Abstract
The effectiveness of vertical bone augmentation was evaluated in the cranial bone of 15 rabbits using a block of deproteinized bovine bone plus 10% porcine collagen (DBBB) and a cortico-cancellous human bone block (CHBB) with recombinant human bone morphogenetic protein-2 (rhBMP-2) in comparison with a guided bone regeneration (GBR) technique. The rabbits were divided into six groups: DBBB alone, DBBB/rhBMP-2, DBBB/membrane, CHBB alone, CHBB/rhBMP-2 and CHBB/membrane groups. After 12 weeks, the rabbits were killed. The CHBB groups showed higher values than the DBBB groups in terms of vertical height, the area of new bone fill and the maintained grafted area. In the CHBB groups, the CHBB/rhBMP-2 group revealed similar results to GBR. This animal study verifies that a CHBB with rhBMP-2 could be an alternative treatment option for vertical bone augmentation.
Vertical ridge augmentation is necessary for orthodontic treatment and implant placement in widely absorbed alveolar bone areas . Surgical methods for vertical ridge augmentation include autogenous bone block grafting, guided bone regeneration (GBR), distraction osteogenesis, and forced tooth eruption, of which autogenous bone block grafting using bone with osteogenic potential is the most reliable method . It has been reported that in vertical ridge augmentation, 0–25% of the grafted autogenous bone is absorbed , and up to 60% is absorbed after connecting implant abutments . GBR using barrier membranes is effective in vertical ridge augmentation, and has shown long-term safety at the time of implant placement . Z itznam et al. and J ovanovic et al. reported that the incidence of wound dehiscence with the exposure of a barrier membrane was 8–40% and that in cases of exposed barrier membranes, the risk of infection increased, while the amount of regenerated bone decreased .
In cases where non-absorbable barrier membranes are used, secondary surgery is needed for the removal of the membrane. Forced tooth eruption can be performed in a limited number of patients, and distraction osteogenesis causes a high incidence of complications in a narrow area limited to 1 or 2 teeth . Many clinical studies have been carried out with signalling molecules, such as recombinant human bone morphogenetic protein (rhBMP), for inducing bone formation to overcome the drawbacks of the procedures for vertical ridge augmentation . rhBMP is a potent signalling molecule that has induced excellent bone formation in experimental and clinical settings . When used alone, its maintenance and continuous release are extremely difficult , so carriers are needed for its maintenance and release . New surgical procedures using various carriers have been tried , but it is difficult to find clinically applicable carriers that have sufficient mechanical strength for vertical ridge augmentation. Among the conventional bone graft materials, a block of deproteinized bovine bone plus 10% porcine collagen (DBBB) and a cortico-cancellous human bone block (CHBB) can be applied in clinical settings; they have multiporous structures for maintenance and release of BMP, mechanical properties for the maintenance of the space, and the ability to solidify to maintain the configuration. Particulate BMP has been used successfully alone or in combination with absorbable or non-absorbable barrier membranes in clinical conditions such as repair of bone defects, maxillary sinus floor augmentation and alveolar bone augmentation.
This study was conducted to assess the effectiveness of vertical bone augmentation in rabbit calvaria using DBBB and CHBB with rhBMP-2 in comparison with GBR.
Material and methods
15 adult New Zealand White rabbits weighing 3.0–3.5 kg were used in this study. All rabbits were acclimatized to the laboratory conditions for 2 weeks. The graft materials and BMP used in the study are shown in Table 1 .
| Materials | Manufacturer |
|---|---|
| A block of deproteinized bovine bone + 10% porcine collagen (DBBB) | Bio-oss collagen ® , Osteohealth Co., USA |
| A dried cortico-cancellous human bone block (CHBB) | Purous Block ® , Zimmer Dental, Carlsbad, CA, USA |
| rhBMP-2 | Cowellmedi Co, Seoul, Korea |
General anaesthesia was induced by i.m. injection of zoletil (Vibac, France; 0.2 ml/kg). Lidocaine HCl (2% xylocaine) mixed with epinephrine at a dilution of 1:80,000 was injected into the operative site. A 6 mm long, 1 mm deep circular groove was made on each side of the exposed calvarium using a trephin bur (Prugo Co, Seoul, Korea). Eight holes were created within the circular groove using a No. 2 round bur to allow blood flow through the bone marrow. A 6 mm diameter, 4 mm long graft bone block was fixed on each side using 1.0 mm diameter, 6 mm long titanium alloy screws (Ace Surgical Supply Co, USA) ( Fig. 1 a) . The graft material was completely covered with polytetrafluoethylene membrane (Ace Surgical Supply Co, USA) a non-absorbable barrier membrane and was fixed using membrane-fixing pins in the DBBB/membrane and CHBB/membrane groups (Dentium Co, Seoul, Korea) ( Fig. 1 b). The stitches were removed on the tenth postoperative day.
