The Effect of Graft Bone Particle Size on Bone Augmentation in a Rabbit Cranial Vertical Augmentation Model: A Microcomputed Tomography Study Kazuhiro Kon, DDS, PhD1/Makoto Shiota, DDS, PhD2/Maho Ozeki, DDS, PhD3/Shohei Kasugai, DDS, PhD4 Purpose: The purpose of this study was to investigate the impact of graft bone particle size on autogenous bone graft augmentation in a vertical augmentation chamber model. Materials and Methods: A total of 12 rabbits were used in this study. The donor bone particles were of different sizes: small (150 to 400 µm), large (1.0 to 2.0 mm), and a mixture comprising equal weights of both large and small bone particles. One type of bone graft material was placed into each of two polytetrafluoroethylene chambers that were implanted in the parietal bone of each rabbit’s cranium. Animals were sacrificed 4 or 8 weeks after the grafting procedure. The recovered samples were analyzed by microcomputed tomography (micro-CT) for quantitative analysis. Total bone volume, bone height, and the distribution of bone structure were calculated by micro-CT. Results: Micro-CT evaluations revealed that the bone grafts performed with large bone particles provided, statistically, the best outcome. Total bone volume and bone height decreased in a time-dependent manner, and there was a statistically significant reduction in total bone volume between 4 and 8 weeks in the group with the mixed bone particle sizes. Conclusion: Within the limitations of the present study, large bone graft particles provided the best preservation of total bone volume and bone height up to 8 weeks after grafting in an animal vertical augmentation model. Int J Oral Maxillofac Implants 2014;29:402–406. doi: 10.11607/ jomi.2804 Key words: animal study, autogenous bone particle size, micro-CT, vertical augmentation graft
W
ithin the past quarter century, dental implants have become reliable treatments for the restoration of missing teeth.1 Complete or partially edentulous patients prefer dental implants because of their predictable and highly satisfying results.2,3 To achieve 1Dental
Resident, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 2Associate Professor, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 3Visiting Lecturer, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 4Professor, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. Correspondence to: Kazuhiro Kon, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Fax: +81-3-5803-5774. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
proper therapeutic outcome, the implant site must have adequate bone volume. However, in many cases, implant placement is affected by alveolar bone resorption after tooth extraction and/or the position of the mandibular canal and maxillary sinus. Therefore, successful bone augmentation procedures are imperative in order to achieve successful restorative implants.4,5 Currently, autogenous bone, allogeneic bone, xenogeneic bone, and synthetic bone are being used as bone grafting materials. Moreover, developments in molecular biology have also allowed the use of specific signal molecules, with or without scaffolds, in regenerative dentistry6,7; currently, the cost of this treatment option is very high. Among the different types of graft materials, autogenous bone is still considered to be the gold standard graft material due to its powerful osteoconductivity and reliable results.8,9 Autogenous bone grafting does have some drawbacks, however. These include, for example, morbidity at the donor site,10 limitations regarding the amount of harvestable bone, and volume reduction of the graft during healing. Volume reduction of the grafted bone is a particular problem during the implant pro-
402 Volume 29, Number 2, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
cedure because it makes implant insertion difficult or warrants additional bone augmentation during the implantation procedure. In long-term follow-up clinical studies, reductions in buccal tissue volumes have been reported after autogenous block bone grafting. This phenomenon was described to be the result of grafted bone resorption.11 Autogenous bone grafts, used for maxillary sinus augmentation, have also been observed to reduce in volume within the first year after surgery.12 The size of bone particle used for augmentation has also been reported to affect outcomes in animal studies, with large-particle, autogenous bone grafts showing better bone augmentation than small-particle grafts.13 However, small-particle grafts showed favorable formation of bone trabeculae. On the basis of these results, mixtures of the two sizes of autogenous bone may provide more effective augmentation by harnessing the advantages of both particle sizes. The purpose of this study was to investigate the volume stability of autogenous bone grafts, composed of either large or small particles or a mixture of both particle sizes, in an experimental model using a vertical augmentation chamber and rabbit cranial bones.
MATERIALS AND METHODS Experimental protocols were approved by the Institutional Committee of Animal Care and Use at Tokyo Medical and Dental University. Japanese male, white rabbits (n = 12), weighing 3.2 to 3.8 kg, were used in this study.
