Minoru Sanda Makoto Shiota Masaki Fujii Kazuhiro Kon Tatsuya Fujimori Shohei Kasugai
Capability of new bone formation with a mixture of hydroxyapatite and beta-tricalcium phosphate granules
Authors’ affiliations: Minoru Sanda, Makoto Shiota, Masaki Fujii, Kazuhiro Kon, Tatsuya Fujimori, Shohei Kasugai, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Key words: animal experiments, biomaterials, bone substitutes, material sciences
Corresponding author: Minoru Sanda Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo, Japan Tel.: +81 35 803 5774 Fax: +81 35 803 5774 e-mail: [email protected]
of the hard tissues volume, new bone formation, and residual graft after 4 and 8 weeks.
Abstract Objectives: The aim of this experimental study was to test a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) granules inserted in cranial defects in rabbits, by the evaluation Material and methods: Two defects of 8 mm diameter were created at the calvarial bone of 24 Japanese white rabbits for a total of 48 defects. Four groups were created: defects filled with a mixture of HA and beta-TCP granules (test A), defects filled with HA alone (test B), defects filled with beta-TCP (test C), and empty defects (control). Hard tissues volume (remaining graft + new bone) was evaluated by l-CT and new bone (NB) and remaining graft (RG) percentages were evaluated by histomorphometry. The animals were sacrificed at 4 or 8 weeks postoperatively. Results: The test groups A, B, and C showed a significant higher total volume compared with controls at 4 and 8 weeks (P < 0.05). Regarding the percentages of NB and RG at 4 and 8 weeks, no significant differences were detected (P > 0.05). When comparing 4 and 8 weeks, test group A showed a significant increase in new bone formation. At both 4 and 8 weeks, no group showed significant differences in NB (P > 0.05). At 8 weeks, test group B had more RG than test group A. Conclusions: The novel mixture could maintain the volume of the grafted area compared with that with intervention, and in a similar way compared with HA.
Date: Accepted 24 July 2014 To cite this article: Sanda M, Shiota M, Fujii M, Kon K, Fujimori T, Kasugai S. Capability of new bone formation with a mixture of hydroxyapatite and beta-tricalcium phosphate granules. Clin. Oral Impl. Res.00, 2014, 1–6 doi: 10.1111/clr.12473
Endosseous dental implants have become one of the most predictable and satisfactory prosthetic treatment alternatives (Albrektsson et al. 1981, 1986; van Steenberghe et al. 1990; Buser et al. 1997). Some patients have an inadequate volume of the alveolar ridge for implant placement or esthetic outcome, therefore requires augmentation procedures prior to implantation. Although autograft is still the gold standard of graft material (Boyne & James 1980), it has certain disadvantages, including donor-site morbidity and limited graft availability. Consequently, synthetic materials have an important role in augmentation procedures (Moore et al. 2001). In terms of synthetic material, hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) are routinely used as bone substitutes in the field of implant dentistry. As HA is scarcely resorbed, the volume of the augmented site can be maintained, but few granules can be replaced by new bone (Ara ujo et al. 2009). Beta-TCP, on the other hand, has higher degradability than HA; therefore, new
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
bone replacement can be expected, but it can be difficult to maintain the volume of the grafted site. To benefit from the advantages of both, some manufacturers and researchers have developed biphasic calcium phosphate (BCP) made from HA and beta-TCP, which has shown some efficacy in several studies (Nery et al. 1992; Zerbo et al. 2004; Lee et al. 2008). However, the effects of the mixture of monophasic granules of HA and beta-TCP are not well documented. Therefore, the purpose of the present experimental work was to test a conventional mixture of HA and beta-TCP granules by the evaluation of the volumetric changes by lCT and the new bone formation and residual graft by histomorphometric analysis.
Material and methods Experimental protocols were approved by the Institutional Committee of Animal Care and Use at Tokyo Medical and Dental University
1
Sanda et al Osteoconductivity of mixture of HA/beta-TCP
(Approval No. 0130230A). We used the following four groups with six samples each: (i) mixture of HA granules and beta-TCP granules in a volume ratio of 1 : 1 (test group A), (ii) pure HA particles (test group B), (iii) pure beta-TCP granules (test group C), and (iv) no graft (control group). All substitutes were fabricated by the same company (BrainBase Co., Tokyo, Japan). One hundred percent purity of both ingredients was verified by X-ray diffraction analysis, with sintering at 1100°C. Both granules have an average particle size of 450 lm (range, 250–1000 lm), which is a cluster of 50-lm particles. The particles of both ingredients contain macropores with an average size of 50 lm (range, 10–100 lm) and 2-lm micropores inside, which are interconnected. Surgical procedure
Twenty-four Japanese male white rabbits weighing 3.0–3.5 kg were employed. Animals were anesthetized preoperatively with an intramuscular injection of 50 mg/kg ketamine (Ketalar; Daiichi Sankyo Inc., Tokyo, Japan) and 25 mg/kg thiopental sodium (Rabonal; Tanabe, Inc., Tokyo, Japan). The surgical area was shaved and disinfected. Then, 1.8 ml of local anesthetic (2% xylocaine/epinephrine 1 : 80,000; Dentsply Sankin, Tokyo, Japan) was injected into the surgical site. The parietal bones were selected as the experimental site. Skin incision and subperiosteal dissection were carried out sagittally between the parietal and frontal bones, and the periosteum was raised. Two noncritical-sized bone defects, each with a diameter of 8 mm, were prepared on both sides of the parietal bone. Six samples each were randomly selected for the three test groups and the control group. Depending on the thickness of the calvarial bone, 0.07–0.1 g of bone substitute was placed into the defect, so that the entire defect was filled and contoured similar to the original bone. During the observation period, all rabbits were given water and standard rabbit feed. Animals were sacrificed at 4 or 8 weeks after surgery with a lethal dose of intravenous thiopental sodium. The entire cranial bone was removed and fixed for 15 days in neutral 10% formalin.