0.5 mg of rhBMP-2 was diluted in 10 ml of injectable distilled water (Jeil Phamaceutical Co, Seoul, Korea) to yield a concentration of 0.05 mg/ml. The DBBB specimens were placed in rhBMP solution for 5 min and were hydrated to the saturated state. A DBBB specimen absorbed approximately 0.12 ml of diluent (6 μg of rhBMP-2 per specimen).
CHBB has a multiporous structure, so the CHBB specimens and diluent were put in a syringe. The plunger of the syringe was pulled to yield negative pressure according to the manufacturer’s instructions, and the specimens were hydrated for 5 min. A CHBB specimen absorbed approximately 0.1 ml of diluent (5 μg of rhBMP-2 per specimen). In the CHBB control group, sterile distilled water (Choog Wae Pharmaceutical Co, Seoul, Korea) was used instead of rhBMP solution.
The right and left calvaria were selected as implantation sites in each rabbit. In the control group, a bone block was applied alone in 5 rabbits (5 implantation sites) for each graft material. In the experimental groups, rhBMp-2 and a barrier membrane were applied in 5 rabbits (5 implantation sites), respectively ( Table 2 ).
| Graft materials | ||
|---|---|---|
| Groups of specimen | DBBB | CHBB |
| Block bone alone (control) | 5 | 5 |
| Block bone/rhBMP-2 | 5 | 5 |
| Block bone/membrane | 5 | 5 |
Histologic preparation and histomorphometric analysis
After the rabbits were sedated with intravenous administration of acepromazine maleate (Samu Median Co, Ltd, Seoul, Korea) at a dose of 2 mg/kg, they were killed by intravenous administration of KCl (Choong Wae Pharmaceutical Co) at a dose of 2 mg/kg. The graft material and the adjacent calvarium were harvested using an 8 mm diameter trephin bur. When the surgical specimens had been fixed with 10% neutral buffered paraformaldehyde (Sigma–Aldrich Co, St. Louis, MO, USA) for more than 1 week, the CHBB specimens were fabricated according to the routine method. The embedded specimens were cut into 400 μm sections using an EXAKT diamond cutter (KULZER EXAKT 300, Germany), with the graft material in the centre. These sections were cut into 30 μm microsections using an EXAKT grinding machine (KULZER EXAKT 400CS). These microsections were stained with haematoxylin and eosin, and the patterns of bone formation were examined under a light microscope. Histomorphometric analysis was performed using SPOT software V 4.9 (Diagnostic Instrument, Inc, USA) under a personal computer-coupled light microscope (Olympus BX, Tokyo, Japan) which was attached to a CCD camera (Polaroid DMC2 Digital Microscope Camera, Polaroid Corporation, Cambridge, MA, USA). The grafted area was examined under a magnification of 12.5× and histomorphometry was performed under a magnification of 40× at non-overlapping sites.
The vertical heights of the residual grafted materials were measured at 12 different sites spaced at equal intervals on each side of the centre of the fixating screw, which were averaged and expressed as the mean ± SD ( Fig. 2 ). The percentages of the area of new bone fill to a given area of 16 mm 2 were measured ( Fig. 3 ).
The percentage of the area of the maintained grafted area to a given area of 16 mm 2 was measured. The maintained grafted area contained the new mineralized tissue, residual graft material and marrow space. The given area was 4 mm in height and 2 mm in width on each side of the centre of the fixing screw ( Fig. 3 ). This area was determined on the basis of the original grafted block dimensions.
Statistical analysis
The data are expressed as the mean ± SD. All statistical analyses were performed using SPSS ver. 12.0 (SPSS Inc, Chicago, IL, USA). Comparisons of the measurements were made using one-way ANOVA between the individual groups. The post hoc test was performed using the Duncan multiple range test. A P value of <0.05 was considered statistically significant.
Results
All groups showed normal patterns of healing with no specific lesions. New bone formation from the calvarium was observed, which was closely connected to the inferior portion of the graft material. In the CHBB control group, a few inflammatory cells were observed. In the DBBB groups, the overall configurations of the graft materials were severely collapsed and the DBBB particles remained unabsorbed around the residual graft materials ( Fig. 4 ). In the CHBB groups, the initial configurations of the grafted area were well preserved, and all of the initial graft materials were absorbed. The grafted area was occupied by new bone and wide bone marrow space ( Fig. 5 ).
The vertical height of the residual graft material
In the DBBB groups, the values of the vertical height of the residual graft material were relatively low except the DBBB/membrane group. In the CHBB groups, the CHBB/membrane group showed the highest values of the vertical height, but the differences were statistically insignificant between the individual groups ( Table 3 ).
| Vertical height | ||
|---|---|---|
| Mean (mm) | SD | |
| DBBB alone (control) | 1.85 | 0.55 |
| DBBB/rhBMP-2 | 1.89* | 0.55 |
| DBBB/membrane | 2.88 † | 0.71 |
| CHBB alone (control) | 4.00 ‡ | 0.34 |
| CHBB/rhBMP-2 | 4.11 ‡ | 0.41 |
| CHBB/membrane | 4.14 ‡ | 0.29 |
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