Surgical Procedures
Animals were anesthetized preoperatively with an intramuscular injection of ketamine (50 mg/kg Ketalar, Sankyo) and sodium thiopental (25 mg/kg Rabonal, Tanabe). Each surgical site was shaved and disinfected. In addition, 1.8 mL of a local anesthetic (2% xylocaine/ epinephrine 1:80,000, Dentsply Sankin) was injected into the surgical sites before the start of surgery. Autogenous bone particles of two sizes were harvested from the tibia. The large-particle bone, which was cuboidal in shape (approximately 1 mm thick × 1 mm wide × 2 mm long, standardized by sieves), was harvested with bone forceps. The small-particle bone, which was comprised of bone debris (diameter, 150 to 400 mm; average, 250 mm), was harvested using a 3.2-mm-diameter trephine bone mill system (K-System, Dentak) under saline cooling. Parietal bone was chosen as the augmentation model site. A skin incision and subperiosteal dissection were carried out, sagittally, between the parietal and frontal bone, and the periosteum was raised. Polytet-
rafluoroethylene chambers (hollow cylinders, 5.0-mm inner width and 3.0-mm high, with outer brims) were fixed with two stainless steel screws (FKG Dentaire) to the parietal bone on the right and left sides. Each animal received two chambers. The bone grafts were standardized by weight. In the separate large-particle and small-particle bone groups, 60 mg of bone was grafted. The same weight (30 mg) of large-particle and small-particle bone was mixed to fabricate a mixed bone graft material. These bone grafts were implanted into each chamber, and the skin flaps were sutured with 3-0 silk. During the observation period, all rabbits were given water and a standard rabbit feed ad libitum. Rabbits were sacrificed at 4 weeks (n = 6) and at 8 weeks (n = 6) with a lethal dose of sodium thiopental. The entire cranial bone was removed and fixed for 10 days in neutral 10% formalin.
Microcomputed Tomography Analysis
Following the fixation period, the grafted regions of the parietal bones were quantified via microcomputed tomography (micro-CT) analysis (SMX-90CT, Shimadzu). The total bone volume and bone height were calculated by determining the number of radiopaque voxels (60-μm cubes) observed in each chamber. Tri/3D-Bon software (RATOC System Engineering) was used to make a three-dimensional (3D) reconstruction from the micro-CT scans. From the 3D dataset, a cylindric region of interest (ROI), with a diameter of 5.00 mm and a height of 3.00 mm, which covered the entire inner volume of the experimental chamber, was selected for analysis. The ROI was carefully placed on the experimental chamber. Mask work was performed using binarized (two-color) images of the bone structure in the ROI, according to host bone intensity values. Then, the region representing bone structure in the chamber was extracted and measured. Total bone volume was calculated by radiopaque voxels in the ROI; the bone height was determined from the distance between the basal host bone and the highest point of the bone fragment in the chamber, measured at the center of the 3.00-mm high part of the chamber in each animal.
Statistical Analysis
At each time point, the total bone volume and bone height, between groups, were statistically compared using one-way analysis of variance (ANOVA) and nonparametric multiple comparisons using the Dunnet T3 test. For each type of bone graft, the results of the measured parameters were compared between time points using the Mann–Whitney test. P values < .05 were considered statistically significant. The statistical analyses were performed using a commercial computer program (PASW Statistics, IBM). The International Journal of Oral & Maxillofacial Implants 403
© 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
a
3,000 μm
b
3,000 μm
c
4,000 μm
Fig 1 Grafted site at 4 weeks; extracted bone structure in blue. (a) In the small-particle bone group, the bone structure demonstrated a trabecular shape. (b) In the large-particle bone group, block-shaped bone structure was observed. (c) In the mixed bone group, block- and trabecular-shaped bone structures were observed in the chamber.
a
4,000 μm
b
3,000 μm
c
4,000 μm
Fig 2 Grafted site at 8 weeks; extracted bone structure in blue. (a) In the small-particle bone group, the bone structure volume in the chamber decreased. Host bone was observed at bottom of the chamber. (b) In the large-particle bone group, block-shaped bone structure was observed in the chamber. (c) In the mixed bone group, the bone structure in the chamber decreased in volume; a few block-shaped bone structures were observed in the chamber.
RESULTS All of the rabbits completed the study without any complications; changes in body weight were not observed over the course of the experiments.
Micro-CT Analysis
Radiologic quantitative analysis was performed by micro-CT. In the small-particle bone group, at 4 weeks, the observed bone structure in the chamber had a trabecular shape (Fig 1a). In the large-particle bone group, block-shaped bone was observed; the grafted large-particle bone had retained its shape to this time point (Fig 1b). In the mixed bone group, both blockand trabecular-shaped bone structures were observed in the chamber (Fig 1c). At 8 weeks, in the small-particle bone group, the bone structure observed in the chamber tended to demonstrate a decreased volume, and host bone was observed in the bottom of the chamber (Fig 2a). In the large-particle bone group, block-shaped bone structures continued to be observed in the chamber; the shape was maintained to this time point (Fig 2b). In the
mixed bone group, the bone structure in the chamber tended to demonstrate a decreased volume, and a few block-shaped bone sections were observed (Fig 2c).
