Abstract
An osteogenic inducer (OI) consisting of dexamethasone, vitamin C, and β-sodium glycerophosphate has the capacity to induce bone formation in vitro. The aim of this study was to assess the efficacy of the application of this OI on extraction socket healing. The bilateral first mandibular premolars were extracted from 75 New Zealand rabbits. Gelatin sponges carrying OI were implanted into the sockets. Sockets undergoing implantation of gelatin sponges alone were also evaluated, as well as non-implantation sockets. Specimens from each group were evaluated radiographically, histologically, and histomorphometrically using haematoxylin–eosin staining. Results showed earlier new bone formation and higher bone quality and quantity in the OI group compared to the other groups, and the differences were significant at 2, 4, 8, and 12 weeks postoperative. The OI significantly reduced the absorption of alveolar bone in terms of height; however, changes in the width were not significantly different between the three groups ( P > 0.05). The OI was shown to have a positive effect on healing of the tooth extraction sockets, was inexpensive, and was convenient to use during the operational procedure; therefore this could represent a promising implant material for human clinical application.
Tooth extraction results in a decrease in alveolar bone volume and density due to the lack of masticatory stimuli. Thus, it may exert a negative effect on subsequent restoration therapy, particularly for implant dentures, which are dependent on the quality and quantity of bone. Furthermore, patients are required to wait for a longer period of time before receiving a denture restoration, which presents aesthetic and functional challenges. A wide range of biological materials have been applied to limit alveolar bone loss and to shorten the healing time after tooth extraction, including allogeneic bone, artificial materials, and growth factors. However, each of these has its limitations, including problems of supply, complex operational procedures, the risk of cross-contamination, and high costs. Furthermore, there are no clear guidelines supported by scientific evidence to indicate the type of biomaterial that should be used. Thus, to compensate for these limitations, it is necessary to find a simple, convenient, and low-cost technique.
Dexamethasone is a popular osteogenic inducer; it has the ability to activate alkaline phosphatase (ALP) in bone marrow mesenchymal stem cells (BMSCs) and to stimulate the formation of calcium deposition and mineralisation nodules. However, high-density dexamethasone prohibits the proliferation of BMSCs and their differentiation into osteoblasts by activating the glucocorticoid receptor on the cell surface. Thus, it is generally acknowledged that the best density range is between 10 −8 and 10 −10 mol/l. Morsczeck et al. have reported that dexamethasone can also induce undifferentiated mesenchymal cells to differentiate into osteoblasts. Vitamin C is known to increase gelatin synthesis, which may result in cell proliferation, formation of calcification, and regulation of ALP activity, thereby initiating calcification and promoting bone formation. Taira et al. reported that there is a synergistic effect between vitamin C and several growth factors, which could increase the effect of other growth factors on cell proliferation. β-Sodium glycerophosphate may provide phosphate ions to osteoblasts, to promote the deposition and calcification in physiological calcium salt and thus accelerate section node calcification.
The combination of dexamethasone, vitamin C, and β-sodium glycerophosphate has been regarded as a typical osteogenic inducer; this has the ability to induce BMSCs to differentiate into osteoblasts in vitro, thereby accelerating bone regeneration. However, the effect of this osteogenic inducer on the healing of tooth extraction sites is unknown. There are many undifferentiated mesenchymal cells in the tooth extraction socket, in addition to a large number of BMSCs, which enter the socket through the blood circulation during the healing process. Thus, the aim of this study was to investigate the effect of this particular osteogenic inducer on alveolar ridge preservation, when applied to fresh extraction sockets in rabbits.
Materials and methods
Animals and experimental methods
This study was approved by the animal care and use committee of the study institution in Luzhou, China. The article was prepared according to the ARRIVE guidelines. Seventy-five male New Zealand White rabbits weighing 2.5–3.0 kg (average 2.8 kg) and aged 8–12 weeks were used in this study; they were kept in individual cages and provided with tap water and food ad libitum. All animals were purchased from the Department of Animal Science Central of Luzhou Medical College (certificate number SYXK (Chuan) 2013-065) and were provided care by experienced and licensed laboratory technicians. They were housed with an inverse 12 h day–night cycle with lights on at 8:00 p.m., in a temperature (22 ± 1 °C) and humidity (55 ± 5%) controlled room. All rabbits had normal physical examination findings prior to their inclusion in the study; thereafter, cone beam computed tomography (CBCT) was performed on the animals. The animals were numbered randomly from 1 to 75.
