Evaluation of the effects of the combination of xenogeneic bone grafts with autologous platelet concentrates prepared using different centrifugation protocols on the treatment of critical-size bone defects in rat calvaria: microtomographic and histomorphometric analyses at a late healing stage

Evaluation of the effects of the combination of xenogeneic bone grafts with autologous platelet concentrates prepared using different centrifugation protocols on the treatment of critical-size bone defects in rat calvaria: microtomographic and histomorphometric analyses at a late healing stage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the effects of the combination of xenogeneic bone grafts with autologous platelet concentrates prepared using different centrifugation protocols on the treatment of critical-size bone defects in rat calvaria: microtomographic and histomorphometric analyses at a late healing stage Wanderson Thalles de Souza Braga, Ulli da Costa Cunha Martins, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6631987/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background This study assessed the impact of xenogeneic bone grafts (XEN), either utilized alone or combined with autologous platelet concentrates (APCs) prepared using three different centrifugation protocols on healing of critical-size defects (CSDs) in rat calvaria. Methods Forty rats were assigned to five groups (n = 8): Control, XEN, XEN + L-PRF, XEN + A-PRF, and XEN + Bio-PRF. Blood concentrates were prepared by centrifuging blood collected via cardiac puncture: L-PRF (~ 700g for 12 minutes), A-PRF (~ 208g for 14 minutes), and Bio-PRF (~ 700g for 8 minutes). Defects (5 mm diameter) in the right parietal bone of each rat were filled with blood clot, XEN or XEN combined with L-PRF, A-PRF, or Bio-PRF. Animals were euthanized 60 days post-surgery and the calvariae were analyzed using histomorphometry and micro-CT. Results Groups XEN-A-PRF, XEN-L-PRF and XEN-BIO-PRF presented higher bone volume (BV) and lower bone porosity (Po) than the control group (p < 0.05). The XEN group did not show different values of VO and Po compared to the control group. Only the XEN + A-PRF and XEN + Bio-PRF groups showed a higher percentage of mature collagen fibers (red color) compared to the XEN and control groups (p < 0.05). The XEN group showed a higher amount of immature collagen fibers (green color) compared to the XEN + A-PRF, XEN + L-PRF, and XEN + Bio-PRF groups (p < 0.05). Conclusions It can be concluded that CSDs treated with XEN combined with A-PRF or Bio-PRF, in late-stage healing assessments, presented higher newly formed bone tissue with greater collagen maturation than those treated with XEN alone; ii) the centrifugation protocol used for preparing APCs seems to be a decisive factor for the quality of the collagen matrix in newly formed bone tissue. Bone regeneration Bone substitutes Blood concentrates Tissue engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background In recent decades, advancements in bioengineering and cellular and molecular biology have led to a better understanding of tissue regeneration mechanisms¹. This scientific progress has enabled the development of new biomaterials and methods that enhance the regeneration of specific tissues, thereby driving the concept and development of tissue engineering [ 1 – 2 ]. The use of autologous platelet concentrates (APCs) with particular focus on leukocyte- and platelet-rich fibrin (L-PRF), produced from the centrifugation of the patient’s own blood, has been extensively studied as a low-cost and clinically easy-to-apply autologous biomaterial to enhance tissue regeneration processes, especially when combined with bone scaffolds such as xenogeneic grafts. These concentrates form a three-dimensional matrix containing growth factors, cytokines, inflammatory cells, and platelets, which, when applied to surgical sites, stimulate tissue regeneration [ 3 – 6 ]. L-PRF is a second-generation blood concentrate first described by Choukroun et al. (2001) [ 7 ]. It is a three-dimensional fibrin network biological scaffold that provides physical support to various cells, such as neutrophils, macrophages, and fibroblasts. Additionally, it serves as a source of cytokines and growth factors, including Platelet-derived growth factor (PDGF), Transforming growth factor-beta (TGF-β), Insulin-like growth factor (IGF-1), Fibroblast growth factor (FGF), Vascular endothelial growth factor (VEGF), angiopoietin, and integrin αvβ3 [ 3 , 8 – 10 ]. As a result, it activates chemotaxis, mesenchymal stem cell differentiation, angiogenesis, and modulation of the immune response. The growth factors released by L-PRF bind to specific tyrosine kinase receptors on the surface of target cells, such as osteoblasts, cementoblasts, and fibroblasts, which may enhance tissue regeneration [ 11 – 13 ]. It has been demonstrated that changes in centrifugation speeds can alter the distribution and quantity of cells in the blood concentrates [ 14 – 17 ]. For instance, reducing centrifugation speeds [ 14 – 15 ] or using horizontal centrifugation result in an increased concentration of cells trapped in the fibrin networks, which could enhance the regenerative effects of the matrices [ 16 – 18 ]. These findings led to the development of low-speed centrifugation protocols for producing Advanced Platelet-Rich Fibrin (A-PRF) [ 14 – 15 ] and Platelet-Rich Fibrin obtained through horizontal centrifugation (Bio-PRF) [ 16 – 18 ]. Despite several in vitro studies described in the literature, only two in vivo studies [ 19 – 20 ] have demonstrated the impact of different protocols for blood concentrate production on bone neoformation. However, these studies assessed the effects of APCs used alone, which complicates the translation of these results to clinical practice, where they are more commonly combined with bone grafts for various reconstructive procedures. Thus, the present study aimed to evaluate the effects of bovine-derived xenogeneic bone grafts (XEN), either used alone or in combination with PRF prepared via three distinct centrifugation protocols, on the healing of critical-size defects (CSDs) created in rat calvaria. Methods Animals Fourty rats ( Rattus norvegicus, albinus , Wistar) aged 14 weeks (3.5 months) with body mass 350 and 450g were used obtained after submission and approval by the Committee on Ethics in the Use of Animals (CEUA) of the School of Dentistry of Ribeirão Preto (FORP) of Universidade de São Paulo (USP), under number 2022.1.244.58.2. All rats were kept in groups of three per cage, each lined with sawdust bedding, under controlled conditions including a 12-hour light/dark cycle and a temperature maintained between 22°C and 24°C, and were fed unrestricted access to a standardized solid diet and water. Wistar rats were randomly divided into five groups (n=8): Control, Xenograft (XEN), XEN + L-PRF, XEN + A-PRF, and XEN + Bio-PRF. Sample size was calculated using GraphPad StatMate 2.0 to ensure 80% statistical power with a significance level of 0.05, based on previous data [19–20]. Anesthesia was induced via inhalation of 4% isoflurane and maintained at 1.5–3%, followed by intramuscular administration of morphine sulfate (8 mg/kg) for analgesia. Blood collection and PRF processing After anesthesia and prior to the creation of the Critical Size Defect (CSD), all animals from all experimental groups (C, XEN, XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF) underwent cardiac puncture (Fig. 1a,b). Approximately 3 mL of blood was obtained using 5 mL syringes (Descarpack, São Paulo, Brazil), followed by centrifugation at three distinct protocols for the preparation of APCs. In the XEN+L-PRF group the blood was processed using the protocol for obtaining Leukocyte- and platelet-rich fibrin (L-PRF) at 2700 rotations per minute (rpm) for 12 minutes (~700g Relative Centrifugal Force [RCFmax]) in an Intra-Spin™ centrifuge (33º rotor angle, 55 mm radius at clot height, 86 mm maximum radius, Intra-Lock® International, Inc., Boca Raton, FL, USA). In the XEN+A-PRF group the blood was processed using the protocol for obtaining A-PRF following the protocol established by Ghanaati et al. (2014) 13 . A-PRF preparation was carried out in the same centrifuge used for L-PRF preparation at 1500 rpm for 14 minutes (~208g RCFmax). In the XEN+Bio-PRF group the blood was processed using the horizontal centrifugation protocol (Bio-PRF) following an adaptation of Miron et al. (2019a) [15]. A horizontal centrifuge (Eppendorf 5702; Germany) was used and the blood was centrifuged for 8 minutes at an RCFmax of 700g. The L-PRF, A-PRF and Bio-PRF clots were collected with kits and specific instruments (Tissue Regeneration Kit and Xpression™ Box, Intra-Lock® International, Inc., Boca Raton, FL, USA) (Fig. 1c-f). Creation of critical size defects (CSDs) The dorsal region of each animal's skull was shaved, followed by local antisepsis. A semilunar incision was made, and a full-thickness flap was then raised towards the posterior. A 5-mm diameter surgical defect was created in the right parietal bone of each animal using a trephine drill (5 mm Trephine Drill, Neodent®, Curitiba, PR, Brazil), mounted on a low-speed contra-angle handpiece set to a constant rotation speed of 1000 rpm, with abundant irrigation using sterile saline solution (Fig. 1g). The defects were filled as follows in each experimental group: C group: Filled only with blood clot (n=8); XEN group: Filled only with XEN (Geistlich Bio-Oss®, Geistlich Pharma AG, Lucerne, Switzerland) (n=8); XEN+L-PRF group: Filled with XEN combined with L-PRF (n=8); XEN+A-PRF group: Filled with XEN combined with A-PRF (n=8); XEN+Bio-PRF group: Filled with XEN combined with Bio-PRF (n=8). The amount of XEN and blood concentrate used in each experimental group was controlled. After defect filling, the surgical sites (Fig. 1h-i) were closed with 4-0 silk sutures to promote primary wound healing (Fig. 1j). Postoperative care included intramuscular Penicillin G-benzathine (Pentabiótico Veterinário Pequeno Porte, Fort Dodge Animal Health®, Campinas, SP, Brazil; 24,000 IU/kg) administered immediately after surgery and every 48 hours for one week. Pain management was provided with daily intramuscular injections of Tramadol hydrochloride 8 mg/kg (Cronidor 2%, Agener União®, Apucarana, PR, Brazil) and Meloxicam 0.2 mg/kg for two days. Euthanasia Sixty days after the creation of the CSDs, the animals were euthanized using inhaled Isoflurane at a concentration of 4–5%. Calvarial specimens, encompassing the original defect site and adjacent tissues, were excised in blocks and fixed in 10% neutral buffered formalin for 24 hours. Analysis by micro-computed tomography (micro-CT) Calvarial samples were scanned using cone-beam micro-computed tomography (micro-CT) with a SkyScan 1172 system (SkyScan N.V., Kontich, Belgium), operating at a high spatial resolution of 10 micrometers (µm). The X-ray source was set to an acceleration voltage of 60 kV and a current of 165 µA to optimize image quality and resolution. Using DataViewer version 1.4.3 software, three-dimensional (3D) images were reconstructed from rotational scans of the samples. A precise region of interest (ROI) was established with a diameter of 5 mm, corresponding exactly to the location of the original CSD. Within this ROI, a volume of