and 3 mm height. Then, the volume of hard tissue including new bone and bone substitute remnants inside the defect was quantified by an analyzing application (TRI/3D BON; RATOC System Engineering Co., Ltd., Tokyo, Japan). Using this software, calculation of hard tissue volume was carried out by computing the radiopaque substance exceeding the constant threshold thought to be the boundary of hard tissue and soft tissue, as determined from histologic analysis. Total volume was calculated by the radiopaque voxels observed in the grafted site. Histologic processing
Samples were dehydrated in ascending grades of ethanol, embedded in acrylic resin (Technovit 7200; Heraeus Kulzer Japan Co., Ltd., Japan), and light cured. Consequently sagittal sections were obtained by the sawing and grinding technique (Exakt; Mesmer, Ost Einbeck, Germany) and stained with the Villanueva Stain. Histologic observations were performed under a light microscope (Biozero; Keyence Corporation of America, Itasca, IL, USA). Histomorphometric analysis
Area ratio of new bone (NB) and residual graft (RG) in the defect area were calculated. Area of the entire defect, new bone, and residual graft were measured using graphic software (Photoshop CS6 Extended software Adobe Systems Incorporated, San Jose, CA, USA). New bone and residual graft ratios were calculated using the following formulas:
new bone ratio = (area of new bone)/(area of the entire defect); residual graft ratio = (area of residual graft)/(area of the entire defect). Statistical analysis
Comparison between the four groups at the same time point regarding new bone formation, residual graft, and micro-CT data was performed using Dunnett’s T3 test. Comparisons between 4 and 8 weeks in each kind of substitute were performed using the Mann– Whitney U-test. All statistical analyses were performed using SPSS Statistics 21 (IBM Japan, Ltd., Tokyo, Japan), with P values of 0.05). At 8 weeks, test group A showed the highest mean value followed by the control group, test group C, and test group B. Residual graft
Fig. 2. Radiolographic hard tissue volume in each group at 4 and 8 weeks. Significant differences were detected between the control group and three test groups at both 4 and 8 weeks. There were no significant differences between the three test groups at each time points. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP; control, empty defect.
Histomorphometric analysis New bone formation
At 4 weeks, percentage of new bone formation was 15.7% 4.80% in test group A, 21.0% 5.60% in test group B, 19.9% 10.15% in test group C, and 16.3% 3.56% in the control group (Fig. 3). At 8 weeks, the percentages of new bone formation were 28.2% 9.95% in test group A, 18.3%
At 4 weeks, the percentage of residual graft was 57.2% 13.53% in test group A, 52.8% 12.28% in test group B, and 45.4% 13.42% in test group C (Fig. 4). At 8 weeks, the percentages were 43.1% 12.64% in test group A, 63.3% 9.02% in test group B, and 36.9% 20.93% in test group C. The mean value was highest in test group A followed by the test groups B and C at 4 weeks. However, there was no significant difference in this value among the three test groups. At 8 weeks, the mean value was the highest in test group B followed by the test groups A and C. The difference was significant only between test groups A and B. Histomorphologic analysis
Beta-TCP particles appeared as collapsed and diffused in both test groups A and B. Such
Table 2. Histological evaluation parameters (mean, median, and standard deviation in %) of new bone formation for every group and time point 4 weeks
Test A (n = 6) Test B (n = 6) Test C (n = 6) Control (n = 6) P-value1
8 weeks
N
Mean
Median
SD
N
Mean
Median
SD
P-value2
6 6 6 6
15.67 21.04 19.94 16.26
16.30 18.88 19.44 15.16 0.413
4.80 5.60 10.15 3.56
6 6 6 6
28.16 18.27 21.78 26.77
26.47 16.15 21.77 26.48 0.269
9.95 7.60 10.72 9.08
0.026 0.485 0.699 0.065
Test A, mixture of HA and beta-TCP; test B, pure HA; test C, pure beta-TCP; control, empty; SD, standard deviation. Area ratio of new bone inside the grafted area in each group at both 4 weeks and 8 weeks. P-value 1: computed between 4 groups in each time point P-value 2: computed between two healing periods.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
degradation was more remarkable at 8 weeks than at 4 weeks (Figs 5–8). Beta-TCP granules degraded to form smaller particles. At 4 weeks, the granules seemed degraded, while at 8 weeks, granules had disappeared to some extent. In some samples, the granules were completely encapsulated by soft tissue, and there was no new bone replacement inside the defect. On the other hand, HA particles showed slight or almost no morphologic changes and retained their form at both 4 and 8 weeks. HA particles were not completely embedded inside the new bone, whereas many betaTCP particles were involved completely inside the bone tissue. In addition, test group B had lesser soft tissue inside the grafted site than that in the other groups. In the control group, new bone formation in the defect area was detected, especially in the later time point. However, the thickness of the new bone is thinner compared with the original one. At 4 weeks, histologic analysis showed no organized lamellated appearance was detected at 4 weeks. At 8 weeks, well-organized lamellated structures were identified. This finding implies progress in the maturation of new bone inside the defect.
