Effect of Different Hydroxyapatite:β-Tricalcium Phosphate Ratios on the Osteoconductivity of Biphasic Calcium Phosphate in the Rabbit Sinus Model Hyun-Chang Lim, DDS, MS1/Ming-Lan Zhang, DDS, MS2/ Jung-Seok Lee, DDS, PhD3/Ui-Won Jung, DDS, PhD4/Seong-Ho Choi, DDS, PhD5 Purpose: This study compared the volume stability and bone-forming capacity of biphasic calcium phosphate (BCP) with a high versus a low ratio of β-tricalcium phosphate (β-TCP) relative to hydroxyapatite (HA), ie, 70:30 vs 30:70, in the rabbit sinus model. Materials and Methods: Bilateral sinus windows were created in eight adult New Zealand white rabbits (2.5 to 3.5 kg); each sinus in each rabbit was assigned to one of two experimental BCP groups according to the HA:β-TCP ratio. One sinus was grafted with BCP with a high ratio of β-TCP (30:70; TCP70), and the contralateral sinus was grafted with BCP with a low ratio of β-TCP (70:30; TCP30). The animals were sacrificed after 2 weeks (n = 4) or 8 weeks (n = 4) of healing. Biopsy specimens were harvested and evaluated histologically, histomorphometrically, and with microcomputed tomography. Results: The bone volume did not differ significantly between the two groups at each healing point, or between 2 and 8 weeks of healing in both groups. The amount of new bone increased significantly between 2 and 8 weeks of healing in both groups, and it did not differ significantly between the TCP30 and TCP70 groups. The residual material was significantly more resorbed in the TCP70 group than in the TCP30 group at both 2 and 8 weeks. In the TCP70 group, a greater number of multinucleated giant cells were observed at both weeks. The bone-to–residual material contact ratio did not differ significantly between the two groups. Conclusion: The volume stability and osteoconductive capacity of BCP with an HA:β-TCP ratio of 30:70 was comparable to that with an HA:β-TCP ratio of 70:30. Thus, within the limitations of this study, it can be argued that BCP with an HA:β-TCP ratio of 30:70 can be successfully used for sinus augmentation. Int J Oral Maxillofac Implants 2015;30:65–72. doi: 10.11607/jomi.3709 Key words: biphasic calcium phosphate, bone regeneration, rabbit sinus model, sinus augmentation
A
utogenous bone has long been considered an ideal material for bone regeneration because of
1Instructor,
Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 2Researcher, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 3Clinical Assistant Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 4 Associate Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 5Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. Correspondence to: Prof Seong-Ho Choi, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, South Korea. Fax: +82-2-3920398. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.
its osteogenic potential, but the amount of tissue that can be harvested from intraoral donor sites is limited, and the procedure is associated with problematic morbidity. In a review on sinus augmentation, Pjetursson et al1 reported a low annual failure rate of rough-surfaced implants placed in bone substitute–augmented sinuses (1.13%). Moreover, because there is no clear evidence that autogenous bone should be chosen over a bone substitute for sinus augmentation,2 various bone substitutes have been used for that procedure. One of the bone substitutes, biphasic calcium phosphate (BCP), was demonstrated to be effective in sinus augmentation. BCP is composed of slowly resorbing hydroxyapatite (HA) and fast-resorbing β-tricalcium phosphate (β-TCP). HA stabilizes the augmented space, and β-TCP provides the space for de novo bone formation, so the HA:β-TCP ratio is an important factor determining its reactivity.3,4 However, the optimum HA:β-TCP ratio has yet to be determined; moreover, it is possible that the optimum HA:β-TCP ratio is site-specific according to the defect type. The most commonly documented HA:β-TCP ratio in BCP for sinus augmentation is 60:40, the resulting outcomes for which are The International Journal of Oral & Maxillofacial Implants 65
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Lim et al
a
b
Fig 1 Clinical photographs of the surgery. (a) Two osteotomies were prepared, and the sinus membranes were carefully elevated. (b) The sinuses were then grafted with BCP with an HA: β-TCP ratio of 70:30 (TCP30) on one side and an HA:β-TCP ratio of 30:70 (TCP70) on the contralateral side.
