1. Animals:
The present research was ethically endorsed by the Animal Care and Welfare Committee of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt. All national and institutional guidelines for animal care and use have been followed throughout the study procedures. All animals have been accommodated and cared for according to the Egyptian animal welfare law (No. 53, 1966).
A total of 27 clinically healthy adult donkeys were used in this study (13 males and 14 females). The animals ranged from 3 to 6 years old (mean=4.4+/-1.05) and weighed from 70 to 120 kg (mean=99.5+/- 15.5). All animals were inspected clinically before the experiment to ensure that they were not lame. They were kept in the Veterinary Teaching Hospital (VTH), Faculty of Veterinary Medicine, Assiut University two weeks before the beginning of work. The food and water were introduced to animals ad libitum. The animals were randomly divided into 3 groups, each with 9 donkeys. Group1: Full-thickness tenotomy of the superficial digital flexor tendon (SDFT) without treatment (control). Group 2: Full-thickness tenotomy of SDFT treated with silver nanoparticles (AgNPs). Group 3: Full-thickness tenotomy of SDFT treated with platelet-rich fibrin (PRF). Animals in each group were subdivided into 3 equal subgroups (n=3). Subgroup 1: Animals were examined clinically and a tendon specimen was harvested after 1 month. Subgroup 2: Animals were examined and samples were obtained after 2 months. Subgroup 3: Animals were examined and tendon samples were harvested after 3 months.
2. Pre-operative preparations
The physiological parameters including temperature, HR, RR, and capillary refilling time were evaluated in all animals. The posture and gait of animals were examined from all views. All the selected donkeys for this study were within normal physiological parameters and free from all external lesions especially those affecting the musculoskeletal system. The donkeys were dewormed using 40 gram/100 K.B.W of piperazine citrate (Piperazine citrate 100%; UCCMA, Nasr City, Cairo, Egypt). The animals were fasted for 8 hours before the operation. The surgical site was prepared by clipping and shaving the hair, washing with soap and water, brushing with a soft brush, and then scrubbing with Povidone Iodine (Betadine®, El-nil Co., Egypt).
General anaesthesia was performed using intravenous administration of 2mg/K.B.W of xylazine Hcl (XYLA-JECT 2%, Adwia Pharmaceuticals Co. 10th of Ramadan city, Egypt) and 3 mg/kg.b.w of ketamine (Ketamine Rotexmedica 50 mg/ml, Arzneimittelwerk GmbH Rotexmedica, Germany). The animals were positioned in a lateral recumbence on the left side. The experimental operations were performed on the right forelimb in all groups of animals. Tourniquet was applied just below the carpal joint to control hemorrhage in the operated limb.
Preparation of silver nanoparticles (AgNPs)
The solution was prepared according to Vigneshwaran et al., (2006) [21]. Briefly, in a 250 ml flask, 1.0 g of soluble starch (El-Gomhouria Co., Egypt) was added to 100 ml of distilled water and heated till complete dissolution. 1 ml of a 100 mM aqueous solution of silver nitrate (GAMMA Laboratory Chemicals., Egypt) was added to the starch solution and shacked well. This mixture was kept in an autoclave at 15 psi pressure, 121°C for 5 minutes. The resulting solution was clear yellow indicating the formation of silver nanoparticles (Figure 1).
The concentration of the resulting solution was estimated using a spectrophotometer (Graphite Furnace Atomic Absorption Model 210VGP) at the Chemistry Department, Faculty of Science, Assuit University. The concentration was 157 µM (157 ppm). The size of the silver nanoparticles was also estimated using transmission electron microscopy (TEM) (JEOL-JEM- 100CX II) at the Electron Microscopy Unit, Assuit University. The size was ~ 7.96 nm to 14.8 nm (figure).
Preparation of platelet-rich fibrin (PRF)
It was prepared according to Choukroun et al., (2000) [22]. Briefly, ten ml of blood is collected from each donkey from the jugular vein and divided into two plain tubes (without anticoagulant). They were immediately centrifuged at a rate of 4000 rpm for 10 min. After centrifugation, the tube consists of three layers. The topmost layer represents acellular PPP (platelet-poor plasma). The PRF clot is in the middle while RBCs are present at the bottom of the test tube. The fibrin clot was collected from the tube and the attached red blood cells were scraped off and discarded (Figure 2).
