Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 23/09/2024 Aceptado:14/11/2024 Publicado: 18/02/2025 hps://doi.org/10.52973/rcfcv-e35520 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Comparison of the Effects of Platelet Rich Fibrin and Platelet Rich Plasma on Experimentally Induced Tendon Injury in Rabbits Treated with Methylprednisolone Comparación de los efectos de la plasma rico en plaquetas y la fibrina rico en plaquetas sobre la lesión tendinosa inducida experimentalmente en conejos tratados con melprednisolona Muharrem Erol 1* , Cengiz Ceylan 1 , Mustafa Usta 2 , Serdar Sargın 3 , Musa Karaman 2 , Fatma Ilhan 2 , Eyup Tolga Akyol 1 1 Balikesir University, Faculty of Veterinary Medicine, Department of Surgery. Balikesir, Türkiye 2 Balikesir University, Faculty of Veterinary Medicine, Department of Pathology. Balikesir, Türkiye 3 Balikesir University, Faculty of Medicine, Department of Orthopedics and Traumatology. Balikesir, Türkiye * Corresponding Author: erolmuharrem@hotmail.com ABSTRACT The aim of this study is to compare the effects of PRP and PRF on tendon injuries treated with methylprednisolone. In this context, the histopathological and immunohistochemical ef- fects of PRP and PRF on tendon healing were evaluated. The study included 30 New Zealand rabbits (15 females and 15 ma- les) with varying weights (1.5-3 kg) and ages (1-3 years). The experimental groups were designated as the steroid control group (Group B, n=10), the steroid+PRP group (Group C, n=10), and the steroid+PRF group (Group D, n=10). Addionally, the control ssues from a simultaneous study served as the emp- ty control group (Group A). On the 14th postoperave day, a secon encompassing the tenotomy site was excised from the area via incision. Healing was observed to be beer in Groups C and D compared to Groups A and B. In Group A, inflamma- tory cell infiltraon, primarily consisng of macrophages and polymorphonuclear leukocytes, along with a small number of lymphocytes and foreign-body giant cells, was higher than in all other groups. Group B exhibited less inflammatory cell in- filtraon compared to Group A. Addionally, hyperemia, he- morrhage, necrosis, new vessel formaon, and fibroblasts of varying shapes and orientaons were noted in Groups A and B. In contrast, cell infiltraon was significantly lower in Groups C and D, while vascularizaon, fibroblast acvity, and collagen density were higher. Collagen fibers were observed more regu- lar bundles forms in Groups C and D compared to the control groups. The findings suggest that the combinaon of PRP or PRF with systemic methylprednisolone can effecvely enhance tendon healing by modulang inflammatory responses, redu- cing adhesions, and supporng the formaon of a well-organi- zed extracellular matrix. Key words: Glucocorcoid; PRP; PRF; tendon healing; rabbit RESUMEN El objevo de este estudio es comparar los efectos de PRP y PRF en lesiones tendinosas tratadas con melprednisolona. En este contexto, se evaluaron los efectos histopatológicos e in- munohistoquímicos de PRP y PRF en la curación de tendones. El estudio incluyó 30 conejos de Nueva Zelanda (15 hembras y 15 machos) con diferentes pesos (1,5-3 kg) y edades (1-3 años). Los grupos experimentales se designaron como el grupo de control con esteroides (Grupo B, n = 10), el grupo esteroides + PRP (Grupo C, n = 10) y el grupo esteroides + PRF (Grupo D, n = 10). Además, los tejidos de control de un estudio simultáneo sirvieron como grupo de control vacío (Grupo A). El día 14 del posoperatorio, se exrpó una sección que abarcaba el sio de la tenotomía del área a través de una incisión. Se observó que la curación fue mejor en los grupos C y D en comparación con los grupos A y B. En el grupo A, la infiltración de células infla- matorias, que consisa principalmente en macrófagos y leu- cocitos polimorfonucleares, junto con una pequeña candad de linfocitos y células gigantes de cuerpo extraño, fue mayor que en todos los demás grupos. El grupo B mostró una me- nor infiltración de células inflamatorias en comparación con el grupo A. Además, se observaron hiperemia, hemorragia, nec- rosis, formación de nuevos vasos y fibroblastos de diferentes formas y orientaciones en los grupos A y B. En contraste, la in- filtración celular fue significavamente menor en los grupos C y D, mientras que la vascularización, la acvidad de los fibrob- lastos y la densidad de colágeno fueron mayores. Se observó que las fibras de colágeno formaban haces más regulares en los grupos C y D en comparación con los grupos de control. Los hallazgos sugieren que la combinación de PRP o PRF con melprednisolona sistémica puede mejorar efecvamente la curación del tendón al modular las respuestas inflamatorias, reducir las adherencias y apoyar la formación de una matriz extracelular bien organizada. Palabras clave: Glucocorcoide; PRP; PRF; curación del tendón; co- nejo 1 of 8