Statistical Analysis
There were significant differences in the total bone volume between the small-particle bone/large-particle bone, small-particle bone/mixed bone, and largeparticle bone/mixed bone grafts at 4 weeks (P < .001, P = .03, P = .009, respectively). Similarly, significant differences were observed between the small-particle bone/large-particle bone and large-particle bone/ mixed bone grafts at 8 weeks (P = .005, P = .003, respectively). Within the mixed bone group, there was also a significant difference in the total bone volume between the 4- and 8-week measurements (P = .004) (Fig 3). Bone height also showed significant differences between the small-particle bone/large-particle bone, small-particle bone/mixed bone, and large-particle bone/mixed bone groups at 4 weeks (P < .001, P < .001, P = .007, respectively). At the 8-week time point, only the small-particle bone/large-particle bone
404 Volume 29, Number 2, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
50
***
** **
40
**
**
30 20 10 0
SB
LB 4 wk
***
3
MIX
SB
LB 8 wk
MIX
Bone height (mm)
Total bone volume (mm3)
* ***
** ***
**
**
2
1
0
SB
LB 4 wk
MIX
SB
LB 8 wk
MIX
Fig 3 Total bone volume calculated by micro-CT. A multiple comparison test revealed statistically significant differences. SB = small-particle bone; LB = large-particle bone; MIX = mixed bone. *P < .05, **P < .01, ***P < .001.
Fig 4 Bone height calculated by micro-CT. A multiple comparison test revealed statistically significant differences. SB = small-particle bone; LB = large-particle bone; MIX = mixed bone. *P < .05, **P < .01, ***P < .001.
and large-particle bone/mixed bone groups demonstrated significant differences (P < .001, P = .001, respectively). Within a single bone graft group, only the mixed bone graft showed a significant difference in bone height between 4 and 8 weeks (P = .004) (Fig 4).
ic materials used after the extraction of third molars. These authors demonstrated the potential application of the micro-CT technique, in conjunction with an in vitro analysis system, for characterizing the morphometric features of bone tissue. The authors also contributed to the use of this technique in studies related to biomaterials and bioscaffolds. The vertical augmentation animal model has previously been used to examine the impact of grafted bone particle size on the stability of bone graft volume and height.13 In that study, histologic observation showed that small-particle bone grafts were almost completely replaced by newly formed vital bone; however, large-particle bone grafts were only modestly integrated with newly formed vital bone in 8 weeks. Micro-CT quantitative analysis revealed that volume reductions associated with autogenous small-particle bone grafts were 48.7%, whereas large-particle bone grafts demonstrated only a 3% volume reduction, 8 weeks after the graft. In addition, the reduction in graft height associated with small-particle grafts was 49%, and that associated with large-particle bone grafts was only 2%. Thus, this model has previously been used to show that large-sized bone particles preserve graft height and volume in autogenous bone grafts much better than do small-sized bone particles. In the present study, the experimental performance of large and small bone particles, as well as a mixture of the two types, was evaluated in an animal model of autogenous bone grafts. The results from the mixed bone group demonstrated that there were significant differences between weeks 4 and 8 with regard to the total bone volume and bone height, suggesting that the mixture of autogenous bone particles was resorbed in a time-dependent manner. In contrast, the large-particle bone showed favorable results at both time points, similar to the results of the previously described study. These results suggest that small bone particles might
DISCUSSION In implant dentistry, autogenous bone has been regarded as the gold standard for bone augmentation.2,4 However, with autogenous grafting, the augmented area shows continuous volume reduction during the healing and follow-up periods.10,11 The stability of the grafted bone has also been shown to vary according to the site from which it was harvested. Donovan et al14 reported that the total bone volume of a graft decreased by 60% when the bone graft material was harvested from the iliac bone. However, when calvarial bone was used, the reduction was only 15%. Another study15 reported that total bone volume reduction following a calvarial bone graft volume was 19.2% after a 1-year follow-up period. Johansson et al11 reported that bone grafts harvested from the iliac bone demonstrated a 49.5% total bone volume reduction in inlay sinus grafts and a 47% reduction in onlay buccal bone grafts. Similarly, other studies have highlighted the vertical instability of grafted iliac bone. For example, atrophied mandibles, which received iliac bone grafts, showed a 36% vertical height reduction.16 These studies indicated that calvarial bone is less prone to volume reductions than is iliac bone, suggesting that the donor material may play a role in the long-term success of an implant. Today, structural evaluation of bone by micro-CT is widely accepted in the field of bone and biomaterials, including dental implants.17,18 Meleo et al19 reported on the micro-CT and histologic analysis of bioceram-
The International Journal of Oral & Maxillofacial Implants 405 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
be resorbed by osteoclast-like cells, early in the healing process. Consequently, new bone formation could not proceed rapidly enough to outpace osteoclastic activity, resulting in decreased total bone volume. To overcome the problem of volume reduction, different types of bone substitutes have been studied. Several studies have reported that bovine bone mineral matrix shows a favorable volume preservation ability.8,20 Osteoinductive proteins have also been reported to have favorable effects on bone augmentation. Jovanovic et al21 reported the potential of osteoinductive proteins with an absorbable collagen sponge carrier. A study examining osteoconductive scaffolds, combined with osteoinductive proteins, has also demonstrated that bone regeneration, induced by recombinant human growth/differentiation factor-5 (rhGDF-5) delivered on a β–tricalcium phosphate (β-TCP) carrier, resulted in significantly more bone area in the group being treated with both rhGDF-5 and β-TCP than in those treated with only β-TCP and autogenous bone.22 The current study showed that large-particle autogenous bone grafts are better able to preserve bone volume and height and that a mixture of different sized autogenous bone particles is not effective in maintaining the augmented bone volume.
