Nanostructure characteristics of three types of platelet-rich fibrin biomaterial: a histological and immunohistochemical study

Background. Platelet-rich fibrin (PRF) is a blood-derived biomaterial that has shown potential in regenerative medicine. The objective of this study was to characterize the structure of fibrin network nanoparticles and cellular components using histological and immunohistochemical techniques. Methods. Three different types of PRF were manufactured: Choukri’s platelet-rich fibrin (Ch-PRF), pure platelet-rich fibrin (P-PRF), and leukocyte platelet-rich fibrin (L-PRF), according to established protocols. The histological structures of the biomaterials were evaluated using hematoxylin and eosin staining. The fibrin network nanostructure was confirmed by Sirius Red staining and immunohistochemical staining with a fibrinogen antibody. Leukocyte components were identified by immunohistochemical staining using CD45 antibody. Results. Histological and immunohistochemical staining of the fibrin network from the PRF biomaterial revealed a natural nanostructure characterized by porous and complex branching networks. The L-PRF and Ch-PRF fibrin networks were delicate and branched, whereas the P-PRF fibrin network displayed thicker bundles of fibers that were sometimes twisted and had noticeable pores. Nonetheless, the proportion of the fibrin network area in all three types of PRF biomaterials was not significantly different. No living cells were found in the P-PRF biomaterials, whereas Ch-PRF and L-PRF contained cells. A large number of red and white blood cells were observed within the Ch-PRF fibrin network, with a non-uniform distribution. The L-PRF biomaterial possesses a uniform structure with a high density of embedded leukocytes. Conclusions. The use of peripheral blood-derived PRF biomaterials, which mimic the natural structure of fibrin nanostructures and living cell components, offers promising possibilities for tissue engineering and regenerative medicine. Additional investigation is necessary to assess the properties of PRF architecture and its practical application in medical treatment.


Introduction
Tissue regeneration engineering is an emerging field that combines cell sources, growth factors, and scaffolds to guide the regeneration of damaged tissues or trigger the body's self-healing potential [1].Biomaterials play a crucial role by providing scaffolds for cell proliferation and tissue repair.To be effective, biomaterials must possess certain critical properties, such as biocompatibility, biodegradability, appropriate mechanical properties, and a porous structure that allows cellular migration and nutrient delivery.Additionally, biomaterials must avoid rejection by the host body and be biodegradable to allow cells to replace the extracellular matrix [1][2][3].
Platelets are essential for tissue repair, as they are the first cell fragments to reach damaged areas and contain large amounts of bioactive proteins [4].Among advanced regenerative agents, autologous platelet concentrates have proven to be an effective and low-cost regenerative modality with the ability to stimulate wound healing processes and accelerate angiogenesis [5,6].Platelet-rich plasma (PRP), a first-generation platelet concentrate, was obtained from centrifuged blood with an anticoagulant [4].PRP is defined as a nanostructured protein because of the nanometric scale of these particles, especially nanometric PRP exosomes (diameter smaller than 50 nm) [4,7].
Platelet-rich fibrin (PRF) biomaterial, the second generation of platelet concentrates, was first described by Choukroun et al in 2001 and has been widely used to accelerate many human tissue regeneration procedures [8].PRF biomaterials are completely autologous substrates and are easy to prepare from whole blood at a minimal expense.Several types of PRF can be prepared using different protocols, enabling versatility in the application of this biomaterial in the regenerative medicine [9,10].In PRF, a fibrin network scaffold is formed by a natural polymerization process, which is similar to the physiological architecture of the fibrin matrix.Moreover, scanning electron microscopy revealed that the nanostructure of the PRF membrane was similar to that of medical gauze, with condensed fibrin strands adhering to each other.The creation of this structure is considered responsible for the slower release of growth factors and entrapment of more platelets inside the matrix [11,12].
Recently, PRF biomaterials have gained increasing interest in the research and clinical applications of regenerative medicine.PRF can be used for different indications to support tissue repair in various fields of medicine for both hard-and soft-tissue regeneration [8,13].One of the innovations in modern dentistry is the application of PRF biomaterial for dental tissue engineering with high success rates in clinical dentistry, specifically in oral and maxillofacial surgery, alveolar bone defect repair, sinus augmentation, and implant stability [14,15].In the field of bone and cartilage regeneration, PRF induces cell proliferation, migration, adhesion, and differentiation, increasing several inflammation-related mediators and further supporting wound healing and tissue regeneration in bone and cartilage [16][17][18].In tendon regeneration, PRF biomaterials are paving the way for new therapeutic strategies that may overcome the limitations of current tendon reconstruction surgical [19][20][21].PRF biomaterials have been used in various dermatological treatments for various skin disorders [22,23].
The available data on the characterization of PRF architecture for tissue regeneration are limited.Therefore, this study aimed to evaluate the histological and immunohistochemical features of three types of PRF to gain insights into the nanostructures of PRF biomaterials.