reportedly highly predictable5–7; however, this in itself is not strong evidence that this is the optimum ratio. Some studies have also demonstrated that other ratios, such as 70:30 and 30:70, can be used successfully for sinus augmentation.8–11 Space maintenance is essential for bone regeneration. A higher HA ratio may be advantageous for volume stability, given that the maxillary sinus is constantly exposed to air pressure and that repneumatization has been reported.12,13 Conversely, a higher HA ratio may result in a reduction in the space available for de novo bone formation, because HA is highly stable and either remains unresorbed or is slowly resorbed over a long time.13 In the long term, the increased amount of vital new bone may strengthen the predictability of sinus augmentation in terms of higher bone-to-implant contact and better load distribution. It was demonstrated in an animal study that β-TCP has similar characteristics to autogenous bone14; accordingly, a higher β-TCP ratio may lead to the production of a greater amount of vital bone. Moreover, the maxillary sinus is reported to have good healing potential,15,16 which may increase the degree of β-TCP resorption. Thus, it can be postulated that for sinus augmentation, the relative amount of HA in BCP can be minimized without disturbing volume stability, and conversely, the relative amount of β-TCP can be maximized to enable greater new bone formation. The aforementioned situation prompted the present study to compare the volume stability and bone-forming capacity between BCP with a higher ratio of β-TCP (HA:β-TCP ratio of 30:70) and a lower ratio of β-TCP (HA:β-TCP ratio of 70:30) in the rabbit sinus model.
MATERIALS AND METHODS Eight adult New Zealand white rabbits (2.5 to 3.5 kg) were used in this study. The protocol for this research was approved by the Institutional Animal Care and Use
Fig 2 Color-coded reconstructed micro-CT image of the grafted sinuses. Blue color, TCP70; red color, TCP30.
Committee, Yonsei Medical Center. In each rabbit, each bilateral sinus window was assigned to one of two experimental groups according to the composition of the grafted BCP (ie, the HA:β-TCP ratio): (1) TCP30 (Osteon I, Genoss; HA:β-TCP = 70:30, porosity = 77%, pore size = 300 to 500 mm) or (2) for the contralateral sinus, TCP70 (Osteon II, Genoss; HA:β-TCP = 30:70, porosity = 70%, pore size = 250 mm).
Surgical Procedure
The animals were anesthetized using an intramuscular injection of ketamine hydrochloride (Ketalar, Yuhan) and xylazine (Rompun, Bayer Korea). The surgical area was shaved, disinfected, and then anesthetized locally with 2% lidocaine (lidocaine HCl, Huons). A skin incision was made along the midsagittal line on the nasal bone, and a full-thickness flap was reflected sufficiently to reveal the lateral antral wall. A trephine bur (outer diameter, 6 mm) was carefully used to create windows under saline irrigation. The bony window was detached, and the sinus membrane was gently elevated. Each sinus was grafted with 0.2 mL of one of the two BCP formulations (ie, Osteon I into one sinus and Osteon II into the contralateral sinus; Fig 1). After grafting, the flaps were sutured with 4-0 Monosyn (B-Braun Aesculap). The animals were sacrificed after either 2 weeks (n = 4) or 8 weeks (n = 4) of healing.
Microcomputed Tomography Analysis
Block sections including the grafted site and the surrounding tissues were harvested and fixed in 10% buffered formalin solution for 10 days. Microcomputed tomography (micro-CT) (SkyScan 1072, SkyScan) images of these sections were obtained at a resolution of 35 mm (achieved using settings of 100 kV and 100 mA). The images were stored in Digital Imaging and Communications in Medicine (DICOM) format and reconstructed in three dimensions using commercially available software (On-Demand3D, CyberMed). The augmented space was color-coded, and the total volume of augmented space was calculated (Fig 2).