CONCLUSIONS Within the limitations of the present study, the authors concluded that autogenous large-particle bone grafts have favorable bone augmentation abilities. Mixtures of large and small bone particles used in the bone grafts of this study demonstrated statistically significant bone volume and height reductions at both 4 and 8 weeks after the grafting procedure in an animal vertical augmentation model.
ACKNOWLEDGMENTs The authors reported no conflicts of interest related to this study.
REFERENCES 1. Adell R, Lekholm U, Rockler B, Branemark PI. A 15-year study of osseointegrated implants in the treatment of the edentulous jaw. Int J Oral Surg 1981;10:387–416. 2. Boronat-Lopez A, Carrillo C, Perrocha M, Perrocha-Diago M. Immediately restored dental implants for partial-arch applications: A study of 12 cases. J Oral Maxillofac Surg 2009;67:195–199. 3. Kammeyer G, Proussaefs P, Lozada J. Conversion of a complete denture to a provisional implant-supported, screw-retained fixed prosthesis for immediate loading of a completely edentulous arch. J Prosthet Dent 2002;87:473–476.
4. Proussaefs P, Lozada J, Kleinman A, Rohrer MD. The use of ramus autogenous block grafts for vertical alveolar ridge augmentation and implant placement: A pilot study. Int J Oral Maxillofac Implants 2002;17:238–248. 5. Klijn RJ, Meijer GJ, Bronkhorst EM, Jansen JA. Sinus floor augmentation surgery using autologous bone grafts from various donor sites: A meta-analysis of the total bone volume. Tissue Eng Part B 2010;16:295–303. 6. Young CS, Ladd PA, Browning CF, et al. Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment (TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP). J Control Release 2009;140:250–255. 7. McKay WF, Peckham SM, Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE (R) Bone Graft). Int Orthop 2007;31:729–734. 8. Moy PK, Lundgren S, Holmes RE. Maxillary sinus augmentation— Histomorphometric analysis of graft materials for maxillary sinus floor augmentation. J Oral Maxillofac Surg 1993;51:857–862. 9. Boyne PJ, James RA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980;38:613–616. 10. Barone A, Santini S, Sbordone L, Crespi R, Covani M. Clinical study of the outcomes and complications associated with maxillary sinus augmentation. Int J Oral Maxillofac Implants 2006;21:81–85. 11. Johansson B, Grepe A, Wannfors K, Hirsch JM. A clinical study of changes in the volume of bone grafts in the atrophic maxilla. Dentomaxillofac Radiol 2001;30:157–161. 12. Galindo-Moreno P, Moreno-Riestra I, Avila G, et al. Effect of an organic bovine bone to autogenous cortical bone ratio upon bone remodeling patterns following maxillary sinus augmentation. Clin Oral Implants Res 2011;22:857–864. 13. Kon K, Shiota M, Ozeki M, Yamashita Y, Kasugai S. Bone augmentation ability of autogenous bone graft particles with different sizes: A histological and micro-computed tomography study. Clin Oral Implants Res 2009;20:1240–1246. 14. Donovan MG, Dickerson NC, Hellstein JW, Hanson LJ. Autologous calvarial and iliac onlay bone-grafts in miniature swine. J Oral and Maxillofac Surg 1993;51:898–903. 15. Smolka W, Eggensperger N, Carollo V, Ozdoba C, Iizuka T. Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation. Clin Oral Implants Res 2006;17:149–155. 16. Verhoeven JW, Cune MS, Terlou M, Zoon M, dePutter C. The combined use of endosteal implants and iliac crest onlay grafts in the severely atrophic mandible: A longitudinal study. Int J Oral Maxillofac Surg 1997;26:351–357. 17. Okanoue Y, Ikeuchi M, Takemasa R, et al. Comparison of in vivo bioactivity and compressive strength of a novel superporous hydroxyapatite with beta-tricalcium phosphates. Arch Orthop Trauma Surg 2012;132:1603–1610. 18. Matsumoto G, Hoshino J, Kinoshita Y, et al. Evaluation of guided bone regeneration with poly(lactic acid-co-glycolic acid-co-εcaprolactone) porous membrane in lateral bone defects of the canine mandible. Int J Oral Maxillofac Implants 2012;27:587–594. 19. Meleo D, Bedini R, Pecci R, Mangione F, Pacifici L. Microtomographic and morphometric characterization of a bioceramic bone substitute in dental implantology. Ann Ist Super Sanita 2012;48:59–64. 20. Urban IA, Nagursky H, Lozada JL. Horizontal ridge augmentation with a resorbable membrane and particulated autogenous bone with or without an organic bovine bone–derived mineral: A prospective case series in 22 patients. Int J Oral Maxillofac Implants 2011;26:404–414. 21. Jovanovic SA, Hunt DR, Bernard GW, Spiekermann H, Wozney JM, Wikesjö UM. Bone reconstruction following implantation of rhBMP-2 and guided bone regeneration in canine alveolar ridge defects. Clin Oral Implants Res 2007;18:224–230. 22. Weng D, Poehling S, Pippig S, et al. The effects of recombinant human growth/differentiation factor-5 (rhGDF-5) on bone regeneration around titanium dental implants in barrier membraneprotected defects: A pilot study in the mandible of beagle dogs. Int J Oral Maxillofac Implants 2009;24:31–37.