Discussion Biphasic calcium phosphate material has been used as a synthetic bone substitute for 20 years. Many studies have proved its validity, which is attributed to the synergistic effect that stable HA maintains the volume of the grafted site and soluble beta-TCP is replaced by new bone. Unlike the biphasic material, the present study used a simple mixture of HA and beta-TCP granules, expecting the same synergistic effect as BCP. The advantages of this material are the simplicity of the combining procedure and the ease of regulating the ratio of the two ingredients. Experimentally, we can separately observe the behavior of each granule type in vivo; however, this is difficult to distinguish in biphasic material. Hydroxyapatite particles were resorbed to a less extent, as expected. Therefore, new bone was formed in the spaces between the HA particles, but all the HA particles were not replaced by new bone. These results are consistent with those other reports that proved HA is nonresorbable. This nature of HA might inhibit soft tissue invasion into the augmented site. In histologic analysis, test groups A and B showed lesser soft tissue ingrowth
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Sanda et al Osteoconductivity of mixture of HA/beta-TCP
Table 3. Histological evaluation parameters (mean, median, and standard deviation in %) of residual graft for every test group and time point 4 weeks
Test A (n = 6) Test B (n = 6) Test C (n = 6) P-value1
8 weeks
N
Mean
Median
SD
N
Mean
Median
SD
P-value2
6 6 6
57.15 52.83 45.43
57.33 53.98 44.49 0.320
13.53 12.28 13.42
6 6 6
43.07 63.28 36.94
45.18 60.50 30.55 0.021
12.64 9.02 20.93
0.180 0.240 0.394
Test A, mixture of HA and beta-TCP; test B, pure HA; test C, pure beta-TCP; SD, standard deviation. Area ratio of residual graft detected inside the defect in three test groups at both 4 weeks and 8 weeks. P-value 1: computed between 4 groups in each time point P-value 2: computed between two healing periods.
into the defect than the TCP group. The region among particles can also affect the extent of soft tissue migration. In a study of BCP use in a dog mandible, Kim et al. (2011) found similar results that the nonresorbable HA part inhibited soft tissue ingrowth into the grafted site.
Fig. 3. Area ratio of new bone inside the grafted area. There were no significant differences between the groups at both 4 and 8 weeks. In comparison between each time point, only test group A shows significant increase in new bone. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP; control, empty defect.
In contrast, beta-TCP granules were biodegradable, predominantly at 8 weeks, whereas HA particles were not. These findings support Koerten and van der Meulen’s experimental findings (1999), which showed betaTCP was more biodegradable than HA and fluorapatite in mice. Lu et al. (2002) also reached the same conclusion in a rabbit tibia model. However, Handschel et al. (2002) reported that there was no resorption of TCP granules grafted in a rat calvarial defect model and that the control group with no substitute showed more new bone formation on histologic analysis. In our experiment, some samples had no beta-TCP granule degradation. Our results suggest that beta-TCP granules have great variability of new bone formation. Histologic analysis showed great variability in soft tissue invasion and new bone formation. von Arx et al. (2001) also reported on guided bone regeneration in a dog mandible and noted that sites augmented with TCP granules and covered with expanded polytetrafluoroethylene membrane exhibited very inconsistent findings with regard to ridge contour and new bone formation. This variability may be
(a)
(b)
Fig. 4. Area ratio of residual graft detected inside the defect. There was a significant difference between test groups A and B at 8 weeks. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP.
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attributed to the quantity of blood supply, the site that is grafted, and the difference in potential for new bone formation of each animal. Using micro-CT analysis, the three substitute groups were compared with respect to volume of hard tissue including bone substitute and new bone formation inside the defect. Although the mean value was the highest in the HA group and the lowest in the TCP group, a significant difference was not detected. Although the ratio of residual graft, as detected using histomorphometric evaluation, in test groups A and B was not significantly different at 4 weeks, the ratio was significantly higher in test group B than in test A group at 8 weeks. This may be because the resorbable part (beta-TCP granules) that was present in test group A had disappeared during the period between 4 and 8 weeks. However, the percentage of beta-TCP remnants was not significantly lower, although we expected that the granules would disappear due to their biodegradability; this may have occurred because of beta-TCP resorption. Lu et al. (1998) and Zerbo et al. (2005) analyzed beta-TCP’s biodegradability and reported that degradation of beta-TCP occurs mainly by chemical dissolution by tissue fluids but the effect by osteoclast. Therefore, beta-TCP granules degraded into the small parts at first; that is, original particles divided into smaller ones and subsequently disappeared. In the present study, the divided particles spread among the newly formed tissue, including both soft and hard tissues, so there was little reduction in their volume, and therefore, no significant differences were observed in the micro-CT results. However, the volume of the grafted
(c)
Fig. 5. Histomorphologic analysis of test group A (mixture of beta-TCP and HA) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). New bone (NB) can be observed between the cranial bone (CB). At a higher magnification, the 8-week samples show typical differences between degraded beta-tricalcium phosphate (beta-TCP) and nondegraded hydroxyapatite (HA) granules (c) (original magnification 920).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Sanda et al Osteoconductivity of mixture of HA/beta-TCP
(a)
(b)
(c)
Fig. 6. Histomorphologic analysis of test group B (HA only) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). New bone (NB) can be seen between the granules of the substitute (c) (original magnification 940). Almost no change in HA granule size is observed. CB, Cranial bone.
