Chitosan-augmented advanced platelet rich fibrin improves the regeneration of superficial digital flexor tendon gap tenorrhaphy in donkeys (Equus Asinus)

Chitosan-augmented advanced platelet rich fibrin improves the regeneration of superficial digital flexor tendon gap tenorrhaphy in donkeys (Equus Asinus) | 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 Chitosan-augmented advanced platelet rich fibrin improves the regeneration of superficial digital flexor tendon gap tenorrhaphy in donkeys (Equus Asinus) Mahmoud Najeb, Alaa Samy, Awad Rizk, Mohamed EL-Henawey, Gamal Karrouf This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9390812/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Tendon healing remains a formidable challenge in equine rehabilitation as injured tendons are healed by a fibrotic scar that exhibits compromised biomechanical characteristics. The objectives were to evaluate the regenerative effectiveness of autologous advanced platelet rich fibrin (A-PRF) and chitosan (Ch) for repairing the superficial digital flexor tendon (SDFT) following gap tenorrhaphy in a prospective, blinded, randomized, controlled experimental study carried out on 18 male donkeys based on clinical, ultrasonographic, and histopathological interpretations. These animals were allocated according to the gap-filled biomaterials into three groups (n = 6 each): the control group, the PRF group, and the PRF/Ch group. Results The addition of A-PRF, both alone or with chitosan, led to significant (P < 0.05) and marked changes compared to those in the control group. Both the PRF and PRF/Ch groups exhibited normal clinical index scores, characterized by a comfortable attitude with neither swelling, pain, nor lameness, after the recovery of normal tendon ultrasonographic characteristics (echogenicity, alignment, thickness, shape, and gliding properties) and histologic features. The addition of chitosan to A-PRF in the PRF/Ch group significantly (P < 0.05) reduced the SDFT thickness, intratendinous edema, inflammatory cell infiltration, and vascularization. Conclusion The combination of both A-PRF and chitosan could represent an innovative and effective approach for enhancing SDFT healing outcomes, offering promising improvements in the field of equine rehabilitation. Platelet-rich Fibrin Superficial digital flexor tendon Chitosan Donkeys Tenorrhaphy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Tendon lacerations in equines present significant challenges, due to the need for restoring the tendon’s structural and functional integrity ( Chen et al., 2018 ). Effective treatment aims to achieve strong repair at the anastomosis site, re-establish normal vascularization, restore tendon microanatomy and functional length, and minimize scar tissue or restrictive adhesions that could impair tendon mechanical properties and function ( Jani et al., 2020 ). Research to enhance and expedite the regeneration of injured tendons has been spurred by this predicament. A method for enhancing the repair process has been developed through tissue engineering ( Ribitsch et al., 2021 ), which employs three main strategies: gene/cell-based, scaffold-based, and growth factor-based approaches ( Ruiz-Alonso et al., 2021 ). Tissue engineering for tendon laceration repair leverages both biological and synthetic scaffolds, each offering unique benefits. Biological scaffolds like hydrogels and decellularized tissues provide a natural environment for healing, while synthetic options offer structural support and integration. The ideal scaffold combines mechanical strength, biocompatibility, and biodegradability to promote effective tendon regeneration without adverse immune responses ( Alshomer et al., 2018 ; Vasiliadis and Katakalos, 2020 ; Ruiz-Alonso et al., 2021 ). Advanced Platelet-rich fibrin (A-PRF) is a platelet concentrate suspended in a small amount of plasma that is rich in growth factors that promote angiogenesis, chemotaxis, cell proliferation, cell differentiation, and collagen production. Furthermore, the fibrin matrix supporting these materials acts as a scaffold and is an essential component of the therapeutic value of PRF. The three-dimensional fibrin network with intrinsic platelet integration offers the best scaffold for cell migration and gradual prolonged release of cytokines ( Barbon et al., 2019 ; Celikten et al., 2024 ). Chitosan is a distinctive biopolymer that displays exceptional properties beyond being biocompatible and biodegradable. Consequently, this polysaccharide is a viable alternative in the biomaterials domain, particularly in applications related to tissue engineering. Chitosan contributes to the repair of soft tissues without adhesion or scar formation during tendon recovery ( Kim et al., 2023 ; Chen et al., 2024 ). Numerous imaging methods, such as scintigraphy, thermography, magnetic resonance imaging, and ultrasonography are employed to assess tendinopathy. However, ultrasonography is a widely used imaging modality for diagnosing and monitoring tendinopathy due to its accessibility, cost-effectiveness, and ability to provide real-time images. It is particularly favored for its ability to assess tendon thickness, detect structural changes, and monitor healing progress ( Fahmy, 2024 ; Hinckley et al., 2024 ). The use of donkeys in this study provides a relevant model for tendon injury and repair, offering high translatability to other equids, such as horses, due to functional and anatomical similarities. The hind limb's superficial digital flexor tendon (SDFT) was selected for its vulnerability to injury in both donkeys and horses. The anatomical and biomechanical properties of the donkey's SDFT make it an ideal model for studying tendon healing and treatments that can benefit both human and animal tendon injuries. Although both platelet-rich fibrin (PRF) and chitosan have been explored in various experimental investigations ( Dietrich et al., 2015 ; Melamed et al., 2015 ; Liao et al., 2017 ; Chen et al., 2018 ; Wong et al., 2020 ; Ibrahim Al Dirawi, 2023 ; Celikten et al., 2024 ; Diaz et al., 2024 ), there is a notable absence of studies documenting their exclusive application as filling biomaterials for gap tenorrhaphy in equines. Consequently, this study aimed to assess the effectiveness of autologous A-PRF alone or in conjunction with chitosan in repairing induced gap tenorrhaphy in a donkey model. The evaluation will involve clinical observations, ultrasonographic examinations, and histopathological analyses to provide comprehensive insights. Materials and Methods Study design In this study, a total of eighteen donkeys were enrolled, and their SDFTs in the right hind limbs were severed at the midpoint of the metatarsal region. Subsequently, gap-tenorrhaphy was performed, creating a full-thickness gap measuring 1 cm in length. Based on the bioscaffold added to the SDFT gap, donkeys were randomly allocated into three experimental groups (n = 6 per group) using a computer-generated randomization list created: the control group, where no biomaterials were added to the SDFT gap; the PRF group, where the SDFT gap was filled with autogenous A-PRF; and the PRF/Ch group, where the SDFT gap was filled with autogenous A-PRF and subsequently wrapped with a chitosan film. Animals The present study involved 18 clinically healthy male donkeys (Equus asinus) with an average age [mean ± standard deviation (SD)] of 34.5 ± 3.4 months and a mean body weight (BW) of 136.5 ± 12.1 kg. The donkeys were housed under consistent environmental conditions and fed a standardized diet consisting of a mixed ration of chopped wheat straw, bran, and whole corn provided twice daily, with ad libitum access to clean water. The general physical parameters, including heart rate, respiration rate, and rectal temperature, were monitored to ensure the overall health of the donkeys. Additionally, detailed clinical and ultrasonographic examinations were performed to assess their suitability for participation in the experiment. The clinical examination involved evaluating lameness, palpation for tenderness, and assessment for signs of swelling. Ultrasonographic evaluation focused on imaging the SDFT to assess its cross-sectional area, fiber alignment, and presence of any lesions or abnormalities. Only healthy donkeys with no pre-existing conditions affecting animal and tendon soundness were included in the study. The animals were housed and managed in straw bedded isolation stables at the Mansoura Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Mansoura University, Egypt. The study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code MU-ACUC (VM.PhD.25.04.45) All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical use of animals in research. Monitoring of Animal Welfare To fulfill institutional and national ethical regulatory requirements, the animals were closely monitored twice daily, both before and after the surgical induction, till the conclusion of the study. A composite welfare scale (Table S1 ) ( Kearney et al., 2022 ) was utilized for this purpose. The scale consisted of scores (ranging from 0 to 4) assigned to each of the following categories: food and water intake, clinical parameters (temperature, heart rate, and respiratory rate), natural behavior, and provoked behavior. Implant Preparation Preparation of A-PRF Eight milliliters of whole blood were collected in plain tubes and centrifuged at 1500 rpm for 14 minutes (RCF = 101×g), resulting in three layers: platelet-poor plasma, a PRF clot, and red blood cells ( Ghanaati et al., 2014 ). The PRF clot was implanted into the tendon defect with care to ensure proper graft-tendon contact. Scanning electron microscopy (SEM) was utilized to investigate the distinctive fibrin network within the A-PRF, and platelets and white blood cells were counted for characterization. For SEM imaging (JSM 6510 LV, JEOL, Japan), fresh A-PRF samples were fixed in 2.5% glutaraldehyde at 4°C for 24 hours, dehydrated through graded ethanol, dried using critical point drying, and coated with a 20 nm gold layer for conductivity. Preparation of chitosan A magnetic stirrer set to 500 rpm for two hours was used to dissolve one gram of chitosan powder (> 85% deacetylation degree, purchased from Meron, India) in fifty milliliters of deionized water with one-and-a-half milliliters of acetic acid at room temperature. To get rid of air bubbles, the chitosan solution was wrapped in cling film and left overnight. The chitosan film was formed by casting the clear solution into a Petri dish and letting it to dry for three days at room temperature. A 3 × 3 cm 2 chitosan film was used to enclose the gap filled with A-PRF in the PRF/Ch group. The characteristic chitosan functional groups were examined using Fourier-transform infrared (FT-IR) spectroscopy in order to characterize the material. Surgical Procedures Preanesthetic preparation guidelines were considered for the animals. Additionally, a broad-spectrum preoperative antibiotic composed of penicillin and streptomycin (Pen and Srept, Norbrook, UK) was injected intramuscularly 2 hours before surgery at a dose rate of 8 mg/kg procaine penicillin with 10 mg/kg dihydrostreptomycin equivalent to 1 ml per 25 kg BW. Through a fixed 14-gauge IV catheter in the jugular vein, donkeys were premedicated with acepromazine maleate (Castran, 15 mg/mL; Interchemie, Netherland) at a dose of 0.05 mg/kg followed by xylazine Hcl (Xyla-Ject, 2%; Adwia, Egypt) at a dose of 1.1 mg/kg with a 20-minute interval between administrations. General anesthesia was induced by IV injection of propofol (Diprivan 1%, Corden pharma, Italy) at a dose of 2 mg/kg BW. Subsequently, double tourniquets were placed above the tarsus to minimize hemorrhage and accomplish intravenous regional analgesia ( Samy et al., 2020 ). This involved a slow infusion of 40 ml of lidocaine HCL (Debocaine 2%; Sigmatec Pharmaceutical Industries Co, Egypt). The depth of anesthesia was continuously assessed, and maintenance was achieved by infusing 0.1% propofol during the operation. Under left lateral recumbency and aseptic conditions, a 7 cm linear skin incision was made over the plantar aspect of the right limb mid-metatarsal region ( Fig. 1 ). The subcutaneous and partenone were carefully incised. Following exposure of the SDFT, a full-thickness tenotomy was performed and a 1 cm gap tenorrhaphy was performed using a bunnell pattern with polypropylene size 1 (Egyprolene, TAISIER-MED, Egypt). In the control group, the tendon gap was left empty. In both PRF and