Ameliorative effect of different mesoporous bioactive glass materials in experimental tibial defects in rats

In the synthesis of MBGs and metal loaded mesoporous bioactive glasses (M-MBGs), the following chemicals were used (analytical purity grade): poly(ethylene glycol)-block-poly(propylene glycol)-block-poly (ethylene glycol) (Pluronic P123, C₄H₅O(C₂H₄O)x(C₃H₆O)y(C₂H₄O)zC₄H₅O₂), tetraethyl orthosilicate (TEOS, Si(OC₂H₅)4), triethyl phosphate (TEP, (C₂H₅O)₃PO), calcium nitrate (Ca(NO₃)₂ 4H₂O), copper nitrate (Cu(NO₃)₂ 6H₂O), zinc nitrate (Zn(NO₃)₂ 6H₂O), nitric acid (HNO₃, 65%), and sodium hydroxide (NaOH). All chemicals were procured from Sigma-Aldrich and used without any further purification. Ultrapure water was used for the preparation of all solutions.

MBGs were synthesized using the hydrothermal synthesis method by making some modifications to the prescription of Anand et al. [18]. In a selection that is typical of manufacturing processes applicable to MBG and M-MBG, P123 was chosen as the structure-directing agent, and TEOS and TEP, Ca(NO₃)₂ 4H₂O, Cu(NO₃)₂ 6H₂O, and Zn(NO₃)₂ 6H₂O were chosen as the sources of Si, P, Ca, Cu, and Zn, respectively. During synthesis, 1 g of P123 was dissolved in 50 mL of a 1.6 M HNO₃ solution at room temperature and mixed for 30 min. Then, 10 mL TEOS was added dropwise and the resulting mixture was kept in an ultrasonic bath for 30 min; TEP and calcium nitrate, at a Ca:Si:P ratio of 33:61:6 by weight (%), were added at intervals of 30 min and kept for 12 h with stirring. During the synthesis of copper loaded glasses, zinc loaded glasses, or both, the same processes as in the synthesis of calcium silicate were followed. While for the synthesis of copper loaded glasses (Cu:Ca:Si:P) and zinc loaded glasses (Zn:Ca:Si:P) separately, the copper nitrate and zinc nitrate were added at the ratio of 3:30:61:6 by weight (%), respectively, for the synthesis of copper zinc loaded glasses (Zn:Cu:Ca:Si:P), the copper nitrate and zinc nitrate were added at the ratio of 1:2:30:61:6 by weight (%) after the addition of calcium nitrate. These prepared mixtures were kept at 100°C for 48 h and then dried at room temperature. After drying, the bioactive materials were subject to a 2-step heat treatment process, consisting of the following: heating to 400°C at a 2°C/min heating rate and maintaining for 2 h at this temperature, and heating to 650°C at a 2°C/min heating rate and calcining for 4 h at this temperature. The products obtained were coded as MBG, Cu-MBG, Zn-MBG, and Cu–Zn-MBG, respectively.

All MBGs were investigated using X-ray diffraction (XRD) to identify the phases formed in the material’s surface before and after immersion in simulated body fluid (SBF). XRD patterns were obtained using a Rigaku Ultima IV diffractometer with Ni filtered CuKα (λ = 0.154,06 nm) radiation in the range of 2θ = 10º–90º, step size 0.020º, 45 kV, and 40 mA. Fourier transformed infrared (FTIR) spectra have been used to identify compounds in MBGs. FTIR spectra were obtained using the PerkinElmer Frontier model spectrometer by applying the KBr pellet technique in the range 4000–400/cm.

The in-vitro bioactivity of the synthesized materials was evaluated based on the formation of hydroxyapatite on their surface during the process of soaking them for 28 d in the SBF (pH 7.4, T = 37°C, and volume/mass ratio corresponding to VSBF/mMBGs = 200 mL/g) prepared according to the procedure proposed by Kokubo and Takadama [19]. Samples soaking in SBF were washed with water after separating from SBF by filtration and dried at 37°C for 24 h. XRD and FTIR analyzes were performed to confirm the formation of hydroxyapatite.

Fifty 5-month-old male Wistar albino rats weighing 300–350 g were obtained from the Experimental Animal Production and Experimental Research Center of Burdur Mehmet Akif Ersoy University (Turkey). All experiments were performed in accordance with the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines 2.0 [21], and the study approved by the Local Ethical Committee on Animal Research of Burdur Mehmet Akif Ersoy University, Turkey (certificate of approval no. 526).

The rats were kept under standard laboratory conditions (humidity 60 ± 5%; temperature 21 ± 2°C; 12:12 h light:dark cycle). All animals were fed with a standard commercial chow diet (Korkuteli Yem) and they had free access to food pellets and water.

The rats were randomly distributed into 5 groups of 10 rats each and simple random sampling was used to select the rats to be assigned to each group. All rats that were comprised in these 5 groups were assigned to either of 2 groups depending on the duration of the experimentation period elapsed, and humanely euthanized at the end of the experiment. Totally 10 subgroups were used for this study. Tibial defects were created at both tibiae’s distal region, and inserted using different types of granules, which were MBG, Cu-MBG, Zn-MBG, or Cu–Zn-MBG. The animals were humanely euthanized on the 15th and 30th days post operation. Sham treatment groups were also constituted, comprised of rats with tibial defects. Totally 10 tibiae were harvested from each group. The procedures applied to the groups are shown in Figure 1 and experimental procedures illustrated in Figure 2.