interest (VOI) measuring 0.5 mm × 5 mm × 5 mm was defined, based on previous methodologies [19-20], to focus the quantitative analysis on the defect area and its immediate surroundings. A trained and calibrated examiner conducted a detailed assessment of the bone microarchitecture within each VOI using CT-Analyzer® software (version 1.13.5.1+, Bruker, Kontich, Belgium). The evaluation included several key structural parameters of each VOI: i) the percentage of the VOI occupied by bone tissue (BV/TV); ii) the percentage of the VOI occupied by bovine bone graft - remaining particles volume (RPV); iii) the percentage of bone porosity within the VOI (BP); iv) the number of trabecular bones present in the VOI (Tb.N); v) the separation of trabeculae in the VOI (Tb.Sp); and vi) the connectivity density between the trabecular structures present in the VOI (Conn.Dn). Histological analysis After Micro-CT scanning, the specimens fixed in formalin and stored in 70% ethanol were washed in running water and subsequently decalcified in a 4% ethylenediaminetetraacetic acid (EDTA) solution for 8 weeks. Following the decalcification process, each specimen was longitudinally divided along the center of the original defect into two parts (A and B), using reference markings filled with amalgam [19-20]. The specimens were then processed and embedded in paraffin. Serial longitudinal sections of 4 µm thickness were cut from the center of the original surgical defect. Two sections from each animal were stained using Hematoxylin and Eosin (HE) staining, and another two sections were stained with Picro-Sirius red. These sections were then subjected to photomicrography using a light microscope. Histomorphometric Analysis Hematoxylin and eosin (HE) staining sections The histomorphometric analysis was conducted using a computer-based image assessment system and specific image acquisition and analysis software (LAS EZ v. 4.1.0, Leica Microsystems®). For each selected histological section, photomicrography was performed using a trinocular light and fluorescence microscope (model DMLB, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany) with a 1.6x objective, coupled with a camera (DFC300FX, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany). In each captured image, the analysis area corresponding to the center of the CSD was defined as the Total Area (TA). Within each TA, the New Bone Area (NBA) and the Remaining Particles Area (RPA) were selected and defined. The TA value was considered as 100% of the analyzed area, and the NBA value was calculated as a percentage of the TA. Each section was also analyzed for the inflammatory profile present and the histopathological characteristics of the newly formed bone, using 10x and 20x objectives. PicroSirius Red staining sections The sections were captured using a trinocular microscope for bright field and fluorescence (model DMLB, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany) under polarized light, with a 1.6x objective. Using ImageJ software, the analysis area corresponding to the center of the defect was delineated, termed as the TA. After determining the TA, the images were subjected to channel selection for red or green, with all other colors being subtracted from the region to analyze the percentage of type I or mature collagen fibers (red color) and type III or immature collagen fibers (green color). The value of TA was considered as 100% of the analyzed area, and the red or green colored area was calculated as a percentage of TA (Fig. 2). It is important to note that any collagen fibers within the defect area that were not part of the trabecular bone, as well as soft tissues and remaining biomaterial particles, were excluded from consideration. Statistical analysis Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., v. 5.01, San Diego, CA, USA), with a significance level set at 5% (p < 0.05). Data were grouped and presented as means with standard deviations. The normality of data distribution was assessed using the Shapiro-Wilk test. The significance of differences between groups for all variables was determined by analysis of variance (ANOVA), followed by the Tukey post-hoc test. Results Analysis by micro-computed tomography (micro-CT) The three-dimensional reconstructions of the calvarial samples are presented in Fig. 3a. The mean values and standard deviations for BV/TV, RPV, Po, Tb.N, Tb.Sp and Conn.Dn across all experimental groups, along with the results of intergroup comparisons, are displayed in Fig. 3b–g. The XEN+A-PRF, XEN+Bio-PRF, and XEN+L-PRF groups showed significantly higher values for BV/TV, Tb.N, and Conn.Dn, and lower values for Po and Tb.Sp compared to the C group (p < 0.05). The XEN group did not differ significantly from the C group in terms of BV/TV and Po; however, it demonstrated significantly higher Tb.N and Conn.Dn values, and lower Tb.Sp values (p < 0.05). No statistically significant differences were found among the XEN, XEN+A-PRF, XEN+Bio-PRF, and XEN+L-PRF groups regarding RPV or any of the other evaluated parameters. Histomorphometric analysis Histopathological analysis Control Group At the defect margins, a limited amount of newly formed bone was detected (Fig. 4a–c), accompanied by osteoblasts positioned around the nascent bone tissue. Most of the defect area was filled with connective tissue rich in collagen fibers aligned parallel to the wound surface. This tissue appeared thinner than the original bone at the defect edges. In some samples, marked bone resorption was evident at the borders. Across all specimens, only a few blood vessels and fibroblasts were observed. Moreover, an inflammatory infiltrate composed of lymphocytes and plasma cells was present throughout the defect site. XEN Group Newly formed bone tissue containing a substantial number of osteocytes was observed near the edges of the surgical defect, as well as surrounding and interspersed among the remaining bone graft particles (Fig. 4d–f). Collagen fibers within the defect area appeared more organized compared to those in the control group. Most of the residual graft particles showed advanced resorption, associated with a dense vascular network and a prominent osteoid matrix. A mild inflammatory infiltrate was also present. XEN+L-PRF Group At the margins of the defects, less bone resorption and a higher number of osteoblasts were observed compared to the XEN and C groups (Fig. 4g–i). In most samples, the newly formed bone tissue contained numerous osteocytes and blood vessels, as well as distinct islands of new bone and residual particles located in the central regions of the defect. Collagen fibers surrounding the defect and residual particles exhibited a more organized arrangement than those seen in the C and XEN groups. Similar to the XEN group, an osteoid matrix was present around the remaining xenograft particles. Notably, this group showed the lowest quantity of residual biomaterial among all groups. Additionally, the presence of blood vessels and newly formed bone throughout the defect area was slightly higher than that observed in the XEN group. XEN+A-PRF Group Similar to the XEN+L-PRF group, the defect borders in this group showed a high density of osteoblasts and reduced bone resorption compared to the C and XEN groups (Fig. 4j–l). The newly formed bone tissue exhibited features comparable to those observed in the XEN+L-PRF group, including a considerable number of osteocytes and abundant vascularization. In some specimens, continuous bone formation extending from the defect margins toward the central region was noted. As seen in the XEN and XEN+L-PRF groups, an osteoid matrix surrounded the residual graft particles. The number of blood vessels and the extent of new bone formation along the defect length were slightly greater than in the XEN group. The quantity of remaining biomaterial particles was lower than in the XEN group but higher than in the XEN+L-PRF and XEN+Bio-PRF groups. A mild inflammatory infiltrate was also observed. XEN+Bio-PRF Group As observed in the XEN+L-PRF and XEN+A-PRF groups, the defect borders in this group showed a high number of osteoblasts and reduced bone resorption compared to the C and XEN groups (Fig. 5m–o). The newly formed bone tissue displayed a substantial osteoid matrix and rich vascularization, consistent with findings in the XEN+L-PRF and XEN+A-PRF groups. In some specimens, continuous bone formation was evident from the margins toward the center of the defect. Similar to the XEN, XEN+L-PRF, and XEN+A-PRF groups, an osteoid matrix was present around residual graft particles. The amount of remaining biomaterial was lower than in the XEN and XEN+A-PRF groups. Additionally, blood vessel density along the defect was slightly higher than that observed in the XEN, XEN+L-PRF, and XEN+A-PRF groups. A mild inflammatory infiltrate was also present. Histometric Analysis of HE Stained Sections The means and standard deviations of NBA and RPA, as well as the results of the intergroup comparisons, are shown in Fig. 5. There were no statistically significant differences in NBA and RPA values between the experimental groups. The XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF groups showed a trend toward higher NBA values compared to the C and XEN groups (p < 0.1). Quantitative analysis of collagen with red or green birefringence By polarizing the collagen fibers stained with PicroSirius Red, it was possible to assess the quality of the collagen in the bone matrix through birefringence and the organization of type I collagen fibers (more organized – reddish color – more mature fibers) and type III collagen fibers (disorganized – greenish color – immature fibers) [21-23]. The means and standard deviations of Red Area (%) and Green Area (%), as well as the results of the intergroup comparisons, are depicted in Fig. 6a-b. Regarding the quantity of mature collagen fibers, only the XEN+Bio-PRF and XEN+A-PRF groups showed significantly higher values compared to the C and XEN groups (p < 0.05). In terms of immature collagen fibers, the C and XEN groups exhibited significantly higher values compared to the XEN+A-PRF and XEN+Bio-PRF groups (p < 0.05). The XEN+L-PRF group had significantly lower values of immature collagen fibers only when compared to the XEN group. Discussion This in vivo study investigated the effects of XEN, used alone or in combination with APCs prepared through three distinct centrifugation protocols (L-PRF, A-PRF, and Bio-PRF), on the healing of CSDs in rat calvaria. While micro-CT and histomorphometric analyses revealed no statistically significant differences in the total volume of newly formed bone among the XEN and XEN + APC groups at 60 days post-operation, notable differences were observed in the collagen composition of the regenerated bone tissue. These variations were directly influenced by the specific centrifugation protocol used to produce the APCs. Understanding and identifying the cellular and molecular mechanisms involved in tissue regeneration is essential for the development of surgical techniques and biomaterials [ 21 – 22 ]. For regeneration to occur, there must be interactions between three main pillars of tissue engineering; one of which is the scaffold, similar to the extracellular matrix, that remains structurally stable after the overlay of soft tissues. An ideal scaffold should