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Tendon injuries can occur for various reasons, such as sports injuries and chronic diseases. Since tradional treatment methods provide limited healing, new treatment approaches are being invesgated. Autologous blood products such as Pla- telet Rich Plasma (PRP) and Platelet Rich Fibrin (PRF) have emer- ged as promising treatment methods for tendon and ligament healing [1 , 2 ,3]. The idea of healing bone and tendon ssue damages in a shorter me and more robustly has directed researchers towards biomaterials. PRF [4 , 5 , 6] and PRP [7 , 8 , 9 , 10] have been used for this purpose. Corcosteroids have the potenal to modulate tendon healing through various immunological ef- fects, whether used as systemic or local treatments. However, this therapeuc approach may adversely impact tendon health due to potenal side effects, somemes even leading to tendon ruptures [11]. Following injury, the inflammatory phase iniates the he- aling process, which is subsequently followed by the prolife- rave and remodeling phases [12]. Corcosteroids exert their effects primarily by promong the resoluon of inflammaon [13]. While they can suppress the inflammatory response [14], they also influence connecve ssue granulaon and the for- maon of a more organized extracellular matrix [15]. Resoluon of inflammaon plays a vital role in the tendon healing process, as prolonged inflammaon can hinder effecve recovery [16]. Thus, the mely resoluon of inflammaon is crucial to facilitate progression to subsequent repair stages, including proliferaon and remodeling. This resoluon process involves a complex in- terplay of mediators such as lipoxins and resolvins, as well as va- rious cellular mechanisms [17]. Addionally, an-inflammatory cytokines like interleukin-10 (IL-10) contribute to inflammaon resoluon by inhibing pro-inflammatory signaling pathways and promong macrophage transion from the M1 to the M2 phenotype [18]. Corcosteroids not only aid in the resoluon of inflammaon but may also support tendon repair by reducing fibroc healing [19]. During this process, reducons in extracellular matrix-as- sociated components such as fibronecn and tenascin C have been observed. Fibronecn rapidly increases post-injury and plays a role in fibrosis [20], while tenascin C facilitates the rec- ruitment, migraon, and differenaon of myofibroblasts, cont- ribung to early stages of myocardial repair [21]. However, pro- longed corcosteroid use may have serious systemic effects on the cardiovascular, gastrointesnal, endocrine, and immune sys- tems. Metabolic effects, such as glucose intolerance and hyper- tension, are frequently reported with long-term corcosteroid therapy [22]. Their immunosuppressive effects can suppress im- mune responses and increase suscepbility to infecons [23]. Furthermore, an associaon between corcosteroid use and an elevated risk of pepc ulcers and gastrointesnal bleeding has been idenfied [24]. Prolonged corcosteroid use may also lead to degenerave changes in tendons and ligaments, alteraons in stress levels, and disrupons in homeostasis [25]. The aim of this study was to compare the effects of PRP and PRF on experimentally induced tendon damage in rabbits (Oryctolagus cuniculus domescus) treated with methylpredni- solone. In this context, the histopathological and immunohisto- chemical effects of PRP and PRF on tendon healing were evalu- ated. The hypothesis of the study was that PRP and PRF would accelerate the healing of tendon damage in animals treated with methylprednisolone and that this effect may differ depen- ding on the treatment method. Although the healing effects of PRP and PRF on various ssue types have been demonstrated, the comparison of their effecveness in tendon damage during methylprednisolone treatment has been the subject of a limi- ted number of studies in the literature. Therefore, this study will make significant contribuons to the exisng literature and will guide clinical applicaons. MATERIALS AND METHODS Animals and study design The study was carried out with the ethical permission of