to a low rate of cell migration and blood vessel formation, namely angiogenesis [1,2,3]. Many efforts have been carried out to develop novel bone graft materials that can enhance the wound healing [4,5,6]. Advanced ceramic materials have been studied considering particle size, chemical composition, porosity, pore size, and absorption rate into the bone [7,8,9,10]. Thus, the enhancement of bone healing is strongly dependent on the osteogenic cell migration, angiogenesis, and biomaterial resorption rate.

Platelet-rich fibrin (PRF) is an autologous biomaterial that contains a high amount of growth factors, platelets, fibrin monomer, fibrinogen, and blood platelets. The use of PRF has been shown to enhance tissue healing in different fields of medicine and dentistry [11, 12]. PRF reveals a dense fibrin network with nano-scale fibers that can act as scaffold for cell proliferation, migration, and differentiation. In addition, PRF acts like a drug delivery system of growth factors leading to an enhancement of neoangiogenesis [12, 13]. The cross-linking of fibrin fibers mechanically stabilizes the architecture of the PRF-based scaffold and therefore such intricate fibrin nanostructure shows extraordinarily viscous-elastic behavior for agglomeration of other materials and surgical application [14, 15].

PRF can be produced like a membrane or a viscous and flowable substance containing a liquid phase by varying methods of centrifugation [12, 16]. The liquid phase of PRF can be achieved at an early time point at low centrifugation speed and using non-glass centrifugation tubes. Such flowable PRF can be injectable using syringes or agglomerated with particulate biomaterials [17,18,19]. Flowable PRF is slowly converted into a viscous fibrin network that agglomerates particulate bone graft materials acting as an autologous fibrin binder (AFB) [19].

The use of PRF can overcome the clinical limitations of particulate bone grafts regarding its handling, bioactivity, and fitting in surgical sites. The combination of chopped PRF membranes, injectable PRF, and particulate graft materials results in manufacturing a bioactive composite block properly designed for bone augmentation [19]. In addition, the graft instability of particulate biomaterials can be avoided due to the fibrin cross-linking of the PRF that decreases the failure risks during bone healing [20]. Particulate graft materials can act as scaffolds and mineralization nuclei for cell migration and angiogenesis [21, 22]. Such bioactive composite block can be produced using a simple technique and therefore the association between PRF with other biomaterials may reduce treatment costs as less bone graft material is needed to fill the bone defect [19, 23]. In addition, the patient experiences less morbidity since no secondary harvesting surgical site is needed. Nevertheless, studies regarding the morphologic characterization of flowable PRF and PRF blocks containing different particulate biomaterials are scarce in literature. In addition, the chemical composition, dimensions, and three-dimensional morphological aspects of the bone substitutes and implant surfaces vary depending on the combination of bioactive materials.

Thus, the present study aimed to evaluate the morphological aspects of an injectable and flowable PRF after mixing with two different particulate hydroxyapatite-based granules, a porous zirconia block, and laser-textured zirconia or titanium surfaces. The present study reveals a detailed assessment of the morphological aspects regarding the use of particulate, textured, and porous biocompatible materials after interaction with platelet rich fibrin. In addition, novel porous zirconia and laser-textured zirconia or titanium surfaces have been examined after mixing with an injectable platelet rich fibrin that can be recommended for enhanced bone healing of large bone defects. It was hypothesized that the combination of particulate bioactive ceramics, porous zirconia, or laser-textured implant surfaces and platelet-rich fibrin revealed morphological and chemical aspects for enhanced bone healing considering the three-dimensional architecture and biological potential of the platelet-rich fibrin and biocompatible surfaces.

Materials and Methods

Preparation of the Bone Substitutes

Two different types of particulate hydroxyapatite (Hap) derivatives were used to produce PRF-based blocks (putty state): deproteinized bovine bone mineral (DBBM) and a synthetic Hap (Fig. 1A–C). Particulate DBBM had a grain size ranging from 125 up to 800 μm (Biograft™, Ossmed, Cantanhede, Portugal) while the synthetic Hap had a grain size ranging from 300 up to 400 μm (Osteogen™, IntraLock, Boca Raton, FL, USA). The chemical composition and grain size of the particulate bioactive materials are given in Table 1.