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Lim et al
Histologic and Histomorphometric Analysis
After obtaining the micro-CT images, the blocks were decalcified in 5% formic acid for 14 days and embedded in paraffin. The sections were serially sliced at a thickness of 5 mm through the middle of the windows in the antral area, and the two central sections were chosen for histologic analysis. The selected sections were stained with hematoxylin-eosin and Masson’s trichrome. Histologic observation and histomorphometric analysis were performed using a stereomicroscope (MZFL III, Leica), a light microscope (DM-LB, Leica), and an automated image-analysis system (Image-Pro Plus, Media Cybernetics). The following parameters were measured for histomorphometric analysis: the total augmented area (TA), the area of newly formed bone (NB), the area of residual material (RM), the area of fibrovascular tissue (FV), and the NB-RM contact ratio. The NB-RM contact ratio was calculated after linear measurement of the perimeter of each residual particle and the contact length between the bone and the particles. The percentages of NB (%NB), RM (%RM), and FV (%FV) were measured and compared at each healing time point (ie, 2 and 8 weeks).
Statistical Analysis
Commercially available statistical software (SPSS 15.0, SPSS) was used for the statistical analysis. The histomorphometric and micro-CT data are presented as mean ± SD values. Paired t tests were used to evaluate the differences between the TCP30 and TCP70 groups at each healing time point. The Student t test was used to compare the parameters at 2 and 8 weeks in each group. The level of statistical significance was set at P < .05. In one animal, the sinus assigned to the TCP70 8-week group was excluded from paired t test analysis because one of its sinuses was severely inflamed, possibly as a result of membrane perforation.
RESULTS Clinical Results
Minor membrane perforation occurred in one sinus in the TCP70 8-week group, as described earlier. The membrane elevation was carefully extended so as to protect the perforated area, and grafting was performed. No special treatment was conducted to repair the perforation. None of the other experimental animals suffered any adverse event.
Micro-CT Analysis
The augmented sinus had an inverse dome-like shape that was elongated in the sagittal direction. The gross morphology of the augmented sinuses was similar in all animals irrespective of the group or healing time. A
Table 1 Total Augmented Volume (Mean ± SD)* TCP30 (mm3)
TCP70 (mm3)
2 wk
212.50 ± 36.28
210.02 ± 21.65
8 wk
188.12 ± 17.54
201.61 ± 56.76
Healing period
*There was no statistically significant difference between the groups at each time point, or between the time points within each group.
well-defined osteotomy was observed at 2 weeks, the appearance of which was maintained at 8 weeks. The BCP particles were uniformly distributed within the augmented space. The volume of total augmentation was calculated from micro-CT images. The total augmented volumes in the TCP30 group were 115.83 ± 11.99 mm3 and 94.35 ± 20.74 mm3 at 2 and 8 weeks, respectively; the corresponding values in the TCP70 group were 114.35 ± 4.08 mm3 and 92.03 ± 36.12 mm3. There was no statistical significance between the groups at each healing time point. There appeared to be a reduction in volume over time, but the difference was not statistically significant. A relatively high standard deviation was noted in both groups at 8 weeks (Table 1).
Histologic Observations
At 2 Weeks. The augmented area was convex in cross section at 2 weeks in both the TCP30 and TCP70 groups. The bone material was well maintained under the elevated sinus membrane in all animals. The window areas were still not healed with bone at this time point; instead, there was a fibrous tissue bridge between opposing window edges. Ingrowth of NB was observed along the surface of the sinus walls near the sinus windows. Very little bonelike tissue was observed in the central area in both groups. New bone had formed adjacent to the sinus membrane and in contact with the graft particles in both groups. The border between the particles and the NB was rougher in the TCP70 group than in the TCP30 group, and a larger number of multinucleated giant cells were observed near the sinus membrane and at the center of the augmented area in the TCP70 group (Figs 3 and 4). At 8 Weeks. The augmented areas in all groups were well maintained at 8 weeks, and the quantity of NB in the augmented areas was increased compared to that at 2 weeks. Although the windows were not still bridged with NB, a substantial amount of new bone had formed between the edges of the window in both the TCP30 and TCP70 groups. One sample in the TCP70 group presented with loss of sinus membrane integrity due to unhealed membrane perforation and significant inflammation throughout almost the entire cavity. The two groups differed in terms of RM. While The International Journal of Oral & Maxillofacial Implants 67
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×100
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Fig 4 Histologic observations in the center and sinus membrane areas in the TCP70 group at 2 weeks. Black arrows indicate osteoclasts. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
×200
RM
RM
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Fig 3 Histologic observations in the center and sinus membrane areas in the TCP30 group at 2 weeks. NB = new bone; RM = residual material; black arrows indicate osteoclasts. (Left) Masson’s trichrome stain, original magnification ×100; (right) hematoxylin-eosin stain, original magnification ×200.
