الفهرس 
ACKNOWLEDGMENT
Bone anatomyOnly 14 pages are availabe for public view
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Abstract
This experimental study aimed to evaluate the potential stimulatory effects of Graphitic carbon nitride (g-C3N4), Graphene oxide (GO), and University Institute of Oslo-66 (UiO-66) scaffolds on the regeneration of critical-sized bone defect separately. The study was divided into three main steps:
Step I: Materials synthesis and characterization:
The g-C3N4, GO, and UiO-66 scaffolds were synthesized and characterized using XRD and TEM.
The g-C3N4 scaffold was prepared by heating of 10 g of melamine in a muffle furnace at 550°C for 5 hours and then cooling down to room temperature. The product was washed with absolute ethanol and distilled deionized water and dried at 80°C for 24 hours. The final product was prepared at a concentration of 1 mg/mL via ultrasonication dispersion overnight.
While GO scaffold was synthesized from graphite. Nitric acid (10 mL) and sulfuric acid (15 mL) were added to 1 g of graphite and soaked in the ice to keep the temperature below 0°C. Next, 3 g of potassium permanganate mixed with them for 12 hours. Hydrogen peroxide was then added to remove the excess permanganate. After that, the product was filtered and washes several times to remove any metals. Finally, GO solution was prepared via ultrasonic dispersion in water.
Regarding the UiO-66 scaffold, it was synthesized from a mixture of ZrOCl2, terephthalic acid, and glacial acetic acid. This mixture was dissolved in DMF and then dispersed through ultrasonication for 30 min, followed by autoclaving at 120 ºC for 24 hours. The product was washed thrice with DMF and ethanol, dried overnight in a hot air oven at 100 ºC.
Step II: In vitro experiments:
The cytotoxicity and hemocompatibility of the different scaffolds were evaluated. To evaluate the cytotoxicity of the different scaffolds, hFOB cells (15 x 103) were placed on a 48-well plate and cultured on the conditioned media of the different scaffolds, normal medium as a negative control, or 20% DMSO as a positive control. The viability of cells was evaluated over 7 days of culture qualitatively and quantitatively with Live/Dead and MTT assays, respectively. While the hemocompatibility of the different scaffolds was evaluated by treating erythrocytes (2 x 109 cells/ mL) with the extracts of the different scaffolds, or SDS as a positive control, while untreated erythrocytes were used as a negative control. The suspension was then collected and centrifuged. The absorbances of the supernatants were examined using spectrophotometer to calculate the hemolysis percentages
The potential of these nanomaterials to stimulate osteoblasts function was also assessed using qPCR analysis. They were added to hFOB cells (10 x 103) in a 48-well plate and incubated at 34 °C in 5% CO2 for 28 days. After 3, 7, 14, 21, and 28 days of in vitro culture, qPCR analysis for Col-I, OC, and OP was performed.
Step III: In vivo bone repair model:
In the present study, the rabbit femur condyle defect model was used to assess the efficacy of different scaffolds in inducing bone regeneration. This experimental study was carried out on 72 clinical healthy, 6 month old, male New Zealand white rabbits. They were randomly divided into 4 groups according to the type of treatment; 18 animals for each; group I: control group, group II: g-C3N4 treated group, group III: GO treated group, and group IV: UiO-66 treated group. The animals in each group were divided into 3 subgroups (6 animals for each subgroup) according to different follow up periods (4th, 8th, and 12th postoperative weeks).
All animals were subjected to food starvation for 8 hours before surgery. General inhalation anesthesia was performed using isoflurane. The experimental operations were performed on the right hindlimb in all groups of animals, whereas the experimental critical sized bone defects (5 mm diameter, 10 mm length) were created using a trephine burr in the center of lateral femoral condyles. These defects are either implanted with different scaffolds or left empty as a control. Postoperative care was achieved by injection of meloxicam and penicillin, once daily for 5 days. Rabbits were subjected to daily clinical observation for any surgical complications.
Radiographical assessment of the operated femurs were performed postoperatively on the 1st day, and in the 4th,8th, and 12th weeks. In addition, CT scanning was conducted in the postoperative 12th week. Also, femur condyles were examined grossly, histologically and histomorphometry, and by qPCR analysis for OC and OP expressions on the postoperative 4th,8th, and 12th weeks.
The results obtained by in vitro and in vivo evaluations revealed that the different scaffolds that used in the current study are cytocompatible, hemocompatible, and upregulate osteoblast marker genes. Also, they could enhance the healing the critical sized bone defect after their implantation. Moreover, the g-C3N4 implanted group showed the best radiographical findings and macro/microscopical images, and the most efficacy for bone healing in terms of new osteoid tissue percentages, new mature bone collagen percentage, biodegradation, and bone related-gene expressions, while the UiO-66 implanted group showed the lowest efficacy among the implanted groups.