improve cellular viability, adhesion, proliferation, cell homing, osteogenic differentiation, and vascularization. Moreover, it should avoid host immune responses, interact with surrounding tissues, maintain three-dimensional stability, and be biologically active [ 23 ]. Bio-Oss® was used in the present study due to its exceptional osteoconductive properties [ 24 ]. This biomaterial is widely recognized as a slow-resorbing xenograft that promotes blood vessel proliferation and the migration of bone cells onto its particles [ 25 ]. However, Bio-Oss does not present osteoinductive potential nor osteogenic properties. The aim in the present study was to combine it with APCs to enhance its properties during bone regeneration processes. APCs provide a slow and gradual release of cytokines and growth factors during a critical period for collagen, fibronectin, and other extracellular matrix components formation that guides tissue regeneration [ 8 , 10 , 13 ]. This gradient of cytokines can primarily attract mesenchymal stem cells (MSCs) from bone marrow and those in regions adjacent to the wound or defect, followed by their differentiation into osteoprogenitor cells. L-PRF, a second-generation blood concentrate, is a biomaterial obtained through the centrifugation of autogenous blood [ 6 – 8 , 11 , 17 ]. Its use in bone regeneration of CSDs in rats has been extensively studied over the past 10 years and has shown satisfactory results in bone neoformation [ 19 – 20 , 26 – 31 ]. The modification of the protocol initially established by Choukroun and colleagues (2001) [ 7 ] focused on changes in RCF, centrifugation time, and introduction of new centrifugation devices has led to the development and emergence of new protocols, such as A-PRF [ 14 ] and Bio-PRF [ 18 ]. These new matrices produced from modified protocols have demonstrated various advantages over the original matrices in various in vitro and in vivo evaluations [ 14 , 16 , 18 , 32 – 33 ]. Indeed, the scientific literature increasingly aims to obtain a blood concentrate matrix with higher mechanical resistance, prolonged degradation time, and greater retention and release rates of growth factors. Few in vivo studies have directly compared the impact of different protocols for blood concentrate preparation on bone regeneration. Using microtomographic and histomorphometric evaluations, Da Silva et al. (2022) [ 19 ] demonstrated that both L-PRF and A-PRF enhanced bone formation in CSDs in rats at 30 days post-surgery. Although no statistically significant differences in the volume of newly formed bone were found between the L-PRF and A-PRF groups, the authors emphasized key findings from qualitative histological assessments and microarchitectural analyses, which revealed distinct characteristics in bone tissue organization and quality among the experimental groups. According to the authors, compared to defects filled with L-PRF, defects treated with A-PRF showed a more significant amount of osteoid matrix and a smaller spacing between bone trabeculae. The authors also argue that studies with longer follow-up periods and immunohistochemical analyses could better explain the obtained results and highlight possible molecular impacts resulting from changes in PRF matrix composition. Sávio and colleagues (2023) [ 20 ] demonstrated through microtomographic, histomorphometric and immunofluorescence analyses that blood concentrate matrices produced from fixed-angle centrifuges (L-PRF and A-PRF) and horizontal centrifugation (H-PRF; Bio-PRF) enhance bone neoformation in CSDs in rat calvaria in evaluations performed at 30 days post-operatively. It was also observed that CSDs treated with Bio-PRF showed higher values of BV/TV, Tb.N, and alizarin precipitation compared to those treated with A-PRF and L-PRF. The authors concluded that Bio-PRF has greater biological potential in the bone neoformation process. Previous studies evaluating different protocols for preparing blood concentrates in bone regeneration have primarily focused on short-term healing periods and did not include the use of bone graft materials. These investigations were largely proof-of-concept studies designed to assess the isolated regenerative potential of APCs in bone formation. In contrast, the present study is the first to assess the combined effects of a XEN with three distinct APC preparation protocols (L-PRF, A-PRF, and Bio-PRF) on bone regeneration in CSDs in rat calvaria during a late-stage healing period. Despite the lack of statistically significant differences in the volume of newly formed bone between defects treated with XEN alone and those treated with XEN plus APCs, histometric analysis revealed a trend toward increased NBA in the XEN + APC groups. This trend, although not significant, suggests a potential additive effect of APCs. The 60-day healing period analyzed in this study represents an advanced phase of bone repair, which may partly explain the absence of significant differences between the groups, as bone remodeling tends to stabilize over time. These findings are consistent with those reported by Oliveira et al. (2015) [ 26 ], who observed significant differences in bone formation between XEN and XEN + L-PRF groups only at the 30-day postoperative mark, but not at 60 days. Similarly, in a study with a shorter healing period, Engler-Pinto et al. (2019) [ 29 ] demonstrated that the combination of XEN and L-PRF resulted in greater bone volume compared to XEN alone in ovariectomized rats. These results collectively suggest that the use of APCs may accelerate early bone healing when compared to XEN alone. However, further studies are needed to determine whether APCs can consistently promote faster or more efficient bone regeneration, especially in the early stages of healing. It is also important to highlight that tissue volume formation does not equate to tissue maturation, as the treated defect may be filled with immature tissue, compromising its functional viability [ 22 ]. The quality of bone tissue is not limited to its mineralization but also includes the quantity and quality of collagen fibers, which are both crucial for bone matrix mineralization and thus for the mechanical quality of bone tissue [ 34 – 35 ]. In the Picrosirius red analysis, the more organized (mature) fibers appear redder, while the more disorganized (immature) fibers exhibit a greener coloration [ 35 – 37 ]. Type I collagen, representing more mature fibers, acts as a support for bone matrix mineralization, directly influencing the microarchitecture of trabecular bone [ 38 ]. In this study, it was observed that only the XEN + A-PRF and XEN + Bio-PRF groups had a higher amount of mature collagen fibers (type I) compared to the XEN group. This result indicates that certain protocols developed with advanced study can potentially better enhance the osteoconductive properties of XEN and that the type of preparation protocol used can provide different organic matrix compositions in newly formed bone tissue. Considering the effects of this organic matrix on the functionality and strength of bone tissue [ 37 ], future studies are important to, for example, longitudinally evaluate the degree of bone remodeling associated with dental implants installed in bone tissue regenerated with XEN combined with blood concentrates produced by different protocols. In a recent study [ 39 ], the outcomes of comparing simply the centrifugation devices was investigated. The overview review article offered deeper insights into the advantages of H-PRF when compared to fixed-angle methods across a wide range of regenerative medical and dental applications. A total of 75 studies were included with 13 studies directly compared horizontal centrifugation to fixed-angle centrifugation for producing PRF, while the remaining 62 studies were non-comparative and focused on expanding the uses and clinical applications of H-PRF. These studies spanned categories such as cell concentrations, fibrin matrix structure, growth factor release, antibacterial and anti-inflammatory properties, and regenerative applications in bone, periodontal, cartilage, skin, hair, regenerative endodontics, corneal defect repair, wound healing, and soft tissue regeneration. Of the studies comparing horizontal to fixed-angle centrifugation, 84.6% favored horizontal centrifugation, while 15.4% found no difference. None of the studies favored fixed-angle centrifugation. Nevertheless, further comparative clinical studies are warranted to further support these findings [ 39 ]. Lastly, it has also been reported that the PRF tubes utilized during the production of PRF likely matters even more so than the centrifugation device itself [ 40 ]. It has been reported that many tube types contain chemical additives or other substances that may either delay or promote or delay the clotting process and strength in the clot [ 41 ]. In the present animal study, the standard 10 mL PRF tubes from each manufacturer could not be utilized owing to the small size of animals and instead 3 mL of blood was drawn into 5 mL tubes containing chemical additives. Therefore, it remains to be clinically observed what effect the tube types from each of the respective manufacturers will have on the final PRF outcomes including their subsequent regenerative potential. In conclusion, this study demonstrated that the centrifugation protocol used for the preparation of APCs was shown to impact the quality of newly formed bone tissue when combined with XEN. Some additional limitations of this study should be considered when analyzing the results. Since the bone defects were not protected by a membrane (barrier), it is not possible to evaluate if the results would be the same under standard guided bone regeneration principles/conditions. The levels of growth factors in the blood concentrates were also not measured in the smaller tube sizes utilized, so a direct correlation between the levels and types of factors present and the obtained bone formation could not be evaluated/estimated. Future studies should address this topic for a better understanding of biological response variability. The use of animals with systemic impairments and on a larger phylogenetic scale should also be considered in future studies to better elucidate the role of APCs in promoting faster bone formation and their optimal benefit in clinical practice. Conclusion Within the limits of this study, it can be concluded that defects treated with XEN combined with A-PRF or Bio-PRF, when evaluated at a later stage of healing, show neoformed bone tissue with greater collagen maturation compared to those treated with XEN alone. Additionally, the type of protocol used for preparing blood concentrates appears to be crucial for the quality of the collagen matrix in the neoformed bone tissue. Abbreviations A-PRF Advanced Platelet-Rich Fibrin APCs Autologous Platelet Concentrates Bio-PRF Horizontal Platelet-Rich Fibrin (Bio-PRF) BP Bone Porosity within the Volume of Interest (VOI) BV Bone Volume BV/TV Bone Tissue (Bone Volume / Total Volume CSDs Critical-Size Defects Conn.Dn Connectivity Density EDTA Ethylenediaminetetraacetic Acid FGF Fibroblast Growth Factor HE Hematoxylin and Eosin IGF-1 Insulin-Like Growth Factor L-PRF Leukocyte- and Platelet-Rich Fibrin Micro-CT Micro-Computed Tomography MSCs Mesenchymal Stem Cells NBA New Bone Area PDGF Platelet-Derived Growth Factor Po Lower Bone Porosity RPA Remaining Particles Area RPV Remaining Particles Volume rpm Rotations Per Minute ROI Region of Interest TA Total Area Tb.N Number of Trabecular Bones Tb.Sp Separation of Trabeculae TGF-β Transforming Growth Factor-Beta VEGF Vascular Endothelial Growth Factor VOI Volume of Interest XEN Bovine-derived xenogeneic bone grafts Declarations Ethical approval and consent to participate All in vivo animal experiments were conducted in compliance with ARRIVE guidelines, and protocols were approved by the Committee on Ethics in the Use of Animals (CEUA) of the School of Dentistry of Ribeirão Preto (FORP), University of São Paulo (USP), under approval number 2022.1.244.58.2. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding This research article received no funds. Authors' contributions Wanderson Thalles de Souza Braga – Conducted experimental analyses, performed a web-based literature review, collected and extracted data, created data visualizations, and drafted both the initial and final versions of the manuscript. Ulli da Costa Cunha Martins, Débora de Souza Ferreira Sávio, and Lucia Moitrel Pequeno da Silva – Performed animal experiments. Maria da Conceição Pereira Saraiva – Conducted statistical data analysis. Flávia Furlaneto, Carlos Fernando Mourão, Richard Miron, and Roberta Okamoto – Contributed to data interpretation and critical revision of the manuscript. Michel Messora – Project administration, validation of results, overall supervision, drafting, and critical revision of the final version of the manuscript. Acknowledgements Not applicable. References Martens W, Bronckaers A, Politis C, Jacobs R, Lambrichts I. Dental stem cells and their promising role in neural regeneration: an update. Clin Oral Investig. 2013;17(9):1969-1983. https://doi.org/10.1007/s00784-013-1062-2 Abdelaziz AG, Nageh H, Abdo SM, et al. A Review of 3D Polymeric Scaffolds for Bone Tissue Engineering: Principles, Fabrication Techniques, Immunomodulatory Roles, and Challenges. Bioengineering (Basel). 2023;10(2):204. https://doi.org/10.3390/bioengineering10020204 Ghanaati S, Herrera-Vizcaino C, Al-Maawi S, et al. Fifteen Years of Platelet Rich Fibrin in Dentistry and Oromaxillofacial Surgery: How High is the Level of Scientific Evidence?. J Oral Implantol. 2018;44(6):471-492. https://doi.org/10.1563/aaid-joi-D-18-00056 Miron RJ, Zucchelli G, Pikos MA, et al. 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Clin Oral Investig. 2020;24(5):1637-1650. https://doi.org/10.1007/s00784-019-03164-4 Ciobanu P, Danciu M, Pascu A, et al. Experimental Study on Rats with Critical-Size Bone Defects Comparing Effects of Autologous Bone Graft, Equine Bone Substitute Bio-Gen® Alone or in Association with Platelet-Rich Fibrin (PRF). Polymers (Basel). 2024;16(11):1502. https://doi.org/10.3390/polym16111502 Fujioka-Kobayashi M, Schaller B, Mourão CFAB, Zhang Y, Sculean A, Miron RJ. Biological characterization of an injectable platelet-rich fibrin mixture consisting of autologous albumin gel and liquid platelet-rich fibrin (Alb-PRF). Platelets. 2021;32(1):74-81. https://doi.org/10.1080/09537104.2020.1747581 Shirakata Y, Sena K, Nakamura T, Shinohara Y, Imafuji T, Setoguchi F, Noguchi K, Kawase T, Miron RJ. Histological evaluation of gingival and intrabony periodontal defects treated with platelet-rich fibrin using different protocols: a canine study. Oral health & preventive dentistry. 2021 Oct 22;19:b2182985. https://doi.org/10.1515/ohpd-2021-2182985 Burr DB. The contribution of the organic matrix to bone's material properties. Bone. 2002;31(1):8-11. https://doi.org/10.1016/S8756-3282(02)00808-4 Munerato MS, Biguetti CC, Parra da Silva RB, et al. Inflammatory response and macrophage polarization using different physicochemical biomaterials for oral and maxillofacial reconstruction. Mater Sci Eng C Mater Biol Appl. 2020;107:110229. https://doi.org/10.1016/j.msec.2019.110229 Lattouf R, Younes R, Lutomski D, et al. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem. 2014;62(10):751-758. https://doi.org/10.1369/0022155414543071 Vivan RR, Mecca CE, Biguetti CC, et al. Experimental maxillary sinus augmentation using a highly bioactive glass ceramic. J Mater Sci Mater Med. 2016;27(2):41. https://doi.org/10.1007/s10856-016-5749-3%5D( Seeman E, Delmas PD. Bone quality--the material and structural basis of bone strength and fragility. N Engl J Med. 2006;354(21):2250-2261. https://doi.org/10.1056/NEJMra053077 Farshidfar N, Apaza Alccayhuaman KA, Estrin NE, et al. Advantages of horizontal centrifugation of platelet‐rich fibrin in regenerative medicine and dentistry. Periodontology 2000. 2025 Mar 27. https://doi.org/10.1111/prd.12521 Miron RJ, Kawase T, Dham A, Zhang Y, Fujioka-Kobayashi M, Sculean A. A technical note on contamination from PRF tubes containing silica and silicone. BMC Oral health. 2021 Dec;21:1-1. https://doi.org/10.1186/s12903-020-01299-w Wei Y, Cheng Y, Wei H, et al. Development of a super-hydrophilic anaerobic tube for the optimization of platelet-rich fibrin. Platelets. 2024 Dec 31;35(1):2316745. https://doi.org/10.1080/09537104.2023.2316745 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6631987","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463176352,"identity":"ade04445-d5cf-4279-8f07-f26e437af3fc","order_by":0,"name":"Wanderson Thalles de Souza Braga","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Wanderson","middleName":"Thalles de Souza","lastName":"Braga","suffix":""},{"id":463176353,"identity":"30890f4d-db05-4779-8acd-3d7f52164566","order_by":1,"name":"Ulli da Costa Cunha Martins","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Ulli","middleName":"da Costa Cunha","lastName":"Martins","suffix":""},{"id":463176354,"identity":"ea42c6e5-d3aa-439d-97f0-bb9dff7fe854","order_by":2,"name":"Débora de Souza Ferreira Sávio","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Débora","middleName":"de Souza Ferreira","lastName":"Sávio","suffix":""},{"id":463176355,"identity":"e591662e-6b58-49d1-8027-711c8a9a705e","order_by":3,"name":"Lucia Moitrel Pequeno da Silva","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"Moitrel Pequeno da","lastName":"Silva","suffix":""},{"id":463176356,"identity":"931ef04c-7e74-458f-a396-eec90da80c0f","order_by":4,"name":"Maria da Conceição Pereira Saraiva","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"da Conceição Pereira","lastName":"Saraiva","suffix":""},{"id":463176357,"identity":"cbee913d-195b-46fe-b252-2b871e5b9d01","order_by":5,"name":"Flávia Furlaneto","email":"","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":false,"prefix":"","firstName":"Flávia","middleName":"","lastName":"Furlaneto","suffix":""},{"id":463176358,"identity":"1df1bfe0-0be2-44b8-925a-c0eaa09c2673","order_by":6,"name":"Carlos Fernando Mourão","email":"","orcid":"","institution":"Tufts University School of Dental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Fernando","lastName":"Mourão","suffix":""},{"id":463176359,"identity":"9f13b4e7-ba46-4b0a-8674-9ab28a347fa4","order_by":7,"name":"Richard Miron","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Miron","suffix":""},{"id":463176360,"identity":"79efd675-2697-4ce2-96d2-5e4aed6f6d49","order_by":8,"name":"Roberta Okamoto","email":"","orcid":"","institution":"Paulista State University – UNESP","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Okamoto","suffix":""},{"id":463176361,"identity":"8a313b34-f2da-4e0c-b62e-a8f629d106b1","order_by":9,"name":"Michel Messora","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFAD9sYHMCYbkVp4DhuQqkUimUgtug3ciQ9//LHJ55/5mPFx5Q6GxPmzG9geV+DRYnaAd7Mxb1ua5YzbycyGZ88wJG64c4Dd8Ax+LdukGRuA/ridf0yysQ2oRSKBTbIBv5btP3/8OWwgf/MwG1jL/BmEtWxj4GE7bGBwgxmipeEGIS2HeTdLA/1iYHgG6JfGMxLGG+4cbDfEq+V478aPwBAzkDt+mPFh4w4b2fmzm489xKeFgRmZw9ggAYwgRrwa0ABYsQQJGkbBKBgFo2BEAAC+Rk2qvalQKwAAAABJRU5ErkJggg==","orcid":"","institution":"University of São Paulo – USP","correspondingAuthor":true,"prefix":"","firstName":"Michel","middleName":"","lastName":"Messora","suffix":""}],"badges":[],"createdAt":"2025-05-10 02:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6631987/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6631987/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83811561,"identity":"384cb104-674b-4002-b401-59de83fa1b8f","added_by":"auto","created_at":"2025-06-03 07:07:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":657608,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental procedures performed. A,shaving and antisepsis of the area; B, Blood collection using the cardiac puncture technique; (c) tube after centrifugation: observe a horizontal interface between the formed clot and the red cell layer; D, collection of Bio-PRF matrix and separation of the red cell layer with scissors; E, obtained matrix; F, matrix obtained after compression in a specific kit; G, osteotomy with a 5 mm diameter trephine drill; H, filling the created defect with XEN associated with the blood concentrate; I, placement of part of the non-perforated blood concentrate matrix covering the defect; J, interrupted simple suture of the flap.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/97163bcb293d22c7020526c5.png"},{"id":83812747,"identity":"b0255980-738a-496a-bc6c-f79e7cfc2e03","added_by":"auto","created_at":"2025-06-03 07:15:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":618843,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrography of histological sections for collagen type I deposition analysis. A, the Total Area (TA) is outlined by the blue line and corresponds to the area of the calvaria where the surgical defect was originally created. The height of the TA corresponds to the average of the height of the right stump (RS) and the left stump (LS). The width of the TA is 5 mm, corresponding to the width of the original defect. Magnification = 1.6x; B, crop of the TA to be analyzed; C, image with only the red tones selected by the ImageJ software; D, marking after automatic threshold selection for counting the percentage of area selected in the color to be analyzed.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/2b4556e402c65c1ccf7124b8.png"},{"id":83812749,"identity":"12766f4c-feae-45d7-96fc-c263ab9a6f9e","added_by":"auto","created_at":"2025-06-03 07:15:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":579306,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-CT of the calvaria. Rendered reconstructions of the Control, XEN, XEN+L-PRF, XEN+A-PRF and XEN+Bio-PRF (A); Micro-CT of the calvariae. Means and standard deviations of BV (BV/TV) (B), RPV (C), BP (D), Tb.N (E), Tb.Sp (F), and Conn.Dn (G) for the groups C, XEN, XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF, with results of comparisons between groups (ANOVA, Tukey, p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/d7c82c85112e95a97b781808.png"},{"id":83812748,"identity":"ce08e469-ac75-4d2f-9a6c-8f8154302152","added_by":"auto","created_at":"2025-06-03 07:15:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1133481,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of histological sections from the area near the original bone stump of each defect; Group C (A and B); Group XEN (D and E); Group XEN+L-PRF (G and H); Group XEN+A-PRF (J and K); Group XEN+Bio-PRF (M and N); NB = newly formed bone; XEN = residual xenograft particle; CT = connective tissue; red arrow= osteoid matrix; black arrow = blood vessels; yellow arrow = margins of the surgically created defect; green arrow = area of inflammatory infiltrate; *Images c, f, i, l, and o represent regions distant from the margin of the surgical defect. Staining: Hematoxylin and Eosin. Original magnification = 10x (A, D, G, J and M); 20x (B, C, E, F, H, L, N and O).