PRF/Ch groups, PRF clots were utilized to fill the gap with particular attention to ensuring graft-tendon contact. In the PRF/Ch group, a 2*2 cm chitosan film was employed to wrap the tendon-PRF-construct. The biomaterials were secured within the SDFT gap by suturing the paratenon. Closure involved routine suturing of both paratenon and subcutaneous tissues using a simple continuous pattern with polyglycolic Acid No 0 (Egysorb, TAISIER-MED, Egypt). The skin was sutured separately and routinely. All surgical procedures were performed under a tourniquet within a limited timeframe, not exceeding 30 minutes. (Fig. ) Postoperative management Following the surgical procedures, surgical wounds were managed. Sterile nonadherent medicated pads were used to cover the surgical wounds. Subsequently, the operated limbs were immobilized for a duration of 6 weeks using a distal limb fiberglass cast (up to hock), maintaining the fetlock angle in a semiflextion position. The preoperative antibiotics (pen and strep) were continued for 5 successive days in combination with gentamicin (Gentacure 10% Pharma swede, Egypt) at a dose rate of 4 mg/kg. Flunixin meglumine as an anti-inflammatory agent was intravenously administered at a dose rate of 1.1 mg/kg (Flamicure 5%, Pharma Swede, Egypt) for 3 successive days. Additionally, on the day of surgery, 1500 IU of antitetanic serum/animal (antitetanic serum, Vacsera, Egypt) was injected. Daily wound dressing was applied through the window within the cast which changed every week till the 7th week, when an extended heel shoe was applied, and a controlled daily exercise regimen of 10 minutes hand walking was applied until the 9th week, followed by 20 minutes of hand walking and 3 minutes of trotting till the end of the experiment. Clinical observation Donkeys were observed daily after surgery to assess skin wound healing and the condition of the applied cast. On the 45th and 90th days, the limb circumference (LC) of the AOI was meticulously measured using a standard measuring tape and compared to the preoperative circumference. Additionally, donkeys were examined for the presence of lameness and discomfort using the modified Equine Utrecht University Scale for Donkeys Composite Pain Assessment ( van Dierendonck et al., 2020 ) (Tables S2 & S3) . Lameness scores were scaled as follow: 0, no lameness; 1, mild lameness; 2, moderate lameness; and 3, severe lameness. Total discomfort scores ranged from 0 for comfort to 1–3 for mild discomfort, 4–6 for moderate discomfort, and 7–9 for sever discomfort. All clinical assessments were blindly conducted by two independent observers (M.N and A.S) throughout the study. Ultrasonographic evaluation Ultrasonographic scanning was carried out at the following time points: prior to surgery (N), two days after surgery (T0), and at 2, 4, 6, 8, 10, and 12 weeks postoperatively. Scans were conducted in both the longitudinal and transverse planes using a 13-MHz linear transducer (CHISON Digital Color Doppler Ultrasound System, iVis 60 EXPERT VET; CHISON Medical Imaging Co., Ltd., China) with setting adjusted to 52 gain, 30 mm depth and 15 focal position. The evaluation included the assessment of the fiber echogenicity score (FES) and fiber alignment score (FAS, Table S4) ( Carlier et al., 2024 ). Additionally, the examination aimed to evaluate the SDFT thickness, shape, position, and presence of intratendinous edema ( Table S5) . For the assessment of peritendinous adhesion, a US video-assisted gliding test was performed at the 12th week PO. This involved frequent flexion and extension of the fetlock joint, with observation of the gliding resistance of the flexor tendons. The test was recorded and saved as a video in cin format. Ultrasound scanning procedures were performed by a single examiner (M.N), while the visual interpretation of the ultrasonographic data was blindly assessed and recorded by three independent observers. Tissue Harvest At the scheduled time points (45 and 90 days postoperatively), donkeys were euthanized using a pentobarbital overdose ( Gaesser et al., 2021 ). The hindlimbs were dissected, and 2 cm sections of the SDFT were harvested from the AOI for histopathological analysis. For comparative evaluation, similarly sized sections were collected from the corresponding region of the contralateral limb, which had not undergone tenotomy (normal). Histopathological evaluation The Movin’s scoring system ( Movin et al., 1997 ) was employed for the quantitative assessment includes: fiber structure, fiber arrangement, rounding of the nuclei, inflammation, increased vascularity and cell density. These variables were quantified using a 0–3 scale, where 0 indicated normal and 3 signified maximal abnormality. Therefore, a perfectly normal tendon received a score of 0, whereas a maximally abnormal tendon received a score of 3 (Table S6) . Three sections were randomly selected from each sample and were blindly evaluated by a pathologist. Statistical analysis Statistical analyses were performed using a statistical software program (SPSS for Windows version 20, USA). The normality of all variable distributions was assessed using the Shapiro-Wilk test. Measurable data, such as age, body weight (BW), and limb circumference, are presented as the mean ± SD and were analyzed using normal probability plots of one-way ANOVA. Nonparametric data are expressed as the median (minimum-maximum) and the homogeneity of groups was assessed using the Kruskal-Wallis nonparametric ANOVA. One-way ANOVA with post-hoc Tuckey and Duncan multiple comparison tests was performed to investigate the impact of various interventions over time. The results were considered significant when P ≤ 0.05. Results Platelet-Rich Fibrin and Chitosan Characterization In the comprehensive characterization of A-PRF, the platelet count analysis yielded notable results compared to the normal platelet count (275000 cells/µL ± 93.5). The A-PRF demonstrated a remarkable increase, documenting a nearly threefold rise (2.7 ± 0.18) from the baseline platelet count. Scanning electron microscope revealed details of the densely packed fibrin network with thin elongated fibers. Notably, a higher concentration of visible cells (platelets and white blood cells) was detected specifically near the red blood clot layer. FT-IR spectroscopy was used to investigate the distinctive functional groups of chitosan in the 500–4000 cm-1 range (Fig. 2). The sample's absorption peaks were observed at 3448, 2921, 2856, 1730, 1638, 1406, 1193, 1027, 892, 811, and 671 cm -1. The hydroxyl group (O-H) vibrations are represented by the broad band at 3448 cm-1 ( Zemlyakova et al., 2019 ). The lower intensity peaks that were observed at 2921 and 2856 cm-1 are attributed to the asymmetric vibrational modes of CH2 group and the usual C–H stretch vibrations, respectively ( Vlăsceanu et al., 2022 ). The carbonyl group (C = O) stretching vibration band appeared at 1730 cm-1, while the absorption band at 1638 cm-1 corresponds to the C = C stretch ( Krishnaveni and Ragunathan, 2015 ). The symmetrical deformation mode of CH3 group was indicated by the peak at 1406 cm -1 ( Vlăsceanu et al., 2022 ). The combined vibrations of the NHCO group may be the cause of the absorption band at 1193 cm -1, which may suggest the presence of amide III in chitosan ( Bahrami Miyanji et al., 2022 ). The C-O stretching of C6 in chitosan is responsible for the absorption peak at 1027 cm-1.The band at 892 cm–1 is attributed to ω(C–H) of the polysaccharide structure ( Negrea et al., 2015 ). The absorption band at 811 cm -1 is assigned to C–H bending out of the plane of the aromatic group ( Vlăsceanu et al., 2022 ). Finally, the band at 671 cm-1 is related to (CO) ring stretching and different ring deformation modes ( Wiercigroch et al., 2017 ). (Fig. 2) Welfare monitoring There were no statistically significant (P > 0.05) time effects found for the increases in the composite welfare score observed across the entire period of the experiment in all groups. No animals were excluded from the analysis. All enrolled donkeys completed the study protocol and were included in the final evaluation. For those donkeys that had slightly increased composite welfare score [5 (4–7)] with a nonsignificant difference (P > 0.05) between all groups, their scores had returned to the normal range by (0–3)72 hours postinduction (Data not shown). Clinical findings Generally, the addition of bioscaffolds to the SDFT gap significantly (P = 0.000-0.016) enhanced the clinical observation parameters compared to those of the control group. Statistical analysis revealed nonsignificant differences (P > 0.05) between the PRF and PRF/Ch groups across all clinical examination parameters (Table S7) . Concerning the limb circumference (LC), both the PRF and PRF/Ch groups nearly maintained a normal LC measurement (12.07 ± 0.11 and 12.03 ± 0.08 cm, respectively) on the 90th day, in contrast to the marked increase in LC observed in the control group (14.50 ± 0.45 cm). Mild to severe pain reactions, as evidenced by palpation of the area of interest and concurrent moderate to severe lameness [score (S) = 2.3 (2–3)] were observed in the control group donkeys. In contrast, animals in both treatment groups showed only mild symptoms at day 45, with complete resolution observed at the end of the study period (Fig. 3). Extreme bad standing postures (S = 2 and 2.5) were significantly (P = 0.005-0.000) observed in the control group donkeys on both the 45th and 90th days compared to the treated groups that exhibited mild weight shifting (S = 0.5) on the 45th day and eventually retained to a normal standing posture by the 90th day. Regarding resting postures, donkeys in the control group were significantly (P = 0.000-0.002) observed laying down on their side with stretched limbs or laying down for more than 50% of the examination time (24 hours) on both evaluation times, in comparison to the treated groups. The treated groups showed attempts to lie down for less than 50% of the examination time (S = 1) on the 45th day, followed by a return to normal resting posture on the 90th day. In terms of the total discomfort scale of the modified Equine Utrecht University Scale for Donkeys Composite Pain Assessment, statistical analysis indicated a significant (P = 0.000- 0.002) decrease in the discomfort scores in the treated groups (S = 0–3) compared to those in the control group (S = 6–8). (Fig. 3) Ultrasonographic findings The longitudinal (LS) and transverse (TS) sonograms of all animals before surgery (N) showed normal US pictures, with the SDFT thickness measuring 0.45 ± 0.05 cm. At T0, the LS showed a complete anechoic segmental defect [Fiber echogenicity score (FES = 3)] with total loss of its striations [Fiber alignment score (FAS = 3)]. Additionally, the control group exhibited excessive fluid content within the SDFT, both proximally and distally to the defect area, in comparison to both the PRF and PRF/Ch groups. Generally, the addition of chitosan and/or A-PRF to the SDFT defect resulted in a significant difference (P < 0.05) in all US assessment scores compared to those of the control group, except for the position at which the SDFT showed medial displacement across all groups and evaluation times (Fig. 4, Tables S8 &S9) . Statistical analysis revealed a nonsignificant (P > 0.05) difference between the results of PRF and PRF/Ch groups, except for the difference in thickness score. The median FES of both treated groups showed significant (P = 0.00) improvement over time. By T6, the FES of both treated groups had nearly returned to normal (echogenicity almost identical to that of the contralateral tendon), while the control group exhibited mild improvement in FES [S = 2 (1–2)]. Complete loss of fiber alignment (FAS = 3) was observed in both treated groups until T4, followed by the detection of linear echoic bands rather than retraction of tendon fibers (FAS = 2) at T6. A significant improvement (P < 0.05) in FAS was observed until T12, with a near restoration of the total fiber alignment. In the control group, the initiation of fiber alignment was noted at T12 (FAS = 2). A gradual increase in the SDFT thickness was observed in all groups till the T4. In contrast to the control group, which exhibited a marked increase in SDFT thickness until the end of the experiment, both treatment groups showed a significant decrease (P > 0.05). in particular the SDFT thickness in the PRF/Ch group nearly returned to the normal level at T12, in contrast to the mild increase in