site may have been reduced because decom-
(a)
(b)
(c)
Fig. 7. Histomorphologic analysis of test group C (beta-TCP only) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). Degraded granules are detected at both time points. Connective tissue ingrowth into the defect also is identified (c) (original magnification 940). CB, Cranial bone; NB, new bone.
(a)
posed small particles were collected into spaces between larger particles. In the mixture group, histologic findings included characteristics of both B and C groups. Although beta-TCP particles also degraded and divided into smaller particles in A group, the total volume of the grafted area did not decrease due to the effect of the HA particles. The amount of new bone formation, evaluated by histomorphometric analysis, was not significantly different between groups at the same time point. However, when focusing on the difference between time points, only the mixture group showed a significant increase in new bone formation. This can be attributed to new bone formation in the space that was caused by beta-TCP degradation and sustained by the HA particles. In the control group, although the volume of hard tissue was significantly lower than that in the other three groups, the extent of new bone formation was similar. Therefore, bone substitute itself did not increase the amount of new bone, which is consistent with results of the alpha-TCP experiment by Kihara et al. (2006). From these results, the mixed material was able to maintain the volume of the grafted area compared with that without intervention, and in a similar way to that with HA. Histologic analysis showed degraded betaTCP particles, especially at 8 weeks. Although there was no significant difference in new bone formation among the three test groups, histologically, we noted beta-TCP granule degradation and new bone replacement. Because the mixed group maintains the volume to the same extent as that with HA alone, new bone replacement would be possible without affecting the volume maintained by HA.
Conclusion
(b)
Despite the limitations of this study, the mixed material could maintain the volume of the grafted site compared with that without intervention. HA particles showed the same potential. Findings from histologic analysis showed degradation of beta-TCP particles, implying that this mixed material may enable new bone replacement.
References Fig. 8. Histomorphologic analysis of the control group at 4 weeks (a) and 8 weeks (b) by Villanueva stain (magnification 910). New bone (NB) formation is detected, but the newly formed bone area is thinner than that in the other groups. CB, Cranial bone.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Albrektsson, T., Br anemark, P.I., Hansson, H.A. & Lindstr€ om, J. (1981) Osseointegrated titanium implants. Requirements for ensuring a long-last-
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ing, direct bone-to-implant anchorage in man. Acta Orthopaedica Scandinavica 52: 155–170. Albrektsson, T., Zarb, G., Worthington, P. & Eriksson, A.R. (1986) The long-term efficacy of currently used dental implants: a review and proposed criteria of success. The International Journal of Oral & Maxillofacial Implants 1: 11–25. Ara ujo, M., Linder, E. & Lindhe, J. (2009) Effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. Clinical Oral Implants Research 20: 1–6. von Arx, T., Cochran, D.L., Hermann, J.S., Schenk, R.K. & Buser, D. (2001) Lateral ridge augmentation using different bone fillers and barrier membrane application. A histologic and histomorphometric pilot study in the canine mandible. Clinical Oral Implants Research 12: 260–269. Boyne, P. & James, R. (1980) Grafting of the maxillary sinus floor with autogenous marrow and bone. Journal of Oral Surgery 38: 613–616. Buser, D., Mericske-Stern, R., Bernard, J.P., Behneke, A., Behneke, N., Hirt, H.P., Belser, U.C. & Lang, N.P. (1997) Long-term evaluation of nonsubmerged ITI implants. Part 1: 8-year life table analysis of a prospective multi-center study with 2359 implants. Clinical Oral Implants Research 8: 161–172. Handschel, J., Wiesmann, H.P., Stratmann, U., Kleinheinz, J., Meyer, U. & Joos, U. (2002) TCP is hardly resorbed and not osteoconductive in a
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non-loading calvarial model. Biomaterials 23: 1689–1695. Kihara, H., Shiota, M., Yamashita, Y. & Kasugai, S. (2006) Biodegradation process of alpha-TCP particles and new bone formation in a rabbit cranial defect model. Journal of Biomedical Materials Research Part B: Applied Biomaterials 79: 284–291. Kim, S., Jung, U.W., Lee, Y.K. & Choi, S.H. (2011) Effects of biphasic calcium phosphate bone substitute on circumferential bone defects around dental implants in dogs. The International Journal of Oral & Maxillofacial Implants 26: 265–273. Koerten, H.K. & van der Meulen, J. (1999) Degradation of calcium phosphate ceramics. Journal of Biomedical Materials Research 44: 78–86. Lee, J.H., Jung, U.W., Kim, C.S., Choi, S.H. & Cho, K.S. (2008) Histologic and clinical evaluation of maxillary sinus augmentation using macroporous biphasic calcium phosphate in human. Clinical Oral Implants Research 19: 767–771. Lu, J., Descamps, M., Dejou, J., Koubi, G., Hardouin, P., Lemaitre, J. & Proust, J.P. (2002) The biodegradation mechanism of calcium phosphate biomaterials in bone. Journal of Biomedical Materials Research 63: 408–412. Lu, J.X., Gallur, A., Flautre, B., Anselme, K., Descamps, M., Thierry, B. & Hardouin, P. (1998) Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical,
