Ectopic osteogenesis effect of antigen-extracted xenogeneic cancellous bone graft with chitosan/rhBMP-2/bFGF sequential sustained-release nanocapsules.

To explore the ectopic osteogenesis effect of sequential sustained release application of recombinant human bone morphogenic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF). Antigen-extracted xenogeneic cancellous bone coupled with growth factor-loaded chitosan nanocapsules were implanted in rats in intramuscular site in accordance with the following experimental pattern: group A: simultaneous burst release of rhBMP-2 and bFGF; group B: simultaneous sustained release of rhBMP-2 and bFGF; group C: preferential burst release of rhBMP-2, then sustained release of bFGF; group D: preferential burst release of bFGF, then sustained release of rhBMP-2; group E: sustained release of rhBMP-2 alone; group F: sustained release of bFGF alone, blank control group G: antigen-extracted xenogeneic cancellous bone graft only; negative control group H: not filled with anything. Specimens were obtained after executing the animals at 2 and 4 weeks for general observation and weighing, calcium content detection, micro-CT scanning and bone parameter measurement analysis, H&E staining, ALP staining and CD34 staining. The materials weight of A-2, B-2, C-2, A-4, B-4, C-4, D-4 and E-4 were significantly higher than that of preoperative materials ( P < 0.05). The concentration of calcium of group B-4 was the highest (414.7 ± 12.03 mg/dl). Micro-CT scanning and bone parameter measurement analysis showed that the values of bone mineral density and trabecular thickness of group A, B, D, E at 4 weeks were both higher than the ones at 2 weeks ( P < 0.05), and both the bone mineral density (367.52 ± 11.64 mg/cc) and the trabecular thickness (126.17 ± 11.36 µm) of group B-4 were the highest. H&E staining showed that a large region of calcified cartilage and haemopoietic tissues were newly formed, especially in group B-4. ALP staining and CD34 staining showed the most positive expression region in group B-4. Therefore, we conclude that simultaneous sustained release of rhBMP-2 and bFGF is the ideal way to release drug, and has better inducement of antigen-extracted xenogeneic cancellous bone graft.

Case History Report: Cone Beam Computed Tomography for Implant Insertion Guidance in the Presence of a Dense Bone Island.

This article describes the use of cone beam computed tomography (CBCT) to diagnose a dense bone island (DBI) to facilitate implant insertion guidance in a patient followed up for 4 years. Suitable image-directed preplanning and periodic review by CBCT scanning is recommended when a jaw DBI is encountered in treatment planning for implant placement.

Synergistic effect of RhBMP-2 and bFGF on ectopic osteogenesis in mice.

OBJECTIVE: To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF). METHODS: 24 KM male mice were randomly divided into 6 groups with 4 mice in each group, namely, Group A (control group), Group B (only treated with collagen), Group C (treated with 2 ng bFGF+collagen), Group D (treated with 4 µ g rhBMP-2+collagen), Group E (treated with 4 µ g rhBMP-2+2 ng bFGF+collagen) and Group F (treated with 4 µ g rhBMP-2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro-CT), three-dimensional reconstruction scan, measurement of bone mineral density (BMD), bone volume fraction (BVF) and trabecular thickness (Tb.Th), as well as histological observation with HE staining and ALP and CD34 immumohistochemical staining were performed. RESULTS: Ectopic osteogenesis was found in Groups D, E and F mice. The difference in concentration of calcium contents was statistically significant between Groups D and E (P<0.05), but insignificant between Groups E and F (P>0.05). Micro-CT and three-dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance. The differences in BMD, BVF and Tb.Th were statistically significant between Groups D and E or F (P<0.01 or <0.05). HE staining showed that in Groups D, E and F, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation in Groups E and F was better than that in Group D. ALP and CD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positive expression in Groups E and F was larger than that in Groups D. CONCLUSIONS: rhBMP-2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.