thickness (S = 1(1–2)) in the PRF group. Intratendinous edema was consistently observed throughout all evaluation times in the control group, whereas in the PRF and PRF/Ch groups, it was present only during the initial six postoperative weeks. The SDFT in the control group exhibited an irregular shape, while both treated groups demonstrated a nearly normal crescent-shaped tendon at T8. The results of the video-assisted US gliding test showed freely movable SDFT without movement restriction in both treated groups, in contrast to the control group where SDFT gliding resistance was observed, albeit with freely movable DDFT (Video.S1). (Fig. 4) Histopathological findings The addition of biological scaffolds to the tendon defects resulted in remarkable histopathological changes ( Figs. 5 – 7 ) , and statistical analysis revealed significant (P = 0.001–0.012) differences in all histopathological assessment scores among the three experimental groups. Approximately the PRF/Ch group restored the normal tendon histology on the 90th day. Moreover, statistical analysis showed no significant difference (P = 0.090-1.000) between the PRF and PRF/Ch groups in any of the histopathological assessment scores, except for the cell density score (P = 0.026) on the 45th day and the inflammatory cell infiltration and angiogenesis scores (P = 0.032) on the 90th day. Additionally, time exerted a positive proportional significant (P = 0.000-0.09) effect on improving these histopathological assessment scores in all groups, except in the control group, where the fiber arrangement and rounding of nuclei remained stable (P > 0.05) (Table S10). Concerning the fiber structure of the repaired tendon at the AOI, treatment with a combination of A-PRF and chitosan (PRF/Ch group) significantly (P = 0.001) restored the continuous long fiber structure characteristic of normal tendon tissue [0 (0–0)] on the 90th day. In the PRF group, the fiber structure was slightly fragmented to normal continuous long fibers [0.5 (0–1)], whereas in the control group, where moderate to severely fragmented tendons were observed [2 (2–3)]. In term of fiber arrangement, treatment with A-PRF either alone (PRF group) or in combination with chitosan (PRF/Ch group) resulted in highly significant (P = 0.003–0.006) restoration of the normal fiber arrangement; on the 45th day, moderate to slightly loose and wavy collagen fibers [PRF: 1.8(1–2); PRF/Ch: 1(1–2)] were observed, meanwhile on the 90th day, slightly loose and wavy to normal compacted and parallel collagen bundles [PRF:1(0–1); PRF/Ch:0(0–1)] were detected. In the control group, nonsignificant (P = 0.733) observations of moderatly loose, wavy and crossed fibers to nonidentified pattern were noted on both the 45th and 90th days [3(2–3) and 2(2–3), respectively]. Similar to the fiber arrangement, both the PRF and PRF/Ch groups a demonstrated a significant improvement (P < 0.05) in restoring the normal shape of tenocyte nuclei. On the 45th day moderate to slight rounded nuclei were observed [1(1–2)], transitioning to normal spindle-shaped nuclei on the 90th day [PRF: 0.25 (0–1); PRF/Ch: 0(0–1)]. In contrast, the control group showed a ≥ 30% increase (score = 1–3) in inflammatory cell infiltration and neovascularization, while the PRF and PRF/Ch groups showed only a 10–20% increase (score 0–1). Compared to the pattern of cell density in the normal tendon, on the 45th day, a slight to moderate increase in cell density was observed in the PRF/Ch group, versus a moderate to severe increase in both control and PRF groups. By the 90th day, the normal cell density pattern [0(0–1)] was significantly restored in the PRF and PRF/Ch groups (P = 0.001). Finally the overall histological score confirmed the superiority of PRF/Ch (0.14 ± 0.35; P = 0.000) over PRF (0.53 ± 0.50) and control (2.1 ± 0.52) groups in restoring the normal histological articheture of the SDFT at 90th day postoperative. (Figs. 5 – 7 ) Discussion Tendon has a limited healing capacity, resulting in the formation of a relatively low-quality fibrotic scar. This scar compromises the tendon mechanical properties, leading to difficulties in regaining the original structure, alignment, and strength ( Chen et al., 2018 ). Given the limitations of existing tendon reconstruction methods, the field of tissue engineering has become crucial for achieving effective tendon regeneration ( Smith, 2022 ). From different tendon tissue engineering modalities, in this study, we selected PRF and chitosan from various tissue engineering modalities, drawing encouragement from studies conducted on tendon repair ( Dietrich et al., 2015 ; Melamed et al., 2015 ; Liao et al., 2017 ; Chen et al., 2018 ; Wong et al., 2020 ; Ibrahim Al Dirawi, 2023 ; Celikten et al., 2024 ; Diaz et al., 2024 ). These studies demonstrated improved tendon regeneration with a substantial restoration of regular collagen structures and function. A-PRF, with its rich content of growth factors and cytokines in a 3D fibrin network, provides an optimal matrix for cell migration and sustained release of cytokines, which promotes early granulation tissue formation, enhances cellular proliferation and differentiation, supports gradual tendon fiber alignment, and contributes to the morphological restoration of the injured tissue ( Liao et al., 2017 ; Gadallah et al., 2022 ). Its autologous nature reduces the risk of immune rejection and complications, making it an ideal biological scaffold for tissue regeneration ( Vrana, 2018 ) . Additionally, A-PRF’s low cost, ease of preparation, and quick processing without specialized equipment made it a practical choice for this study. Chitosan is a biopolymer with beneficial properties like biodegradability, antibacterial activity, and muco-adhesivity, making it an ideal candidate for tissue engineering ( Gupta et al., 2024 ). Its structure, similar to glycosaminoglycans and hyaluronic acid, enhances cell migration, nutrient diffusion, and collagen production ( Deepthi et al., 2016 ). Chitosan also supports scarless healing of soft tissues and prevents adhesion formation during tendon healing ( Chen et al., 2015 ). Its biocompatibility minimizes the risk of immune rejection, making it a favorable choice for this study ( Jennings and Bumgardner, 2016 ; Brebels and Mignon, 2022 ). All the results of this study demonstrated the clear superiority of both treatment groups (PRF and PRF/Ch) over the control group. This superiority can be attributed to the well-established regenerative potential of PRF ( Wong et al., 2020 ) as well as the anti-adhesive properties of chitosan ( Fakhraei et al., 2022 ). Clinically, this was reflected in the normalization of clinical index scores in the PRF and PRF/Ch groups, with complete resolution of swelling, lameness, and discomfort responses by the end of the study indicating reduced inflammatory responses and early onset of tendon regeneration. These clinical improvements are consistent with the ultrasonographic and histological findings, providing structural confirmation of tissue repair and regeneration. Additionally, the results obviously establish the superiority of the PRF/Ch combination over the sole A-PRF in stimulating tendon gap healing. This was demonstrated by greater and faster reductions in SDFT thickness, inflammation, along with reduced peritendinous adhesions and less intratendinous edema. Rodríguez-Vázquez et al. ( 2015 ) ( Rodríguez-Vázquez et al., 2015 ) supported these findings by identifying chitosan as an ideal polymer for delivering bioactive compounds. They proposed that the synergy between chitosan derivatives and growth factors results in a sustained and prolonged release of these molecules, which may explain the enhanced healing observed in the PRF/Ch group. Tenorrhaphy is the preferred method for treating lacerated tendons in equines due to its ability to promote stronger, more mature tissue repair while preserving tendon function and improving prognosis ( Giacchi and McMaster, 2021 ). To better simulate the clinical challenges encountered in the field, such as tendon end retraction and irregular cuts, gap tenorrhaphy was chosen for this study, providing a more accurate reflection of real-world cases. In this study, during the 2nd week (inflammatory phase), ultrasonography revealed severe abnormal FES in all groups, likely due to intratendinous edema, hemorrhage, and inflammatory exudate, consistent with findings reported by several studies ( Hiramatsu et al., 2018 ; Montague et al., 2025 ). By the 4th week, echogenic tissue appeared within the AOI in both treatment groups, signifies the onset of the proliferative phase marked by fibroblast activity and extracellular matrix production ( Thomopoulos et al., 2015 ; Schulze-Tanzil et al., 2022 ). From the 6th week until the end of the study, ultrasonographic assessment indicated early healing of the tendon gap in both treatment groups, displaying normal FES and FAS. These findings were supported by histological analysis, which revealed a significantly restored tendon architecture in the PRF and PRF/Ch groups compared to the control, in line with previous studies ( Melamed et al., 2015 ; Wong et al., 2020 ). Histopathological and ultrasonographic evaluations consistently showed ongoing inflammation and intratendinous edema in the control group throughout the study. This was characterized by marked vascular permeability, neovascularization, infiltration of inflammatory cells, and fluid accumulation (intratendinous edema) within the tendon. In contrast, both the PRF and PRF/Ch groups exhibited signs of inflammation and edema primarily during the early inflammatory phase, which significantly subsided at later stages. This improvement can be attributed to the potent and prolonged anti-inflammatory effects of both chitosan ( Yang et al., 2010 )d PRF ( Zhang et al., 2020 ). These findings were in agreement with clinical measurements of limb circumference, where differences in tendon thickness were significantly influenced by factors such as intratendinous edema, inflammatory exudate, extracellular matrix production, scar tissue, and granulation tissue formation ( Nichols et al., 2019 ; Arvind and Huang, 2021 ; Chartier et al., 2021 ). The prevention of adhesion during tendon repair is critical for successful healing, and the significant differences in peritendinous adhesion observed between the control and treated groups highlight the positive impact of the applied treatments. Chen et al. ( 2015 ) ( Chen et al., 2015 ) reported that chitosan possesses unique properties enabling close contact with tendon surfaces, which promotes tissue regeneration and prevents adhesion to surrounding tissues. Additionally, the effectiveness of PRF in minimizing peritendinous adhesions has also been supported by recent experimental study on donkeys, in which the application of PRF significantly reduced adhesion formation and enhanced tendon gliding function during SDFT repair ( Abdelhakiem et al., 2023 ). This was clearly demonstrated during the ultrasonographic gliding test, where donkeys in the control group showed restricted SDFT movement due to thick adhesions with the overlying skin, while those in the PRF and PRF/Ch groups displayed normal tendon gliding without restriction. The integration of a video-assisted gliding test provided a valuable dynamic assessment of tendon functionality, adding practical insight into the therapeutic efficacy of the treatments. In this study, a correlation between clinical, ultrasound, and histological findings was observed, supporting the formation of scar tissue in the control group as opposed to the restoration of normal tendon architecture in the treated groups. The control group showed increased limb circumference clinically, and ultrasound revealed irregular tendon shape, disorganized fibers (FAS), and increased tendon thickness, which was further confirmed histologically by tendon fiber disarrangement. In contrast, the treated groups (PRF and PRF/Ch) demonstrated nearly normal limb circumference, restored crescent-shaped tendon morphology, aligned fibers on ultrasound (FAS), and normal tendon thickness, with histological analysis confirming well-organized fiber structure. These findings collectively reinforce the beneficial effects of the treatments in promoting tendon regeneration and preventing scar tissue formation, in contrast to the control group, where the tendon healing was characterized by scar tissue. Conclusion This study revealed the positive impact of A-PRF on tendon healing in gap tenorrhaphy, while