and medullar bone sites in rabbits. Journal of Biomedical Materials Research 42: 357–367. Moore, W.R., Graves, S.E. & Bain, G.I. (2001) Synthetic bone graft substitutes. ANZ Journal of Surgery 71: 354–361. Nery, E.B., LeGeros, R.Z., Lynch, K.L. & Lee, K. (1992) Tissue response to biphasic calcium phosphate ceramic with different ratios of HA/b-TCP in periodontal osseous defects. Journal of Periodontology 63: 729–735. van Steenberghe, D., Lekholm, U., Bolender, C., Folmer, T., Henry, P., Herrmann, I., Higuchi, K., Laney, W., Linden, U. & Astrand, P. (1990) Applicability of osseointegrated oral implants in the rehabilitation of partial edentulism: a prospective multi-center study on 558 fixtures. The International Journal of Oral & Maxillofacial Implants 5: 272–281. Zerbo, I.R., Bronckers, A.L., de Lange, G. & Burger, E.H. (2005) Localisation of osteogenic and osteoclastic cells in porous beta-tricalcium phosphate particles used for human maxillary sinus floor elevation. Biomaterials 26: 1445–1451. Zerbo, I.R., Zijderveld, S.A., de Boer, A., Bronckers, A.L., de Lange, G., ten Bruggenkate, C.M. & Burger, E.H. (2004) Histomorphometry of human sinus floor augmentation using a porous a tricalcium phosphate: a prospective study. Clinical Oral Implants Research 15: 724–732.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Capability of new bone formation with a mixture of hydroxyapatite and beta-tricalcium phosphate granules
Authors’ affiliations: Minoru Sanda, Makoto Shiota, Masaki Fujii, Kazuhiro Kon, Tatsuya Fujimori, Shohei Kasugai, Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Key words: animal experiments, biomaterials, bone substitutes, material sciences
Corresponding author: Minoru Sanda Department of Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo, Japan Tel.: +81 35 803 5774 Fax: +81 35 803 5774 e-mail: [email protected]
of the hard tissues volume, new bone formation, and residual graft after 4 and 8 weeks.
Abstract Objectives: The aim of this experimental study was to test a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) granules inserted in cranial defects in rabbits, by the evaluation Material and methods: Two defects of 8 mm diameter were created at the calvarial bone of 24 Japanese white rabbits for a total of 48 defects. Four groups were created: defects filled with a mixture of HA and beta-TCP granules (test A), defects filled with HA alone (test B), defects filled with beta-TCP (test C), and empty defects (control). Hard tissues volume (remaining graft + new bone) was evaluated by l-CT and new bone (NB) and remaining graft (RG) percentages were evaluated by histomorphometry. The animals were sacrificed at 4 or 8 weeks postoperatively. Results: The test groups A, B, and C showed a significant higher total volume compared with controls at 4 and 8 weeks (P < 0.05). Regarding the percentages of NB and RG at 4 and 8 weeks, no significant differences were detected (P > 0.05). When comparing 4 and 8 weeks, test group A showed a significant increase in new bone formation. At both 4 and 8 weeks, no group showed significant differences in NB (P > 0.05). At 8 weeks, test group B had more RG than test group A. Conclusions: The novel mixture could maintain the volume of the grafted area compared with that with intervention, and in a similar way compared with HA.
Date: Accepted 24 July 2014 To cite this article: Sanda M, Shiota M, Fujii M, Kon K, Fujimori T, Kasugai S. Capability of new bone formation with a mixture of hydroxyapatite and beta-tricalcium phosphate granules. Clin. Oral Impl. Res.00, 2014, 1–6 doi: 10.1111/clr.12473
Endosseous dental implants have become one of the most predictable and satisfactory prosthetic treatment alternatives (Albrektsson et al. 1981, 1986; van Steenberghe et al. 1990; Buser et al. 1997). Some patients have an inadequate volume of the alveolar ridge for implant placement or esthetic outcome, therefore requires augmentation procedures prior to implantation. Although autograft is still the gold standard of graft material (Boyne & James 1980), it has certain disadvantages, including donor-site morbidity and limited graft availability. Consequently, synthetic materials have an important role in augmentation procedures (Moore et al. 2001). In terms of synthetic material, hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) are routinely used as bone substitutes in the field of implant dentistry. As HA is scarcely resorbed, the volume of the augmented site can be maintained, but few granules can be replaced by new bone (Ara ujo et al. 2009). Beta-TCP, on the other hand, has higher degradability than HA; therefore, new
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
bone replacement can be expected, but it can be difficult to maintain the volume of the grafted site. To benefit from the advantages of both, some manufacturers and researchers have developed biphasic calcium phosphate (BCP) made from HA and beta-TCP, which has shown some efficacy in several studies (Nery et al. 1992; Zerbo et al. 2004; Lee et al. 2008). However, the effects of the mixture of monophasic granules of HA and beta-TCP are not well documented. Therefore, the purpose of the present experimental work was to test a conventional mixture of HA and beta-TCP granules by the evaluation of the volumetric changes by lCT and the new bone formation and residual graft by histomorphometric analysis.