Fig. 1
figure 1

A Photographic image of the hydroxyapatite (Hap) and B SEM image of the deproteinized bovine bone mineral (DBBM). C PRF block involving Hap. D Photographic and E SEM image of the porous YTZP. F Photographic image of the PRF/YTZP block. G Photographic and H SEM image of the laser-textured YTZP. I Coating of the YTZP with i-PRF for J clinical procedure or K SEM analyses

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Table 1 Details on the particulate bone graft materials
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Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) spray-dried powder (TZ-3YSB-E™, TOSOH Co., Tokyo, Japan) with high purity (99% Y-TZP) was used in this study. According to the supplier, the powder had a maximum particle size of 120 µm and spherical agglomerates with an average size of 60 μm. A zirconia suspension was prepared according to the method described in previous studies [24, 25]. The suspension was prepared by adding 20 ml distilled water to 30 g zirconia powder. Then, the mixture was milled in a planetary ball mill (PM100, Retsch) at 400 rpm for 80 min to promote a powder deagglomeration and slurry homogenization. Afterwards, 1 wt% dispersant (Duramax 3005, Angene, UK) was added to 60 wt% Y-TZP powder to achieve the required pseudoplastic and thixotropic rheological behaviour for the replica procedure. The particle size distribution of the zirconia was measured in a laser diffraction equipment (Mastersizer 2000, Malvern, UK). For the development of the optimal processing route, porous specimens with interconnected pores were produced using PU sponges of 60 ppi (10 mm × 10 mm × 10 mm) as temporary templates. The PU sponges were manually impregnated with the ceramic suspension without clogging the pores and kept in a chamber at 100 °C for 20 min. A heat treatment with the usual parameters for sintering such material (5 °C/min up to 1500 °C for 120 min) was performed in a conventional high-temperature furnace to eliminate PU and consolidate the porous structures. At the end of the thermal treatment, fully sintered monolithic Y-TZP porous structures with interconnected pores were produced (Fig. 1D and E) although the percentage and morphological aspects of the interconnected pores changed when compared to the PU sponge. Such details were evaluated by further microscopic and chemical analyses. Also, the zirconia phase was not entirely maintained after the thermal treatment.

Preparation of Laser-Textured Implant Surfaces

Zirconia discs were prepared by cold compression for laser-texturization. The compaction of the 3 mol% Y-TZP spray-dried powder (TZ-3YSB-E™, TOSOH Co., Tokyo, Japan) was performed on a steel cylindrical mold with an internal diameter of 8 mm and 30 mm of height, with a pressure of 200 MPa for 30 s. After that, the pressure was released evenly to produce green zirconia compacts with 8 mm of diameter and 5 mm of thickness. The texture of zirconia discs was performed using Nd:YAG laser (OEM Plus, SISMA, Italy), with an output power of 6W, a spot size of 3 μm and a pulse width of approximately 35 ns. A focusing unit containing a fused quartz lens with a nominal focal length of 160 mm, a wavelength of 1.064 μm and a maximum pulse energy of 0.3 mJ/pulse was used. Surface texturing was carried out at room environment and then a jet of air braided was used to remove any debris produced over laser processing. Two strategies regarding line patterning  (8 and 16 lines pattern) and different laser parameters (laser power and number of laser passages) were adopted to assess their influence on the morphological aspects of the surfaces (Fig. 1G and H). Such patterns were designed  by computer-aided design (CAD) and then textured on the zirconia surfaces. Properties of implant materials are shown in Table 2.

Table 2 Properties of the zirconia and titanium specimens
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Also, commercially pure titanium (cp Ti) grade 4 specimens with 8 mm diameter and 5 mm thickness were cut and laser textured using a Nd:YAG laser (OEM Plus, Italy). As on the zirconia disks, a focusing unit containing a fused quartz lens with a nominal focal length of 160 mm, a wavelength of 1.064 μm and a maximum pulse energy of 0.3 mJ/pulse was used at argon atmosphere. The duration of scanning process was about 6 min in each experiment. Different laser parameters were tested to produce the above-mentioned textured lines. Specimens were then ultrasonically cleaned with acetone to remove any debris generated over Nd:YAG laser texturing.