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Fig 5 Histologic observation of the center and sinus membrane areas in the TCP30 group at 8 weeks. Yellow arrows indicate bone-forming fronts; stars, reversal line. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
Fig 6 Histologic observation of the center and sinus membrane areas in the TCP70 at 8 weeks. Black arrows indicate osteoclasts; yellow stars, reversal line. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
in the TCP30 group the RM maintained its structure well, with slowly resorbing characteristics at its surface, the RM in the TCP70 group was relatively reduced and partitioned. In both groups, substantial new bone formation was observed in both the central and sinus membrane areas. The NB was in close contact with BCP particles in both groups, and reversal lines were observed. Numerous osteocytes were observed in the NB. The NB in the TCP30 group was bridged along the surface of the BCP particles, forming a linear bony outline partially surrounding the particles. The NB in the TCP70 group exhibited not only a linear pattern but also a sprouting pattern, producing small islands, and tended to be embedded in soft tissue more than in the TCP30 group. The borders of the NB against particles were rougher in TCP70 than in TCP30. In the TCP70
group, the number of multinucleated giant cells in the center and near the sinus membrane was reduced, but still higher compared to the TCP30 group (Figs 5 and 6).
Histometric Analysis
The histometric findings are summarized in Tables 2 and 3 and Fig 7. Although both the TA and the area of RM appeared to have decreased over the observation period in both groups, the differences were not statistically significant. There was a statistically significant increase in the area of NB in both groups over the observation period. Neither TA nor the area of NB differed significantly between the TCP30 and TCP70 groups at both 2 and 8 weeks. The area of RM was significantly smaller in the TCP70 group than in the TCP30 group at both healing
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Lim et al
Table 2 Total Augmented Area, Area of Newly Formed Bone, and Area of Residual Material TA (mm2) Healing time
Area of NB (mm2) TCP30
Area of RM (mm2)
TCP30
TCP70
TCP70
TCP30
TCP70
2 wk
16.32 ± 4.03
14.46 ± 1.55
0.91 ± 0.26
0.89 ± 0.28
6.46 ± 0.72
4.40 ± 0.32*
8 wk
14.43 ± 2.81
13.06 ± 3.71
3.11 ± 1.28†
3.06 ± 0.89†
5.67 ± 0.85
2.66 ± 0.58*
*Statistically significant difference compared to corresponding TCP30 group. †Statistically significant difference compared to corresponding 2-week group.
Table 3 NB-RM Contact Ratio (%) TCP30
TCP70
2 wk
9.63 ± 3.23
4.44 ± 2.78
8 wk
39.33 ± 9.43
39.49 ± 6.12
There was no statistically significant difference between the groups at each time point, or between the time points within each group.
100%
FV RM NB
80%
60%
40%
40%
20%
20% TCP30
TCP70
FV RM NB
80%
60%
0% a
100%
0% b
TCP30
TCP70
Fig 7 Percentages of NB, RM, and fibrovascular tissue (FV) in the augmented area at (a) 2 and (b) 8 weeks after surgery in the TCP30 and TCP70 groups.
time points. Although %NB did not differ significantly between both groups at each healing time, 2 and 8 weeks, %RM was greater at 8 weeks than at 2 weeks. The NB-RM contact ratio was found to have increased significantly in both groups over time. There was no statistically significant difference in this ratio between the TCP30 and TCP70 groups at either 2 or 8 weeks.
