Introduction
Autologous platelet concentrates have become valuable tools in regenerative medicine due to their capacity to release growth factors at supraphysiological concentrations and to stimulate tissue healing (Miron et al.., 2019; Fujioka, 2020). Among them, platelet-rich fibrin (PRF) is considered a second-generation autologous biomaterial, obtained through centrifugation of whole blood without anticoagulants. PRF generates a three-dimensional fibrin matrix that acts as a biodegradable scaffold, harboring platelets and leukocytes, and supporting angiogenesis and tissue regeneration

(Soares et al.., 2020; Graciani et al.., 2023). In human dentistry, PRF has been successfully used for periodontal defects and regenerative procedures (Fujioka-Kobayashi et al.., 2017; Miron et al.., 2019). In veterinary medicine, particularly in veterinary dentistry, its application remains poorly standardized (Soares et al.., 2020). Technical parameters such as g-force, centrifugation time, and rotor type have been shown to directly influence clot characteristics (Miron et al.., 2023). Recently, Farshidfar et al.. (2025) demonstrated that horizontal centrifugation results in a more homogeneous cellular distribution and greater release of growth factors compared to fixed-angle rotors. In dogs, Caterino et al.. (2022) standardized production protocols for L-PRF, confirming that methodology significantly impacts macroscopic and histological properties. The hypothesis of this study is that centrifugation force modifies the physical and histological characteristics of canine PRF, allowing the identification of an optimal protocol for application in veterinary dentistry.

Methodology
Blood samples were collected from four clinically healthy dogs (>15 kg) in plastic tubes without anticoagulants. Samples were centrifuged immediately for 8 minutes at 200 g, 300 g, 400 g, and 500 g using a Thermo Electron Corporation IEC CL30R centrifuge, equipped with a horizontal rotor (90°) and set at 18 °C. Clot mass was measured, and morphometric analysis was performed using ImageJ software. Histological processing: clots were fixed in 10% formalin and processed using routine techniques. Representative slides were selected and digitized with a Motic Easy Scan One® slide scanner. Image analysis: digitized preparations were evaluated using Motic DS Assistant® (VM V1 Viewer 2.0). Each clot was divided into three zones (apical, middle, basal) and nine quadrants per zone. A total of 27 standardized areas of 50,000 µm² were analyzed per clot (figure 1). Cell counting: cellular remnants were defined as ~5 µm eosinophilic spheroidal structures. Counting was performed with ImageJ (Cell Counter), by three independent observers, blinded and in triplicate, with averaged results. Statistical analysis: normality was assessed by the Shapiro-Wilk test. A two-way ANOVA with Tukey’s post hoc test was performed, considering g-force and clot zone as factors.

Results
Leukocytes were occasionally identified in only three PRF clots, while absent in the remaining samples. Cellular remnants, defined as spheroidal eosinophilic structures of approximately 5 µm in diameter, were consistently observed across all clots. Descriptive statistics revealed no significant effect of clot region (p > 0.05), indicating a relatively uniform distribution of remnants along the PRF structure. Two-way ANOVA demonstrated a significant main effect of centrifugation force on cellular remnant counts (F = 6.41, p = 0.003), whereas neither clot region nor the interaction between force and region were significant. Post hoc Tukey’s test confirmed that clots obtained at 200 g contained significantly more remnants than those produced at 500 g (p = 0.0017), and that 400 g also yielded higher counts compared to 500 g (p < 0.05). A trend for increased remnants at 300 g versus 500 g was observed, although this contrast did not reach statistical significance after correction. No differences were found between 200 g and 300 g or between 300 g and 400 g (figure 2). Overall, centrifugation at 200–300 g produced clots with the highest and most consistent cellular content, together with favorable macroscopic features (length, consistency, weight), supporting their application as optimal protocols for canine PRF preparation in veterinary dentistry.