Antigen-extracted xenogeneic cancellous bone graft with recombinant human bone morphogenetic protein-2 enhances bone regeneration in repair of mandibular defect in rabbits.

The effects of a large-piece of xenogeneic bone that was separated from healthy pigs as a scaffold for the repair of a mandibular defect was investigated, and the applicability of antigen-extracted xenogeneic cancellous bone (AXCB) soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) in bone defect repair was assessed. Mandibular defects were created in 48 New Zealand rabbits, and the animals were randomly divided into four groups, in which the mandibular defects were grafted with AXCB, AXCB soaked with rhBMP-2, and autograft bone, or left blank. An equal number of animals from each group were classified into three time points (4, 8, and 12 weeks) after surgery for gross pathological observation, hematoxylin and eosin (H and E) staining, radiographic examination, and bone density measurement. H and E staining revealed that the area percentage of bone regeneration in the group of the AXCB/rhBMP-2 graft was 27.72 ± 4.68, 53.90 ± 21.92, and 77.35 ± 9.83 at 4 weeks, 8 weeks, and 12 weeks, respectively. These results were better than those of the autogenous bone graft, suggesting that the group of the AXCB/rhBMP-2 graft achieved a good osteogenic effect. With regard to the AXCB graft without rhBMP-2, the area percentage of bone regeneration was only 14.03 ± 5.02, 28.49 ± 11.35, and 53.90 ± 21.92. Therefore, the osteogenic effect of the AXCB/rhBMP-2 graft was demonstrated to have the best effect. In the group of the AXCB/rhBMP-2 graft, the area percentage of bone regeneration increased, and the implanted materials were gradually degraded and replaced by autogenous bone regeneration over time. We conclude that the AXCB graft soaked with rhBMP-2 showed good osteogenic effect in the repair of bone defects and good biocompatibility. AXCB serves as a good carrier of rhBMP-2, which promotes bone formation.

Effect of rhBMP-2 sustained-release nanocapsules on the ectopic osteogenesis process in Sprague-Dawley rats.

OBJECTIVE: To explore the effect of sustained-release recombinant human bone morphogenetic protein-2 (rhBMP-2) on ectopic osteogenesis in the muscle pouches of rats through preparing rhBMP-2 sustained-release capsules by wrapping morphogenesis protein bones-2 (BMP-2) using chitosan nanoparticles, and compositing collagen materials. METHODS: Twenty four Sprague-Dawley rats were randomly divided into four groups with six rats in each group, that is Group A (control group), Group B (only treated with collagen), Group C (rhBMP-2+collagen treated group) and Group D (rhBMP-2/cs+collagen treated group). The composite materials for each group were implanted in the bilateral peroneal muscle pouches in rats. The peroneal muscles were only separated without implanting any materials in control group. Rats were sacrificed 2 weeks and 4 weeks post treatment and samples were cut off for general observation, Micro CT scans and histological observation. RESULTS: General observation showed no new bone formation in Groups A and B mice, while new bones were formed in Groups C and D mice. Two weeks after treatment Micro CT scans showed that The bone volume fraction (BVF), trabecular thickness (Tb.Th), bone mineral density (BMD) in Group C mice were all higher than that in Group D (P<0.05). At the fourth week, the BVF, Tb.Th and BMD were significantly higher than that at the second week (P<0.01). CONCLUSIONS: The slow-release effect of rhBMP-2/cs sustained-release capsules can significantly promote ectopic osteogenesis. Its bone formation effect is better than that of rhBMP-2 burst-release group.

Accelerating bone generation and bone mineralization in the Interparietal sutures of rats using an rhBMP-2/ACS composite after rapid expansion.