Material and methods Experimental protocols were approved by the Institutional Committee of Animal Care and Use at Tokyo Medical and Dental University
1
Sanda et al Osteoconductivity of mixture of HA/beta-TCP
(Approval No. 0130230A). We used the following four groups with six samples each: (i) mixture of HA granules and beta-TCP granules in a volume ratio of 1 : 1 (test group A), (ii) pure HA particles (test group B), (iii) pure beta-TCP granules (test group C), and (iv) no graft (control group). All substitutes were fabricated by the same company (BrainBase Co., Tokyo, Japan). One hundred percent purity of both ingredients was verified by X-ray diffraction analysis, with sintering at 1100°C. Both granules have an average particle size of 450 lm (range, 250–1000 lm), which is a cluster of 50-lm particles. The particles of both ingredients contain macropores with an average size of 50 lm (range, 10–100 lm) and 2-lm micropores inside, which are interconnected. Surgical procedure
Twenty-four Japanese male white rabbits weighing 3.0–3.5 kg were employed. Animals were anesthetized preoperatively with an intramuscular injection of 50 mg/kg ketamine (Ketalar; Daiichi Sankyo Inc., Tokyo, Japan) and 25 mg/kg thiopental sodium (Rabonal; Tanabe, Inc., Tokyo, Japan). The surgical area was shaved and disinfected. Then, 1.8 ml of local anesthetic (2% xylocaine/epinephrine 1 : 80,000; Dentsply Sankin, Tokyo, Japan) was injected into the surgical site. The parietal bones were selected as the experimental site. Skin incision and subperiosteal dissection were carried out sagittally between the parietal and frontal bones, and the periosteum was raised. Two noncritical-sized bone defects, each with a diameter of 8 mm, were prepared on both sides of the parietal bone. Six samples each were randomly selected for the three test groups and the control group. Depending on the thickness of the calvarial bone, 0.07–0.1 g of bone substitute was placed into the defect, so that the entire defect was filled and contoured similar to the original bone. During the observation period, all rabbits were given water and standard rabbit feed. Animals were sacrificed at 4 or 8 weeks after surgery with a lethal dose of intravenous thiopental sodium. The entire cranial bone was removed and fixed for 15 days in neutral 10% formalin.
and 3 mm height. Then, the volume of hard tissue including new bone and bone substitute remnants inside the defect was quantified by an analyzing application (TRI/3D BON; RATOC System Engineering Co., Ltd., Tokyo, Japan). Using this software, calculation of hard tissue volume was carried out by computing the radiopaque substance exceeding the constant threshold thought to be the boundary of hard tissue and soft tissue, as determined from histologic analysis. Total volume was calculated by the radiopaque voxels observed in the grafted site. Histologic processing
Samples were dehydrated in ascending grades of ethanol, embedded in acrylic resin (Technovit 7200; Heraeus Kulzer Japan Co., Ltd., Japan), and light cured. Consequently sagittal sections were obtained by the sawing and grinding technique (Exakt; Mesmer, Ost Einbeck, Germany) and stained with the Villanueva Stain. Histologic observations were performed under a light microscope (Biozero; Keyence Corporation of America, Itasca, IL, USA). Histomorphometric analysis
Area ratio of new bone (NB) and residual graft (RG) in the defect area were calculated. Area of the entire defect, new bone, and residual graft were measured using graphic software (Photoshop CS6 Extended software Adobe Systems Incorporated, San Jose, CA, USA). New bone and residual graft ratios were calculated using the following formulas:
new bone ratio = (area of new bone)/(area of the entire defect); residual graft ratio = (area of residual graft)/(area of the entire defect). Statistical analysis
Comparison between the four groups at the same time point regarding new bone formation, residual graft, and micro-CT data was performed using Dunnett’s T3 test. Comparisons between 4 and 8 weeks in each kind of substitute were performed using the Mann– Whitney U-test. All statistical analyses were performed using SPSS Statistics 21 (IBM Japan, Ltd., Tokyo, Japan), with P values of 0.05). At 8 weeks, test group A showed the highest mean value followed by the control group, test group C, and test group B. Residual graft
Fig. 2. Radiolographic hard tissue volume in each group at 4 and 8 weeks. Significant differences were detected between the control group and three test groups at both 4 and 8 weeks. There were no significant differences between the three test groups at each time points. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP; control, empty defect.