406 Volume 29, Number 2, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
W
ithin the past quarter century, dental implants have become reliable treatments for the restoration of missing teeth.1 Complete or partially edentulous patients prefer dental implants because of their predictable and highly satisfying results.2,3 To achieve 1Dental
Resident, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 2Associate Professor, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 3Visiting Lecturer, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. 4Professor, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan. Correspondence to: Kazuhiro Kon, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Fax: +81-3-5803-5774. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
proper therapeutic outcome, the implant site must have adequate bone volume. However, in many cases, implant placement is affected by alveolar bone resorption after tooth extraction and/or the position of the mandibular canal and maxillary sinus. Therefore, successful bone augmentation procedures are imperative in order to achieve successful restorative implants.4,5 Currently, autogenous bone, allogeneic bone, xenogeneic bone, and synthetic bone are being used as bone grafting materials. Moreover, developments in molecular biology have also allowed the use of specific signal molecules, with or without scaffolds, in regenerative dentistry6,7; currently, the cost of this treatment option is very high. Among the different types of graft materials, autogenous bone is still considered to be the gold standard graft material due to its powerful osteoconductivity and reliable results.8,9 Autogenous bone grafting does have some drawbacks, however. These include, for example, morbidity at the donor site,10 limitations regarding the amount of harvestable bone, and volume reduction of the graft during healing. Volume reduction of the grafted bone is a particular problem during the implant pro-
402 Volume 29, Number 2, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
cedure because it makes implant insertion difficult or warrants additional bone augmentation during the implantation procedure. In long-term follow-up clinical studies, reductions in buccal tissue volumes have been reported after autogenous block bone grafting. This phenomenon was described to be the result of grafted bone resorption.11 Autogenous bone grafts, used for maxillary sinus augmentation, have also been observed to reduce in volume within the first year after surgery.12 The size of bone particle used for augmentation has also been reported to affect outcomes in animal studies, with large-particle, autogenous bone grafts showing better bone augmentation than small-particle grafts.13 However, small-particle grafts showed favorable formation of bone trabeculae. On the basis of these results, mixtures of the two sizes of autogenous bone may provide more effective augmentation by harnessing the advantages of both particle sizes. The purpose of this study was to investigate the volume stability of autogenous bone grafts, composed of either large or small particles or a mixture of both particle sizes, in an experimental model using a vertical augmentation chamber and rabbit cranial bones.
MATERIALS AND METHODS Experimental protocols were approved by the Institutional Committee of Animal Care and Use at Tokyo Medical and Dental University. Japanese male, white rabbits (n = 12), weighing 3.2 to 3.8 kg, were used in this study.