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/65bd3cff670deb9a1ced9a3c.png"},{"id":83811564,"identity":"af3c13e7-8b71-4ecc-8dfe-961c9d19b385","added_by":"auto","created_at":"2025-06-03 07:07:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103810,"visible":true,"origin":"","legend":"\u003cp\u003eMeans and standard deviations of NBA (A) and RPA (B) for the groups C, XEN, XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF, with results of comparisons between groups. No statistically significant differences were observed in either parameter among the experimental groups (ANOVA, Tukey, p\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/135da823060f060d04caa893.png"},{"id":83811558,"identity":"162715c1-7537-4ff2-861d-66713931423f","added_by":"auto","created_at":"2025-06-03 07:07:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134306,"visible":true,"origin":"","legend":"\u003cp\u003eMeans and standard deviations for Red Area (%) (A) and Green Area (%) (B) for the groups C, XEN, XEN+A-PRF, XEN+Bio-PRF, and XEN+L-PRF, with the results of the comparisons between groups (ANOVA, Tukey, p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/1da33f4de5114edba94fa1f7.png"},{"id":91656527,"identity":"c9cfe13b-73a8-4be1-9ca1-6bf1932ce5f9","added_by":"auto","created_at":"2025-09-18 18:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5018873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6631987/v1/66164a5f-c760-48d4-9335-875c47553af8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the effects of the combination of xenogeneic bone grafts with autologous platelet concentrates prepared using different centrifugation protocols on the treatment of critical-size bone defects in rat calvaria: microtomographic and histomorphometric analyses at a late healing stage","fulltext":[{"header":"Background","content":"\u003cp\u003eIn recent decades, advancements in bioengineering and cellular and molecular biology have led to a better understanding of tissue regeneration mechanisms\u0026sup1;. This scientific progress has enabled the development of new biomaterials and methods that enhance the regeneration of specific tissues, thereby driving the concept and development of tissue engineering [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe use of autologous platelet concentrates (APCs) with particular focus on leukocyte- and platelet-rich fibrin (L-PRF), produced from the centrifugation of the patient\u0026rsquo;s own blood, has been extensively studied as a low-cost and clinically easy-to-apply autologous biomaterial to enhance tissue regeneration processes, especially when combined with bone scaffolds such as xenogeneic grafts. These concentrates form a three-dimensional matrix containing growth factors, cytokines, inflammatory cells, and platelets, which, when applied to surgical sites, stimulate tissue regeneration [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eL-PRF is a second-generation blood concentrate first described by Choukroun et al. (2001) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is a three-dimensional fibrin network biological scaffold that provides physical support to various cells, such as neutrophils, macrophages, and fibroblasts. Additionally, it serves as a source of cytokines and growth factors, including Platelet-derived growth factor (PDGF), Transforming growth factor-beta (TGF-β), Insulin-like growth factor (IGF-1), Fibroblast growth factor (FGF), Vascular endothelial growth factor (VEGF), angiopoietin, and integrin αvβ3 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As a result, it activates chemotaxis, mesenchymal stem cell differentiation, angiogenesis, and modulation of the immune response. The growth factors released by L-PRF bind to specific tyrosine kinase receptors on the surface of target cells, such as osteoblasts, cementoblasts, and fibroblasts, which may enhance tissue regeneration [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been demonstrated that changes in centrifugation speeds can alter the distribution and quantity of cells in the blood concentrates [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For instance, reducing centrifugation speeds [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] or using horizontal centrifugation result in an increased concentration of cells trapped in the fibrin networks, which could enhance the regenerative effects of the matrices [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings led to the development of low-speed centrifugation protocols for producing Advanced Platelet-Rich Fibrin (A-PRF) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and Platelet-Rich Fibrin obtained through horizontal centrifugation (Bio-PRF) [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite several in vitro studies described in the literature, only two in vivo studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] have demonstrated the impact of different protocols for blood concentrate production on bone neoformation. However, these studies assessed the effects of APCs used alone, which complicates the translation of these results to clinical practice, where they are more commonly combined with bone grafts for various reconstructive procedures. Thus, the present study aimed to evaluate the effects of bovine-derived xenogeneic bone grafts (XEN), either used alone or in combination with PRF prepared via three distinct centrifugation protocols, on the healing of critical-size defects (CSDs) created in rat calvaria.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFourty rats (\u003cem\u003eRattus norvegicus, albinus\u003c/em\u003e, Wistar) aged 14 weeks (3.5 months) with body mass 350 and 450g were used obtained after submission and approval by the Committee on Ethics in the Use of Animals (CEUA) of the School of Dentistry of Ribeir\u0026atilde;o Preto (FORP) of Universidade de S\u0026atilde;o Paulo (USP), under number 2022.1.244.58.2. All rats were kept in groups of three per cage, each lined with sawdust bedding, under controlled conditions including a 12-hour light/dark cycle and a temperature maintained between 22\u0026deg;C and 24\u0026deg;C, and were fed unrestricted access to a standardized solid diet and water.\u003c/p\u003e\n\u003cp\u003eWistar rats were randomly divided into five groups (n=8): Control, Xenograft (XEN), XEN + L-PRF, XEN + A-PRF, and XEN + Bio-PRF. Sample size was calculated using GraphPad StatMate 2.0 to ensure 80% statistical power with a significance level of 0.05, based on previous data [19\u0026ndash;20]. Anesthesia was induced via inhalation of 4% isoflurane and maintained at 1.5\u0026ndash;3%, followed by intramuscular administration of morphine sulfate (8 mg/kg) for analgesia.\u003c/p\u003e\n\u003ch2\u003eBlood collection and PRF processing\u003c/h2\u003e\n\u003cp\u003eAfter anesthesia and prior to the creation of the Critical Size Defect (CSD), all animals from all experimental groups (C, XEN, XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF) underwent cardiac puncture (Fig. 1a,b). Approximately 3 mL of blood was obtained using 5 mL syringes (Descarpack, S\u0026atilde;o Paulo, Brazil), followed by centrifugation at three distinct protocols for the preparation of APCs. In the XEN+L-PRF group the blood was processed using the protocol for obtaining Leukocyte- and platelet-rich fibrin (L-PRF) at 2700 rotations per minute (rpm) for 12 minutes (~700g Relative Centrifugal Force [RCFmax]) in an Intra-Spin\u0026trade; centrifuge (33\u0026ordm; rotor angle, 55 mm radius at clot height, 86 mm maximum radius, Intra-Lock\u0026reg; International, Inc., Boca Raton, FL, USA). In the XEN+A-PRF group the blood was processed using the protocol for obtaining A-PRF following the protocol established by Ghanaati et al. (2014)\u003csup\u003e13\u003c/sup\u003e. A-PRF preparation was carried out in the same centrifuge used for L-PRF preparation at 1500 rpm for 14 minutes (~208g RCFmax). In the XEN+Bio-PRF group the blood was processed using the horizontal centrifugation protocol (Bio-PRF) following an adaptation of Miron et al. (2019a) [15]. A horizontal centrifuge (Eppendorf 5702; Germany) was used and the blood was centrifuged for 8 minutes at an RCFmax of 700g. The L-PRF, A-PRF and Bio-PRF clots were collected with kits and specific instruments (Tissue Regeneration Kit and Xpression\u0026trade; Box, Intra-Lock\u0026reg; International, Inc., Boca Raton, FL, USA) (Fig. 1c-f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCreation of critical size defects (CSDs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dorsal region of each animal\u0026apos;s skull was shaved, followed by local antisepsis. A semilunar incision was made, and a full-thickness flap was then raised towards the posterior. A 5-mm diameter surgical defect was created in the right parietal bone of each animal using a trephine drill (5 mm Trephine Drill, Neodent\u0026reg;, Curitiba, PR, Brazil), mounted on a low-speed contra-angle handpiece set to a constant rotation speed of 1000 rpm, with abundant irrigation using sterile saline solution (Fig. 1g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe defects were filled as follows in each experimental group:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eC group: Filled only with blood clot (n=8);\u003c/li\u003e\n \u003cli\u003eXEN group: Filled only with XEN (Geistlich Bio-Oss\u0026reg;, Geistlich Pharma AG, Lucerne, Switzerland) (n=8);\u003c/li\u003e\n \u003cli\u003eXEN+L-PRF group: Filled with XEN combined with L-PRF (n=8);\u003c/li\u003e\n \u003cli\u003eXEN+A-PRF group: Filled with XEN combined with A-PRF (n=8);\u003c/li\u003e\n \u003cli\u003eXEN+Bio-PRF group: Filled with XEN combined with Bio-PRF (n=8).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe amount of XEN and blood concentrate used in each experimental group was controlled. After defect filling, the surgical sites (Fig. 1h-i) were closed with 4-0 silk sutures to promote primary wound healing (Fig. 1j). Postoperative care included intramuscular Penicillin G-benzathine (Pentabi\u0026oacute;tico Veterin\u0026aacute;rio Pequeno Porte, Fort Dodge Animal Health\u0026reg;, Campinas, SP, Brazil; 24,000 IU/kg) administered immediately after surgery and every 48 hours for one week. Pain management was provided with daily intramuscular injections of Tramadol hydrochloride 8 mg/kg (Cronidor 2%, Agener Uni\u0026atilde;o\u0026reg;, Apucarana, PR, Brazil) and Meloxicam 0.2 mg/kg for two days.\u003c/p\u003e\n\u003ch2\u003eEuthanasia\u003c/h2\u003e\n\u003cp\u003eSixty days after the creation of the CSDs, the animals were euthanized using inhaled Isoflurane at a concentration of 4\u0026ndash;5%. Calvarial specimens, encompassing the original defect site and adjacent tissues, were excised in blocks and fixed in 10% neutral buffered formalin for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis by micro-computed tomography (micro-CT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCalvarial samples were scanned using cone-beam micro-computed tomography (micro-CT) with a SkyScan 1172 system (SkyScan N.V., Kontich, Belgium), operating at a high spatial resolution of 10 micrometers (\u0026micro;m). The X-ray source was set to an acceleration voltage of 60 kV and a current of 165 \u0026micro;A to optimize image quality and resolution. Using DataViewer version 1.4.3 software, three-dimensional (3D) images were reconstructed from rotational scans of the samples.