Preparation of Flowable PRF and PRF Block

Peripheral venous blood was collected from three healthy, nonsmoker, men volunteers (age range 30, 32, and 34 years old), as seen in Figs. 1 and 2. A total of 78 ml blood was collected in eight tubes from volunteers within  18 ml in two three plastic tubes without anticoagulants or silica coating (VACUETTE™, Greiner Bio-one GmbH, Kremsmünster, Austria) while 18 ml blood was harvested in six silica-coated tubes (BD Vacutainer Serum Becton Dickinson, Franklin Lakes, NJ, USA). Blood retrieval was performed by a nurse at the Federal University of Santa Catarina (UFSC). The use of human blood was approved by the Human Research Ethics Committee at the Federal University of Santa Catarina (CEPSHUFSC), cod. 2229.085, that is in accordance with the Helsinki declaration of 1964. The volunteers signed the informed consent prior to inclusion in the study since the purpose of the project was established.

Fig. 2
figure 2

A Blood harvesting from the voluneteer followed by B centrifugation. C Tubes containing three different phases of the centrifuged blood. D L-PRF clot harvesting. E Injectable PRF. F Hydroxyapatite (Hap) granules and G Hap-based PRF block. H Porous zirconia and I zirconia-based PRF block

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The harvested blood was immediately centrifuged using a high-quality table centrifuge at 2700 rpm (408 g) and at room temperature for 3 min, according to the IntraSpin protocol (IntraSpin™, IntraLock™, Boca Raton, FL, USA). After 3 min centrifugation, 3 plastic tubes were removed from the centrifuge and the flowable PRF (upper yellow liquid phase) was immediately harvested using sterile syringes. Six silica-coated tubes remained under centrifugation for 9 min over a period of 12 min centrifugation [18, 19].

After 12 min centrifugation in silica-coated tubes, three phases can be noted: red blood corpuscles at the bottom of the tube, platelet-poor plasma (PPP) on the top, and an intermediate layer called “buffy coat” (yellow layer) where most leucocytes and platelets are concentrated [26]. Fibrin clots were harvested from the tubes using tweezers and the red blood cells were separated from the fibrin clot by using a periosteal elevator. Red blood corpuscles and PPP were discarded and then a yellow PRF clot was harvested from the middle of each tube. PRF clot was then compressed into a membrane using a proper apparatus (Xpress kitTM, IntraLock, Boca Raton, FL, USA) to avoid any damage to the cells. The PRF membranes were cut using a surgical trephine and divided into smaller cylindric fragments with 1 × 1 mm. The PRF fragments were added to 0.5 g particulate graft materials at a 50:50 ratio. Then, the flowable injectable PRF was added and stirred gently for ± 10 s while shaping it to the desired PRF block form regarding particulate. The injectable PRF was injected onto the porous zirconia blocks until immersion while the laser-textured surfaces were coated with the injectable PRF.

Scanning Electron Microscopy (SEM)