the addition of chitosan further enhanced and accelerated the regeneration of the SDFTs. Spontaneous tendon healing is characterized by irregular scar tissue formation, extensive peritendinous adhesion, marked inflammation, severe lameness, and pain. A-PRF application in situ during gap tenorrhaphy stimulates tendon regeneration, nearly restoring normal ultrasonographic and histologic features and improving clinical index scores. The inclusion of chitosan in A-PRF clots potentiates its effect and greatly enhances and accelerates SDFT regeneration, as indicated by a greater and faster reduction in SDFT thickness, a reduction in peritendinous adhesion, and a decrease in intratendinous edema and inflammation, suggesting that A-PRF and chitosan combination is a promising approach for improving the quality and speed of tendon healing compared to spontaneous healing processes. Study limitations Despite promising results, the study had several limitations. The small sample size (n = 6 per group) may limit the generalizability of the findings. While donkeys served as a relevant model for tendon repair, they may not fully replicate human tendon healing, particularly in chronic or complex injuries. The short duration of the study restricted long-term healing assessments, and more advanced imaging or molecular analyses could have enhanced the findings. Abbreviations Platelet-rich fibrin (PRF) A-Platelet-rich fibrin (A-PRF) Chitosan (Ch) Superficial digital flexor tendon (SDFT) Superficial digital flexor tendons (SDFTs) Ultrasound (US) Longitudinal sonogram (LS) Transverse sonogram (TS) Fiber echogenicity score (FES) Fiber alignment score (FAS) Limb circumference (LC) Area of interest (AOI) Postoperative (PO) Declarations Acknowledgments The authors would like to thank Prof. Dr. Sabry El-Khodery for his help and technical support. Author contribution to the study MN: Study design, experimental operations, statistical analysis, and manuscript writing. AS: Statistical analysis, manuscript writing and editing. AR: Study design, surgical procedures, and manuscript editing. ME: Preparation, characterization, and discussion of chitosan film results. GK: Study design and manuscript writing editing, Supervision and final revisions of the manuscript. Funding No funding was obtained for this study. Availability of data and materials The raw data associated with this study are available upon request. Please contact the corresponding author [Gamal Karrouf] at [ [email protected] ]. The tables supporting the findings are available as Supplementary tables file. The video material is available as Supplementary video file (video-S1). Ethics approval and consent to participate The ethical conduct of the study was ensured through adherence to established guidelines and regulations. Approval for all experiments was obtained from the Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, Egypt. The study protocol received ethical clearance, and the approval was documented with registration number (Approval Code: Ph.D./101). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests Author details 1Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt. 2 Physics Department, Faculty of Science, Mansoura University, Mansoura, 35516, Egypt. 3 Physics Department, Faculty of Science, New Mansoura University, New Mansoura, Egypt. References Abdelhakiem, M.A., Hussein, A., Seleim, S.M., Abdelbaset, A.E., Abd-Elkareem, M. (2023). Silver nanoparticles and platelet-rich fibrin accelerate tendon healing in donkey. Sci. Rep. 13, 3421. https://doi.org/10.1038/s41598‑023‑30543‑w Alshomer, F., Chaves, C., Kalaskar, D. (2018). Advances in tendon and ligament tissue engineering: materials perspective. J. Mater. 2018, 17. https://doi.org/10.1155/2018/9868151 Arvind, V., Huang, A.H. (2021). Reparative and maladaptive inflammation in tendon healing. Front. Bioeng. 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A skin incision was performed over the mid-plantar aspect of the metatarsus (A), and the SDFT (white arrow) was approached after the paratenon incision (yellow arrow) (B). A cm gap tenorrhaphy in the SDFT (C). The SDFT gap was filled with A-PRF (yellow arrow) and a chitosan film (black arrow) was used to enclose it (D). Finally, the paratenon was sutured (white arrow) (E), and a fiberglass with a window facing the surgical site (F) was made.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/3f0c29fa5f47a1261bc2da07.png"},{"id":108491566,"identity":"e32e802d-409f-4b8e-aef1-63bc40318a71","added_by":"auto","created_at":"2026-05-05 09:54:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":423106,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum of the chitosan film.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/3fa593b90f671abce320cbf9.png"},{"id":108231871,"identity":"923673ed-a734-41c3-9a67-3ee5a10ae7f7","added_by":"auto","created_at":"2026-04-30 17:50:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":521354,"visible":true,"origin":"","legend":"\u003cp\u003eResults of clinical assessment scores between the different groups at two different evaluation times. Superscripts (a, b) indicate statistically significant differences over time within the same group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/70dbf3c4187e92512f0ed294.png"},{"id":108491275,"identity":"8d7a7128-e2ba-456c-8c6c-28e61b7a68ff","added_by":"auto","created_at":"2026-05-05 09:53:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":429946,"visible":true,"origin":"","legend":"\u003cp\u003eResults of ultronographic assessment scores between the different groups at two different evaluation times.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/945816f73c7eb9d17cd81372.png"},{"id":108231873,"identity":"e1ac151a-66d5-4c01-bd13-6a020c758ea1","added_by":"auto","created_at":"2026-04-30 17:50:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8798449,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic pictures of the AOI of the Control group (A, B, and C\u0026amp;D), PRF group (E \u0026amp;F) and PRF/Ch group (G\u0026amp;H) on the 45\u003csup\u003eth\u003c/sup\u003e day showing: A \u0026amp; B: irregularly arranged collagen bundles, numerous leukocyte cells infiltration (arrowheads) and neovascularization (red arrow). C\u0026amp;D: Increased cell density (*) consisting of inactive fibrocytes (black arrows) with few active fibroblasts (dashed black arrow) running in various directions. The collagen fibers do not form densely packed bundles. (E \u0026amp; F) Low regularly arranged immature collagen fibers forming sparsely packed bundles, few leukocytic cells infiltration (arrowheads) and neovascularization (red arrow). Number of active fibroblasts (dashed black arrows) increased with the presence of some inactive fibrocytes (black arrows). (G \u0026amp;H) More regularly arranged immature collagen fibers forming densely packed bundles, few leukocytic cells infiltration (arrowheads) and neovascularization (red arrow) are shown. Higher numbers of active fibroblasts (dashed black arrows) are seen with presence of some inactive fibrocytes (black arrows). H\u0026amp;E Low magnification X: 100 bar 100 µm and high magnification X: 400 bar 50 µm.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/33ba708aa0fd8adbb22c8cb0.png"},{"id":108491957,"identity":"cecf3a6c-0ee4-46ea-89ee-af05847c8454","added_by":"auto","created_at":"2026-05-05 09:56:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9273212,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic pictures of the AOI from the control group (A\u0026amp;B), PRF group (C \u0026amp;D) and PRF/Ch group (E\u0026amp;F) on the 90th day. A \u0026amp; B show irregularly arranged collagen bundles, numerous leukocytic cells infiltration (arrowheads) and neovascularization (red arrow). Most of the tendon cells are active fibroblasts (dashed black arrows) with few inactive fibrocytes (black arrow). The collagen fibers do not form densely packed bundles. C \u0026amp;D show regularly arranged mature collagen fibers forming densely packed bundles, few blood vessels (red arrow). Most of the tendon cells are inactive fibrocytes (black arrows) with few active fibroblasts (dashed black arrow). E\u0026amp;F show an organized tissue consisting of regularly arranged mature collagen fibers forming densely packed bundles, few blood vessels (red arrow), decreased cell density consisting mainly of fibrocytes (black arrows) characterizing a completely healed tissue. H\u0026amp;E Low magnification X: 100 bar 100 µm and high magnification X: 400 bar 50 µm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/7f64a0a1eae73a7767beb624.png"},{"id":108231875,"identity":"a8c31bfa-d149-4963-8757-b67d5a7f9629","added_by":"auto","created_at":"2026-04-30 17:50:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":969840,"visible":true,"origin":"","legend":"\u003cp\u003eResults of histopathological assessment scores between the different groups at two different evaluation times.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/d2b3d70115db80c2e428506c.png"},{"id":108494681,"identity":"23f66660-a3ab-46c6-8b87-8b76fe176c1b","added_by":"auto","created_at":"2026-05-05 10:06:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28156198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/ac579ff9-f080-4602-bfdc-ad4f44d3d3b0.pdf"},{"id":108231876,"identity":"bf641967-1627-49de-9b36-f6e25533df98","added_by":"auto","created_at":"2026-04-30 17:50:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":51816,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytablesofmanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-9390812/v1/85959d4cd3e0c20960ce57ca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan-augmented advanced platelet rich fibrin improves the regeneration of superficial digital flexor tendon gap tenorrhaphy in donkeys (Equus Asinus)","fulltext":[{"header":"Background","content":"\u003cp\u003eTendon lacerations in equines present significant challenges, due to the need for restoring the tendon\u0026rsquo;s structural and functional integrity \u003cb\u003e(\u003c/b\u003eChen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Effective treatment aims to achieve strong repair at the anastomosis site, re-establish normal vascularization, restore tendon microanatomy and functional length, and minimize scar tissue or restrictive adhesions that could impair tendon mechanical properties and function \u003cb\u003e(\u003c/b\u003eJani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Research to enhance and expedite the regeneration of injured tendons has been spurred by this predicament. A method for enhancing the repair process has been developed through tissue engineering \u003cb\u003e(\u003c/b\u003eRibitsch et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which employs three main strategies: gene/cell-based, scaffold-based, and growth factor-based approaches \u003cb\u003e(\u003c/b\u003eRuiz-Alonso et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTissue engineering for tendon laceration repair leverages both biological and synthetic scaffolds, each offering unique benefits. Biological scaffolds like hydrogels and decellularized tissues provide a natural environment for healing, while synthetic options offer structural support and integration. The ideal scaffold combines mechanical strength, biocompatibility, and biodegradability to promote effective tendon regeneration without adverse immune responses \u003cb\u003e(\u003c/b\u003eAlshomer et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vasiliadis and Katakalos, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ruiz-Alonso et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdvanced Platelet-rich fibrin (A-PRF) is a platelet concentrate suspended in a small amount of plasma that is rich in growth factors that promote angiogenesis, chemotaxis, cell proliferation, cell differentiation, and collagen production. Furthermore, the fibrin matrix supporting these materials acts as a scaffold and is an essential component of the therapeutic value of PRF. The three-dimensional fibrin network with intrinsic platelet integration offers the best scaffold for cell migration and gradual prolonged release of cytokines \u003cb\u003e(\u003c/b\u003eBarbon et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Celikten et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chitosan is a distinctive biopolymer that displays exceptional properties beyond being biocompatible and biodegradable. Consequently, this polysaccharide is a viable alternative in the biomaterials domain, particularly in applications related to tissue engineering. Chitosan contributes to the repair of soft tissues without adhesion or scar formation during tendon recovery \u003cb\u003e(\u003c/b\u003eKim et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNumerous imaging methods, such as scintigraphy, thermography, magnetic resonance imaging, and ultrasonography are employed to assess tendinopathy. However, ultrasonography is a widely used imaging modality for diagnosing and monitoring tendinopathy due to its accessibility, cost-effectiveness, and ability to provide real-time images. It is particularly favored for its ability to assess tendon thickness, detect structural changes, and monitor healing progress \u003cb\u003e(\u003c/b\u003eFahmy, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hinckley et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of donkeys in this study provides a relevant model for tendon injury and repair, offering high translatability to other equids, such as horses, due to functional and anatomical similarities. The hind limb's superficial digital flexor tendon (SDFT) was selected for its vulnerability to injury in both donkeys and horses. The anatomical and biomechanical properties of the donkey's SDFT make it an ideal model for studying tendon healing and treatments that can benefit both human and animal tendon injuries.