Histomorphometric analysis New bone formation
At 4 weeks, percentage of new bone formation was 15.7% 4.80% in test group A, 21.0% 5.60% in test group B, 19.9% 10.15% in test group C, and 16.3% 3.56% in the control group (Fig. 3). At 8 weeks, the percentages of new bone formation were 28.2% 9.95% in test group A, 18.3%
At 4 weeks, the percentage of residual graft was 57.2% 13.53% in test group A, 52.8% 12.28% in test group B, and 45.4% 13.42% in test group C (Fig. 4). At 8 weeks, the percentages were 43.1% 12.64% in test group A, 63.3% 9.02% in test group B, and 36.9% 20.93% in test group C. The mean value was highest in test group A followed by the test groups B and C at 4 weeks. However, there was no significant difference in this value among the three test groups. At 8 weeks, the mean value was the highest in test group B followed by the test groups A and C. The difference was significant only between test groups A and B. Histomorphologic analysis
Beta-TCP particles appeared as collapsed and diffused in both test groups A and B. Such
Table 2. Histological evaluation parameters (mean, median, and standard deviation in %) of new bone formation for every group and time point 4 weeks
Test A (n = 6) Test B (n = 6) Test C (n = 6) Control (n = 6) P-value1
8 weeks
N
Mean
Median
SD
N
Mean
Median
SD
P-value2
6 6 6 6
15.67 21.04 19.94 16.26
16.30 18.88 19.44 15.16 0.413
4.80 5.60 10.15 3.56
6 6 6 6
28.16 18.27 21.78 26.77
26.47 16.15 21.77 26.48 0.269
9.95 7.60 10.72 9.08
0.026 0.485 0.699 0.065
Test A, mixture of HA and beta-TCP; test B, pure HA; test C, pure beta-TCP; control, empty; SD, standard deviation. Area ratio of new bone inside the grafted area in each group at both 4 weeks and 8 weeks. P-value 1: computed between 4 groups in each time point P-value 2: computed between two healing periods.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
degradation was more remarkable at 8 weeks than at 4 weeks (Figs 5–8). Beta-TCP granules degraded to form smaller particles. At 4 weeks, the granules seemed degraded, while at 8 weeks, granules had disappeared to some extent. In some samples, the granules were completely encapsulated by soft tissue, and there was no new bone replacement inside the defect. On the other hand, HA particles showed slight or almost no morphologic changes and retained their form at both 4 and 8 weeks. HA particles were not completely embedded inside the new bone, whereas many betaTCP particles were involved completely inside the bone tissue. In addition, test group B had lesser soft tissue inside the grafted site than that in the other groups. In the control group, new bone formation in the defect area was detected, especially in the later time point. However, the thickness of the new bone is thinner compared with the original one. At 4 weeks, histologic analysis showed no organized lamellated appearance was detected at 4 weeks. At 8 weeks, well-organized lamellated structures were identified. This finding implies progress in the maturation of new bone inside the defect.
Discussion Biphasic calcium phosphate material has been used as a synthetic bone substitute for 20 years. Many studies have proved its validity, which is attributed to the synergistic effect that stable HA maintains the volume of the grafted site and soluble beta-TCP is replaced by new bone. Unlike the biphasic material, the present study used a simple mixture of HA and beta-TCP granules, expecting the same synergistic effect as BCP. The advantages of this material are the simplicity of the combining procedure and the ease of regulating the ratio of the two ingredients. Experimentally, we can separately observe the behavior of each granule type in vivo; however, this is difficult to distinguish in biphasic material. Hydroxyapatite particles were resorbed to a less extent, as expected. Therefore, new bone was formed in the spaces between the HA particles, but all the HA particles were not replaced by new bone. These results are consistent with those other reports that proved HA is nonresorbable. This nature of HA might inhibit soft tissue invasion into the augmented site. In histologic analysis, test groups A and B showed lesser soft tissue ingrowth
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Sanda et al Osteoconductivity of mixture of HA/beta-TCP
Table 3. Histological evaluation parameters (mean, median, and standard deviation in %) of residual graft for every test group and time point 4 weeks
Test A (n = 6) Test B (n = 6) Test C (n = 6) P-value1
8 weeks
N
Mean
Median
SD
N
Mean
Median
SD
P-value2
6 6 6
57.15 52.83 45.43
57.33 53.98 44.49 0.320
13.53 12.28 13.42
6 6 6
43.07 63.28 36.94
45.18 60.50 30.55 0.021
12.64 9.02 20.93
0.180 0.240 0.394
Test A, mixture of HA and beta-TCP; test B, pure HA; test C, pure beta-TCP; SD, standard deviation. Area ratio of residual graft detected inside the defect in three test groups at both 4 weeks and 8 weeks. P-value 1: computed between 4 groups in each time point P-value 2: computed between two healing periods.
into the defect than the TCP group. The region among particles can also affect the extent of soft tissue migration. In a study of BCP use in a dog mandible, Kim et al. (2011) found similar results that the nonresorbable HA part inhibited soft tissue ingrowth into the grafted site.
Fig. 3. Area ratio of new bone inside the grafted area. There were no significant differences between the groups at both 4 and 8 weeks. In comparison between each time point, only test group A shows significant increase in new bone. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP; control, empty defect.