Animals
The animal experimental protocol followed a standardized procedure approved by the Animal Ethics Committee of Hue University (Certificate No. HU VN0010), which involved the use of ten adult male New Zealand rabbits (10-15 weeks old and weighing 2-2.5 kg) for PRF preparation.Peripheral blood was collected from each rabbit via the central ear artery using a 10 ml syringe to create three types of PRF: Choukroun's platelet-rich fibrin (Ch-PRF), leukocyte platelet-rich fibrin (L-PRF), and pure platelet-rich fibrin (P-PRF).All the experimental animals were provided with the same feeding and care conditions.
Preparation of choukroun's platelet-rich fibrin Ch-PRF was prepared according to the protocol described by Choukroun [24].Rabbit blood was collected from the periphery and immediately transferred into a 15 ml polypropylene centrifuge tube without anticoagulant.The tube was then centrifuged at 2700 rpm for 12 min in a tabletop centrifuge.After centrifugation, blood in the tube was separated into three layers: platelet-poor plasma, platelet-rich fibrin, and red blood cells.The upper plasma serum was aspirated using a pipette and the Ch-PRF clot was gently pulled out using tweezers (figure 1(a)).
Preparation of pure platelet-rich fibrin and leukocyte platelet-rich fibrin The preparation of P-PRF and L-PRF biomaterials followed O'Connell's protocol, which involves a two-step centrifugation procedure [25].First, 9 ml of rabbit blood was collected in tubes containing EDTA, and the tubes were gently tilted to ensure prompt mixing with the anticoagulant.The blood was then transferred into a 15 ml polypropylene tube and centrifuged at 2000 rpm for 5 min.Following the first centrifugation, the blood was separated into three layers: an upper layer of plasma, platelets, and white blood cells; a middle layer of mostly white blood cells; and a bottom layer of red blood cells.The upper and middle layers were transferred to a new tube and centrifuged at 4000 rpm for 5 min.After the second centrifugation, the sample was separated into plasma and white blood cell pellets.The upper plasma was discarded and the lower plasma, known as platelet-rich plasma (PRP), was transferred into a new tube without a white blood cell pellet.Fibrin polymerization was then performed by adding calcium chloride (10%) to a final concentration of 0.1% in the plasma to produce the P-PRF biomaterial (figure 1

(b)).
To create the L-PRF biomaterial, a two-step centrifugation process similar to that used for P-PRF was performed.During this process, the white blood cell pellet was resuspended in platelet-rich plasma before calcium chloride was added for fibrin polymerization (figure 1(b)).

Tissues sample processing
Tissue processing was performed according to the protocol of the Department of Pathology at the Hue University of Medicine and Pharmacy Hospital.Subsequently, the obtained PRF samples were fixed in neutralbuffered formalin for 48 h and then processed using a Shandon Citadel 1000 Tissue Processor, which was automatically operated.The processed samples were paraffin-embedded and sliced into 5 μm sections.These sections were then transferred onto gelatin-coated slides and subjected to histological staining.
Hematoxylin and eosin staining H&E staining was performed according to the protocol described by Schmitz et al [26].PRF slides were deparaffinized by immersion in xylene, followed by rehydration with a series of ethanol and water.Subsequently, the slides were stained with hematoxylin and eosin and examined under a light microscope at magnifications of 1000-6300 to observe the histological characteristics.

Picro-sirius red staining
The fibrin architecture was identified using Picro-Sirius Red staining as previously described [27].For staining, PRF slides were deparaffinized, hydrated, treated with an appropriate concentration of Picrosirius Red solution (Abcam, ab246832), and incubated for 60 min.Finally, the stained slides were mounted on resin medium.

Statistical analysis
Statistical analyses were performed using the PASW Statistics 18 software (SPSS Inc., Chicago, IL, USA).For data with a normal distribution, the Student's t-test was used to determine the statistical significance between the means of two independent groups.The nonparametric Mann-Whitney U test was used for data without normal distribution.Values are expressed as the mean ± standard deviation (SD), and a p-value of less than 0.05 was considered statistically significant.