For SEM analysis in triplicate, three drops of the flowable i-PRF (each one within 40 µl) were applied onto each biomaterial specimens (n = 9). On the PRF blocks, specimens (n = 9) were placed onto titanium-based stubs for coupling to the SEM chamber. The SEM preparation protocol was adapted from the study of Varela et al. (2018) [18]. The injectable PRF was exposed to the specimens for 20 min for the achievement of the fibrin fibers’ cross-linking followed by washing three times in distilled water for 5 min. On the retention of PRF over the biomaterials for SEM analyses, specimens were immersed in a solution containing 2.5% glutaraldehyde and 2.5% paraformaldehyde for 30 min and therefore they were washed three times in PBS for 5 min. Then, the dehydration of PRF was achieved by immersing in a gradually series (25, 50, 75, 95, and 100%) of ethanol solutions. At last, samples were air-dried at room temperature for 5 min and then placed in a vacuum oven at 37 °C for 2 h. After dehydration, specimens embedded with PRF were sputter-coated with AuPd thin films and analyzed using two scanning electron microscopes (SEM, Vega 3, TESCAN, Brno, Czech Republic; and JSM-6010 LV, JEOL, Japan) equipped with energy dispersive X-ray spectroscopy (EDX). The morphological analysis of the fibrin fiber’s network and the bioactive block or coating was performed by SEM on secondary (SE) or backscattered electrons (BSE) mode at an accelerated voltage of 15 kV. SEM images were recorded at magnification ranging from × 30 up to × 20,000 at three different areas for each sample (n = 9). The chemical composition of the materials was evaluated by EDX following standard guidelines and other previous studies [18]. The chemical composition was acquired by rearranging the oxygen content to estimate the weight percentage of elements regarding the most stable stoichiometric arrangement. The dimensions and morphological aspects of the PRF components and materials were analyzed using the ImageJ 1.51 software (NIH, Bethesda, Maryland, USA).

Histomorphometry Analysis

For histological analyses, six samples of 2 ml flowable i-PRF (total of three samples per volunteer) were transferred to separate cell-culture well plates. After 20 min of fibrin cross-linking, 2 ml DMEM was placed onto the PRF samples and incubated in 5% CO2 and 37% CO2. Samples were harvested at 1 h time point and fixed in 10% buffered formaldehyde [18]. After fixation, samples were dehydrated in increasing ethanol series and embedded in paraffin. Then, 3-μm cross-section thin slices were produced and stained using the hematoxylin–eosin staining protocol. Three slides per sample were independently evaluated by two blinded and well-trained researchers in histomorphometry using light microscopy (MC 80 DX™ digital camera—Axioskop 2 plus™, Zeiss, Jena, Germany).

Results

The morphological aspects of the DBBM granules free of PRF are shown in Fig. 3A.

Fig. 3
figure 3

Scanning electron microscope (SEM) images showing the morphological aspects of flowable PRF mixed with the Hap or DBBM granules. A–C SEM images of the DBBM granules and their B macro- and C micro-scale pores. Fibrin-network and PRF components in contact with D–F DBBM and G–I Hap granules. Blood platelets (yellow arrows), red cells (red arrows), and leukocytes (white arrows) embedded in the fibrin network

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SEM images revealed a mean size of DBBM granules ranging from 150 up to 800 µm DBBM granules. Also, SEM images showed irregular morphological aspects with macro-scale pores ranging between 50 and 460 μm in diameter (Fig. 3B). The mean size of the micro-scale pores was around 110 μm (× 500 original magnification). Micro-scale pores ranged from approximately 330 up to 670 nm in diameter (Fig. 3C). The dimensions of Hap granules ranged from 200 up to 400 μm and pores were not detected on the surface.

After mixing with PRF, the fibrin network was noticeable embedding the DBBM and Hap granules and blood cells. The highest distance among DBBM granules was recorded around 730 µm while Hap blocks showed the distance among Hap granules at around 585 µm. The lowest distance among DBBM granules was recorded around 143 µm while Hap granules showed the distance among Hap granules at around 98 µm. A dense fibrin mesh was detected covering the particles that supported the agglomeration of DBBM or Hap granules (Fig. 3D–I). PRF bundles contained clusters of platelets, red blood cells, and some leukocytes which retained the components of the block assembled (Fig. 3D). Blood platelets are shown by yellow arrows while red cells are indicated with red arrows and leukocytes are pointed out with white arrows (Fig. 3D–H).

Energy dispersive X-ray spectroscopy (EDX) showed the following weight percentage of elements in the flowable PRF free of biomaterials: 50.6% C, 15.6% O, 12% N, 1.5% S, 1.0% Ti, and 0.8% Na. The specimen containing the PRF block with the synthetic Hap showed a weight percentage of 23.6% Ca and 12.3% P. Therefore, the weight percentage of elements in the specimens containing the PRF block with the DBBM revealed 8.1% Ca and 3.3% P.