DISCUSSION The balance between the slowly resorbing HA and fast-resorbing β-TCP in BCP is thought to determine the amount of new bone formation at graft sites. The stability of HA acts to maintain the augmented space, while β-TCP promotes bone formation within that space. Some authors have labeled β-TCP a temporary bone replacement.17 The maxillary sinus has strong potential for bone regeneration, and this potential can be maximized with the use of active biomaterials. It was thus hypothesized that increasing the β-TCP ratio would not harm the volume stability while increasing
the amount of vital bone. In the present study, increasing the β-TCP ratio to 70% (ie, TCP70) did not impede the volume stability at the graft site; however, it also did not increase the amount of NB compared to TCP30. It has been reported that β-TCP has similar characteristics to autogenous bone in terms of its new bone formation and resorption properties. Jensen et al14 demonstrated that β-TCP grafting with an expanded polytetrafluoroethylene membrane results in substantial new bone formation at 8 weeks in the mandibles of minipigs, which is not statistically different to that achieved with autogenous bone grafts (57% vs 55%), and that the β-TCP and autogenous bone particles had almost completely resorbed by 4 weeks. In sinus augmentation, β-TCP also yielded comparable bone formation to autogenous bone. Szabo et al18 demonstrated that the percentage of NB in sinuses grafted with β-TCP was comparable to that in sinuses grafted with autogenous bone (36% vs 38%). However, the reported performance of β-TCP appears to be somewhat contradictory. Von Arx et al19 reported that the amount of NB obtained with pure phased TCP and barrier membrane was inconsistent The International Journal of Oral & Maxillofacial Implants 69
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Lim et al
in a lateral-ridge augmentation model, which may indicate that the degradation of TCP may be too fast to support bone formation. Similar inconsistencies were also reported in sinus grafting. Zijderveld et al17 reported a low percentage of NB in lateral-core biopsy samples in β-TCP-grafted sinuses (17% ± 5%) compared to autogenous bone–grafted sinuses (41% ± 10%). In another study, Zijderveld et al20 demonstrated a significant resorption of β-TCP 5 years after sinus augmentation. The graft height reduced 57.8% to 62.2% in comparison to the originally augmented amount. Rapid replacement or resorption of biomaterials may render them unsuitable for sinus grafting or where long-term scaffolding is required in the graft. The findings for HA in BCP reciprocates the inconsistent outcomes of β-TCP, suggesting that BCP with a low HA ratio may provide both volume stability and sufficient bone formation. In a long-term study on costal defects in sheep, the resorption and bone formation achieved with an HA:β-TCP ratio of 30:70 did not differ significantly from that obtained with a ratio of 50:50; however, the 30:70 ratio resulted in better integration in the bone-remodeling process, implying that a higher β-TCP ratio in BCP may accelerate bone formation.21 In a study evaluating the efficacy of an HA:β-TCP ratio of 30:70 in the human maxillary sinus, the NB, RM, and marrow spaces were found to be comparable to those of a study using a ratio of 60:40.11,22 Indeed, the 30:70 ratio was found to perform well even in lateral ridge augmentation; the BCP particles were well integrated with the NB, and the dental implants exhibited a high bone-implant contact ratio with the regenerated bone.23,24 However, the amount of NB in the present study did not differ significantly between the TCP70 and TCP30 groups at each healing time interval. This finding may be explained by the osteoconductive mechanism of BCP and the cellular response to the HA:β-TCP ratio. Dissolution of BCP apatite crystals in the superficial layer is known to facilitate reprecipitation of an apatite layer, which leads to bone formation on the surface of the BCP particles.25 Furthermore, physiochemical dissolution during the resorption of calcium phosphate releases calcium ions into the grafted area, and the resulting elevated level of calcium ions may act as a reservoir that promotes bone formation away from the grafted particles.26 Conversely, the content of TCP has been reported to be inversely correlated with the degree of viable cell adhesion. Higher concentrations of TCP decrease the attachment of osteoblasts and monocytes to the BCP particles,27,28 which is in agreement with the smaller bone islands observed in the present TCP70 group. Furthermore, an increased number of multinucleated giant cells were observed adjacent to the sinus membrane in the TCP70 group at
