Cone-Beam Computed Tomography-Assisted Evaluation of the Bone Regenerative Potential of Modulated Sol-Gel-Synthesized 45S5 Bioglass Intended for Alveolar Bone Regeneration.

Objectives: The objective of the study was to evaluate the in vitro cell compatibility and in vivo regenerative potential of 45S5 Bioglass (45S5-BG)-based bone graft implanted in critical-size defects (CSD) created at rat calvaria using cone-beam computed tomography (CBCT). Materials and
Methods: In vitro cell compatibility of 45S5-BG was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. For in vivo experiments, CSD of diameter 6 mm was created in the parietal bone and was treated with 45S5-BG bone graft in the test group (Group B), while the control group (Group A) CSD remained empty. Rats were euthanized at the 4th and 8th postsurgical weeks, and CBCT analysis was done for samples. The grayscale value in VGi and the selected region of interest (ROI, in mm) of CSD diameter were calculated.
Results: In vitro cytotoxicity analysis of 45S5-BG showed that cell viability of more than 70% as compared to the control confirmed cell compatibility. CBCT analysis of CSD confirmed a significant increase in VGi (P < 0.001) and reduction in ROI of CSD (P < 0.001) from the 4th and 8th weeks in the test group as compared to the control. Conclusions: In vitro cytotoxicity analysis confirmed cell compatibility of 45S5-BG bone graft and CBCT analysis revealed its bone regenerative potential. Copyright:

INTRODUCTION

Approximately 200,0000 bone grafts-assisted surgeries are performed every year globally, and out of these, 700,000 involved maxillofacial bone repairs.[1] Although autografts remained as a gold standard, they are associated with donor-site morbidity. Ideal requirements of bone grafts are not attained by autografts, allografts, and xenografts.[2] Alloplasts which can be tailored to possess the ideal qualities of bone grafts got a wider application in periodontal regenerative procedures.[3] 45S5 Bioglass (45S5-BG) (45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5), hereafter denoted as 45S5-BG bone graft, has been implanted in 1.500000 recipients.[4] Commercially available 45S5-BG was synthesized by conventional melt-quenching technique, whereas sol–gel technique enabled the synthesis of bioglass with enhanced regenerative potential.[5] The gold standard for imaging critical-sized defects (CSD) within a bone of animal models is microcomputed tomography (μ-CT).[6] However recently, cone-beam CT (CBCT) is considered as accurate for evaluation of the bone structures in the maxillofacial region.[7] The aim of the present study is to investigate the in vitro cell compatibility and CBCT-assisted in vivo regenerative potential of sol–gel-synthesized 45S5-BG bone grafts.

MATERIALS AND METHODS

45S5-BG was prepared by sol–gel route as per the protocol reported earlier in our research work.[8]

In vitro cell viability assay

The Saos-2 cell line was used to evaluate the cell compatibility of 45S5-BG using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with Novabone and 10% dimethyl sulfoxide (DMSO) as a positive and negative control, respectively, whereas cell alone served as the control. For the in vitro cytotoxic analysis of 45S5-BG (100 μg/ml), Novabone (100 μg/ml), 10% DMSO, and Saos-2 cells were incubated in Dulbecco's Modified Eagle's Medium. MTT (5 mg/ml) reagent was pipetted to the wells at 24 h, 48 h, and 72 h, and absorbance was read at 570 nm in a microplate reader. The results were calculated in percentage of cell viability with standard deviation (SD) value.

In vivo experiments

Twenty male Sprague–Dawley rats of age 8–10 weeks and of weight 250–350 g were included in the study. Animal studies were performed after obtaining consent from the institutional animal ethics committee, and experiments were conducted in strict adherence to the CPCSEA (No. 602/PO/Re/S/2002/CPCSEA) guidelines. Anesthesia, surgical procedures, postsurgical care, and animal sacrifice were all performed under standard aseptic conditions.

Surgical procedures

All surgical procedures were performed under ketamine hydrochloride (35–50 mg/kg) intramuscular along with xylazine hydrochloride (5–10 mg/kg) to attain anesthesia. A linear midsagittal scalp incision of 2 cm was performed to expose the underlying bone. On the dorsal part of the parietal bone, CSD was created using dental trephine of diameter 6 mm [Figure 1a] attached to physiodispenser (Nobel BioCare, Sweden), operated at 10,000 rpm with saline irrigation. Control group CSD (Group A) bone defect remained empty, as shown in Figure 1b, while the CSD in the test group (Group B) was loaded (2.5 mg) with 45S5-BG, as shown in Figure 1c. Surgical sites were closed using 3-0 silk sutures, as shown in Figure 1d. Rats were euthanized in a carbon dioxide chamber at the 4th and 8th weeks after surgery, and specimens with CSD were fixed in 10% neutral buffered formalin solution for 1 day.