Surgical Procedures
Animals were anesthetized preoperatively with an intramuscular injection of ketamine (50 mg/kg Ketalar, Sankyo) and sodium thiopental (25 mg/kg Rabonal, Tanabe). Each surgical site was shaved and disinfected. In addition, 1.8 mL of a local anesthetic (2% xylocaine/ epinephrine 1:80,000, Dentsply Sankin) was injected into the surgical sites before the start of surgery. Autogenous bone particles of two sizes were harvested from the tibia. The large-particle bone, which was cuboidal in shape (approximately 1 mm thick × 1 mm wide × 2 mm long, standardized by sieves), was harvested with bone forceps. The small-particle bone, which was comprised of bone debris (diameter, 150 to 400 mm; average, 250 mm), was harvested using a 3.2-mm-diameter trephine bone mill system (K-System, Dentak) under saline cooling. Parietal bone was chosen as the augmentation model site. A skin incision and subperiosteal dissection were carried out, sagittally, between the parietal and frontal bone, and the periosteum was raised. Polytet-
rafluoroethylene chambers (hollow cylinders, 5.0-mm inner width and 3.0-mm high, with outer brims) were fixed with two stainless steel screws (FKG Dentaire) to the parietal bone on the right and left sides. Each animal received two chambers. The bone grafts were standardized by weight. In the separate large-particle and small-particle bone groups, 60 mg of bone was grafted. The same weight (30 mg) of large-particle and small-particle bone was mixed to fabricate a mixed bone graft material. These bone grafts were implanted into each chamber, and the skin flaps were sutured with 3-0 silk. During the observation period, all rabbits were given water and a standard rabbit feed ad libitum. Rabbits were sacrificed at 4 weeks (n = 6) and at 8 weeks (n = 6) with a lethal dose of sodium thiopental. The entire cranial bone was removed and fixed for 10 days in neutral 10% formalin.
Microcomputed Tomography Analysis
Following the fixation period, the grafted regions of the parietal bones were quantified via microcomputed tomography (micro-CT) analysis (SMX-90CT, Shimadzu). The total bone volume and bone height were calculated by determining the number of radiopaque voxels (60-μm cubes) observed in each chamber. Tri/3D-Bon software (RATOC System Engineering) was used to make a three-dimensional (3D) reconstruction from the micro-CT scans. From the 3D dataset, a cylindric region of interest (ROI), with a diameter of 5.00 mm and a height of 3.00 mm, which covered the entire inner volume of the experimental chamber, was selected for analysis. The ROI was carefully placed on the experimental chamber. Mask work was performed using binarized (two-color) images of the bone structure in the ROI, according to host bone intensity values. Then, the region representing bone structure in the chamber was extracted and measured. Total bone volume was calculated by radiopaque voxels in the ROI; the bone height was determined from the distance between the basal host bone and the highest point of the bone fragment in the chamber, measured at the center of the 3.00-mm high part of the chamber in each animal.
Statistical Analysis
At each time point, the total bone volume and bone height, between groups, were statistically compared using one-way analysis of variance (ANOVA) and nonparametric multiple comparisons using the Dunnet T3 test. For each type of bone graft, the results of the measured parameters were compared between time points using the Mann–Whitney test. P values < .05 were considered statistically significant. The statistical analyses were performed using a commercial computer program (PASW Statistics, IBM). The International Journal of Oral & Maxillofacial Implants 403
© 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
Kon et al
a
3,000 μm
b
3,000 μm
c
4,000 μm
Fig 1 Grafted site at 4 weeks; extracted bone structure in blue. (a) In the small-particle bone group, the bone structure demonstrated a trabecular shape. (b) In the large-particle bone group, block-shaped bone structure was observed. (c) In the mixed bone group, block- and trabecular-shaped bone structures were observed in the chamber.
a
4,000 μm
b
3,000 μm
c
4,000 μm
Fig 2 Grafted site at 8 weeks; extracted bone structure in blue. (a) In the small-particle bone group, the bone structure volume in the chamber decreased. Host bone was observed at bottom of the chamber. (b) In the large-particle bone group, block-shaped bone structure was observed in the chamber. (c) In the mixed bone group, the bone structure in the chamber decreased in volume; a few block-shaped bone structures were observed in the chamber.
RESULTS All of the rabbits completed the study without any complications; changes in body weight were not observed over the course of the experiments.
Micro-CT Analysis
Radiologic quantitative analysis was performed by micro-CT. In the small-particle bone group, at 4 weeks, the observed bone structure in the chamber had a trabecular shape (Fig 1a). In the large-particle bone group, block-shaped bone was observed; the grafted large-particle bone had retained its shape to this time point (Fig 1b). In the mixed bone group, both blockand trabecular-shaped bone structures were observed in the chamber (Fig 1c). At 8 weeks, in the small-particle bone group, the bone structure observed in the chamber tended to demonstrate a decreased volume, and host bone was observed in the bottom of the chamber (Fig 2a). In the large-particle bone group, block-shaped bone structures continued to be observed in the chamber; the shape was maintained to this time point (Fig 2b). In the
mixed bone group, the bone structure in the chamber tended to demonstrate a decreased volume, and a few block-shaped bone sections were observed (Fig 2c).