As seen in Fig. 4A and B, SEM images revealed the network of the Y-TZP struts leading to an interconnected macro-scale porous structure. The surface of the zirconia struts also revealed pores at micro-scale (Fig. 4C). PRF components were detected covering the zirconia surface and embedding the porous structure, as seen in Fig. 4F. A dense fibrin network was the major component of the PRF as noted in Fig. 4D and E. The mean thickness of the fibrin clusters was recorded at 389 nm while the area occupied by fibers was measured at 49.55% . Leukocytes were detected entrapped in the fibrin mesh (Fig. 4F).

Fig. 4
figure 4

SEM images of a porous zirconia block A–C free of PRF and D–F covered with PRF. E A dense fibrin layer and F the network of fibrin filaments. F Leukocytes and blood platelet covered by PRF (× 2,000 magnification)

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SEM images of the laser-textured zirconia or titanium surfaces are shown in Figs. 5 and 6.

Fig. 5
figure 5

SEM images of the laser-textured zirconia surfaces A, B free of PRF and C–F covered with PRF. Fibrin network embedded with D leukocytes and E, F blood platelets

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Fig. 6
figure 6

SEM images of the laser-textured titanium surfaces A, B free of PRF and C–F covered with PRF. B Micro-scale peaks and vallers onto the titanium surfaces. Fibrin network embedding D, F leukocytes and F blood platelets

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The morphological aspects of both laser-structured surfaces are beneficial for the flowing of the i-PRF and a high micro-scale retention of the fibrin network enriched with platelets and leukocytes (Figs. 5 and 6). The flowable injectable PRF entirely covered pillars, grooves, peaks, and valleys of both laser-textured surfaces (Figs. 5 and 6). Also, the laser-textured titanium surfaces shown micro-scale irregularities (Fig. 6B) that increase the surface area and retention of the fibrin network and PRF components (Fig. 6C–F). Blood cells can also be seen trapped into the fibrin network on the laser-textured surfaces (Fig. 5 and 6).

As seen in Fig. 7, platelet-rich fibrin mesh surrounded by leukocytes (mainly lymphocytes) and some erythrocytes are also evidenced by histomorphometry. The blood cells presented typical characteristics such as membrane integrity and homochromatism. That indicated absence of atypia and maintenance of morphological aspects. Fibrin was arranged in thin, tortuous, and filaments that occasionally formed eosinophilic clusters compatible with fibrin clumps and platelets.

Fig. 7
figure 7

Representative histomorphometry images of injectable PRF. Numerous leukocytes (arrows) and fibrin clumps (stars) associated with platelet aggregation. (Scanner blades Panoramic Viewer – H&E, × 400)

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Discussion

This study described the morphologic aspects of an injectable platelet-rich fibrin (i-PRF) after mixing with particulate bioactive ceramics, porous zirconia, laser-textured zirconia or titanium surfaces. The chemical composition and dimensions of the particulate bioactive ceramics promoted a complete agglomeration with the i-PRF that is beneficial for an enhanced bone healing process. Also, laser-textured zirconia or titanium surfaces revealed retentive areas for i-PRF and for later adhesion of proteins and osteogenic cells when placed into bone defects. Thus, the findings of the present study validate the hypothesis on the three-dimensional architecture and biological potential of the PRF blocks with bioactive ceramics or PRF-coated implant surfaces.