Figure 1

(a) Six mm trephine attached to the surgical handpiece. (b) Critical-sized defect of 6 mm created on the calvarial bone for control group (Group A). (c) Test group (Group B) implanted with 45S5 Bioglass. (d) Postoperative view of the surgical site

Cone-beam computed tomography imaging

All harvested samples (20) were mounted in cylindrical plastic tubes (3.5 cm diameter and 7 cm height) with 10% neutral buffered formalin and were scanned using a CBCT imaging unit (MyRay Hyperion X5 CBCT, Seoul, Korea). Exposure settings were 90 kV, 6.3 mA, 8 cm × 8 cm high-resolution field of view (FOV) (voxel size 0.127 mm), and 11 sec of exposure time. Tubes where the specimens were mounted were aligned in the middle of the selected FOV. The thickness of the slice was up to 0.5 mm and ten slices were obtained from each specimen for measurement. Using the measuring tool provided by the software (CS 3D Imaging Software 3.2.9), the measurements were done in coronal and sagittal slices for grayscale value (VGi) and in coronal slices for defect size in millimeters (mm). A digital tool was used to select an area corresponding to CSD in each group, and these were selected as the region of interest (ROI). The mean grayscale value and reduction in defect size were calculated from each group at the 4th and 8th weeks.

Statistical analysis

Data were presented as the mean ± SD. P < 0.05 was considered statistically significant. The Wilcoxon signed-rank test was also performed for comparison of time points within each group. The significance of multiple comparisons was adjusted using the Bonferroni method.

RESULTS

The cell viability of 45S5-BG at 24, 48, and 72 h are 92.522 ± 3.45, 95.226 ± 4.113, and 96.226 ± 4.987, respectively, while the Novabone (positive control) at 24, 48, and 72 h exhibited cell viability of 93.462 ± 5.382, 94.991 ± 2.474, and 94.885 ± 5.117, respectively. The 10% DMSO (negative control) at 24, 48, and 72 h exhibited cell viability of 21.336 ± 3.119, 19.226 ± 2.114, and 23.116 ± 1.936, respectively, as shown in Figure 2.

Figure 2

Shows the cell viability of Saos-2 cells against 45S5 Bioglass using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for 24 h (a), 48 h (b), and 72 h (c)

Bone regenerative potential of 45S5 Bioglass in vivo

The control group (Group A) showed a mean VGi of 434.12 ± 22.657 and 79.64 ± 16.76 at the 4th and 8th weeks, respectively, while the test group (Group B) showed a mean VGi of 1958.17 ± 21.456 and 2898.19 ± 23.94 at the 4th and 8th weeks after surgery, which is significantly higher than the control group (P < 0.001) [Figure 3 and Table 1].

Figure 3

Coronal and sagittal view by cone-beam computed tomography image of ROI: 4th-week control group (a and b) and test group (c and d); 8th-week control group (e and f) and test group (g and h)

Table 1

The mean grayscale value (VGi) for the control group (Group A) and test group (Group B) at 4th and 8th weeks after surgery

	4th weeks (mean value)	8th weeks (mean value)	P
Control (Group A)	434. 12±22.657	779.64±16.76	<0.001
Test (Group B)	1958.17±21.456	2898.19±23.94

The control group showed a mean ROI of 5.23 ± 0.923 mm and 4.53 ± 0.76 mm at the 4th and 8th week, respectively, while the test group showed a mean ROI of 3.54 ± 0.14 mm and 1.44 ± 0.112 mm at the 4th and 8th weeks after surgery which is a statistically significant reduction (P < 0.001) in ROI as compared to the control group [Figure 3 and Table 2].

Table 2

Mean of critical-sized defect value of the control group (Group A) and test group (Group B) at 4th and 8th weeks after surgery

	4th weeks (mean value)	8th weeks (mean value)	P
Control (Group A) (mm)	5.23±0.923	4.53±0.76	<0.001
Test (Group B) (mm)	3.54±0.14	1.44±0.112

DISCUSSION

45S5-BG application in alveolar bone defects proved to prevent epithelial migration and promoted alveolar bone regeneration.[9] Biomaterials capable of eliciting specific cellular responses when they come into contact with tissue fluids are considered to be bioactive.[10] Bioactive glass can elicit cellular responses at graft and host tissue interface and has been categorized as Class A material.[11] Sol–gel-synthesized bioglass has higher bioactivity and regenerative potential as compared to conventional melt-derived bioglass of similar composition.[12] Low concentrations of 45S5-BG showed bactericidal properties at concentrations nontoxic to human osteoblasts, making them suitable for periodontal regeneration.[13] The 45S5 BG showed no toxicity against L929 cell line as compared to commercially available bioglass (NovaBone® Dental, USA) at 24, 48 and 72 hour and the percentage of viable cells are more than 70% (Figure 2). Hence, according to the ISO 10993-1:2009 the 45S5 BG is cell compatible.[14]

In vivo studies showed that incorporation of 45S5-BG in CSD resulted in significantly higher VGi in the test group as compared to the control group at 4th and 8th weeks [Figure 3 and Table 1], indicating higher new bone formation at a faster rate in CSD during healing. The test group showed a statistically significant (P < 0.001) [Figure 3 and Table 2] reduction in the CSD size as compared to the control at 4th and 8th weeks, indicating centripetal bone regeneration, which was in accordance with the previous study.[15]

The study showed that the presence of 45S5-BG in CSD caused a faster reduction in the volume of the bone defect. Dissolution products of 45S5-BG will accelerate osteoblast gene expression leading to bone regeneration.[16] Bioglass got bone-bonding property and regenerative cell attachment due to the development of hydroxycarbonate apatite layer when it comes into contact with tissue.[17]

MTT results showed the in vitro cell compatible property of 45S5-BG, and CBCT imaging confirmed new bone formation validating its osteoconductive behavior, demonstrating the potential of 45S5-BG bone graft to be used for alveolar bone regeneration material.

Financial support and sponsorship

The study was funded by the authors.

Conflicts of interest

There are no conflicts of interest.