In contrast, beta-TCP granules were biodegradable, predominantly at 8 weeks, whereas HA particles were not. These findings support Koerten and van der Meulen’s experimental findings (1999), which showed betaTCP was more biodegradable than HA and fluorapatite in mice. Lu et al. (2002) also reached the same conclusion in a rabbit tibia model. However, Handschel et al. (2002) reported that there was no resorption of TCP granules grafted in a rat calvarial defect model and that the control group with no substitute showed more new bone formation on histologic analysis. In our experiment, some samples had no beta-TCP granule degradation. Our results suggest that beta-TCP granules have great variability of new bone formation. Histologic analysis showed great variability in soft tissue invasion and new bone formation. von Arx et al. (2001) also reported on guided bone regeneration in a dog mandible and noted that sites augmented with TCP granules and covered with expanded polytetrafluoroethylene membrane exhibited very inconsistent findings with regard to ridge contour and new bone formation. This variability may be
(a)
(b)
Fig. 4. Area ratio of residual graft detected inside the defect. There was a significant difference between test groups A and B at 8 weeks. Test group A, mixture of HA and beta-TCP; test group B, pure HA; test group C, pure beta-TCP.
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attributed to the quantity of blood supply, the site that is grafted, and the difference in potential for new bone formation of each animal. Using micro-CT analysis, the three substitute groups were compared with respect to volume of hard tissue including bone substitute and new bone formation inside the defect. Although the mean value was the highest in the HA group and the lowest in the TCP group, a significant difference was not detected. Although the ratio of residual graft, as detected using histomorphometric evaluation, in test groups A and B was not significantly different at 4 weeks, the ratio was significantly higher in test group B than in test A group at 8 weeks. This may be because the resorbable part (beta-TCP granules) that was present in test group A had disappeared during the period between 4 and 8 weeks. However, the percentage of beta-TCP remnants was not significantly lower, although we expected that the granules would disappear due to their biodegradability; this may have occurred because of beta-TCP resorption. Lu et al. (1998) and Zerbo et al. (2005) analyzed beta-TCP’s biodegradability and reported that degradation of beta-TCP occurs mainly by chemical dissolution by tissue fluids but the effect by osteoclast. Therefore, beta-TCP granules degraded into the small parts at first; that is, original particles divided into smaller ones and subsequently disappeared. In the present study, the divided particles spread among the newly formed tissue, including both soft and hard tissues, so there was little reduction in their volume, and therefore, no significant differences were observed in the micro-CT results. However, the volume of the grafted
(c)
Fig. 5. Histomorphologic analysis of test group A (mixture of beta-TCP and HA) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). New bone (NB) can be observed between the cranial bone (CB). At a higher magnification, the 8-week samples show typical differences between degraded beta-tricalcium phosphate (beta-TCP) and nondegraded hydroxyapatite (HA) granules (c) (original magnification 920).
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Sanda et al Osteoconductivity of mixture of HA/beta-TCP
(a)
(b)
(c)
Fig. 6. Histomorphologic analysis of test group B (HA only) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). New bone (NB) can be seen between the granules of the substitute (c) (original magnification 940). Almost no change in HA granule size is observed. CB, Cranial bone.
site may have been reduced because decom-
(a)
(b)
(c)
Fig. 7. Histomorphologic analysis of test group C (beta-TCP only) at 4 weeks (a) and 8 weeks (b) by Villanueva staining (original magnification 910). Degraded granules are detected at both time points. Connective tissue ingrowth into the defect also is identified (c) (original magnification 940). CB, Cranial bone; NB, new bone.
(a)
posed small particles were collected into spaces between larger particles. In the mixture group, histologic findings included characteristics of both B and C groups. Although beta-TCP particles also degraded and divided into smaller particles in A group, the total volume of the grafted area did not decrease due to the effect of the HA particles. The amount of new bone formation, evaluated by histomorphometric analysis, was not significantly different between groups at the same time point. However, when focusing on the difference between time points, only the mixture group showed a significant increase in new bone formation. This can be attributed to new bone formation in the space that was caused by beta-TCP degradation and sustained by the HA particles. In the control group, although the volume of hard tissue was significantly lower than that in the other three groups, the extent of new bone formation was similar. Therefore, bone substitute itself did not increase the amount of new bone, which is consistent with results of the alpha-TCP experiment by Kihara et al. (2006). From these results, the mixed material was able to maintain the volume of the grafted area compared with that without intervention, and in a similar way to that with HA. Histologic analysis showed degraded betaTCP particles, especially at 8 weeks. Although there was no significant difference in new bone formation among the three test groups, histologically, we noted beta-TCP granule degradation and new bone replacement. Because the mixed group maintains the volume to the same extent as that with HA alone, new bone replacement would be possible without affecting the volume maintained by HA.
Conclusion
(b)
Despite the limitations of this study, the mixed material could maintain the volume of the grafted site compared with that without intervention. HA particles showed the same potential. Findings from histologic analysis showed degradation of beta-TCP particles, implying that this mixed material may enable new bone replacement.
References Fig. 8. Histomorphologic analysis of the control group at 4 weeks (a) and 8 weeks (b) by Villanueva stain (magnification 910). New bone (NB) formation is detected, but the newly formed bone area is thinner than that in the other groups. CB, Cranial bone.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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