Result
Heterogeneity distribution of cells and fibrin nanostructure in Ch-PRF To obtain Ch-PRF according to the original Choukroun guidelines, centrifugation was performed, which resulted in the separation of three distinct fractions: a small part of platelet-poor plasma (PPP) at the top, platelet-rich fibrin (PRF) in the middle, and erythrocytes at the bottom (figure 1(a) and figure 2).Ch-PRF was isolated by decanting the soluble PPP fraction and mechanically removing it from the erythrocyte fraction.The histological and structural properties of different parts of Ch-PRF were analyzed, as shown in figure 2. Hematoxylin and Eosin staining of Ch-PRF revealed a non-uniform distribution of fibrin network and cells.The upper region of the Ch-PRF biomaterial closest to the plasma layer exhibited a less compact fibrin network and a lack of cellular constituents (figure 2(a)).The middle region of the Ch-PRF biomaterial contained leukocytes and erythrocytes encapsulated in the fibrin bundle matrix (figures 2(b)-(d)).The bottom portion closest to the erythrocyte layer, referred to as the buffy coat, contains many leukocytes and erythrocytes enclosed within a compact fibrin structure (figure 2(e)).
Sirius red staining was used to identify the fibrin network in the Ch-PRF biomaterial and distinguish it from other structures [27].As shown in figure 3(b), the staining results revealed porous networks of bundles with very thin fibrin fibers.The presence of erythrocytes and leukocytes inside the fibrin network was also noted, and their distribution depended on the location of Ch-PRF (figures 3(a) and (b)).
The immunohistochemical features of Ch-PRF are depicted in figures 4(a) and (b) through immunostaining with fibrinogen antibody, which marks the fibrin network, and CD45 antibody, which identifies common leukocyte antigens, respectively.The fibrin network of the biomaterial exhibited strong positive staining for the fibrinogen antibody, with complex structures featuring thin fibers that formed branching networks and entrapped leukocytes and erythrocytes (figure 4(a)).The embedded CD45-positive leukocytes were uniformly distributed (figure 4(b)).
Homogeneous distribution of fibrin nanostructure in P-PRF P-PRF was created by centrifuging the peripheral blood with an anticoagulant and then polymerizing it with calcium chloride (figure 1(b)).Histological characteristics of the biomaterial were examined using H&E and Picro Sirius Red staining, which revealed a porous histological nanostructure with a uniform distribution and no leukocytes or erythrocytes in the entire region.The fibrin bundles in the P-PRF scaffold were thicker than those in the Ch-PRF and L-PRF scaffolds (figure 3).Immunostaining of the fibrin network in P-PRF showed thick bundles of fibers with large pores.The fibrin bundles in the P-PRF biomaterial were thicker and more organized than those in the cell-encapsulated biomaterials (Ch-PRF and L-PRF).No CD45-positive leukocytes were present in any of the P-PRF biomaterials (figure 4(d)).

Homogeneous distribution of cells and fibrin nanostructure in L-PRF
The L-PRF biomaterial was produced through a two-step centrifugation process, similar to the P-PRF procedure.However, the leukocyte pellet was suspended before fibrin polymerization with calcium chloride (as shown in figure 1(b)).Histological staining revealed that the L-PRF biomaterial had a uniform structure with leukocytes and a few erythrocytes embedded within fibrin networks throughout the biomaterial.The L-PRF biomaterial contained a higher number of leukocytes than the Ch-PRF, but a lower number of erythrocytes.The fibrin networks in the L-PRF biomaterial were porous and complex, similar to the architecture of P-PRF, but the fibrin bundles were thinner than those observed in P-PRF ( figure 3(e) and (f)).
The evaluation of the architecture and nanostructure of the L-PRF material was further carried out by immunostaining, as shown in figures 4(e) and (f).The distribution of CD45-positive leukocytes in the L-PRF  fibrin network was uniform (figure 4(f)).The fibrin network exhibited positive staining for fibrinogen in the L-PRF material, featuring thin and branching fibrin fibers, similar to the Ch-PRF fibrin network.The area fraction of the fibrin network in the three types of PRF biomaterials derived from peripheral blood (Ch-PRF, P-PRF, and L-PRF) was not significantly different (67.8 ± 12.7, 67.4 ± 8.6, and 72.2 ± 12.3%, respectively), as shown in figure 4(g).