Erciyes University Animal Experiments Local Ethics Commiee (EUHADYEK, Number: 17/018). The study included 30 New Zealand rabbits (Oryctolagus cu- niculus) (15 females and 15 males) with varying weights (1.5- 3 kg) and ages (1-3 years). The rabbits were divided into three equal groups, each consisng of 10 rabbits (5 males and 5 fe- males). Prior to surgery, all animals underwent roune clinical examinaon, and only healthy rabbits were included in the study. The experimental design consisted of a steroid control group (Group B), a steroid+PRP group (Group C), and a ste- roid+PRF group (Group D). Addionally, control ssues from a simultaneous project conducted at Balikesir University Scienfic Research Projects (Project number: 2017/132) were used as the empty control group (Group A). In all animals, IV catheters were inserted into the ear vein to facilitate intravenous medical applicaons when necessary and to obtain blood samples for PRP and PRF preparaon. Prior to the surgical procedure, 5 mL of blood was collected from the PRP group for PRP preparaon, while 8 mL of blood was collect- ed from the PRF group for PRF preparaon. Preparaon of PRP PRP was prepared using a specialized PRP kit (EasyPRP® Kit 10, Neotec Biotechnology, Türkiye). Inially, 0.5 mL of sodium citrate as an ancoagulant (PPS Natrium Citricum 3.13%, Medi- Pac, Germany) was drawn into the kit’s 10mL syringe. Subse- quently, 4.5 mL of blood was collected from each rabbit into the syringe, bringing the total volume to 5 mL. The lower chamber of the syringe was inserted, and the syringe was gently shaken to mix the ancoagulant with the blood. The syringe was then placed in a centrifuge (DMO412, Onilab, USA) and centrifuged at 1200G for 5 min. Aſter the first centrifugaon, the red blood cells that had accumulated in the lower chamber were removed, and a second centrifugaon was performed at 1200G for 10 min. Following the second centrifugaon, three disnct layers were formed in the syringe: PRP, PPP (Platelet-Poor Plasma), and the buffy coat at the boom. The stopper of the syringe was then removed, and the PRP applicaon syringe (3 mL) with a luer lock p provided in the kit was aached. The buffy coat and PRP por- on were drawn into the 3 mL applicaon syringe, and the PRP was prepared for use. Preparaon of PRF Blood collected in tubes without ancoagulant was immedi- ately centrifuged at 400G for 10 min [26]. Aſter centrifugaon, three layers formed in the tube: red blood cells at the boom, a 2 of 8

Comparison of the Effects of Platelet Rich Fibrin and Platelet Rich Plasma / Erol et. al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico fibrin clot (PRF) in the middle, and acellular plasma (PPP) at the top. The PRF clot was carefully removed and placed on sterile gauze. Gentle pressure was applied to flaen the clot, making it ready for use (FIG. 1). FIGURE 1. The preparaon of Platelet Rich Fibrin (PRF) for use in the experiment Surgical Procedure In all groups, anesthesia was induced with medetomidine HCl (0.3 mg/kg, IM, Domitor, Pfizer, Germany) and ketamine HCl (30 mg/kg, IM, Alfamine, Alfasan, Netherlands). Anesthesia was then maintained using sevoflurane (Sevorane, Abbvie, USA) at a concentraon of 4% in 100% oxygen. Under anesthesia, the right Achilles tendon region was prepared for surgery. A skin incision was made to access the Achilles tendon and the paratenon membrane by dissecng the subcutaneous connecve ssue. The tendon was exposed by incising the paratenon membrane. The Achilles tendon of the gastrocnemius muscle was carefully isolated, and a tenotomy was performed. Following the tenotomy, the tendon ends were approximated and sutured using the modified Kessler technique [27]. The paratenon membrane was closed with 4-0 monofila- ment absorbable suture material (PDSII, Ethicon®, USA). In the PRP group, 0.5-1 mL of prepared PRP was injected between the paratenon and the tendon. In the PRF group, the prepared PRF was wrapped around the tendon to cover the incision site. Aſter the paratenon membrane was closed, the subcutaneous connecve ssue and skin incision were rounely sutured using 3-0 absorbable mulfilament suture material (Vicryl, Ethicon®, USA), compleng the procedure. Post-operave Procedure Following the surgery, all rabbits received anbioc (Penicil- lin, 11,000 IU/kg, IM, Ieciline, IE Ulagay, Türkiye) for 5 days (d) and methylprednisolone (4 mg/kg, IM, Prednol-L amp, Mustafa Nevzat, Türkiye) for 14 d. Addionally, a protecve bandage was applied to the surgical