Statistical Analysis
There were significant differences in the total bone volume between the small-particle bone/large-particle bone, small-particle bone/mixed bone, and largeparticle bone/mixed bone grafts at 4 weeks (P < .001, P = .03, P = .009, respectively). Similarly, significant differences were observed between the small-particle bone/large-particle bone and large-particle bone/ mixed bone grafts at 8 weeks (P = .005, P = .003, respectively). Within the mixed bone group, there was also a significant difference in the total bone volume between the 4- and 8-week measurements (P = .004) (Fig 3). Bone height also showed significant differences between the small-particle bone/large-particle bone, small-particle bone/mixed bone, and large-particle bone/mixed bone groups at 4 weeks (P < .001, P < .001, P = .007, respectively). At the 8-week time point, only the small-particle bone/large-particle bone
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Kon et al
50
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40
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30 20 10 0
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Fig 3 Total bone volume calculated by micro-CT. A multiple comparison test revealed statistically significant differences. SB = small-particle bone; LB = large-particle bone; MIX = mixed bone. *P < .05, **P < .01, ***P < .001.
Fig 4 Bone height calculated by micro-CT. A multiple comparison test revealed statistically significant differences. SB = small-particle bone; LB = large-particle bone; MIX = mixed bone. *P < .05, **P < .01, ***P < .001.
and large-particle bone/mixed bone groups demonstrated significant differences (P < .001, P = .001, respectively). Within a single bone graft group, only the mixed bone graft showed a significant difference in bone height between 4 and 8 weeks (P = .004) (Fig 4).
ic materials used after the extraction of third molars. These authors demonstrated the potential application of the micro-CT technique, in conjunction with an in vitro analysis system, for characterizing the morphometric features of bone tissue. The authors also contributed to the use of this technique in studies related to biomaterials and bioscaffolds. The vertical augmentation animal model has previously been used to examine the impact of grafted bone particle size on the stability of bone graft volume and height.13 In that study, histologic observation showed that small-particle bone grafts were almost completely replaced by newly formed vital bone; however, large-particle bone grafts were only modestly integrated with newly formed vital bone in 8 weeks. Micro-CT quantitative analysis revealed that volume reductions associated with autogenous small-particle bone grafts were 48.7%, whereas large-particle bone grafts demonstrated only a 3% volume reduction, 8 weeks after the graft. In addition, the reduction in graft height associated with small-particle grafts was 49%, and that associated with large-particle bone grafts was only 2%. Thus, this model has previously been used to show that large-sized bone particles preserve graft height and volume in autogenous bone grafts much better than do small-sized bone particles. In the present study, the experimental performance of large and small bone particles, as well as a mixture of the two types, was evaluated in an animal model of autogenous bone grafts. The results from the mixed bone group demonstrated that there were significant differences between weeks 4 and 8 with regard to the total bone volume and bone height, suggesting that the mixture of autogenous bone particles was resorbed in a time-dependent manner. In contrast, the large-particle bone showed favorable results at both time points, similar to the results of the previously described study. These results suggest that small bone particles might
DISCUSSION In implant dentistry, autogenous bone has been regarded as the gold standard for bone augmentation.2,4 However, with autogenous grafting, the augmented area shows continuous volume reduction during the healing and follow-up periods.10,11 The stability of the grafted bone has also been shown to vary according to the site from which it was harvested. Donovan et al14 reported that the total bone volume of a graft decreased by 60% when the bone graft material was harvested from the iliac bone. However, when calvarial bone was used, the reduction was only 15%. Another study15 reported that total bone volume reduction following a calvarial bone graft volume was 19.2% after a 1-year follow-up period. Johansson et al11 reported that bone grafts harvested from the iliac bone demonstrated a 49.5% total bone volume reduction in inlay sinus grafts and a 47% reduction in onlay buccal bone grafts. Similarly, other studies have highlighted the vertical instability of grafted iliac bone. For example, atrophied mandibles, which received iliac bone grafts, showed a 36% vertical height reduction.16 These studies indicated that calvarial bone is less prone to volume reductions than is iliac bone, suggesting that the donor material may play a role in the long-term success of an implant. Today, structural evaluation of bone by micro-CT is widely accepted in the field of bone and biomaterials, including dental implants.17,18 Meleo et al19 reported on the micro-CT and histologic analysis of bioceram-
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Kon et al
be resorbed by osteoclast-like cells, early in the healing process. Consequently, new bone formation could not proceed rapidly enough to outpace osteoclastic activity, resulting in decreased total bone volume. To overcome the problem of volume reduction, different types of bone substitutes have been studied. Several studies have reported that bovine bone mineral matrix shows a favorable volume preservation ability.8,20 Osteoinductive proteins have also been reported to have favorable effects on bone augmentation. Jovanovic et al21 reported the potential of osteoinductive proteins with an absorbable collagen sponge carrier. A study examining osteoconductive scaffolds, combined with osteoinductive proteins, has also demonstrated that bone regeneration, induced by recombinant human growth/differentiation factor-5 (rhGDF-5) delivered on a β–tricalcium phosphate (β-TCP) carrier, resulted in significantly more bone area in the group being treated with both rhGDF-5 and β-TCP than in those treated with only β-TCP and autogenous bone.22 The current study showed that large-particle autogenous bone grafts are better able to preserve bone volume and height and that a mixture of different sized autogenous bone particles is not effective in maintaining the augmented bone volume.