This study aims to investigate the effects of rhBMP-2/ACS composite on bone regeneration and mineralization during expansion of the interparietal suture in rats. Forty 10-week-old Sprague-Dawley rats were divided into four groups (n=10). The first group (intact group) did not receive any intervention. The second group (expansion control group) received an expansion force of 60 g. The remaining two groups received an expansion force of 60 g and were implanted with an atelo-type I absorbable collagen sponge and rhBMP-2/ACS composite positioned on the suture beneath the periosteum. The relapse, relapse ratio, relevant bone remodelling, and calcium and osteocalcin contents were evaluated. Bone regeneration in the interparietal suture was estimated by the histological method. The osteocalcin content was measured by radioimmunoassay, and the calcium content was measured by atomic absorption spectrophotometry. Bone regeneration was more active in the suture after application of the expansion force compared with that of the suture without any intervention. Bone bridges formed in the rhBMP-2/collagen composite group. Both osteocalcin and calcium content were higher in the rhBMP-2/collagen composite group than in the other three groups (P<0.01). The relapse ratio in the rhBMP-2/collagen group was much lower than that in the other two expansion groups (P<0.01). RhBMP-2/ACS composite can promote bone regeneration and bone mineralization in the expanded suture and decrease the relapse ratio. Thus, the rhBMP-2/ACS composite may be therapeutically beneficial to the inhibition of relapse and shortening of the retention period during rapid expansion.

Application of ultrasonic inspection in monitoring dynamic healing of mandibular fracture in rabbit model.

OBJECTIVE: To investigate the feasibility of ultrasonic diagnosis for monitoring fracture healing. METHODS: Thirty rabbit models with fraction of mandible body were established by surgically removing partial lower jawbone. At the 1st, 2nd, 4th, 6th, 8th and 12th week after the operation, they were examined by X-ray and ultrasound, respectively. All detection results were scored according to a generally accepted standard. Spearman rank correlation analysis was conducted to explore the relationship between the results of the two inspection methods. RESULTS: In each healing stage, the results of the ultrasonic inspection were basically consistent with those of the X-ray examination, as supported by a Spearman rank correlation coefficient of 0.892 (P<0.001). CONCLUSIONS: Non-invasive ultrasonic inspection can be used instead of X-ray examination to monitor and diagnose fracture healing.

[Study on osteogenic ability of chitosan/beta-tricalcium phosphate scaffold combined with human bone morphogenetic protein].

OBJECTIVE: Using chitosan (CS)/beta-tricalcium phosphate (TCP)/recombinant human bone morphogenetic protein (rhBMP)-2 for the reconstruction of rabbits' mandible defect, to prove the feasibility of CS/beta-TCP as an injectable bone tissue engineering scaffold material. METHODS: Twenty-four New Zealand white rabbits were randomized into 4 groups on average: Experimental group 1 embedding CS/beta-TCP/rhBMP-2, experimental group 2 embedding CS/ beta-TCP, control group 1 embedding autograft bone group, control group 2 embedding nothing. At 2, 4 and 8 weeks after surgery, all rabbits were executed group by group. The new bone growth situations were observed with hematoxylin-eosin staining and immunofluorescence microscopy, the bone mineral density was detected by bone sonometers. RESULTS: After 2, 4, 8 weeks, there was significant difference among the areas of bone regeneration of all groups. The effect of experimental group 1 was better than experimental group 2. There was significant difference at different times, the areas of bone regeneration was gradually increased with time. The area of stained yellow in experimental group 1 was larger, the area of stained red was smaller. The quantities of bone density in experimental group 1 at every time after surgery were significantly higher than experimental group 1 and control group 2, but had no statistical significance with control group 1. CONCLUSION: CS/beta-TCP/rhBMP-2 has good biocompatibility, degradability and the capacity of guided and inducing osteogenesis. CS/beta-TCP as a good injection of carrier could become a promising carrier for rhBMP-2 and potential new degradable biological material for repairing bone defect in clinical application.

[Pulsed Nd:YAG laser-aided debonding for removing the metal brackets].