site for 7 d. At the end of this period, the bandage was removed, the wounds were inspected, and the animals were returned to their cages without reapplicaon of the bandage. Collecon of Tissue Samples On the 14th postoperave d, all rabbits underwent a second surgery following the same anesthesia and operave protocols. A ssue secon encompassing the previous tenotomy site was excised and fixed in 10% buffered formalin soluon. The tendon ends were then reaached using the modified Kessler method, and the skin and subcutaneous connecve ssue were rounely sutured. Histopathological and Immunohistochemical Evaluaon Tendon ssues were fixed in 10% neutral buffered formalin soluon, rounely processed, and embedded in paraffin blocks. Five-micron secons were cut from the paraffin blocks using a microtome (RM2245, Leica, Germany), and stained with he- matoxylin-eosin and Masson’s Trichrome for histopathological analysis. Tendon healing was assessed using a light microscope (Eclipse Ni, Nikon, Japan). Immunostaining was performed using the Avidin-Bion-Im- munoperoxidase complex method to assess factors influencing fibroblast acvaon and collagen producon during wound healing. Five-micron thick secons from paraffin-embedded tendon ssues were mounted on poly-L-lysine coated slides and dried overnight in an oven. Following deparaffinizaon, the secons were washed in PBS (Phosphate Buffer Soluon) three mes, with 5-min intervals between washes, and incubated (WiseVen, Wisd, France) with 0.001% trypsin at 37˚C for 30 min to unmask angenic receptors. Aſter washing in PBS three mes, the secons were incubated in 3% hydrogen peroxide-methanol soluon for 20 min to inhibit endogenous peroxidase acvity. The secons were washed again in PBS and incubated with non-immune goat serum for 30 min at room temperature to block non-specific staining. Without further washing, secons were incubated with primary anbodies for 1 h at 37°C, target- ing inducible nitric oxide synthase (iNOS, 1:100, ThermoFisher, PA5-16855), nitrotyrosine (AB5411, 1:500, Millipore, USA), βFGF (rabbit polyclonal; 1:200, AB8880, Abcam, Cambridge, UK), VEGF (Mouse monoclonal; 1:200, Abcam, AB1316, Cambridge, UK), and TGFβ1 (AB190503, 1:200, Abcam). Following the incubaon, the secons were washed three mes in PBS and incubated with a bionylated secondary an- body for 30 min. Aſter further washing in PBS, the secons were treated with peroxidase-coupled streptavidin for 30 min. The secons were then washed three mes in PBS and incubat- ed with 3.3-diaminobenzidine tetrahydrochloride-H2O2 (DAB) soluon as the chromogen. Aſter rinsing in dislled water, the secons were counterstained with Mayer’s Hematoxylin. Im- munostaining intensity was evaluated in five disnct areas of each secon under high magnificaon using a light microscope (Eclipse Ni, Nikon, Japan), with staining scored as none (-), low (+), moderate (++), and intense (+++). RESULTS AND DISCUSSIONS In the study, healing was found to be superior in Groups C and D compared to Groups A and B. In Group A, inflammato- ry cell infiltraon, predominantly comprising macrophages and polymorphonuclear leukocytes, along with a small number of lymphocytes and foreign-body giant cells, was greater than in all other groups (FIG. 2a). Group B exhibited reduced inflammatory cell infiltraon compared to Group A (FIG. 2b). Addionally, hy- peremia, hemorrhage, necrosis, neovascularizaon, and fibro- blasts of varying shapes and orientaons were observed in both Groups A and B. In contrast, Groups C and D showed significantly lower levels of cell infiltraon, with increased vascularizaon, fibroblast acvity, and collagen density. Collagen fibers in these groups formed more regular bundles compared to the control groups (FIGS. 2c and 2d). 3 of 8

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 2. a: Inflammatory cell infiltraon predominantly consisng of macrophages, neutrophils, a small number of lymphocytes, foreign-body giant cells, and areas of hemor- rhage (Group A), b: Hemorrhage accompanied by macrophage and neutrophil infiltraon (Group B), c: Increased connecve ssue and new vessel formaon (Group C), d: Regularly arranged connecve ssue bundles (Group D) (Hematoxylin-Eosin stain) In Masson’s Trichrome staining, used to visualize connecve ssue, collagen structures of varying thickness and irregular ori- entaon were observed in the control groups. In contrast, the study groups exhibited collagen fibers that were more regularly aligned and parallel to one another (FIG. 3). FIGURE 3. a: Hemorrhage and irregular collagen fibers (Group A), b: Hemorrhage and ir- regular collagen fibers (Group B), c: Regular connecve ssue cells and collagen (Group C), d: Regular connecve ssue bundles and collagen (Group D) (Hematoxylin-Eosin stain) The immunohistochemical staining scores for TGF, VEGF, FGF-β, iNOS, and nitrotyrosine observed in the study are pre- sented in TABLE I. While the immunoreacvity of TGF and VEGF was similar in the study groups, their intensity was found to be less severe compared to the control group. TGF immunostaining was parcularly intense in macrophages and was present in the cytoplasm of neutrophils, fibroblasts, and vascular endothelial cells (FIG. 4). Cells staining posively for VEGF were primarily vascular endothelial cells, macrophages, and fibroblasts. Intense FGF-β immunostaining was observed across all groups (FIG. 5), predominantly in fibroblasts, with weaker staining in the cyto- plasm of macrophages (FIG. 6). iNOS and nitrotyrosine immu- nostaining were most intense in Group A, moderate in Group B, and similar in the study groups. In Group A, both iNOS and ni- trotyrosine staining were strongly present in macrophages and neutrophils, with moderate staining in connecve ssue. In the other three groups, staining was moderate in the cytoplasm of macrophages, fibroblasts, and vascular endothelial cells (FIGS. 7 and 8). TABLE I. Immunohistochemical staining scores according to groups TGF VEGF FGF-β iNOS Nitrotyrosine Grup A (Control) +++ +++ +++ +++ +++ Grup B (Steroid) +++ +++ +++ ++ ++ Grup C (Steroid+PRP) ++ ++ +++ ++ ++ Grup D (Steroid+PRF) ++ ++ +++ ++ ++ low (+), moderate (++), intense (+++) FIGURE 4. Severe (a, b) and moderate (c, d) TGF-β immunoreacvity in the cytoplasm of fibroblasts and macrophages (Immunohistochemistry, IHC) FIGURE 5. Severe (a, b) and moderate (c, d) VEGF immunoreacvity in the cytoplasm of fibroblasts, macrophages, and vascular endothelial cells (IHC) 4 of 8

Comparison of the Effects of Platelet Rich Fibrin and Platelet Rich Plasma / Erol et. al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 6. Severe FGF-β immunoreacvity in the cytoplasm of fibroblasts and macropha- ges (a, b, c, d) (IHC) FIGURE 7. Severe (a) and moderate (b, c, d) iNOS immunoreacvity in the cytoplasm of fibroblasts, neutrophils, and macrophages (IHC) FIGURE 8. Severe (a) and moderate (b, c, d) Nitrotyrosine immunoreacon in the cytop- lasm of fibroblasts, neutrophils, and macrophages (IHC) Rabbits serve as an effecve model due to their availability, appropriately sized tendons that allow for surgical manipula- on, and the ease of sample examinaon. Furthermore, rabbit flexor tendons are similar to human tendons in terms of diam- eter and the presence of a synovial sheath [28]. In the present study, rabbits were selected to enable more accurate evaluaon of the intervenons and collected samples, which proved ad- vantageous and consistent with the literature. Systemic corcosteroids have been studied in rats with Achilles tendon incisions, with the ming of administraon be- ing evaluated. When steroids were administered between the 5th and 9th postoperave days, they suppressed inflammaon during this period and posively impacted tendon healing by the 12th d [29]. Another study conducted on rats also demon- strated that systemic dexamethasone treatment administered between d 7 and 11 could enhance Achilles tendon healing [30]. Moreover, when the treatment was extended into the prolifera- ve/early remodeling phase, there was a more pronounced im- provement in material properes. Although late administraon of systemic corcosteroids is recommended in studies, it has been reported that addional research is needed to obtain fur- ther insights into mechanisms potenally mediated by systemic effects as well as local effects specific to tendon cells and matrix. In the present study, steroids were applied for 14 days postop- eravely and evaluated. Histopathological healing was superior in the steroid-only group compared to the control group, with a notable decrease in inflammatory cell infiltraon. Consistent with the literature, steroid use reduced inflammatory cell infil- traon and showed posive effects on tendon healing. However, the extent to which the ming of steroid administraon influ- enced these results remains unclear. While the steroid’s posive effects on healing throughout the process were evident, further studies are needed to determine the opmal ming and dura- on of steroid