Experimental design and sample size calculation

The current study was performed on a total of 54 male, pathogen-free, albino rats weighting 180–200 g. All experimental procedures were performed according to the protocol that obtained its approval from the Ethical Committee of Faculty of Dentistry, Mansoura University, Egypt with number A11030821. In a light-controlled room, rats were kept at 22 °C constant temperature with a 12:12-h light–dark cycle and a relative humidity of 65–70%. Animals received commercial diet and water. All rats were monitored for at least 1 week preoperatively. They were randomly allocated into 3 groups (n = 18); group I (control group) where mandibular bone defect was left empty, group II where mandibular bone defect was grafted with HANPs, and group III where mandibular bone defect was grafted with AgNPs in a gel form. The group size was based on power analysis based on our published data (histologic analysis) with a 5% significance level and a power of 95%. Based on previous publications [13, 14], 6 animals/group/time point were normally sufficient.

Materials formulation

Rat bone marrow mesenchymal stem cell line (BMMSCs) was purchased from Cyagen OriCell-RAWMX-01001. The cell line was already characterized according to the manufacture instructions.

Synthesis of HANPs

HANPs were prepared by chemical precipitation using calcium nitrate tetrahydrate (Samchun Chemicals, Seoul, Korea) and diammonium hydrogen phosphate (Samchun Chemicals, Seoul, Korea) according to the method described by Mondal, 2016 [15]. The synthesized HANPs underwent dryness at 70 ◦C and calcination at 600 ◦C for 1 h in the air atmosphere.

Synthesis of AgNPs hydrogel

AgNPs were synthesized by photo irradiation method [16]. AgNPs-loaded hydrogels were fabricated. Briefly, 3% w/v concentrated aqueous solution of gelatin mixed at 400 rpm for 1 h at 40 °C. Following cooling to the room temperature, filtration of the solution was performed using 0.22 µm syringe filter, then it was combined with 1% w/v concentrated silver nitrate solution with stirring at 400 rpm for 10 min. The mixture was exposed to 354 nm long UV waves for 1h (JENAWAY6305, UK spectrophotometer).

Characterization of nanoparticlesTransmission electron microscope (TEM)

The size and morphologic properties of HANPs and AgNPs were characterized by TEM (TEM, JEOL 1200EX, Japan) operating at 200 kV. The NPs were dropped on the carbon-coated 400-mesh copper TEM grid and underwent complete dryness prior to TEM examination.

MTT assay for cell viability

The cell viability was determined with a Vybrant® MTT cell proliferation assay kit. In brief, BMMSCs were seeded in a 96-well cell culture plate. After 24–48 h, different nanomaterial concentrations (5, 10, 25, 50, 100 µM) were applied to the culture medium of BMMSCs and then underwent incubation in the CO2 incubator for 72 h. A negative control of medium without cells was used as a blank. The medium was discarded and replaced with a culture medium that was freshly prepared without phenol red. MTT reagent was added to the cells which underwent incubation at 37 °C for 2–4 h. Dimethyl sulfoxide was added to each well to dissolve in soluble forming formazan. Finally, mixing of all samples was done and absorbance was determined at 570 nm (BioTek ELx 800) (HIGHLAND PARK, US).

Surgical procedure

Prior to each surgery, animal body weight was measured. The surgery was carried out under sterile and aseptic conditions. First, the rat was anaesthetized using a mixture of ketamine (Virvac, Nice, France) (40–90 mg/kg) and xylazine (Rompum, Bayer, Leverkusen, Germany) (5–10 mg/kg) [17]. Then the skin on the right side of the mandible was shaved and then disinfected using a sterile cotton pellet wet with povidone-iodine 10% (Betadine®) (Fig. 1).