\u003c/p\u003e \u003cp\u003eAlthough both platelet-rich fibrin (PRF) and chitosan have been explored in various experimental investigations \u003cb\u003e(\u003c/b\u003eDietrich et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Melamed et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Liao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; \u003cb\u003eIbrahim Al\u003c/b\u003e Dirawi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Celikten et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Diaz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), there is a notable absence of studies documenting their exclusive application as filling biomaterials for gap tenorrhaphy in equines. Consequently, this study aimed to assess the effectiveness of autologous A-PRF alone or in conjunction with chitosan in repairing induced gap tenorrhaphy in a donkey model. The evaluation will involve clinical observations, ultrasonographic examinations, and histopathological analyses to provide comprehensive insights.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eIn this study, a total of eighteen donkeys were enrolled, and their SDFTs in the right hind limbs were severed at the midpoint of the metatarsal region. Subsequently, gap-tenorrhaphy was performed, creating a full-thickness gap measuring 1 cm in length. Based on the bioscaffold added to the SDFT gap, donkeys were randomly allocated into three experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group) using a computer-generated randomization list created: the control group, where no biomaterials were added to the SDFT gap; the PRF group, where the SDFT gap was filled with autogenous A-PRF; and the PRF/Ch group, where the SDFT gap was filled with autogenous A-PRF and subsequently wrapped with a chitosan film.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eThe present study involved 18 clinically healthy male donkeys (Equus asinus) with an average age [mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD)] of 34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 months and a mean body weight (BW) of 136.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 kg. The donkeys were housed under consistent environmental conditions and fed a standardized diet consisting of a mixed ration of chopped wheat straw, bran, and whole corn provided twice daily, with ad libitum access to clean water. The general physical parameters, including heart rate, respiration rate, and rectal temperature, were monitored to ensure the overall health of the donkeys. Additionally, detailed clinical and ultrasonographic examinations were performed to assess their suitability for participation in the experiment. The clinical examination involved evaluating lameness, palpation for tenderness, and assessment for signs of swelling. Ultrasonographic evaluation focused on imaging the SDFT to assess its cross-sectional area, fiber alignment, and presence of any lesions or abnormalities. Only healthy donkeys with no pre-existing conditions affecting animal and tendon soundness were included in the study. The animals were housed and managed in straw bedded isolation stables at the Mansoura Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Mansoura University, Egypt. The study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code MU-ACUC (VM.PhD.25.04.45) All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical use of animals in research.\u003c/p\u003e\n\u003ch3\u003eMonitoring of Animal Welfare\u003c/h3\u003e\n\u003cp\u003eTo fulfill institutional and national ethical regulatory requirements, the animals were closely monitored twice daily, both before and after the surgical induction, till the conclusion of the study. A composite welfare scale \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (\u003c/b\u003eKearney et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) was utilized for this purpose. The scale consisted of scores (ranging from 0 to 4) assigned to each of the following categories: food and water intake, clinical parameters (temperature, heart rate, and respiratory rate), natural behavior, and provoked behavior.\u003cb\u003eImplant Preparation\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of A-PRF\u003c/h3\u003e\n\u003cp\u003eEight milliliters of whole blood were collected in plain tubes and centrifuged at 1500 rpm for 14 minutes (RCF\u0026thinsp;=\u0026thinsp;101\u0026times;g), resulting in three layers: platelet-poor plasma, a PRF clot, and red blood cells \u003cb\u003e(\u003c/b\u003eGhanaati et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The PRF clot was implanted into the tendon defect with care to ensure proper graft-tendon contact. Scanning electron microscopy (SEM) was utilized to investigate the distinctive fibrin network within the A-PRF, and platelets and white blood cells were counted for characterization. For SEM imaging (JSM 6510 LV, JEOL, Japan), fresh A-PRF samples were fixed in 2.5% glutaraldehyde at 4\u0026deg;C for 24 hours, dehydrated through graded ethanol, dried using critical point drying, and coated with a 20 nm gold layer for conductivity.\u003c/p\u003e\n\u003ch3\u003ePreparation of chitosan\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA magnetic stirrer set to 500 rpm for two hours was used to dissolve one gram of chitosan powder (\u0026gt;\u0026thinsp;85% deacetylation degree, purchased from Meron, India) in fifty milliliters of deionized water with one-and-a-half milliliters of acetic acid at room temperature. To get rid of air bubbles, the chitosan solution was wrapped in cling film and left overnight. The chitosan film was formed by casting the clear solution into a Petri dish and letting it to dry for three days at room temperature. A 3 \u0026times; 3 cm\u003csup\u003e2\u003c/sup\u003e chitosan film was used to enclose the gap filled with A-PRF in the PRF/Ch group. The characteristic chitosan functional groups were examined using Fourier-transform infrared (FT-IR) spectroscopy in order to characterize the material.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSurgical Procedures\u003c/h2\u003e \u003cp\u003ePreanesthetic preparation guidelines were considered for the animals. Additionally, a broad-spectrum preoperative antibiotic composed of penicillin and streptomycin (Pen and Srept, Norbrook, UK) was injected intramuscularly 2 hours before surgery at a dose rate of 8 mg/kg procaine penicillin with 10 mg/kg dihydrostreptomycin equivalent to 1 ml per 25 kg BW. Through a fixed 14-gauge IV catheter in the jugular vein, donkeys were premedicated with acepromazine maleate (Castran, 15 mg/mL; Interchemie, Netherland) at a dose of 0.05 mg/kg followed by xylazine Hcl (Xyla-Ject, 2%; Adwia, Egypt) at a dose of 1.1 mg/kg with a 20-minute interval between administrations. General anesthesia was induced by IV injection of propofol (Diprivan 1%, Corden pharma, Italy) at a dose of 2 mg/kg BW. Subsequently, double tourniquets were placed above the tarsus to minimize hemorrhage and accomplish intravenous regional analgesia \u003cb\u003e(\u003c/b\u003eSamy et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This involved a slow infusion of 40 ml of lidocaine HCL (Debocaine 2%; Sigmatec Pharmaceutical Industries Co, Egypt). The depth of anesthesia was continuously assessed, and maintenance was achieved by infusing 0.1% propofol during the operation.\u003c/p\u003e \u003cp\u003eUnder left lateral recumbency and aseptic conditions, a 7 cm linear skin incision was made over the plantar aspect of the right limb mid-metatarsal region \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The subcutaneous and partenone were carefully incised. Following exposure of the SDFT, a full-thickness tenotomy was performed and a 1 cm gap tenorrhaphy was performed using a bunnell pattern with polypropylene size 1 (Egyprolene, TAISIER-MED, Egypt). In the control group, the tendon gap was left empty. In both PRF and PRF/Ch groups, PRF clots were utilized to fill the gap with particular attention to ensuring graft-tendon contact. In the PRF/Ch group, a 2*2 cm chitosan film was employed to wrap the tendon-PRF-construct. The biomaterials were secured within the SDFT gap by suturing the paratenon. Closure involved routine suturing of both paratenon and subcutaneous tissues using a simple continuous pattern with polyglycolic Acid No 0 (Egysorb, TAISIER-MED, Egypt). The skin was sutured separately and routinely. All surgical procedures were performed under a tourniquet within a limited timeframe, not exceeding 30 minutes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e(Fig. )\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePostoperative management\u003c/h2\u003e \u003cp\u003eFollowing the surgical procedures, surgical wounds were managed. Sterile nonadherent medicated pads were used to cover the surgical wounds. Subsequently, the operated limbs were immobilized for a duration of 6 weeks using a distal limb fiberglass cast (up to hock), maintaining the fetlock angle in a semiflextion position.\u003c/p\u003e \u003cp\u003eThe preoperative antibiotics (pen and strep) were continued for 5 successive days in combination with gentamicin (Gentacure 10% Pharma swede, Egypt) at a dose rate of 4 mg/kg. Flunixin meglumine as an anti-inflammatory agent was intravenously administered at a dose rate of 1.1 mg/kg (Flamicure 5%, Pharma Swede, Egypt) for 3 successive days. Additionally, on the day of surgery, 1500 IU of antitetanic serum/animal (antitetanic serum, Vacsera, Egypt) was injected.