Discussion
The field of biomaterials in tissue regeneration engineering involves the creation of biological substitutes that can replicate native tissues and repair damaged tissues.This interdisciplinary field aims to develop materials with mechanical stability, porosity, and degradation rates that can be controlled.These materials can address the challenges associated with tissue-regenerative medicine [29].Various natural and synthetic polymers, as well as their hybrid combinations, have been utilized in tissue engineering.These materials are designed to mimic the structure of native tissues and provide controlled mechanical stability, porosity, and degradation rates [30][31][32].
Recent studies have established that extracellular matrix (ECM) scaffolds are promising materials for tissue engineering, owing to their excellent biocompatibility, flexibility, and plasticity.These scaffolds offer the opportunity to create microenvironments that regulate cell behavior and function, making them attractive biomimetic materials [33][34][35].A fibrin matrix embedded with cells, proteins, and growth factors is also used as an ECM scaffold in regenerative medicine because of its structural and mechanical properties [36][37][38].
PRF is a biomaterial made from extracellular matrix components, primarily fibrin, which functions as a scaffolding material for tissue repair.This scaffolding material supports the recruitment and differentiation of platelets, leukocytes, stem cells, and immune cytokines, thereby offering a biomimetic environment for tissue regeneration [9].In addition to its role in tissue repair, PRF also stores and releases beneficial substances and cues, such as cytokines, growth factors, and blood proteins, which aid in the healing process.PRF has been employed in dentistry and medicine as a surgical aid for tissue repair [39].
Architecturally and nanostructurally interconnected fibrin networks in PRF biomaterials can be visualized using various techniques, such as hematoxylin and eosin staining, atomic force microscopy, confocal laser scanning microscopy, and scanning electron microscopy [40,41].Scanning electron microscopy (SEM) micrographs of PRF revealed a polymerized interconnected fibrin network and a large population of living cells, including lymphocytes, neutrophils, monocytes, platelets, erythrocytes, and stem cells [39,42].The PRF fibrin matrix, a natural polymeric three-dimensional network formed after fibrin polymerization, allows for increased entrapment of circulating cytokines and growth factors as well as prolonged release of these molecules into the extracellular matrix [11,43].The PRF biomaterial generated using Choukroun's method has a heterogeneous structure divided into three distinct regions.Scanning electron microscope micrographs showed that the upper region had a mature structure of fibrin networks with pore sizes ranging from 0.1 to 1.0 μm and a lack of cell constituents.In the intermediate part, a few visible platelets meshed within the fibrin network.The bottom region had immature fibrin networks meshed with fibrin bundles.The largest fraction of platelets and leukocytes is trapped in three-dimensional fibrin networks [43,44].
In this study, three types of PRF biomaterials generated from the peripheral blood were analyzed using histological and immunohistochemical staining techniques.The Ch-PRF biomaterial displayed a nonuniform fibrin structure and cell distribution.The fibrin networks were complex, consisting of very thin fibers that formed branching networks and were found to contain many erythrocytes and leukocytes, depending on the region of Ch-PRF.On the other hand, the P-PRF biomaterial exhibited a uniform distribution with a porous fibrin network structure consisting of thick bundles of fibers with large pores and the absence of leukocytes and erythrocytes in all regions.The L-PRF biomaterial had a homogeneous structure similar to that of P-PRF, but with many leukocytes and a few erythrocytes meshed within fibrin networks.The fibrin network of L-PRF was thin and branched, similar to that of Ch-PRF, and the area fraction of the fibrin network in the three types of PRF biomaterials from peripheral blood (Ch-PRF, P-PRP, and L-PRF) was not significantly different.
Three types of PRF biomaterials from peripheral blood (Ch-PRF, P-PRP, and L-PRF) are suitable for regenerative medicine.Ch-PRF is manufactured using a quick and simple one-step centrifugation process that can be performed in small clinical settings.Many dental clinics use Ch-PRF for dental reconstruction, but its heterogeneous structure can lead to varying regeneration efficiencies.P-PRP and L-PRF have a uniform fibrin network structure, allowing them to be combined with other ingredients, such as cell sources or artificial bone powder, to create biomaterials with high regeneration potential.The two-step fabrication process for P-PRP and L-PRF ensured uniform distribution of the fibrin scaffold.
Based on the analysis of histological and immunohistochemical characteristics, we observed that the three types of platelet-rich fibrin biomaterials obtained from peripheral blood exhibited natural nanostructures of the fibrin network.Both Ch-PRF and L-PRF displayed cells within the fibrin scaffold.The P-PRP and L-PRF fibrin networks had a more uniform structure than Ch-PRF.However, the area fraction of the fibrin network in the three types of PRF biomaterials was not significantly different.These findings suggest that PRF biomaterials generated from peripheral blood have the potential for use in tissue engineering and regenerative medicine.