Fig. 1

Photographs showing step-by step surgical procedure for creating the CSD in the mandible. E showing the exact location of the CSD in rat’s mandible

A submandibular incision was made through the skin, subcutaneous tissue, and masseter muscle paralleling the inferior border of the mandible. The buccal surface of the mandible was exposed with an elevator, and 5 × 5 mm and 1 mm depth critical defects were created in the body of the mandible in the right side in each animal (Fig. 1E). Defects were created with a trephine bur (with inner diameter 5 mm and outer diameter 6 mm (3i, Palm Beach Gardens, FL, USA) in a slow-speed dental drill (NSK, latch bur, Tokyo, Japan). To prevent over-heating, cooling with saline was dropped onto the contact point between the bur and bone. A periodontal probe was used to measure the bone defect to ensure standardized size [18]. After bone defect formation, it was filled with the tested nano materials (15 mg of powder HANPs, while AgNPs was used as a gel form and 25 µl was used to fill the defect). Soft tissue above the defect was closed with 4–0 absorbable Vicryl sutures (Ethicon, Johonson & Johonson, Somerville, NJ, USA), a sterile cotton pellet wet with Betadine® was used to disinfect the operation site.

Post-operative care

After recovery from anesthesia, rats were placed in clean, dry recovery cages, each animal was kept on 150 mg/kg of body weight amoxicillin IM twice daily (E-MOX® 250 mg, Egyptian International Pharmaceutical Industries, Ramadan, Egypt) and analgesics (Voltaren 75 mg/3 mL Novartis, Giza, Egypt) for 3 days [19].

Euthanasia

Six rats from each group were euthanized after 3, 14, 21 days post-surgically using overdose of halothane. Then their mandibles were harvested for histological assessment.

Specimen processing and histological assessment

Once specimen have been harvested and cleaned carefully from any adherent soft tissues, they were fixed in 10% neutral-buffered formalin for 48 h. Specimens were then washed under running tap water, then they were decalcified using 10% EDTA solution. After confirming adequate decalcification, segments of interest were trimmed, then processed and embedded in wax blocks for histological assessment [20].

Hematoxylin and eosin stain (H&E)

For routine assessment of the bone structure, hematoxylin dye that stains nucleic acid in the cell nucleus a purplish blue and eosin an acidic dye that stains proteins in the cytoplasm and extracellular matrix pink were used.

Immunohistochemistry (IHC)

Using a conventional streptavidin–biotin immunoperoxidase approach, collagen-I antibody (COL-I; Cat# NB600-408, Novus Biologicals) and tumor necrotic factor-alpha (TNF-alpha; Cat# Ab307164, Abcam) were used for the immunohistochemical examination. Staining procedures were performed as sections were deparaffinized in xylene and hydrated with gradient ethanol ethyl alcohol. The enzyme recovery was performed in EDTA over 3 min in a pressure cooker. Blocking of endogenous peroxidase with H2O2 (10 min) was followed by the blocking of non-specific binding (20 min) in BSA/PBS. Incubation of slides was done overnight at pre-diluted concentrations (1:100) of the specific antibodies according to the manufacturer instructions.

Image analysis

After excluding the rats that showed complications, a total of 50 tissue slides were prepared from the control and experimental groups; 17 for control group, 16 for HANPs-, and 17 for AgNPs-treated groups. Six slides were prepared for control and experimental groups at 3 days, five tissue slides were prepared at 7 days and while at 21 days, five tissue slides prepared for HANPs and six tissue slides for either control or AgNPs-treated groups. Four randomly selected areas in the middle of each CSD were captured at 100 × magnification. Then twenty to twenty-four images for each group per time point were used for analysis. The ratio of newly formed bone to the total defect area in the images was calculated. Image analysis was carried out using the Intel® Core I7® based computer and VideoTest Morphology® software (Version 5.0, Russia). It features a specific built-in routine for area and percentage area measurements. Images were stained with COL-I and TNF-alpha where the positive reaction appeared as brown color.

Statistical analysis

Each defect was used as a statistical unit and one defect was prepared in each rat; thus, six statistical units were assigned for each group at three different time points (n = 18). Data were expressed as mean ± SEM. Data were analyzed by SPSS with one-way ANOVA and Tukey’s post hoc test. Significance levels were set at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The threshold of significance was set at 5% level.