applicaon to maximize its benefits. The use of local corcosteroids is controversial [31], as they can have both posive [32] and negave [33 , 34 , 35 , 36] effects on tendons when used in isolaon. In the present study, corco- steroids were administered systemically, and no literature was found addressing their systemic use in combinaon with PRP or PRF. It was thought that PRP or PRF applicaon could provide posive outcomes in paents requiring systemic corcosteroids while migang the negave effects of local corcosteroid use on tenocyte viability. The effecveness of PRF in healing Achilles tendon injuries in rabbits, reported that the PRF group exhibited more organized collagen fibers and reduced vascularity compared to the con- trol group [37]. Similarly, a significant increase was found in fi- broblast density in groups treated with PRF or saline two weeks aſter tenotomy [38]. They also noted reduced inflammatory cell infiltraon in the PRF-treated tenotomy group, though collagen fibrils were of similar density and irregularly distributed. In con- trast, Sen et al. observed no stascally significant difference in healing between the PRP-treated and untreated groups in rabbits’ Achilles tendons 28 days post-tenotomy [39]. Dietrich et al. reported that the PRP and control groups showed similar cellularity on day 14, with the PRP group exhibing acve gran- ulaon ssue and large hemorrhagic areas [40]. Vascularizaon was more developed in the PRF group, with reduced inflamma- tory cell infiltraon and no granuloma formaon, whereas the PRP group displayed dense inflammatory infiltraon rich in poly- morphonuclear and mononuclear cells. Due to containing a pro- poronal combinaon of mulple growth factors, PRP and PRF can accelerate healing through synergisc effects by promong cell proliferaon and differenaon [41]. 5 of 8

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Inducible nitric oxide synthase has been shown to play an important role in tendon healing [29]. In the present study, iNOS expression was severe in the control group and moderate in the other groups. Some studies have reported that iNOS levels peak between the 4th and 14th d, followed by a decline aſter the 14th d [29 , 30]. In the present study, it was suggested that healing progressed more rapidly in the PRP and PRF groups, while the moderate iNOS expression observed in the steroid group was likely due to immunosuppression. Synthesized iNOS rapidly reacts with superoxide to form per- oxynitrite, a potent oxidant. Due to its short half-life, measuring peroxynitrite levels in ssues is challenging. Consequently, ni- trotyrosine is used as a marker for nitrosave stress. In the pres- ent study, nitrotyrosine expression was moderate in the PRP, PRF, and steroid-treated groups, but severe in the control group. This indicates that the applicaon of PRP and PRF reduced nitro- save stress. Previous studies have reported the overexpression of iNOS, nNOS, and eNOS mRNAs in a rat tendon degeneraon model [30 , 31]. In the present study, nitrosave ssue damage was assessed by measuring nitrotyrosine expression. However, as only iNOS immunostaining was performed to assess NO-related damage, it was not possible to determine which NOS isoform was responsible. Further detailed studies are required to clarify this issue. CONCLUSION This present study, which evaluated the effects of systemic methylprednisolone combined with PRP or PRF applicaons on tendon healing in a controlled rabbit model. The findings sug- gest that the combinaon of PRP or PRF with systemic corco- steroids can effecvely enhance tendon healing by modulang inflammatory responses, reducing adhesions, and supporng the formaon of a well-organized extracellular matrix. Systemic corcosteroid administraon may provide a balanced an-in- flammatory effect, counteracng the adverse impacts observed with local steroid applicaons, such as decreased collagen syn- thesis and cellular acvity. Overall, the results indicate that sys- temic corcosteroids in conjuncon with PRP or PRF could be a viable therapeuc strategy for tendon injuries. However, further research is necessary to determine the opmal ming, dosage, and concentraon for maximizing therapeuc efficacy of cor- costeroid. ACKNOWLEDGMENT This research was supported by Balikesir University Sci- enfic Research Projects Coordinator (BAP) (Project number: 2017/126). Conflict of interest The authors declare there is no conflict of interest. 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