In this study, the DBBM granules revealed dimensions ranging from 150 up to 800 µm that were entirely agglomerated by the i-PRF in the presence of leukocytes and blood platelets (Fig. 3). The distribution of small and lager granules provided a high surface area and spaces for the retention of the i-PRF [27]. Particle size distribution of ordinary bone graft materials is reported in literature ranging from 250 μm to 2 mm [1, 9, 10] although granules with a 100–300 μm size have showed higher osteoconductive potential when compared with 1–2 mm granules [28]. DBBM granules revealed macro- and micro-scale pores that increase the surface area for the interaction with i-PRF. The synthetic Hap granules used in the present study did not possess a porosity such that found in the DBBM. Thus, macro-/microstructured DBBM reveals a high osteoconductivity and bioresorbablity [29, 30], due to the higher contact area when compared to ordinary dense Hap granules leading to high success rate for different surgical approaches [8, 31]. However, the Hap granules can induce the formation of an apatite coating, that stimulates the adsorption of proteins and proliferation of osteogenic cells [32,33,34,35,36]. Regarding the selection of particulate bone graft materials, attention should be paid to the morphologic aspects such as the size and porosity of granules for adequate resorption rate over the bone healing [24, 25, 33]. On mixing bone substitutes with PRF, spaces among granules can be occupied by fibrin fibers and blood cells rich in growth factors that can enhance bone healing. On the bone healing process, the migration of cells and formation of blood vessels prior to bone formation also depends on the distance of the granules or else on the size of macro-scale pores.

As seen in Fig. 4, the porous zirconia structure was filled with a flowable platelet-rich fibrin although some regions were free of PRF. The monolithic zirconia blocks (free of PRF) showed a high porosity and the diameter of pores were proper for the angiogenesis, cell response, and bone healing. The 3-D open cell (interconnected pores) structures show the most interesting design for bone tissue engineering applications due to their similar structure to the trabecular bone. Leukocytes, red blood cells, platelets were entrapped into a dense fibrin network and therefore the morphological aspects of the PRF corroborates the findings from previous studies [11, 15, 23]. Previous studies reported that a porosity above 70% is beneficial to osteogenic cells’ differentiation and, therefore, a macro-scale pore size ranging from 100 up to 400 μm promotes cell ingrowth and angiogenesis into the porous structure [1,2,3,4, 7, 8, 10, 24,25,26, 37, 38]. In addition, the size of the pores enhances the transferring of nutrients and oxygen among the cells. Pores at micro-scale (1–50 μm) provide an increase in the wettability of the porous structure as well as the adhesion of proteins and cells [1, 26, 39]. Blood fluid flow, cell migration, and angiogenesis also depend also on the interconnectivity among the pores at different macro- and micro-scale thereby establishing a 3D-vascular network [10, 25, 40]. In vivo studies reported a faster bone formation into porous structures with a higher porosity. New bone ingrowth started by lining the surfaces and gradually filling the entire pores’ volume from the periphery of the porous structures towards the core [7, 24, 37]. Radiographic examination showed clear boundaries of surrounding bone to zirconia interfaces in previous studies. After healing time, the bone to zirconia porous region reveal a transition zone due to the gradual deposition and ingrowth of bone tissue [1, 24].

In this study, laser-textured zirconia or titanium surfaces showed retentive morphological aspects for combination with the i-PRF. Additionally, laser-textured titanium surfaces revealed micro-scale pores that increase the surface area and consequent adhesion of the i-PRF. The retentive area also can retain the i-PRF into the valleys over the implant placement in bone defects. That plays a key role since the remnant i-PRF stimulate the migration, proliferation, and differentiation of osteogenic cells. Such flowable PRF revealed a dense fibrils’ network of nano-scale fibrin fibrils and clusters associated with the activation of blood platelets, as seen in Figs. 5 and 6. Regarding the mixing with particulate ceramics, the fibrinogen was cross-linked into fibrin within a few minutes and trapped the biomaterial into a fibrin mesh containing platelets and leukocytes, forming a PRF block with the particulate bioactive ceramics (Fig. 3). The cross-linking between the fibrin fibers mechanically stabilizes the architecture of the fibrin-based scaffold. As reported in literature, the intricate nanostructured fibrin network shows an extraordinarily elastic mechanical and biological behavior that avoids the graft instability of particulate biomaterials, thus decreasing the risks of failure over the bone healing [14, 15, 20, 41]. Thus, biomaterial embedding PRF supports the nucleation and accumulation of the newly formed bone matrix, thus achieving the mechanical behavior required over first steps of healing. In fact, particulate graft materials serve as a space maintainer over the bone healing process.