OBJECTIVE: To explore the possibility of pulsed Nd:YAG laser-aided debonding for removing orthodontic metal brackets and to compare the method with the conventional mechanical debonding method. METHODS: Fifty healthy premolars extracted for orthodontic purpose were randomly divided into five groups (10 teeth in each group). There were four experimental groups and one control group. Every tooth was bonded with bracket. Laser was used to irradiate the teeth in experimental groups with different electric currents (13.0, 13.5, 14.0, 14.5 A). During the irradiation, the brackets received 4.9 N of force until the brackets off. The time needed for debonding and the temperature change of the pulp cavity were recorded. The teeth in control group were debonded using mechanical method. The adhesive remnant index (ARI) was calculated using stereomicroscope and imagetool software. All samples were examined with a scanning electron microscope. RESULTS: The time taken between the four groups were (67.70 ± 7.18), (35.90 ± 4.28), (24.90 ± 3.76), (6.90 ± 2.33) s, highly statistical difference was found in the time needed for debonding (P < 0.01). The temperature in the pulp cavity among the four groups were (20.97 ± 3.10), (12.75 ± 3.14), (8.99 ± 2.47), (2.91 ± 1.88)°C, and statistical differences were found in temperature change of the pulp cavity (P < 0.05). ARI of three experimental groups and the control group were (8.55 ± 5.02)%, (15.42 ± 7.37)%, (5.55 ± 3.79)%, (13.72 ± 6.69)%, and (74.36 ± 29.44)%. The enamel surface of the control group was coarse with deep scratchs. The enamel surface was smooth and clean in the experimental groups. CONCLUSIONS: Pulsed Nd:YAG laser-aided debonding for removing metal brackets was feasible. Laser-aided debonding was better than conventional mechanical debonding method. The method reduced the damage to the enamel surface.

Applied anatomic site study of palatal anchorage implants using cone beam computed tomography.

AIM: The purpose of this study was to conduct quantitative research on bone height and bone mineral density of palatal implant sites for implantation, and to provide reference sites for safe and stable palatal implants. METHODOLOGY: Three-dimensional reformatting images were reconstructed by cone beam computed tomography (CBCT) in 34 patients, aged 18 to 35 years, using EZ Implant software. Bone height was measured at 20 sites of interest on the palate. Bone mineral density was measured at the 10 sites with the highest implantation rate, classified using K-mean cluster analysis based on bone height and bone mineral density. RESULTS: According to the cluster analysis, 10 sites were classified into three clusters. Significant differences in bone height and bone mineral density were detected between these three clusters (P<0.05). The greatest bone height was obtained in cluster 2, followed by cluster 1 and cluster 3. The highest bone mineral density was found in cluster 3, followed by cluster 1 and cluster 2. CONCLUSION: CBCT plays an important role in pre-surgical treatment planning. CBCT is helpful in identifying safe and stable implantation sites for palatal anchorage.

[Evaluation of the implant sites of palatal implants using cone beam computed tomography].

OBJECTIVE: To investigate the vertical bone height and the bone density of the palate for implants placement using cone beam CT(CBCT) and to provide references to the safe and stable placement of palatal implants. METHODS: Three-dimensional reformatting images were reconstructed with the selected CBCT scanning data of 34 patients aged 18 to 35 yeras, by means of EZ implant software. The vertical bone height was measured at 20 interesting sites of palate. Bone density was measured at 10 sites that could support 3.0 mm long implants. The data of the vertical bone height and bone density were analyzed by K-means cluster analysis. RESULTS: According to the cluster analysis results, the 10 sites were classified into 3 clusters. There were statistical differences among these three clusters in bone height and bone density (P < 0.05). The LSD result showed that the greatest mean value of vertical bone height was obtained in cluster 2, followed by cluster 1 and cluster 3; the highest bone density was founded in cluster 3, followed by cluster 1 and cluster 2. CONCLUSIONS: Evaluation of the sites for palatal implant placement with cone beam CT would be helpful in safe and stable implantation.