\u003c/p\u003e\n\u003cp\u003eA precise region of interest (ROI) was established with a diameter of 5 mm, corresponding exactly to the location of the original CSD. Within this ROI, a volume of interest (VOI) measuring 0.5 mm \u0026times; 5 mm \u0026times; 5 mm was defined, based on previous methodologies [19-20], to focus the quantitative analysis on the defect area and its immediate surroundings.\u003c/p\u003e\n\u003cp\u003eA trained and calibrated examiner conducted a detailed assessment of the bone microarchitecture within each VOI using CT-Analyzer\u0026reg; software (version 1.13.5.1+, Bruker, Kontich, Belgium). The evaluation included several key structural parameters of each VOI: i) the percentage of the VOI occupied by bone tissue (BV/TV); ii) the percentage of the VOI occupied by bovine bone graft - remaining particles volume (RPV); iii) the percentage of bone porosity within the VOI (BP); iv) the number of trabecular bones present in the VOI (Tb.N); v) the separation of trabeculae in the VOI (Tb.Sp); and vi) the connectivity density between the trabecular structures present in the VOI (Conn.Dn).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter Micro-CT scanning, the specimens fixed in formalin and stored in 70% ethanol were washed in running water and subsequently decalcified in a 4% ethylenediaminetetraacetic acid (EDTA) solution for 8 weeks. Following the decalcification process, each specimen was longitudinally divided along the center of the original defect into two parts (A and B), using reference markings filled with amalgam [19-20]. The specimens were then processed and embedded in paraffin. Serial longitudinal sections of 4 \u0026micro;m thickness were cut from the center of the original surgical defect. Two sections from each animal were stained using Hematoxylin and Eosin (HE) staining, and another two sections were stained with Picro-Sirius red. These sections were then subjected to photomicrography using a light microscope.\u003c/p\u003e\n\u003ch2\u003eHistomorphometric Analysis\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHematoxylin and eosin (HE) staining sections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe histomorphometric analysis was conducted using a computer-based image assessment system and specific image acquisition and analysis software (LAS EZ v. 4.1.0, Leica Microsystems\u0026reg;). For each selected histological section, photomicrography was performed using a trinocular light and fluorescence microscope (model DMLB, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany) with a 1.6x objective, coupled with a camera (DFC300FX, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany).\u003c/p\u003e\n\u003cp\u003eIn each captured image, the analysis area corresponding to the center of the CSD was defined as the Total Area (TA). Within each TA, the New Bone Area (NBA) and the Remaining Particles Area (RPA) were selected and defined. The TA value was considered as 100% of the analyzed area, and the NBA value was calculated as a percentage of the TA.\u003c/p\u003e\n\u003cp\u003eEach section was also analyzed for the inflammatory profile present and the histopathological characteristics of the newly formed bone, using 10x and 20x objectives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePicroSirius Red staining sections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sections were captured using a trinocular microscope for bright field and fluorescence (model DMLB, Leica Microsystems GmbH, Wetzlar, Heidelberg, Germany) under polarized light, with a 1.6x objective. Using ImageJ software, the analysis area corresponding to the center of the defect was delineated, termed as the TA. After determining the TA, the images were subjected to channel selection for red or green, with all other colors being subtracted from the region to analyze the percentage of type I or mature collagen fibers (red color) and type III or immature collagen fibers (green color). The value of TA was considered as 100% of the analyzed area, and the red or green colored area was calculated as a percentage of TA (Fig. 2). It is important to note that any collagen fibers within the defect area that were not part of the trabecular bone, as well as soft tissues and remaining biomaterial particles, were excluded from consideration.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., v. 5.01, San Diego, CA, USA), with a significance level set at 5% (p \u0026lt; 0.05). Data were grouped and presented as means with standard deviations. The normality of data distribution was assessed using the Shapiro-Wilk test. The significance of differences between groups for all variables was determined by analysis of variance (ANOVA), followed by the Tukey post-hoc test.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAnalysis by micro-computed tomography (micro-CT)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe three-dimensional reconstructions of the calvarial samples are presented in Fig. 3a. The mean values and standard deviations for BV/TV, RPV, Po, Tb.N, Tb.Sp and Conn.Dn across all experimental groups, along with the results of intergroup comparisons, are displayed in Fig. 3b\u0026ndash;g. The XEN+A-PRF, XEN+Bio-PRF, and XEN+L-PRF groups showed significantly higher values for BV/TV, Tb.N, and Conn.Dn, and lower values for Po and Tb.Sp compared to the C group (p \u0026lt; 0.05). The XEN group did not differ significantly from the C group in terms of BV/TV and Po; however, it demonstrated significantly higher Tb.N and Conn.Dn values, and lower Tb.Sp values (p \u0026lt; 0.05). No statistically significant differences were found among the XEN, XEN+A-PRF, XEN+Bio-PRF, and XEN+L-PRF groups regarding RPV or any of the other evaluated parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistomorphometric analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistopathological analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eControl Group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the defect margins, a limited amount of newly formed bone was detected (Fig. 4a\u0026ndash;c), accompanied by osteoblasts positioned around the nascent bone tissue. Most of the defect area was filled with connective tissue rich in collagen fibers aligned parallel to the wound surface. This tissue appeared thinner than the original bone at the defect edges. In some samples, marked bone resorption was evident at the borders. Across all specimens, only a few blood vessels and fibroblasts were observed. Moreover, an inflammatory infiltrate composed of lymphocytes and plasma cells was present throughout the defect site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eXEN Group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNewly formed bone tissue containing a substantial number of osteocytes was observed near the edges of the surgical defect, as well as surrounding and interspersed among the remaining bone graft particles (Fig. 4d\u0026ndash;f). Collagen fibers within the defect area appeared more organized compared to those in the control group. Most of the residual graft particles showed advanced resorption, associated with a dense vascular network and a prominent osteoid matrix. A mild inflammatory infiltrate was also present.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eXEN+L-PRF Group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the margins of the defects, less bone resorption and a higher number of osteoblasts were observed compared to the XEN and C groups (Fig. 4g\u0026ndash;i). In most samples, the newly formed bone tissue contained numerous osteocytes and blood vessels, as well as distinct islands of new bone and residual particles located in the central regions of the defect. Collagen fibers surrounding the defect and residual particles exhibited a more organized arrangement than those seen in the C and XEN groups. Similar to the XEN group, an osteoid matrix was present around the remaining xenograft particles. Notably, this group showed the lowest quantity of residual biomaterial among all groups. Additionally, the presence of blood vessels and newly formed bone throughout the defect area was slightly higher than that observed in the XEN group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eXEN+A-PRF Group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilar to the XEN+L-PRF group, the defect borders in this group showed a high density of osteoblasts and reduced bone resorption compared to the C and XEN groups (Fig. 4j\u0026ndash;l). The newly formed bone tissue exhibited features comparable to those observed in the XEN+L-PRF group, including a considerable number of osteocytes and abundant vascularization. In some specimens, continuous bone formation extending from the defect margins toward the central region was noted. As seen in the XEN and XEN+L-PRF groups, an osteoid matrix surrounded the residual graft particles. The number of blood vessels and the extent of new bone formation along the defect length were slightly greater than in the XEN group. The quantity of remaining biomaterial particles was lower than in the XEN group but higher than in the XEN+L-PRF and XEN+Bio-PRF groups. A mild inflammatory infiltrate was also observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eXEN+Bio-PRF Group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs observed in the XEN+L-PRF and XEN+A-PRF groups, the defect borders in this group showed a high number of osteoblasts and reduced bone resorption compared to the C and XEN groups (Fig. 5m\u0026ndash;o). The newly formed bone tissue displayed a substantial osteoid matrix and rich vascularization, consistent with findings in the XEN+L-PRF and XEN+A-PRF groups. In some specimens, continuous bone formation was evident from the margins toward the center of the defect. Similar to the XEN, XEN+L-PRF, and XEN+A-PRF groups, an osteoid matrix was present around residual graft particles. The amount of remaining biomaterial was lower than in the XEN and XEN+A-PRF groups. Additionally, blood vessel density along the defect was slightly higher than that observed in the XEN, XEN+L-PRF, and XEN+A-PRF groups. A mild inflammatory infiltrate was also present.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistometric Analysis of HE Stained Sections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe means and standard deviations of NBA and RPA, as well as the results of the intergroup comparisons, are shown in Fig. 5. There were no statistically significant differences in NBA and RPA values between the experimental groups. The XEN+L-PRF, XEN+A-PRF, and XEN+Bio-PRF groups showed a trend toward higher NBA values compared to the C and XEN groups (p \u0026lt; 0.