All rabbits underwent the same surgical procedures under ear marginal vein anaesthesia with 30 mg/kg sodium pentobarbital (Sigma, St. Louis, MO, USA). The bilateral first lower premolars were extracted atraumatically by luxation and adapted forceps, after which the alveolar bone was compressed to reset. For rabbits numbered 1 to 50, a 3 mm × 3 mm × 7 mm absorbable gelatin sponge (Jinling Pharmaceutical Company, Nanjing, China), which conformed to the tooth extraction socket, was soaked with approximately 75 mg of osteogenic inducer and placed into the right side socket (OI group). A neat gelatin sponge was placed into the left side socket (GS group). The sockets of rabbit’s numbered 51 to 75 had no material implanted (NI group). The osteogenic inducer consisted of 10 −8 mol/l dexamethasone (Sanjinshenhe, Sichuan, China), 50 mg/l vitamin C (Kangteneng Pharmaceutical Co. Ltd, Sichuan, China), and 10 mmol/l β-sodium glycerophosphate (Sigma, St. Louis, MO, USA). Overlapping hermetic sutures were performed to protect the operation sites and then CBCT was performed. An intramuscular injection of penicillin (800,000 units three times daily) was administered to all animals for 3 days postoperatively to prevent infection. After 7 days, the animals’ diet was changed from a semi-liquid to a normal diet. Fifteen rabbits (10 of those numbered 1–50 and five of those numbered 51–75) were euthanized by aeroembolism at 1, 2, 4, 8, and 12 weeks after tooth extraction. Their heads were removed and a CBCT was performed immediately.
Radiographic examination
Two CBCT scans were obtained for each animal (CBCT I and CBCT II); one was taken after tooth extraction and the other after euthanasia. All of the tomograms were obtained using a Kodak 9500 CBCT scanner (Carestream Health, Rochester, NY, USA) by the same operator, with the following settings: exposure at 5.0 mA and 120 kV for 9.6 s and axial slice thickness 0.2 mm. Each rabbit head was placed in an upright position in the CBCT machine with a bracket to orient the Frankfort horizontal plane parallel to the floor; a sagittally positioned cursor overlapped the median sagittal plane. The CBCT scans were obtained and analysed by an oral radiologist at the study institution in Luzhou who was blinded to the different treatment groups. Images were processed and analysed using OnDemand3D software (Cybermed, Seoul, Korea). Bone mineral density (BMD) and the alveolar bone width (ABW) and alveolar bone height (ABH) were measured on three different slices. These procedures were repeated three times for each slice. The mean value of the nine measurements was calculated for each socket.
Measurement of the BMD of new bone
The pixel value was used to estimate the BMD, as reported previously. Because all of the graft materials used in this study exhibited radiolucency, the relative size and degree of the radiopaque area following surgery was correlated with the degree of calcification at the extraction site.
Measurements were performed on cross-sectional slices (thickness 1 mm) at 4 mm, 6 mm, and 8 mm above the inferior border of the mandible, representing the apical, median, and coronal third of the socket, respectively ( Fig. 1 A). Using the image tool software, a 5-mm perimeter rectangular region of interest (ROI) was randomly selected from each slice ( Fig. 1 B).
Measurement of the absorption of the alveolar bone width and height
Alveolar absorption reaches a stable stage and bone remodelling is generally completed after 12 weeks of healing. Thus, the absorption of the ABW and ABH was assessed in the rabbits at 12 weeks. The absorption of ABW equals the alveolar width in CBCT I minus that in CBCT II. This was measured on cross-sectional slices (the same as for the measurement of the BMD), and the maximum width on each slice was recorded ( Fig. 1 C). The absorption of ABH equals the alveolar height in CBCT I minus that in CBCT II. The height was measured on three oblique sagittal slices: the buccal tangent plane of the lower second premolar and third premolar roots, the lingual tangent plane of the lower second premolar and third premolar roots, and the middle plane between the former two slices ( Fig. 1 D). The vertical distance, which was parallel to the long axis of the adjacent tooth between the inferior border of the mandible and alveolar apex, was recorded ( Fig. 1 E).
Histological and histomorphometric examination
Specimens were separated individually with a diamond saw and immediately fixed in 10% formaldehyde for 48 h. The samples were demineralized in 10% disodium ethylenediaminetetraacetic acid solution (North Tianyi Chemical Reagent Co. Ltd., Tianjin, China) for 2 weeks, rehydrated, embedded in paraffin (Paraplast; Kendall Healthcare, Mansfield, MA, USA), serially sectioned (5 μm) in the mesiodistal direction, and stained with haematoxylin and eosin. Three non-continuous slices in the median region of the alveolar socket, with a 50-μm interval, were evaluated under a light microscope (Olympus BX50; Olympus Corporation, Tokyo, Japan); images were captured with a digital camera (Canon EOS 60D; Canon Inc., Tokyo, Japan) and processed using Image-Pro Plus 6.0 software (IPP 6.0; Media Cybernetics Inc., Rockville, MD, USA).
For the histomorphometric analysis, five microscopic images were selected at random from each slice and measured an average of three times at 200× magnification. The bone volume fraction, which is the percentage of bone volume to tissue volume (BV/TV), corresponding to the percentage of bone in the area examined, was recorded and compared. The mean value of 15 microscopic images was calculated for each socket. All histological evaluations were performed by the same technician, who was blinded to the treatment conditions.