\u003c/p\u003e \u003cp\u003eDaily wound dressing was applied through the window within the cast which changed every week till the 7th week, when an extended heel shoe was applied, and a controlled daily exercise regimen of 10 minutes hand walking was applied until the 9th week, followed by 20 minutes of hand walking and 3 minutes of trotting till the end of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical observation\u003c/h2\u003e \u003cp\u003eDonkeys were observed daily after surgery to assess skin wound healing and the condition of the applied cast. On the 45th and 90th days, the limb circumference (LC) of the AOI was meticulously measured using a standard measuring tape and compared to the preoperative circumference. Additionally, donkeys were examined for the presence of lameness and discomfort using the modified Equine Utrecht University Scale for Donkeys Composite Pain Assessment \u003cb\u003e(\u003c/b\u003evan Dierendonck et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) \u003cb\u003e(Tables S2 \u0026amp; S3)\u003c/b\u003e. Lameness scores were scaled as follow: 0, no lameness; 1, mild lameness; 2, moderate lameness; and 3, severe lameness. Total discomfort scores ranged from 0 for comfort to 1\u0026ndash;3 for mild discomfort, 4\u0026ndash;6 for moderate discomfort, and 7\u0026ndash;9 for sever discomfort. All clinical assessments were blindly conducted by two independent observers (M.N and A.S) throughout the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eUltrasonographic evaluation\u003c/h2\u003e \u003cp\u003eUltrasonographic scanning was carried out at the following time points: prior to surgery (N), two days after surgery (T0), and at 2, 4, 6, 8, 10, and 12 weeks postoperatively. Scans were conducted in both the longitudinal and transverse planes using a 13-MHz linear transducer (CHISON Digital Color Doppler Ultrasound System, iVis 60 EXPERT VET; CHISON Medical Imaging Co., Ltd., China) with setting adjusted to 52 gain, 30 mm depth and 15 focal position. The evaluation included the assessment of the fiber echogenicity score (FES) and fiber alignment score (FAS, \u003cb\u003eTable S4) (\u003c/b\u003eCarlier et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, the examination aimed to evaluate the SDFT thickness, shape, position, and presence of intratendinous edema (\u003cb\u003eTable S5)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFor the assessment of peritendinous adhesion, a US video-assisted gliding test was performed at the 12th week PO. This involved frequent flexion and extension of the fetlock joint, with observation of the gliding resistance of the flexor tendons. The test was recorded and saved as a video in cin format. Ultrasound scanning procedures were performed by a single examiner (M.N), while the visual interpretation of the ultrasonographic data was blindly assessed and recorded by three independent observers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissue Harvest\u003c/h2\u003e \u003cp\u003eAt the scheduled time points (45 and 90 days postoperatively), donkeys were euthanized using a pentobarbital overdose \u003cb\u003e(\u003c/b\u003eGaesser et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The hindlimbs were dissected, and 2 cm sections of the SDFT were harvested from the AOI for histopathological analysis. For comparative evaluation, similarly sized sections were collected from the corresponding region of the contralateral limb, which had not undergone tenotomy (normal).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological evaluation\u003c/h2\u003e \u003cp\u003eThe Movin\u0026rsquo;s scoring system \u003cb\u003e(\u003c/b\u003eMovin et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) was employed for the quantitative assessment includes: fiber structure, fiber arrangement, rounding of the nuclei, inflammation, increased vascularity and cell density. These variables were quantified using a 0\u0026ndash;3 scale, where 0 indicated normal and 3 signified maximal abnormality. Therefore, a perfectly normal tendon received a score of 0, whereas a maximally abnormal tendon received a score of 3 \u003cb\u003e(Table S6)\u003c/b\u003e. Three sections were randomly selected from each sample and were blindly evaluated by a pathologist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using a statistical software program (SPSS for Windows version 20, USA). The normality of all variable distributions was assessed using the Shapiro-Wilk test. Measurable data, such as age, body weight (BW), and limb circumference, are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were analyzed using normal probability plots of one-way ANOVA. Nonparametric data are expressed as the median (minimum-maximum) and the homogeneity of groups was assessed using the Kruskal-Wallis nonparametric ANOVA. One-way ANOVA with post-hoc Tuckey and Duncan multiple comparison tests was performed to investigate the impact of various interventions over time. The results were considered significant when P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePlatelet-Rich Fibrin and Chitosan Characterization\u003c/h2\u003e \u003cp\u003eIn the comprehensive characterization of A-PRF, the platelet count analysis yielded notable results compared to the normal platelet count (275000 cells/\u0026micro;L\u0026thinsp;\u0026plusmn;\u0026thinsp;93.5). The A-PRF demonstrated a remarkable increase, documenting a nearly threefold rise (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18) from the baseline platelet count. Scanning electron microscope revealed details of the densely packed fibrin network with thin elongated fibers. Notably, a higher concentration of visible cells (platelets and white blood cells) was detected specifically near the red blood clot layer.\u003c/p\u003e \u003cp\u003eFT-IR spectroscopy was used to investigate the distinctive functional groups of chitosan in the 500\u0026ndash;4000 cm-1 range \u003cb\u003e(Fig.\u0026nbsp;2).\u003c/b\u003e The sample's absorption peaks were observed at 3448, 2921, 2856, 1730, 1638, 1406, 1193, 1027, 892, 811, and 671 cm -1. The hydroxyl group (O-H) vibrations are represented by the broad band at 3448 cm-1 \u003cb\u003e(\u003c/b\u003eZemlyakova et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The lower intensity peaks that were observed at 2921 and 2856 cm-1 are attributed to the asymmetric vibrational modes of CH2 group and the usual C\u0026ndash;H stretch vibrations, respectively \u003cb\u003e(\u003c/b\u003eVlăsceanu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The carbonyl group (C\u0026thinsp;=\u0026thinsp;O) stretching vibration band appeared at 1730 cm-1, while the absorption band at 1638 cm-1 corresponds to the C\u0026thinsp;=\u0026thinsp;C stretch \u003cb\u003e(\u003c/b\u003eKrishnaveni and Ragunathan, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The symmetrical deformation mode of CH3 group was indicated by the peak at 1406 cm -1\u003cb\u003e(\u003c/b\u003eVlăsceanu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The combined vibrations of the NHCO group may be the cause of the absorption band at 1193 cm -1, which may suggest the presence of amide III in chitosan \u003cb\u003e(\u003c/b\u003eBahrami Miyanji et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The C-O stretching of C6 in chitosan is responsible for the absorption peak at 1027 cm-1.The band at 892 cm\u0026ndash;1 is attributed to ω(C\u0026ndash;H) of the polysaccharide structure \u003cb\u003e(\u003c/b\u003eNegrea et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The absorption band at 811 cm -1 is assigned to C\u0026ndash;H bending out of the plane of the aromatic group \u003cb\u003e(\u003c/b\u003eVlăsceanu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Finally, the band at 671 cm-1 is related to (CO) ring stretching and different ring deformation modes \u003cb\u003e(\u003c/b\u003eWiercigroch et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e(Fig.\u0026nbsp;2)\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eWelfare monitoring\u003c/h2\u003e \u003cp\u003eThere were no statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) time effects found for the increases in the composite welfare score observed across the entire period of the experiment in all groups. No animals were excluded from the analysis. All enrolled donkeys completed the study protocol and were included in the final evaluation. For those donkeys that had slightly increased composite welfare score [5 (4\u0026ndash;7)] with a nonsignificant difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between all groups, their scores had returned to the normal range by (0\u0026ndash;3)72 hours postinduction (Data not shown).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eClinical findings\u003c/h2\u003e \u003cp\u003eGenerally, the addition of bioscaffolds to the SDFT gap significantly (P\u0026thinsp;=\u0026thinsp;0.000-0.016) enhanced the clinical observation parameters compared to those of the control group. Statistical analysis revealed nonsignificant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the PRF and PRF/Ch groups across all clinical examination parameters \u003cb\u003e(Table S7)\u003c/b\u003e. Concerning the limb circumference (LC), both the PRF and PRF/Ch groups nearly maintained a normal LC measurement (12.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 and 12.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 cm, respectively) on the 90th day, in contrast to the marked increase in LC observed in the control group (14.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 cm). Mild to severe pain reactions, as evidenced by palpation of the area of interest and concurrent moderate to severe lameness [score (S)\u0026thinsp;=\u0026thinsp;2.3 (2\u0026ndash;3)] were observed in the control group donkeys. In contrast, animals in both treatment groups showed only mild symptoms at day 45, with complete resolution observed at the end of the study period \u003cb\u003e(Fig.\u0026nbsp;3).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExtreme bad standing postures (S\u0026thinsp;=\u0026thinsp;2 and 2.5) were significantly (P\u0026thinsp;=\u0026thinsp;0.005-0.000) observed in the control group donkeys on both the 45th and 90th days compared to the treated groups that exhibited mild weight shifting (S\u0026thinsp;=\u0026thinsp;0.5) on the 45th day and eventually retained to a normal standing posture by the 90th day. Regarding resting postures, donkeys in the control group were significantly (P\u0026thinsp;=\u0026thinsp;0.000-0.002) observed laying down on their side with stretched limbs or laying down for more than 50% of the examination time (24 hours) on both evaluation times, in comparison to the treated groups. The treated groups showed attempts to lie down for less than 50% of the examination time (S\u0026thinsp;=\u0026thinsp;1) on the 45th day, followed by a return to normal resting posture on the 90th day. In terms of the total discomfort scale of the modified Equine Utrecht University Scale for Donkeys Composite Pain Assessment, statistical analysis indicated a significant (P\u0026thinsp;=\u0026thinsp;0.000- 0.002) decrease in the discomfort scores in the treated groups (S\u0026thinsp;=\u0026thinsp;0\u0026ndash;3) compared to those in the control group (S\u0026thinsp;=\u0026thinsp;6\u0026ndash;8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e(Fig.\u0026nbsp;3)\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003eUltrasonographic findings\u003c/h2\u003e \u003cp\u003eThe longitudinal (LS) and transverse (TS) sonograms of all animals before surgery (N) showed normal US pictures, with the SDFT thickness measuring 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cm. At T0, the LS showed a complete anechoic segmental defect [Fiber echogenicity score (FES\u0026thinsp;=\u0026thinsp;3)] with total loss of its striations [Fiber alignment score (FAS\u0026thinsp;=\u0026thinsp;3)]. Additionally, the control group exhibited excessive fluid content within the SDFT, both proximally and distally to the defect area, in comparison to both the PRF and PRF/Ch groups.\u003c/p\u003e \u003cp\u003eGenerally, the addition of chitosan and/or A-PRF to the SDFT defect resulted in a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all US assessment scores compared to those of the control group, except for the position at which the SDFT showed medial displacement across all groups and evaluation times \u003cb\u003e(Fig.\u0026nbsp;4, Tables S8 \u0026amp;S9)\u003c/b\u003e. Statistical analysis revealed a nonsignificant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) difference between the results of PRF and PRF/Ch groups, except for the difference in thickness score. The median FES of both treated groups showed significant (P\u0026thinsp;=\u0026thinsp;0.00) improvement over time. By T6, the FES of both treated groups had nearly returned to normal (echogenicity almost identical to that of the contralateral tendon), while the control group exhibited mild improvement in FES [S\u0026thinsp;=\u0026thinsp;2 (1\u0026ndash;2)]. Complete loss of fiber alignment (FAS\u0026thinsp;=\u0026thinsp;3) was observed in both treated groups until T4, followed by the detection of linear echoic bands rather than retraction of tendon fibers (FAS\u0026thinsp;=\u0026thinsp;2) at T6. A significant improvement (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in FAS was observed until T12, with a near restoration of the total fiber alignment. In the control group, the initiation of fiber alignment was noted at T12 (FAS\u0026thinsp;=\u0026thinsp;2).\u003c/p\u003e \u003cp\u003eA gradual increase in the SDFT thickness was observed in all groups till the T4. In contrast to the control group, which exhibited a marked increase in SDFT thickness until the end of the experiment, both treatment groups showed a significant decrease (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). in particular the SDFT thickness in the PRF/Ch group nearly returned to the normal level at T12, in contrast to the mild increase in thickness (S\u0026thinsp;=\u0026thinsp;1(1\u0026ndash;2)) in the PRF group.