3. Surgical technique:
Group 1: (full-thickness tenotomy of SDFT without treatment) (Control group). SDFT was exposed through a linear skin incision at the lateral aspect of the mid-metacarpal region. The edges of the surgical wound were grasped using Allis forceps. The paratenon was dissected with scissors to identify the SDFT. Full-thickness tenotomy was performed using a scalpel. The two ends of SDFT were sutured with a double locking loop suture pattern according to Easley et al., (1990) [23] using No. 2-0 nylon suture material (Ethicon/ India). The paratenon was closed using a simple interrupted suture pattern and 3-0 polyglactin 910 (Egysorb; Taisier-Med). The subcutaneous tissue was sutured continuously using 3-0 Egysorb. The skin was closed in a simple interrupted pattern using braided silk no. 0 (Ethicon/ India). The operated limb was kept in a cast using a combination of a splint and plaster of Paris bandage from the hoof to above the carpal joint for 1, 2 and 3 months in subgroups 1, 2 and 3 respectively.
Group 2: (Full-thickness tenotomy of SDFT treated with AgNPs). Full-thickness tenotomy of superficial flexor tendon was performed, likewise the animals of group 1 except for the addition of 1 ml of AGNPS into the sutured tendon (beneath the paratenon at and around the line of suturing).
Group 3: (Full-thickness tenotomy of SDFT treated with PRF). Full-thickness tenotomy of superficial flexor tendon was performed as group 1, but PRF was applied locally at the seat of SDFT suture, before the suturing of paratenon.
4. Post-operative care
The animals had been received Pencillin-dihydrosterptomycin (Pen-Strep, Norbrook; 1ml / 25 kg I/M every day for 5 consecutive days) and Flunixine-meglumine (Flunixine; Norbrook; 1ml /45kg I/V daily for 3 successive days). The animals were kept in the stable. At 2 weeks postoperatively they were hand-walked for 10 minutes every day. Hand walking was gradually increased in frequency and duration during the next weeks.
5. Clinical evaluation:
All animals were kept under complete observation after the operation. Animals were examined for lameness at 1, 2, and 3 months after the operation. Lameness was graded according to the American Association of Equine Practitioners (AAEP) on a scale of 0–5. Score 0: No lameness. Score 1: Lameness is difficult to be observed and is not consistently apparent, regardless of circumstances (e.g. circling, inclines, hard surface, etc.). Score 2: Lameness is difficult to be observed at a walk or when trotting in a straight line but consistently apparent under certain circumstances (e.g. weight-carrying, circling, inclines, hard surface, etc.). Score 3: Lameness is consistently observable at a trot under all circumstances. Score 4: Lameness is obvious at a walk. Score 5: Lameness produces minimal weight bearing in motion and/or at rest or a complete inability to move. The degree of lameness was determined at 1, 2 and 3 months in different subgroups.
6. Gross evaluation
The healed SDFT was examined in its seat after the incision of the skin of anesthetized animals. The inclusion criteria for evaluation were tendon shape, size, adhesion, and color. A visual system score was used to evaluate the changes in the healed tendon. The symbols +, ++, and +++ were used to refer to mild, moderate, and severe changes respectively.
7. Histopathological samples
The samples which were harvested for histopathological examination were: 1) Normal tendons from normal animals which had not been included in the study and had not been subjected to tenotomy (control negative). 2) Tendons those were subjected to tenotomy and suturing without the addition of any adjuvant (control positive) at 1, 2, and 3 months post-operation. 3) Tendons those were subjected to tenotomy and suturing with the addition of silver nanoparticles (AgNPs group) at 1, 2, and 3 months after surgery. 4) Tendons those were subjected to tenotomy with the addition of Platelet-rich fibrin (PRF group) at 1, 2, and 3 months after the operation.
8. Histopathological examination:
The freshly excised tendon specimens were fixed in neutral buffered formalin 10% solution for 24 hours at room temperature, dehydrated through ascending series of ethanol and cleared in methyl benzoate then embedded in paraffin wax. 5 um paraffin sections were prepared and stained with the following histological stains: 1) Hematoxylin and Eosin for general histopathological examination [24]. 2) Crossmon's trichrome technique for differentiation between mature and immature collagen fibers [25]. 3) Periodic acid Schiff (PAS) technique for demonstration of neutral mucopolysaccharides [26].
9. Statistical analysis:
The obtained data were statistically analyzed using statistical package for the Social Sciences for Windows (SPSS, version 21 (2016), Chicago, IL, USA). Univariate analysis of variances (ANOVA) with HSD–Tukey post hock multiple comparison test was used to determine the effect of time for each group and to determine the difference between groups at each time point. The lameness score data were analyzed in each group at different time intervals after surgery (one, two, and three months) using one-way ANOVA to determine the effect of time on the lameness score and also the effect of treatment on the lameness score. One-way ANOVA was also used for the detection of the differences in the lameness score between different groups (treatments) at one, two, and three months. While two-way ANOVA was used to detect the effect of the time and treatment on the lameness score. The data were expressed as mean± SE. Differences were considered significant when P < 0.05.