CONCLUSIONS Within the limitations of the present study, the authors concluded that autogenous large-particle bone grafts have favorable bone augmentation abilities. Mixtures of large and small bone particles used in the bone grafts of this study demonstrated statistically significant bone volume and height reductions at both 4 and 8 weeks after the grafting procedure in an animal vertical augmentation model.
ACKNOWLEDGMENTs The authors reported no conflicts of interest related to this study.
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4. Proussaefs P, Lozada J, Kleinman A, Rohrer MD. The use of ramus autogenous block grafts for vertical alveolar ridge augmentation and implant placement: A pilot study. Int J Oral Maxillofac Implants 2002;17:238–248. 5. Klijn RJ, Meijer GJ, Bronkhorst EM, Jansen JA. Sinus floor augmentation surgery using autologous bone grafts from various donor sites: A meta-analysis of the total bone volume. Tissue Eng Part B 2010;16:295–303. 6. Young CS, Ladd PA, Browning CF, et al. Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment (TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP). J Control Release 2009;140:250–255. 7. McKay WF, Peckham SM, Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE (R) Bone Graft). Int Orthop 2007;31:729–734. 8. Moy PK, Lundgren S, Holmes RE. Maxillary sinus augmentation— Histomorphometric analysis of graft materials for maxillary sinus floor augmentation. J Oral Maxillofac Surg 1993;51:857–862. 9. Boyne PJ, James RA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980;38:613–616. 10. Barone A, Santini S, Sbordone L, Crespi R, Covani M. Clinical study of the outcomes and complications associated with maxillary sinus augmentation. Int J Oral Maxillofac Implants 2006;21:81–85. 11. Johansson B, Grepe A, Wannfors K, Hirsch JM. A clinical study of changes in the volume of bone grafts in the atrophic maxilla. Dentomaxillofac Radiol 2001;30:157–161. 12. Galindo-Moreno P, Moreno-Riestra I, Avila G, et al. Effect of an organic bovine bone to autogenous cortical bone ratio upon bone remodeling patterns following maxillary sinus augmentation. Clin Oral Implants Res 2011;22:857–864. 13. Kon K, Shiota M, Ozeki M, Yamashita Y, Kasugai S. Bone augmentation ability of autogenous bone graft particles with different sizes: A histological and micro-computed tomography study. Clin Oral Implants Res 2009;20:1240–1246. 14. Donovan MG, Dickerson NC, Hellstein JW, Hanson LJ. Autologous calvarial and iliac onlay bone-grafts in miniature swine. J Oral and Maxillofac Surg 1993;51:898–903. 15. Smolka W, Eggensperger N, Carollo V, Ozdoba C, Iizuka T. Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation. Clin Oral Implants Res 2006;17:149–155. 16. Verhoeven JW, Cune MS, Terlou M, Zoon M, dePutter C. The combined use of endosteal implants and iliac crest onlay grafts in the severely atrophic mandible: A longitudinal study. Int J Oral Maxillofac Surg 1997;26:351–357. 17. Okanoue Y, Ikeuchi M, Takemasa R, et al. Comparison of in vivo bioactivity and compressive strength of a novel superporous hydroxyapatite with beta-tricalcium phosphates. Arch Orthop Trauma Surg 2012;132:1603–1610. 18. Matsumoto G, Hoshino J, Kinoshita Y, et al. Evaluation of guided bone regeneration with poly(lactic acid-co-glycolic acid-co-εcaprolactone) porous membrane in lateral bone defects of the canine mandible. Int J Oral Maxillofac Implants 2012;27:587–594. 19. Meleo D, Bedini R, Pecci R, Mangione F, Pacifici L. Microtomographic and morphometric characterization of a bioceramic bone substitute in dental implantology. Ann Ist Super Sanita 2012;48:59–64. 20. Urban IA, Nagursky H, Lozada JL. Horizontal ridge augmentation with a resorbable membrane and particulated autogenous bone with or without an organic bovine bone–derived mineral: A prospective case series in 22 patients. Int J Oral Maxillofac Implants 2011;26:404–414. 21. Jovanovic SA, Hunt DR, Bernard GW, Spiekermann H, Wozney JM, Wikesjö UM. Bone reconstruction following implantation of rhBMP-2 and guided bone regeneration in canine alveolar ridge defects. Clin Oral Implants Res 2007;18:224–230. 22. Weng D, Poehling S, Pippig S, et al. The effects of recombinant human growth/differentiation factor-5 (rhGDF-5) on bone regeneration around titanium dental implants in barrier membraneprotected defects: A pilot study in the mandible of beagle dogs. Int J Oral Maxillofac Implants 2009;24:31–37.
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