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analysis of collagen with red or green birefringence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy polarizing the collagen fibers stained with PicroSirius Red, it was possible to assess the quality of the collagen in the bone matrix through birefringence and the organization of type I collagen fibers (more organized \u0026ndash; reddish color \u0026ndash; more mature fibers) and type III collagen fibers (disorganized \u0026ndash; greenish color \u0026ndash; immature fibers) [21-23]. The means and standard deviations of Red Area (%) and Green Area (%), as well as the results of the intergroup comparisons, are depicted in Fig. 6a-b.\u003c/p\u003e\n\u003cp\u003eRegarding the quantity of mature collagen fibers, only the XEN+Bio-PRF and XEN+A-PRF groups showed significantly higher values compared to the C and XEN groups (p \u0026lt; 0.05). In terms of immature collagen fibers, the C and XEN groups exhibited significantly higher values compared to the XEN+A-PRF and XEN+Bio-PRF groups (p \u0026lt; 0.05). The XEN+L-PRF group had significantly lower values of immature collagen fibers only when compared to the XEN group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis in vivo study investigated the effects of XEN, used alone or in combination with APCs prepared through three distinct centrifugation protocols (L-PRF, A-PRF, and Bio-PRF), on the healing of CSDs in rat calvaria. While micro-CT and histomorphometric analyses revealed no statistically significant differences in the total volume of newly formed bone among the XEN and XEN\u0026thinsp;+\u0026thinsp;APC groups at 60 days post-operation, notable differences were observed in the collagen composition of the regenerated bone tissue. These variations were directly influenced by the specific centrifugation protocol used to produce the APCs.\u003c/p\u003e \u003cp\u003eUnderstanding and identifying the cellular and molecular mechanisms involved in tissue regeneration is essential for the development of surgical techniques and biomaterials [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For regeneration to occur, there must be interactions between three main pillars of tissue engineering; one of which is the scaffold, similar to the extracellular matrix, that remains structurally stable after the overlay of soft tissues. An ideal scaffold should improve cellular viability, adhesion, proliferation, cell homing, osteogenic differentiation, and vascularization. Moreover, it should avoid host immune responses, interact with surrounding tissues, maintain three-dimensional stability, and be biologically active [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Bio-Oss\u0026reg; was used in the present study due to its exceptional osteoconductive properties [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This biomaterial is widely recognized as a slow-resorbing xenograft that promotes blood vessel proliferation and the migration of bone cells onto its particles [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, Bio-Oss does not present osteoinductive potential nor osteogenic properties. The aim in the present study was to combine it with APCs to enhance its properties during bone regeneration processes. APCs provide a slow and gradual release of cytokines and growth factors during a critical period for collagen, fibronectin, and other extracellular matrix components formation that guides tissue regeneration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This gradient of cytokines can primarily attract mesenchymal stem cells (MSCs) from bone marrow and those in regions adjacent to the wound or defect, followed by their differentiation into osteoprogenitor cells.\u003c/p\u003e \u003cp\u003eL-PRF, a second-generation blood concentrate, is a biomaterial obtained through the centrifugation of autogenous blood [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Its use in bone regeneration of CSDs in rats has been extensively studied over the past 10 years and has shown satisfactory results in bone neoformation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The modification of the protocol initially established by Choukroun and colleagues (2001) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] focused on changes in RCF, centrifugation time, and introduction of new centrifugation devices has led to the development and emergence of new protocols, such as A-PRF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Bio-PRF [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These new matrices produced from modified protocols have demonstrated various advantages over the original matrices in various in vitro and in vivo evaluations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Indeed, the scientific literature increasingly aims to obtain a blood concentrate matrix with higher mechanical resistance, prolonged degradation time, and greater retention and release rates of growth factors.\u003c/p\u003e \u003cp\u003eFew in vivo studies have directly compared the impact of different protocols for blood concentrate preparation on bone regeneration. Using microtomographic and histomorphometric evaluations, Da Silva et al. (2022) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] demonstrated that both L-PRF and A-PRF enhanced bone formation in CSDs in rats at 30 days post-surgery. Although no statistically significant differences in the volume of newly formed bone were found between the L-PRF and A-PRF groups, the authors emphasized key findings from qualitative histological assessments and microarchitectural analyses, which revealed distinct characteristics in bone tissue organization and quality among the experimental groups.\u003c/p\u003e \u003cp\u003eAccording to the authors, compared to defects filled with L-PRF, defects treated with A-PRF showed a more significant amount of osteoid matrix and a smaller spacing between bone trabeculae. The authors also argue that studies with longer follow-up periods and immunohistochemical analyses could better explain the obtained results and highlight possible molecular impacts resulting from changes in PRF matrix composition. S\u0026aacute;vio and colleagues (2023) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] demonstrated through microtomographic, histomorphometric and immunofluorescence analyses that blood concentrate matrices produced from fixed-angle centrifuges (L-PRF and A-PRF) and horizontal centrifugation (H-PRF; Bio-PRF) enhance bone neoformation in CSDs in rat calvaria in evaluations performed at 30 days post-operatively. It was also observed that CSDs treated with Bio-PRF showed higher values of BV/TV, Tb.N, and alizarin precipitation compared to those treated with A-PRF and L-PRF. The authors concluded that Bio-PRF has greater biological potential in the bone neoformation process.\u003c/p\u003e \u003cp\u003ePrevious studies evaluating different protocols for preparing blood concentrates in bone regeneration have primarily focused on short-term healing periods and did not include the use of bone graft materials. These investigations were largely proof-of-concept studies designed to assess the isolated regenerative potential of APCs in bone formation. In contrast, the present study is the first to assess the combined effects of a XEN with three distinct APC preparation protocols (L-PRF, A-PRF, and Bio-PRF) on bone regeneration in CSDs in rat calvaria during a late-stage healing period. Despite the lack of statistically significant differences in the volume of newly formed bone between defects treated with XEN alone and those treated with XEN plus APCs, histometric analysis revealed a trend toward increased NBA in the XEN\u0026thinsp;+\u0026thinsp;APC groups. This trend, although not significant, suggests a potential additive effect of APCs. The 60-day healing period analyzed in this study represents an advanced phase of bone repair, which may partly explain the absence of significant differences between the groups, as bone remodeling tends to stabilize over time. These findings are consistent with those reported by Oliveira et al. (2015) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who observed significant differences in bone formation between XEN and XEN\u0026thinsp;+\u0026thinsp;L-PRF groups only at the 30-day postoperative mark, but not at 60 days. Similarly, in a study with a shorter healing period, Engler-Pinto et al. (2019) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] demonstrated that the combination of XEN and L-PRF resulted in greater bone volume compared to XEN alone in ovariectomized rats. These results collectively suggest that the use of APCs may accelerate early bone healing when compared to XEN alone. However, further studies are needed to determine whether APCs can consistently promote faster or more efficient bone regeneration, especially in the early stages of healing.\u003c/p\u003e \u003cp\u003eIt is also important to highlight that tissue volume formation does not equate to tissue maturation, as the treated defect may be filled with immature tissue, compromising its functional viability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The quality of bone tissue is not limited to its mineralization but also includes the quantity and quality of collagen fibers, which are both crucial for bone matrix mineralization and thus for the mechanical quality of bone tissue [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the Picrosirius red analysis, the more organized (mature) fibers appear redder, while the more disorganized (immature) fibers exhibit a greener coloration [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Type I collagen, representing more mature fibers, acts as a support for bone matrix mineralization, directly influencing the microarchitecture of trabecular bone [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, it was observed that only the XEN\u0026thinsp;+\u0026thinsp;A-PRF and XEN\u0026thinsp;+\u0026thinsp;Bio-PRF groups had a higher amount of mature collagen fibers (type I) compared to the XEN group. This result indicates that certain protocols developed with advanced study can potentially better enhance the osteoconductive properties of XEN and that the type of preparation protocol used can provide different organic matrix compositions in newly formed bone tissue. Considering the effects of this organic matrix on the functionality and strength of bone tissue [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], future studies are important to, for example, longitudinally evaluate the degree of bone remodeling associated with dental implants installed in bone tissue regenerated with XEN combined with blood concentrates produced by different protocols.