The PRF block reveals a dense fibrin network with nano-scale fibers that can act as a scaffold for cell proliferation, migration, and differentiation and for delivery of growth factors, leading to an enhancement of neoangiogenesis [21, 22]. Fibrin also contains binding sites for integrins, growth factors, and other extracellular matrix components including fibronectin, that provides molecular signals to direct cell function [23, 42]. Varela et al. (2018) also studied the fibrin network of flowable PRF by field emission gun scanning electron microscopy (FEG-SEM). The previous study reported fibrin fibers with a thickness ranging from 20 up to 200 nm and with an average diameter of 90 nm [18]. A porous fibrin structure in PRF has shown to enable rapid vascularization and host cell penetration, while a dense fibrin structure is rather resistant to host cell penetration and vascularization [38]. Changes in fibrin network pattern are dependent on the choice of the centrifugation protocol. For instance, Mamajiwala et al. (2019) found denser fibrin network patterns in PRF membranes produced at 3500 rpm for 15 min when compared to the ones formed at 1400 rpm for 8 min. That means a lowering speed and time leads to a less dense fibrin network pattern [40].

In the present study, a significant content of red blood cells, leukocytes, and platelets was still present in the flowable PRF samples. Therefore, SEM images and a cell counting analysis were useful to inspect blood cells contained within the flowable PRF samples. Thus, the yellow liquid was harvested from the resultant liquid phase (region) above the red blood cells phase once the harvesting handle method can vary depending on the clinical operator. In previous studies, a higher number of red blood cells, leukocytes, and platelets were recorded because the yellow liquid was harvested as near as possible to the blood cell region [18, 37]. Varela et al. also reported a higher content of platelets and leukocytes in the composition of an injectable PRF when compared to those samples from peripheral blood [18]. Also, Castro et al. noted that approximately 88% platelets and 70% leukocytes were still present in the injectable PRF from an initial blood sample [37]. Despite findings, some studies cannot be compared since different protocols for preparation of flowable PRF were adopted, thus different final products were produced. Different viscosity, cell content, and physiological aspects of platelet-rich fibrin can occur using specific protocols and centrifuges. Several factors related to the centrifuge has been reported in previous studies such as the radius inclination and vibration amplitude of the tube holders [23, 42]. An increase in temperature occurs on vibration of the tube holders that negatively affect the morphological and biological integrity of the platelet-rich fibrin. In addition, various centrifugation forces can be implemented on producing PRF [23, 42]. Consequently, findings on a protocol may not be compared or inferred to other protocols [23, 42].

Conclusions

Considering the limitations of an in vitro study, the following concluding remarks are drawn:

  • The use of an injectable PRF improves handling and fitting of particulate graft materials and fragile porous blocks in surgical sites. The bioactive composite block produced by combining an flowable injectable PRF and particulate graft or porous materials is a promising approach for bone augmentation.

  • The injectable PRF showed a dense fibrin network which covered the whole surface of bone substitute particles leading to the agglomeration of granules. Also, the injectable PRF filled the spaces in porous zirconia blocks and coated the rough surfaces of laser-textured zirconia or titanium. The fibrin bundles contained clusters of platelets, red blood cells, and some leukocytes forming of assembly of biological and bioactive materials.

  • Histomorphometric analyses of the flowable PRF revealed a fibrin mesh arranged in thin and eosinophilic filaments that occasionally formed clusters compatible with fibrin clumps and platelets. The fibrin network was surrounded by leukocytes (mainly lymphocytes) and some erythrocytes. The cells showed typical characteristics such as membrane integrity and homochromatism, thus indicating absence of atypia and maintenance of morphological aspects.

  • Attention should be taken when selecting the bone graft substitute, porous structures, or textured implants since biological data on the combination of biomaterials and PRF are lacking in literature. Also, different guidelines for preparation of PRF are available that should be combined with specific bioactive ceramics, zirconia, or titanium surfaces. Future studies should compare the effectiveness of different materials to be mixed with PRF and evaluate the healing process after using the bioactive composite block.