Statistics
Data were analysed using IBM SPSS Statistics for Windows, version 19.0 software (IBM Corp., Armonk, NY, USA). Categorical variables were presented as the number and percentage, and continuous variables were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with the Student–Newman–Keuls (SNK) comparison test was used to explore the differences in the absorption of the ABW and ABH among the three treatment groups. For the BMD and BV/TV, factorial design ANOVA was used to evaluate the effect of time and treatment group. Time consisted of five levels and the treatment group included three levels (OI, GS, and NI groups). If a significant time by group interaction was found, the values at the same time point were compared using one-way ANOVA with the SNK comparison test. The significance level was set at 0.05.
Results
Clinical results
No infections or other complications occurred during the postoperative phase. Sutures were removed after a 3-week healing period, without any gingival inflammation.
Radiographic analysis
The BMD increased with time in all of the groups ( Table 1 ). Significantly higher BMD values were observed in the sockets receiving osteogenic inducer (OI group) than in those in the GS and NI groups. The results of factorial design ANOVA showed the interaction between the two variables (time and treatment group) to be statistically significant ( F = 2.959, P = 0.004). Therefore, the different treatment factors at the same time point were compared. These results indicated that the BMD in the OI group was significantly higher compared to the other groups at the different time points ( P < 0.05), except at 1 week ( P > 0.05). There was no statistically significant difference between the GS group and NI group ( P > 0.05).
| Time | n | OI group | GS group | NI group | F | P -value |
|---|---|---|---|---|---|---|
| 1 week | 10 | 159.86 ± 9.63 | 156.41 ± 8.22 | 152.93 ± 4.41 | 2.005 | 0.154 |
| 2 weeks | 10 | 211.54 ± 17.36 a | 176.59 ± 10.80 b | 173.16 ± 4.85 | 30.651 | <0.001 |
| 4 weeks | 10 | 256.55 ± 22.00 a | 210.02 ± 14.26 b | 204.74 ± 7.02 | 33.102 | <0.001 |
| 8 weeks | 10 | 348.37 ± 17.06 a | 325.26 ± 20.24 b | 315.40 ± 14.58 | 9.408 | 0.001 |
| 12 weeks | 10 | 510.65 ± 31.15 a | 473.90 ± 38.22 b | 465.72 ± 13.11 | 6.599 | 0.005 |
a Statistically significant difference compared to the GS and NI groups ( P < 0.05).
b No statistically significant difference compared to the NI group ( P > 0.05).
Absorption of the ABW and ABH is shown in Table 2 . The OI group showed the lowest ABW absorption value (0.20 ± 0.10 mm), followed by the GS group (0.22 ± 0.09 mm). However, there were no significant differences in ABW absorption between the three groups ( P > 0.05). Changes in the ABH in the OI group were significantly lower than those in the other two groups ( P < 0.05). The difference in absorption of the ABH between the GS and NI groups was not significant ( P > 0.05).
| Group | n | Absorption of ABW (mm) a | Absorption of ABH (mm) |
|---|---|---|---|
| OI | 10 | 0.20 ± 0.10 | 1.36 ± 0.15 b |
| GS | 10 | 0.22 ± 0.09 | 1.81 ± 0.16 c |
| NI | 10 | 0.23 ± 0.08 | 1.86 ± 0.14 |
| F | 0.286 | 33.481 | |
| P -value | 0.753 | <0.001 |
a No statistically significant differences among the three groups in the absorption of ABW ( P = 0.753).
b Statistically significant difference compared to the GS and NI groups ( P < 0.001).
c No statistically significant difference compared to the NI group ( P = 0.396).
Histological and histomorphometric analysis
Similar results were observed in the GS and NI groups, but much faster new bone formation was observed in the sockets in the OI group compared to the other groups ( Figs 2 and 3 ). The gelatin sponge was only observed in sockets at 1 week. In the OI group, there were numerous fibroblasts and blood vessels in the material gaps at 1 week. In contrast, obvious infiltration of inflammatory cells and partially engorged capillaries were observed in the GS and NI groups. At 2 weeks, the OI group had a large number of fibroblasts and local bone matrix deposition, while fresh granulation tissue including many inflammatory cells and a few fibroblasts were observed in the other groups. At 4 weeks, the sockets of the OI group were completely filled with fibrous tissue and active osteoblasts, and osteoids were observed in some regions. In contrast, more fibrous tissue and dotted bone islands were observed in the other groups. At 8 weeks, the OI sockets were filled with new trabecular bone; some of these sockets had mature lamellar bone. In addition, there were some Haversian canals. In the other groups, the maturity of the new bone was lower. At 12 weeks, the OI sockets were filled with mature bone, which had a high level of calcification. In contrast, the new bone showed a degree of maturation in the GS and NI groups, and lamellar bone was observed in some areas.