Discussion
Our findings support the “low-speed centrifugation concept”, which proposes that lower centrifugal forces better preserve cellular components and may enhance growth factor release (Miron et al.., 2020). The higher abundance of cellular remnants at 200 g, and to a lesser extent at 300–400 g, reinforces this hypothesis. Conversely, 500 g markedly reduced cellular content, suggesting that higher forces may impair the biological potential of PRF. Recent literature highlights the importance of rotor design: horizontal centrifugation generates more homogeneous and functional clots compared to fixed-angle systems (Miron et al.., 2023; Farshidfar et al.., 2025). In veterinary medicine, Caterino et al.. (2022) emphasized the need to standardize protocols in dogs to ensure reproducible and clinically useful PRF. In this context, our data indicate that centrifugation at 200 g for 8 minutes provides an optimal balance of physical and histological characteristics.
The non-significant trend observed at 300 g versus 500 g suggests that additional replicates might confirm a broader effective range (200–300 g), further supporting its clinical translation in veterinary dentistry.

Conclusions
Centrifugation at 200–300 g for 8 minutes represents an optimal range for canine PRF preparation, producing clots with favorable macroscopic properties and a uniform distribution of cellular remnants. These characteristics support its potential clinical application in veterinary dentistry, particularly for regenerative procedures. Future studies should assess the biological activity and clinical performance of PRF obtained under these conditions to validate its use and contribute to the standardization of regenerative techniques in veterinary practice.

References
• Caterino, C., Della Valle, G., Aragosa, F., De Biase, D., Ferrara, G., Lamagna, F., & Fatone, G. (2022). Production Protocol Standardisation, Macroscopic and Histological Evaluation, and Growth Factor Quantification of Canine Leukocyte- and Platelet-Rich Fibrin Membranes. Frontiers in Veterinary Science, 9, 861255. https://doi.org/10.3389/fvets.2022.861255
• Farshidfar, N., Apaza Alccayhuaman, K. A., Estrin, N. E., Ahmad, P., Sculean, A., Zhang, Y., & Miron, R. J. (2025). Advantages of horizontal centrifugation of PRF and the impact of heat-compression on F-PRF and extended-PRF. Periodontology 2000. Online ahead of print. https://doi.org/10.1111/prd.12625
• Fujioka-Kobayashi, M., Kono, M., Katagiri, H., Schaller, B., Zhang, Y., Sculean, A., & Miron, R. J. (2021). Histological comparison of Platelet rich fibrin clots prepared by fixed-angle versus horizontal centrifugation. Platelets, 32(3), 413-419. https://doi.org/10.1080/09537104.2020.1754382
• Fujioka-Kobayashi, M., Miron, R. J., Hernandez, M., Kandalam, U., Zhang, Y., & Choukroun, J. (2017). Optimized Platelet-Rich Fibrin With the Low-Speed Concept: Growth Factor Release, Biocompatibility, and Cellular Response. Journal of Periodontology, 88(1), 112-121. https://doi.org/10.1902/jop.2016.160443
• Graciani, J. C. A. O. R., Rahal, S. C., Silva, W. M., Moroz, I., Fonseca-Alves, C. E., Govoni, V. M., & Kano, W. T. (2023). Histological, Immunohistochemical, Biomechanical, and Wettability Evaluations of the Leukocyte- and Platelet-Rich Fibrin Membranes Derived from Canine Blood. Journal of Veterinary Dentistry, 40(3), 212–219. https://doi.org/10.1177/08987564231152594
• Miron, R. J., Chai, J., Fujioka-Kobayashi, M., Sculean, A., & Zhang, Y. (2020). Evaluation of 24 protocols for the production of platelet-rich fibrin. BMC Oral Health, 20(1), 310. https://doi.org/10.1186/s12903-020-01299-w
• Miron, R. J., Fujioka-Kobayashi, M., Bishara, M., Zhang, Y., Hernandez, M., & Choukroun, J. (2017). Platelet-Rich Fibrin and Soft Tissue Wound Healing: A Systematic Review. Tissue Engineering Part B: Reviews, 23(1), 83-99. https://doi.org/10.1089/ten.teb.2016.0233
• Miron, R. J., Fujioka-Kobayashi, M., Sculean, A., & Zhang, Y. (2024). Optimization of platelet-rich fibrin. Periodontology 2000, 94(1), 79–91. https://doi.org/10.1111/prd.12521
• Soares, C. S., Babo, P. S., Faria, S., Pires, M. A., & Carvalho, P. P. (2021). Standardized Platelet-Rich
• Fibrin (PRF) from canine and feline origin: An analysis on its secretome pattern and architectural structure. Cytokine, 148, 155695. https://doi.org/10.1016/j/cyto.2021.155695