Twenty-four male New Zealand white rabbits with an age of 6.0 ± 0.3 months and weight of 2.5 ± 0.29 Kg were used. A 12 mm defect was created in the diaphysis of the ulnae in one limb while the contralateral limb was kept intact in all experimental rabbits. Then the rabbits were classified into three experimental groups (n=8): control group; (no materials were added), platelets rich fibrin group (PRF group); defects were filled with autogenous PRF) and combination group (PRF/ZnONPs group); defects were filled with both PRF and ZnONPs). This study was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, Egypt, and registration number (M/6).

Autogenous PRF was prepared from each rabbit as mentioned by Choukroun et al. 7 technique, where 3 mL of whole blood was obtained after aseptic preparation from the ear central or lateral veins of each rabbit in a 4 ml plain tube thereafter the blood was centrifuged immediately at a rate of 3000 rpm for 10 min till separation into three distinct layers. The fibrin clot in the middle layer (Figure 1), is considered the leukocyte-rich PRF which is carefully taken by non-toothed tissue forceps and separated from the RBCs layer with avoidance of the PRF membrane rupture.

In this study, ZnONPs, milky dispersion (Sigma-Aldrich chemicals, Nasr City, Cairo, Egypt) with particles size of <100 nm, the concentration of 20 wt. % (in H2O), the density of 1.7 ±0.1 g/mL (at 25 °C) and pH of 7.5±1.5 were purchased and kept in a dark place at room temperature. The dispersion was diluted to 0.2% using 0.9% normal saline. In the PRF/ZnONPs group, 0.1 ml of ZnONPs 2% (ZnONPs density of 1.72± 0.1 mg) was injected carefully into the PRF clot membrane using an insulin syringe. The ZnONPs dose was selected according to our unpublished pilot study performed before this study.

General anesthesia was achieved using an IM³ injection of a combination of 7.5 mg kg^-1 xylazine (20 mg/ml; Xylaject; ADWIA Co, Egypt) and 35 mg kg^-1 ketamine hydrochloride (Ketamine 50 mg /10 ml, Rotexmedica, Germany). On lateral recumbency, the ulnar shaft is approached by a craniolateral incision in the antebrachial region. Exposing the right ulna was done by incising the antebrachial fascia between the ulnaris lateralis muscle and the lateral digital extensor muscle and retracting the muscles. Then, a 12-mm defect was osteotomized from the shaft of the ulna using a low-speed electric drill (APT, China) with a 1.5 ml diameter drill bit.  In the control group, the defect was left empty. While in the PRF group, a PRF clot was used to fill up the defects. In the PRF/ZnONPs group, 0.1 ml of ZnONPs dispersion 2% (2 mg ZnONPs) was inoculated carefully into the PRF clot membrane using an insulin syringe. The subcutis and the skin were closed by the routine manner using 3-0 vicryl (ETHICON, USA) and 3-0 silk (ETHICON, USA), respectively. A protective bandage was applied and then all rabbits were allowed to recover in an incubator warmed to 37-40 °C until fully awake. Afterward, they were moved to their cage. All animals received meloxicam (meloxicam 15 mg, AMRIYA pharmaceutical company, Egypt) at a dose rate of 0.6 mg/kg for 7 days. The antibiotic regimen included Cefotaxime sodium (Cefotaxime sodium, EIPICO pharmaceutical company, Egypt) at a dose rate of 50 mg/kg bodyweight for 5 days. The incisions were dressed daily with povidone-iodine and bivatracin topical antibiotic spray (Neomycin sulfate 165000 IU, Bacitracin Zinc 12500 IU, and Aerosol Hydrocarbon to 150 ml, ECAP, Egypt). The feeding condition, weight, body temperature, and breathing were investigated daily.

Note:

³Intramuscular

Standardized mediolateral and anteroposterior radiographs were performed immediately after the operation and subsequently on the 1^st and the 2^nd postoperative months at 40 kV, 100 mA, 10 mAs, and 80 cm FFD⁴ using X-ray machine, Poskom Co., Korea. General anesthesia of rabbits was performed before radiological examination by administration of a combination of 5 mg kg^-1 xylazine (20 mg/ml; Xylaject) and 35 mg kg^-1 ketamine hydrochloride (Ketamine 50 mg /10 ml), injected IM in one syringe.

Note:

⁴Focal Film Distance

Compared to the immediate postoperative radiographs, healing of the defect was determined by evaluating the radiographs in each group at the 1^st and the 2^nd postoperative months for bridging the gap, and the healing capacity of each treatment. The healing score was measured by different independent operators using a ruler to measure the percentage of newly formed tissue in the defect depending on the apparent greyscale. Our healing score was designed as follows, score 1 = less than 25% healing, score 2 = 25-50% healing, score 3 = 50-75% healing, and score 4=75-100% healing. Bridging of the gap is considered present if any newly formed growth was found to connect between the two extremities of the defect on X-ray films. Otherwise, gap bridging is considered absent. The higher healing score with gap bridging was considered to have more new bone formation.

Statistical analyses were performed according to Elsaied et al. 2020 and Samy et al. 2020a, b 212223 using SPSS (version 25.0, Inc, USA). Gap bridging results were expressed as frequency while healing scores were expressed as median (minimum-maximum). To determine the effect of time, and group on the healing score, Kruskal-Wallis nonparametric ANOVA test was used meanwhile chi-square was used with gap bridging frequencies. For all analyses, P ≤ 0.05 was considered significant.