\u003c/p\u003e \u003cp\u003eIn a recent study [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the outcomes of comparing simply the centrifugation devices was investigated. The overview review article offered deeper insights into the advantages of H-PRF when compared to fixed-angle methods across a wide range of regenerative medical and dental applications. A total of 75 studies were included with 13 studies directly compared horizontal centrifugation to fixed-angle centrifugation for producing PRF, while the remaining 62 studies were non-comparative and focused on expanding the uses and clinical applications of H-PRF. These studies spanned categories such as cell concentrations, fibrin matrix structure, growth factor release, antibacterial and anti-inflammatory properties, and regenerative applications in bone, periodontal, cartilage, skin, hair, regenerative endodontics, corneal defect repair, wound healing, and soft tissue regeneration. Of the studies comparing horizontal to fixed-angle centrifugation, 84.6% favored horizontal centrifugation, while 15.4% found no difference. None of the studies favored fixed-angle centrifugation. Nevertheless, further comparative clinical studies are warranted to further support these findings [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLastly, it has also been reported that the PRF tubes utilized during the production of PRF likely matters even more so than the centrifugation device itself [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It has been reported that many tube types contain chemical additives or other substances that may either delay or promote or delay the clotting process and strength in the clot [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the present animal study, the standard 10 mL PRF tubes from each manufacturer could not be utilized owing to the small size of animals and instead 3 mL of blood was drawn into 5 mL tubes containing chemical additives. Therefore, it remains to be clinically observed what effect the tube types from each of the respective manufacturers will have on the final PRF outcomes including their subsequent regenerative potential.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated that the centrifugation protocol used for the preparation of APCs was shown to impact the quality of newly formed bone tissue when combined with XEN. Some additional limitations of this study should be considered when analyzing the results. Since the bone defects were not protected by a membrane (barrier), it is not possible to evaluate if the results would be the same under standard guided bone regeneration principles/conditions. The levels of growth factors in the blood concentrates were also not measured in the smaller tube sizes utilized, so a direct correlation between the levels and types of factors present and the obtained bone formation could not be evaluated/estimated. Future studies should address this topic for a better understanding of biological response variability. The use of animals with systemic impairments and on a larger phylogenetic scale should also be considered in future studies to better elucidate the role of APCs in promoting faster bone formation and their optimal benefit in clinical practice.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the limits of this study, it can be concluded that defects treated with XEN combined with A-PRF or Bio-PRF, when evaluated at a later stage of healing, show neoformed bone tissue with greater collagen maturation compared to those treated with XEN alone. Additionally, the type of protocol used for preparing blood concentrates appears to be crucial for the quality of the collagen matrix in the neoformed bone tissue.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eA-PRF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Advanced Platelet-Rich Fibrin\u003c/p\u003e\n\u003cp\u003eAPCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Autologous Platelet Concentrates\u003c/p\u003e\n\u003cp\u003eBio-PRF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Horizontal Platelet-Rich Fibrin (Bio-PRF)\u003c/p\u003e\n\u003cp\u003eBP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone Porosity within the Volume of Interest (VOI)\u003c/p\u003e\n\u003cp\u003eBV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone Volume\u003c/p\u003e\n\u003cp\u003eBV/TV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone Tissue (Bone Volume / Total Volume\u003c/p\u003e\n\u003cp\u003eCSDs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Critical-Size Defects\u003c/p\u003e\n\u003cp\u003eConn.Dn\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Connectivity Density\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEDTA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eFGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fibroblast Growth Factor\u003c/p\u003e\n\u003cp\u003eHE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eIGF-1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Insulin-Like Growth Factor\u003c/p\u003e\n\u003cp\u003eL-PRF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Leukocyte- and Platelet-Rich Fibrin\u003c/p\u003e\n\u003cp\u003eMicro-CT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Micro-Computed Tomography\u003c/p\u003e\n\u003cp\u003eMSCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mesenchymal Stem Cells\u003c/p\u003e\n\u003cp\u003eNBA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;New Bone Area\u003c/p\u003e\n\u003cp\u003ePDGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Platelet-Derived Growth Factor\u003c/p\u003e\n\u003cp\u003ePo\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Lower Bone Porosity\u003c/p\u003e\n\u003cp\u003eRPA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Remaining Particles Area\u003c/p\u003e\n\u003cp\u003eRPV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Remaining Particles Volume\u003c/p\u003e\n\u003cp\u003erpm\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Rotations Per Minute\u003c/p\u003e\n\u003cp\u003eROI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Region of Interest\u003c/p\u003e\n\u003cp\u003eTA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Total Area\u003c/p\u003e\n\u003cp\u003eTb.N\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Number of Trabecular Bones\u003c/p\u003e\n\u003cp\u003eTb.Sp\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Separation of Trabeculae\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Transforming Growth Factor-Beta\u003c/p\u003e\n\u003cp\u003eVEGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Vascular Endothelial Growth Factor\u003c/p\u003e\n\u003cp\u003eVOI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Volume of Interest\u003c/p\u003e\n\u003cp\u003eXEN \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bovine-derived xenogeneic bone grafts\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll in vivo animal experiments were conducted in compliance with ARRIVE guidelines, and protocols were approved by the Committee on Ethics in the Use of Animals (CEUA) of the School of Dentistry of Ribeirão Preto (FORP), University of São Paulo (USP), under approval number 2022.1.244.58.2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research article received no funds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWanderson Thalles de Souza Braga – Conducted experimental analyses, performed a web-based literature review, collected and extracted data, created data visualizations, and drafted both the initial and final versions of the manuscript.\u003c/p\u003e\n\u003cp\u003eUlli da Costa Cunha Martins, Débora de Souza Ferreira Sávio, and Lucia Moitrel Pequeno da Silva – Performed animal experiments.\u003c/p\u003e\n\u003cp\u003eMaria da Conceição Pereira Saraiva – Conducted statistical data analysis.\u003c/p\u003e\n\u003cp\u003eFlávia Furlaneto, Carlos Fernando Mourão, Richard Miron, and Roberta Okamoto – Contributed to data interpretation and critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eMichel Messora – Project administration, validation of results, overall supervision, drafting, and critical revision of the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMartens W, Bronckaers A, Politis C, Jacobs R, Lambrichts I. 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Periodontology 2000. 2025 Mar 27. https://doi.org/10.1111/prd.12521\u003c/li\u003e\n\u003cli\u003eMiron RJ, Kawase T, Dham A, Zhang Y, Fujioka-Kobayashi M, Sculean A. A technical note on contamination from PRF tubes containing silica and silicone. BMC Oral health. 2021 Dec;21:1-1. https://doi.org/10.1186/s12903-020-01299-w\u003c/li\u003e\n\u003cli\u003eWei Y, Cheng Y, Wei H, et al. Development of a super-hydrophilic anaerobic tube for the optimization of platelet-rich fibrin. Platelets. 2024 Dec 31;35(1):2316745. https://doi.org/10.1080/09537104.2023.2316745\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bone regeneration, Bone substitutes, Blood concentrates, Tissue engineering","lastPublishedDoi":"10.21203/rs.3.rs-6631987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6631987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study assessed the impact of xenogeneic bone grafts (XEN), either utilized alone or combined with autologous platelet concentrates (APCs) prepared using three different centrifugation protocols on healing of critical-size defects (CSDs) in rat calvaria.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eForty rats were assigned to five groups (n\u0026thinsp;=\u0026thinsp;8): Control, XEN, XEN\u0026thinsp;+\u0026thinsp;L-PRF, XEN\u0026thinsp;+\u0026thinsp;A-PRF, and XEN\u0026thinsp;+\u0026thinsp;Bio-PRF. Blood concentrates were prepared by centrifuging blood collected via cardiac puncture: L-PRF (~\u0026thinsp;700g for 12 minutes), A-PRF (~\u0026thinsp;208g for 14 minutes), and Bio-PRF (~\u0026thinsp;700g for 8 minutes). Defects (5 mm diameter) in the right parietal bone of each rat were filled with blood clot, XEN or XEN combined with L-PRF, A-PRF, or Bio-PRF. Animals were euthanized 60 days post-surgery and the calvariae were analyzed using histomorphometry and micro-CT.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGroups XEN-A-PRF, XEN-L-PRF and XEN-BIO-PRF presented higher bone volume (BV) and lower bone porosity (Po) than the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The XEN group did not show different values of VO and Po compared to the control group. Only the XEN\u0026thinsp;+\u0026thinsp;A-PRF and XEN\u0026thinsp;+\u0026thinsp;Bio-PRF groups showed a higher percentage of mature collagen fibers (red color) compared to the XEN and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The XEN group showed a higher amount of immature collagen fibers (green color) compared to the XEN\u0026thinsp;+\u0026thinsp;A-PRF, XEN\u0026thinsp;+\u0026thinsp;L-PRF, and XEN\u0026thinsp;+\u0026thinsp;Bio-PRF groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIt can be concluded that CSDs treated with XEN combined with A-PRF or Bio-PRF, in late-stage healing assessments, presented higher newly formed bone tissue with greater collagen maturation than those treated with XEN alone; ii) the centrifugation protocol used for preparing APCs seems to be a decisive factor for the quality of the collagen matrix in newly formed bone tissue.\u003c/p\u003e","manuscriptTitle":"Evaluation of the effects of the combination of xenogeneic bone grafts with autologous platelet concentrates prepared using different centrifugation protocols on the treatment of critical-size bone defects in rat calvaria: microtomographic and histomorphometric analyses at a late healing stage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 07:07:11","doi":"10.21203/rs.3.rs-6631987/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3a6f843-651b-4919-8066-a278c54efb07","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-18T18:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 07:07:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6631987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6631987","identity":"rs-6631987","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}