Figure 1.

Figure 2.

Surgery was performed under general anesthesia, administering a mixture of xylazine HCl (Vetaxyl, Vet-Agro®, 20 mg/mL) 12 mg/kg and ketamine (Vetaketam, Vet-Agro®, 100 mg/mL) 75 mg/kg intraperitoneally. After preparation of the site for aseptic surgery, a 2 cm longitudinal skin incision was made overlying the anteromedial part of the proximal tibia medial section of the tibia metaphysis of both legs. The overlying muscle was separated by blunt dissection. The periosteum was mechanically stripped from the proximal tibiae. A 3 mm monocortical hole defect was perpendicularly drilled using a 10,000 rpm cylindrical burr. The standardized hole was unilateral, 3 mm in diameter and 2 mm deep, and was flushed with a copious amount of saline during drilling (Figure 3). After the drill hole defect was filled in appropriate groups, the soft tissue was closed with 5–0 absorbable sutures (Vicryl Rapide, Ethicon® Polyglactin 910) using the simple interrupted suture technique.

Figure 3.

After the last stitch, enrofloxacin 10 mg/kg (Vicryl Rapide, Ethicon® Polyglactin 910) as an antibiotic and meloxicam 1 mg/kg (Maxicam, Sanovel®) as an analgesic were administered subcutaneously. Animal surgical procedures were performed by the same surgeon to maintain uniformity.

All the rats were weighed and then anesthetized by intraperitoneal injection of 90 mg/kg ketamine (Alfamin, Alfasan, IBV) and 10 mg/kg xylazine (Alfazin, Alfasan, IBV). The animals were humanely euthanized by exsanguination. For confirming death, lack of pulse, breathing, corneal reflex, response to firm toe pinch, inability to hear respiratory sounds and heart beat by use of a stethoscope, and graying of the mucous membranes were checked. Then, tibiae were bilaterally removed at 15 d and 30 d after the surgical procedures. After removal of soft tissues, tibiae including the implanted material were fixed in a 10% neutral-buffered formalin solution. After 2 d, the fixated tibial samples were decalcified in a 5% formic acid solution for 1 week, and then tissues were routinely processed by an automatic tissue processor equipment (Leica ASP300S; Leica Microsystems) and embedded in paraffin. Then, 5 μm serial sections were taken from each sample using a rotary microtome (Leica RM 2155) and one of the sections from each rat stained with hematoxylin and eosin (HE) for light microscopy analysis.

The area of total augmentation (TA; mm²) and the area of NB (mm²) were measured [22]. The numbers of osteoclasts and osteoblasts on the lesioned alveolar bone were counted in an area 1.23 mm² (×400 magnification) [23]. The lesioned areas were analyzed in each rat using a light microscope (Olympus CX41). The Database Manual CellSens Life Science Imaging Software System (Olympus Corporation) was used for histomorphological analysis. Histopathological analyses were performed by a pathologist blinded to the group.

Selected sections were stained to demonstrate the presence of bone morphogenetic protein 2 (Anti-BMP2 antibody [ab14933], Abcam), collagen 1 (Collagen 1 antibody [ab34710], Abcam), osteocalcin (OST) (Anti-osteocalcin antibody [ab93876], Abcam), and vascular endothelial growth factor (VEGF [C-1] antibody, sc-7269, Santa Cruz Biotechnology, Inc.) using the streptavidin–biotin peroxidase technique (SBPT), according to the manufacturer’s instructions. All antibodies used a 1/100 dilution. The Mouse and Rabbit Specific horseradish peroxidase / 3,3′-diaminobenzidine (HRP/DAB) Detection Kit – Micropolymer (ab236466) was used as the secondary antibody, and 3,3′-diaminobenzidine was used as chromogen. The primary antibody step was omitted in negative controls. Seven serial sections were prepared and examined for each rat, and 2 areas of each section were examined. All the slides were analyzed for immunopositivity, and a semiquantitative analysis was carried out as detailed later. Samples were analyzed by examining 4 different sections in each sample, which were then scored from 0 to 3, according to the intensity of staining (0, absence of staining; 1, slight; 2, medium; and 3, marked).

After the conventional microscopic examination, computer-assisted histomorphometric measurements and immunohistochemical scoring were obtained using an automated image analysis system (Olympus CX41). The Database Manual CellSens Life Science Imaging Software System (Olympus Corporation) was used for evaluation of the lesioned area.

The sample size was calculated using the G Power 3.1.9.7 program [24] using the following parameters: effect size = 0.6, alpha = 0.05, expected power (1-beta) = 0.80, and number of groups = 5. Although there were 40 samples in total to begin with, it was determined that it would be necessary to account for the probability of loss during the experiment, and accordingly, the choice was made to include 2 additional animals in each group; thus, the total number of samples for use in the analyses amounted to 5 × 10 = 50.

The one-way analysis of variance test was used to determine significant differences between the groups. To determine differences between groups in the histomorphic data analyses, the Duncan multiple comparison test was used. The Kruskal–Wallis test was used to identify whether there were any notable differences in the groups’ immunohistochemical analyses. Pairwise comparisons using the Dwass–Steel–Critchlow–Flinger formula were performed to identify differences across groups. Calculations were made using the SPSS 15.0 program. P < 0.05 was set as the level of significance.