During the current study, no serious complications and death were observed and the healing took place in all surgical wounds by the first intention. Clinical wellness was observed in all rabbits as early as the second postoperative day. Compared to the postoperative radiographs, the addition of PRF and ZnONPs to segmental ulnar CSD of 12-mm length resulted in obvious radiographic changes (Table 1, Figure 2), and the statistical analysis showed a significant difference in the healing score between the three experimental groups (P = 0.000) at all-time points (P = 0.000- 0.003) during the study.

Concerning bone healing in the region of interest, treatment of CSD by adding PRF either alone (PRF group) or in combination with ZnONPs (PRF/ZnONPs group) resulted in a highly significant (P = 0.000) increase in the healing score of CSD versus the Control group. On the 30^th postoperative day, the healing score was 3.0 (2.0- 3.0) and 4.0 (4.0- 4.0) in PRF and PRF/ZnONPs groups respectively versus 2.0 (1.0- 2.0) in the Control group. On the 60^th postoperative day, complete filling of the CSD represented by 0.4 healing scores was observed in both treatment groups, versus 2.0 healing score and incomplete filling in the Control group.

In terms of bridging callus, there was a significant difference between times within a group (P = 0.000- 0.001) but a non-significant difference between groups within time. On the 30^th day, 62.5% of rabbits in both treatment groups showed gap bridging versus no bridging in the control group with a significant difference (P = 0.018) between groups within this time point. At the end of the study, 75% of the control group rabbits started to show some sort of bridging callus meanwhile all rabbits of both PRF and PRF/ZnONPs groups exhibited complete bridging of the gap.

Comparing the radiographic density of the healed bone, both treatment groups exhibited higher bone density compared to the Control group. In all rabbits of the combination group, maximal density was early obtained on the 30^th postoperative day.

Regarding long bone CSD, diaphysis is the most common site of involvement.69% of all fractures associated with bone loss reported in the Edinburgh Trauma Unit were involving the diaphysis, with the rest having either loss of metaphyseal bone or the articular surface, or both 1. The rabbit ulna or radius defect (between 10 and 20-mm) is often used in scientific research as it is of relatively low cost and does not require any fixation owing to the support offered by the adjacent bone due to the fibro-osseous connection between these two bones 24. This is why the ulnar diaphyseal segmental CSD without internal fixation is decided to be used and evaluated as a surgical model in this study.

PRF is a fibrin clot in which cytokines, platelets and cells are trapped and then released slowly within a certain time 25. The use of PRF in clinical field includes many advantages such as a simple preparation technique without the need for external biochemical additives to the blood. This makes this preparation strictly autogenous which prevents tissue reactions or rejection. Additionally, it provides more tensile strength and adhesiveness properties for clot stabilization. Therefore, using it as an autologous scaffold for periosteal cells and osteoblasts has shown to be able to promote higher cell proliferation and metabolic activity 1026

Most studies involved the use of PRF for soft tissue repair, while the use of its fibrin-based material solely as a filling material for large bone defects remains controversial 27. Positive results were reported by Ozdemir et al. 28 for using PRF in combination with titanium barriers including increased newly formed bone quality and enhanced bone formation rate, attributed to the concentration of growth factors. In a study by Singh et al. 29, PRF was shown to promote bone and soft tissue healing in 40 surgical areas in humans. Kim et al. 30 also reported increased bone mineral density and bone volume after 6 weeks in calvaria bone defects of rabbits that were treated with PRF. 

PRF has been solely used as a filling for the ulnar segmental CSD in this experiment without the addition of any bone substitutes as the fibrin matrix can be considered a scaffold. Good filling of the defect appeared in the radiographs as early as the first month and then filling continued till the end of the study where the defect was completely bridged and filled with newly formed bone and the highest healing score was achieved by the end of the second month. The low density of the bridging bone was noticed at the early stage of healing but then it becomes denser overtime.

In-vitro investigation of the possibility to improve the degradation and mechanical properties of TCP⁵ scaffolds by the addition of ZnONPs has been successfully done by Feng et al. 31. An optimal amount of ZnONPs (2.5 wt. %) was found to highly improve mechanical properties (compressive strength and fracture toughness) and bioactivity of the scaffolds. Once cultured on the surface of the composite scaffolds, The attachment and proliferation ability of osteoblast-like human cells were better than for the control sample. Moreover, after incubation of the optimized scaffolds in SBF⁶ for twenty-eight days, a layer resembling bone apatite was successfully obtained. Zn²⁺ ions are well-known to stimulate bone formation and mineralization and to have an active role in the proliferation of osteoblastic cells. These properties were contributed to the observed osteoconduction (guiding bone growth) and osteoinduction (formation of new bone tissue) which is assumed to be achieved by the released Zn ions from the TCP-ZnO composite scaffold 3233

Note:

⁵Tri-calcium phosphate

⁶Simulated body fluid

Similar findings were reported also by Fielding et al. 34 , who designed a novel 3D-printed TCP scaffold incorporating 0.25 wt. % ZnONPs and confirmed the boosting effects of Zn ions towards osteoblast cells. In addition, these findings were further demonstrated in vivo from the same research group 35 where the scaffolds were completely infiltrated by the newly formed bony tissue after three months postoperatively.

Similarly, the findings of this study support the results of the studies mentioned above. Our scaffold was a PRF incorporating 0.2% ZnONPs. Inoculating the specifically used dose of ZnONPs into the PRF matrix enclosed by its membrane has lowered the rate of Zinc ions release along with the release of growth factors naturally comprising the PRF fibrin matrix and this prolonged the ZnONPs osteogenic action. Statistical analyses showed a significant increase in the healing score in the PRF/ZnONPs group over the other two groups. Also, the use of ZnONPs has accomplished the highest bone density along with the study which proves that it enhances the PRF mineralization capacity and thus the newly formed bone density.