\u003c/p\u003e \u003cp\u003eIntratendinous edema was consistently observed throughout all evaluation times in the control group, whereas in the PRF and PRF/Ch groups, it was present only during the initial six postoperative weeks. The SDFT in the control group exhibited an irregular shape, while both treated groups demonstrated a nearly normal crescent-shaped tendon at T8. The results of the video-assisted US gliding test showed freely movable SDFT without movement restriction in both treated groups, in contrast to the control group where SDFT gliding resistance was observed, albeit with freely movable DDFT \u003cb\u003e(Video.S1).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e(Fig.\u0026nbsp;4)\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003ch2\u003eHistopathological findings\u003c/h2\u003e \u003cp\u003eThe addition of biological scaffolds to the tendon defects resulted in remarkable histopathological changes \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, and statistical analysis revealed significant (P\u0026thinsp;=\u0026thinsp;0.001\u0026ndash;0.012) differences in all histopathological assessment scores among the three experimental groups. Approximately the PRF/Ch group restored the normal tendon histology on the 90th day. Moreover, statistical analysis showed no significant difference (P\u0026thinsp;=\u0026thinsp;0.090-1.000) between the PRF and PRF/Ch groups in any of the histopathological assessment scores, except for the cell density score (P\u0026thinsp;=\u0026thinsp;0.026) on the 45th day and the inflammatory cell infiltration and angiogenesis scores (P\u0026thinsp;=\u0026thinsp;0.032) on the 90th day. Additionally, time exerted a positive proportional significant (P\u0026thinsp;=\u0026thinsp;0.000-0.09) effect on improving these histopathological assessment scores in all groups, except in the control group, where the fiber arrangement and rounding of nuclei remained stable (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(Table S10).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConcerning the fiber structure of the repaired tendon at the AOI, treatment with a combination of A-PRF and chitosan (PRF/Ch group) significantly (P\u0026thinsp;=\u0026thinsp;0.001) restored the continuous long fiber structure characteristic of normal tendon tissue [0 (0\u0026ndash;0)] on the 90th day. In the PRF group, the fiber structure was slightly fragmented to normal continuous long fibers [0.5 (0\u0026ndash;1)], whereas in the control group, where moderate to severely fragmented tendons were observed [2 (2\u0026ndash;3)]. In term of fiber arrangement, treatment with A-PRF either alone (PRF group) or in combination with chitosan (PRF/Ch group) resulted in highly significant (P\u0026thinsp;=\u0026thinsp;0.003\u0026ndash;0.006) restoration of the normal fiber arrangement; on the 45th day, moderate to slightly loose and wavy collagen fibers [PRF: 1.8(1\u0026ndash;2); PRF/Ch: 1(1\u0026ndash;2)] were observed, meanwhile on the 90th day, slightly loose and wavy to normal compacted and parallel collagen bundles [PRF:1(0\u0026ndash;1); PRF/Ch:0(0\u0026ndash;1)] were detected. In the control group, nonsignificant (P\u0026thinsp;=\u0026thinsp;0.733) observations of moderatly loose, wavy and crossed fibers to nonidentified pattern were noted on both the 45th and 90th days [3(2\u0026ndash;3) and 2(2\u0026ndash;3), respectively].\u003c/p\u003e \u003cp\u003eSimilar to the fiber arrangement, both the PRF and PRF/Ch groups a demonstrated a significant improvement (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in restoring the normal shape of tenocyte nuclei. On the 45th day moderate to slight rounded nuclei were observed [1(1\u0026ndash;2)], transitioning to normal spindle-shaped nuclei on the 90th day [PRF: 0.25 (0\u0026ndash;1); PRF/Ch: 0(0\u0026ndash;1)]. In contrast, the control group showed a\u0026thinsp;\u0026ge;\u0026thinsp;30% increase (score\u0026thinsp;=\u0026thinsp;1\u0026ndash;3) in inflammatory cell infiltration and neovascularization, while the PRF and PRF/Ch groups showed only a 10\u0026ndash;20% increase (score 0\u0026ndash;1). Compared to the pattern of cell density in the normal tendon, on the 45th day, a slight to moderate increase in cell density was observed in the PRF/Ch group, versus a moderate to severe increase in both control and PRF groups. By the 90th day, the normal cell density pattern [0(0\u0026ndash;1)] was significantly restored in the PRF and PRF/Ch groups (P\u0026thinsp;=\u0026thinsp;0.001). Finally the overall histological score confirmed the superiority of PRF/Ch (0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35; P\u0026thinsp;=\u0026thinsp;0.000) over PRF (0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50) and control (2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52) groups in restoring the normal histological articheture of the SDFT at 90th day postoperative.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e(Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/h2\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTendon has a limited healing capacity, resulting in the formation of a relatively low-quality fibrotic scar. This scar compromises the tendon mechanical properties, leading to difficulties in regaining the original structure, alignment, and strength \u003cb\u003e(\u003c/b\u003eChen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Given the limitations of existing tendon reconstruction methods, the field of tissue engineering has become crucial for achieving effective tendon regeneration \u003cb\u003e(\u003c/b\u003eSmith, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e From different tendon tissue engineering modalities, in this study, we selected PRF and chitosan from various tissue engineering modalities, drawing encouragement from studies conducted on tendon repair \u003cb\u003e(\u003c/b\u003eDietrich et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Melamed et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Liao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; \u003cb\u003eIbrahim Al\u003c/b\u003e Dirawi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Celikten et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Diaz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These studies demonstrated improved tendon regeneration with a substantial restoration of regular collagen structures and function. A-PRF, with its rich content of growth factors and cytokines in a 3D fibrin network, provides an optimal matrix for cell migration and sustained release of cytokines, which promotes early granulation tissue formation, enhances cellular proliferation and differentiation, supports gradual tendon fiber alignment, and contributes to the morphological restoration of the injured tissue \u003cb\u003e(\u003c/b\u003eLiao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gadallah et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Its autologous nature reduces the risk of immune rejection and complications, making it an ideal biological scaffold for tissue regeneration \u003cb\u003e(\u003c/b\u003eVrana, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Additionally, A-PRF\u0026rsquo;s low cost, ease of preparation, and quick processing without specialized equipment made it a practical choice for this study. Chitosan is a biopolymer with beneficial properties like biodegradability, antibacterial activity, and muco-adhesivity, making it an ideal candidate for tissue engineering \u003cb\u003e(\u003c/b\u003eGupta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its structure, similar to glycosaminoglycans and hyaluronic acid, enhances cell migration, nutrient diffusion, and collagen production \u003cb\u003e(\u003c/b\u003eDeepthi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Chitosan also supports scarless healing of soft tissues and prevents adhesion formation during tendon healing \u003cb\u003e(\u003c/b\u003eChen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Its biocompatibility minimizes the risk of immune rejection, making it a favorable choice for this study \u003cb\u003e(\u003c/b\u003eJennings and Bumgardner, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Brebels and Mignon, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll the results of this study demonstrated the clear superiority of both treatment groups (PRF and PRF/Ch) over the control group. This superiority can be attributed to the well-established regenerative potential of PRF \u003cb\u003e(\u003c/b\u003eWong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) as well as the anti-adhesive properties of chitosan \u003cb\u003e(\u003c/b\u003eFakhraei et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Clinically, this was reflected in the normalization of clinical index scores in the PRF and PRF/Ch groups, with complete resolution of swelling, lameness, and discomfort responses by the end of the study indicating reduced inflammatory responses and early onset of tendon regeneration. These clinical improvements are consistent with the ultrasonographic and histological findings, providing structural confirmation of tissue repair and regeneration. Additionally, the results obviously establish the superiority of the PRF/Ch combination over the sole A-PRF in stimulating tendon gap healing. This was demonstrated by greater and faster reductions in SDFT thickness, inflammation, along with reduced peritendinous adhesions and less intratendinous edema. Rodr\u0026iacute;guez-V\u0026aacute;zquez et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eRodr\u0026iacute;guez-V\u0026aacute;zquez et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) supported these findings by identifying chitosan as an ideal polymer for delivering bioactive compounds. They proposed that the synergy between chitosan derivatives and growth factors results in a sustained and prolonged release of these molecules, which may explain the enhanced healing observed in the PRF/Ch group.\u003c/p\u003e \u003cp\u003eTenorrhaphy is the preferred method for treating lacerated tendons in equines due to its ability to promote stronger, more mature tissue repair while preserving tendon function and improving prognosis \u003cb\u003e(\u003c/b\u003eGiacchi and McMaster, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e To better simulate the clinical challenges encountered in the field, such as tendon end retraction and irregular cuts, gap tenorrhaphy was chosen for this study, providing a more accurate reflection of real-world cases.\u003c/p\u003e \u003cp\u003eIn this study, during the 2nd week (inflammatory phase), ultrasonography revealed severe abnormal FES in all groups, likely due to intratendinous edema, hemorrhage, and inflammatory exudate, consistent with findings reported by several studies \u003cb\u003e(\u003c/b\u003eHiramatsu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Montague et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). By the 4th week, echogenic tissue appeared within the AOI in both treatment groups, signifies the onset of the proliferative phase marked by fibroblast activity and extracellular matrix production \u003cb\u003e(\u003c/b\u003eThomopoulos et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Schulze-Tanzil et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). From the 6th week until the end of the study, ultrasonographic assessment indicated early healing of the tendon gap in both treatment groups, displaying normal FES and FAS. These findings were supported by histological analysis, which revealed a significantly restored tendon architecture in the PRF and PRF/Ch groups compared to the control, in line with previous studies \u003cb\u003e(\u003c/b\u003eMelamed et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathological and ultrasonographic evaluations consistently showed ongoing inflammation and intratendinous edema in the control group throughout the study. This was characterized by marked vascular permeability, neovascularization, infiltration of inflammatory cells, and fluid accumulation (intratendinous edema) within the tendon. In contrast, both the PRF and PRF/Ch groups exhibited signs of inflammation and edema primarily during the early inflammatory phase, which significantly subsided at later stages. This improvement can be attributed to the potent and prolonged anti-inflammatory effects of both chitosan \u003cb\u003e(\u003c/b\u003eYang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)d PRF \u003cb\u003e(\u003c/b\u003eZhang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings were in agreement with clinical measurements of limb circumference, where differences in tendon thickness were significantly influenced by factors such as intratendinous edema, inflammatory exudate, extracellular matrix production, scar tissue, and granulation tissue formation \u003cb\u003e(\u003c/b\u003eNichols et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Arvind and Huang, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chartier et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe prevention of adhesion during tendon repair is critical for successful healing, and the significant differences in peritendinous adhesion observed between the control and treated groups highlight the positive impact of the applied treatments. Chen et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eChen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that chitosan possesses unique properties enabling close contact with tendon surfaces, which promotes tissue regeneration and prevents adhesion to surrounding tissues. Additionally, the effectiveness of PRF in minimizing peritendinous adhesions has also been supported by recent experimental study on donkeys, in which the application of PRF significantly reduced adhesion formation and enhanced tendon gliding function during SDFT repair \u003cb\u003e(\u003c/b\u003eAbdelhakiem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This was clearly demonstrated during the ultrasonographic gliding test, where donkeys in the control group showed restricted SDFT movement due to thick adhesions with the overlying skin, while those in the PRF and PRF/Ch groups displayed normal tendon gliding without restriction. The integration of a video-assisted gliding test provided a valuable dynamic assessment of tendon functionality, adding practical insight into the therapeutic efficacy of the treatments.\u003c/p\u003e \u003cp\u003eIn this study, a correlation between clinical, ultrasound, and histological findings was observed, supporting the formation of scar tissue in the control group as opposed to the restoration of normal tendon architecture in the treated groups. The control group showed increased limb circumference clinically, and ultrasound revealed irregular tendon shape, disorganized fibers (FAS), and increased tendon thickness, which was further confirmed histologically by tendon fiber disarrangement. In contrast, the treated groups (PRF and PRF/Ch) demonstrated nearly normal limb circumference, restored crescent-shaped tendon morphology, aligned fibers on ultrasound (FAS), and normal tendon thickness, with histological analysis confirming well-organized fiber structure. These findings collectively reinforce the beneficial effects of the treatments in promoting tendon regeneration and preventing scar tissue formation, in contrast to the control group, where the tendon healing was characterized by scar tissue.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed the positive impact of A-PRF on tendon healing in gap tenorrhaphy, while the addition of chitosan further enhanced and accelerated the regeneration of the SDFTs. Spontaneous tendon healing is characterized by irregular scar tissue formation, extensive peritendinous adhesion, marked inflammation, severe lameness, and pain.\u003c/p\u003e \u003cp\u003eA-PRF application in situ during gap tenorrhaphy stimulates tendon regeneration, nearly restoring normal ultrasonographic and histologic features and improving clinical index scores.\u003c/p\u003e \u003cp\u003eThe inclusion of chitosan in A-PRF clots potentiates its effect and greatly enhances and accelerates SDFT regeneration, as indicated by a greater and faster reduction in SDFT thickness, a reduction in peritendinous adhesion, and a decrease in intratendinous edema and inflammation, suggesting that A-PRF and chitosan combination is a promising approach for improving the quality and speed of tendon healing compared to spontaneous healing processes.\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitations\u003c/h2\u003e \u003cp\u003eDespite promising results, the study had several limitations. The small sample size (n\u0026thinsp;=\u0026thinsp;6 per group) may limit the generalizability of the findings. While donkeys served as a relevant model for tendon repair, they may not fully replicate human tendon healing, particularly in chronic or complex injuries. The short duration of the study restricted long-term healing assessments, and more advanced imaging or molecular analyses could have enhanced the findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePlatelet-rich fibrin (PRF)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A-Platelet-rich fibrin (A-PRF)\u003c/p\u003e\n\u003cp\u003eChitosan (Ch)\u003c/p\u003e\n\u003cp\u003eSuperficial digital flexor tendon (SDFT)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuperficial digital flexor tendons (SDFTs)\u003c/p\u003e\n\u003cp\u003eUltrasound (US)\u003c/p\u003e\n\u003cp\u003eLongitudinal sonogram (LS)\u003c/p\u003e\n\u003cp\u003eTransverse sonogram (TS) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFiber echogenicity score (FES)\u003c/p\u003e\n\u003cp\u003eFiber alignment score (FAS)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLimb circumference (LC)\u003c/p\u003e\n\u003cp\u003eArea of interest (AOI)\u003c/p\u003e\n\u003cp\u003ePostoperative (PO)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Prof. Dr. Sabry El-Khodery for his help and technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution to the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN: Study design, experimental operations, statistical analysis, and manuscript writing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAS: Statistical analysis, manuscript writing and editing. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAR: Study design, surgical procedures, and manuscript editing.\u003c/p\u003e\n\u003cp\u003eME: Preparation, characterization, and discussion of chitosan film results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGK: Study design and manuscript writing editing, Supervision and final revisions of the manuscript. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was obtained for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data associated with this study are available upon request. Please contact the corresponding author [Gamal Karrouf] at [[email protected]].\u003c/p\u003e\n\u003cp\u003eThe tables supporting the findings are available as Supplementary tables file.\u003c/p\u003e\n\u003cp\u003eThe video material is available as Supplementary video file (video-S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ethical conduct of the study was ensured through adherence to established guidelines and regulations. Approval for all experiments was obtained from the Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, Egypt. The study protocol received ethical clearance, and the approval was documented with registration number (Approval Code: Ph.D./101).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors declare that they have no competing interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2 Physics Department, Faculty of Science, Mansoura University, Mansoura, 35516, Egypt.\u003c/p\u003e\n\u003cp\u003e3 Physics Department, Faculty of Science, New Mansoura University, New Mansoura, Egypt.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAbdelhakiem, M.A., Hussein, A., Seleim, S.M., Abdelbaset, A.E., Abd-Elkareem, M. 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(2019).\u003c/strong\u003e The infrared spectroscopy of chitosan films doped with silver and gold nanoparticles. J. Polym. Eng. 39, 415-421. https://doi.org/10.1515/polyeng-2018-0356\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eZhang, J., Yin, C., Zhao, Q., Zhao, Z., Wang, J., Miron, R.J., Zhang, Y. (2020).\u003c/strong\u003e Anti‐inflammation effects of injectable platelet‐rich fibrin via macrophages and dendritic cells. J. Biomed. Mater. Res. A. 108, 61-68. https://doi.org/10.1002/jbm.a.36792\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"irish-veterinary-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Irish Veterinary Journal](https://irishvetjournal.biomedcentral.com/)","snPcode":"13620","submissionUrl":"https://submission.springernature.com/new-submission/13620/3?","title":"Irish Veterinary Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Platelet-rich Fibrin, Superficial digital flexor tendon, Chitosan, Donkeys, Tenorrhaphy","lastPublishedDoi":"10.21203/rs.3.rs-9390812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9390812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTendon healing remains a formidable challenge in equine rehabilitation as injured tendons are healed by a fibrotic scar that exhibits compromised biomechanical characteristics. The objectives were to evaluate the regenerative effectiveness of autologous advanced platelet rich fibrin (A-PRF) and chitosan (Ch) for repairing the superficial digital flexor tendon (SDFT) following gap tenorrhaphy in a prospective, blinded, randomized, controlled experimental study carried out on 18 male donkeys based on clinical, ultrasonographic, and histopathological interpretations. These animals were allocated according to the gap-filled biomaterials into three groups (n\u0026thinsp;=\u0026thinsp;6 each): the control group, the PRF group, and the PRF/Ch group.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe addition of A-PRF, both alone or with chitosan, led to significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and marked changes compared to those in the control group. Both the PRF and PRF/Ch groups exhibited normal clinical index scores, characterized by a comfortable attitude with neither swelling, pain, nor lameness, after the recovery of normal tendon ultrasonographic characteristics (echogenicity, alignment, thickness, shape, and gliding properties) and histologic features. The addition of chitosan to A-PRF in the PRF/Ch group significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced the SDFT thickness, intratendinous edema, inflammatory cell infiltration, and vascularization.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe combination of both A-PRF and chitosan could represent an innovative and effective approach for enhancing SDFT healing outcomes, offering promising improvements in the field of equine rehabilitation.\u003c/p\u003e","manuscriptTitle":"Chitosan-augmented advanced platelet rich fibrin improves the regeneration of superficial digital flexor tendon gap tenorrhaphy in donkeys (Equus Asinus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 17:50:12","doi":"10.21203/rs.3.rs-9390812/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"211144081844103601473712463225128171392","date":"2026-05-14T07:04:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318519016950919765984338194588508885337","date":"2026-05-12T23:58:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T12:06:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221416022721894300331979456661987144340","date":"2026-04-24T14:00:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T13:47:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T14:45:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-13T14:45:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Irish Veterinary Journal","date":"2026-04-11T23:15:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"irish-veterinary-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Irish Veterinary Journal](https://irishvetjournal.biomedcentral.com/)","snPcode":"13620","submissionUrl":"https://submission.springernature.com/new-submission/13620/3?","title":"Irish Veterinary Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"89e28ff2-855a-409e-913b-80260b5a2c9d","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"211144081844103601473712463225128171392","date":"2026-05-14T07:04:14+00:00","index":49,"fulltext":""},{"type":"reviewerAgreed","content":"318519016950919765984338194588508885337","date":"2026-05-12T23:58:46+00:00","index":48,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T12:06:41+00:00","index":43,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T17:50:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 17:50:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9390812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9390812","identity":"rs-9390812","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}