2 weeks. The rapid dissolution of TCP in BCP may provoke an inflammatory reaction in the initial phase, thus delaying wound healing, even though multinucleated osteoclasts are related to bone formation via their coupling with osteoblasts. Likewise, Hong et al29 observed an increased number of multinucleated osteoclasts in β-TCP– and BCP-grafted (HA:β-TCP ratio of 60:40) extraction sockets, and a pronounced inflammatory reaction at the initial phase. The rabbit sinus model is generally considered to be appropriate for evaluating bone regeneration due to its similarity to the human sinus. However, the healing conditions differ between these two species. The bony area in the rabbit sinus, where the osteotomy is made, is not the sinus floor, but the roof. Accordingly, the graft is ultimately located on the membrane.30 The difference in the final graft position between the rabbit and human sinuses may negatively contribute to new bone formation with TCP70, which means that the ventilation of the sinus and subsequent micromotion of the membrane may jeopardize the bone-forming activity in the graft. In particular, the lower HA content in TCP70 may result in insufficient stability and increase the susceptibility to micromotion relative to BCP with a higher HA content. Statistically significant new bone formation was observed in both BCP groups over time. Lambert et al13 evaluated the percentage of new bone in the augmented area with BCP in a rabbit sinus model. At 1 week, 5 weeks, and 6 months of healing, the percentage of new bone increased significantly, being 0.3% ± 0.1%, 12.9% ± 2.2%, and 21.5% ± 3.8%, respectively. In the present study, %NB at 8 weeks was comparable to that obtained by Lambert et al at 6 months (24% and 23% in the TCP30 and TCP70 groups, respectively). Significant resorption of graft material over time was observed in the TCP70 group, in agreement with earlier animal studies using β-TCP.14,19,31 The stability of the augmented volume is an important factor for the long-term survival of dental implants. In the present study, the sinuses augmented with TCP70 were also well maintained in terms of volume and area throughout the observation period. This might indicate that the augmentation can be maintained with a low proportion of HA in BCP. However, it should be noted that the resorption increased the FV component. Furthermore, it remains to be established whether the increased FV can be stable over the long term, and whether the tissue has the potential to become new bone. One of the parameters of interest after grafting procedures is the NB-RM contact ratio. A high contact ratio infers a stable network of bone and biomaterial, which can be interpreted as strong osteoconductivity. In the present study, an NB-RM contact ratio of almost 40% was calculated for both groups, with no statistically
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significant difference between them, indicating that the integration potency with new bone tissue of the BCPs with two different HA:β-TCP ratios is very similar. Lambert et al13 found that the bone–graft particles contact ratio was lower in BCP-grafted sinuses than in bovine HA–grafted sinuses; those authors speculated that the increase in particles and FV in the BCP could be a buffer against air pressure in the sinus. Jensen et al14 stated that the ideal biomaterial for maxillary sinus grafting is not necessarily an ideal material for other types of bony deficiency, which in turn means that different types of defects may have different biomaterial requirements.4 In the pursuit of preferable biomaterials for sinus grafting, the authors of the present study evaluated BCPs with different compositions. The volume stability and osteoconductive capacity of the BCP with an HA:β-TCP ratio of 30:70 was comparable to that with an HA:β-TCP ratio of 70:30. Additional studies involving greater numbers of experimental animals and extended healing periods are required to confirm these findings.
CONCLUSION The BCP with a high β-TCP ratio did not increase the amount of NB in the rabbit sinus at 8 weeks compared to that with a low β-TCP ratio. However, the high β-TCP ratio did not impair volume stability. Consequently, within the limitations of this study, it can be concluded that BCP with an HA:β-TCP ratio of 30:70 can be successfully used for sinus augmentation. Additional studies are required to confirm the stability and the destiny of the increased amount of FV observed in association with the BCP with the high β-TCP ratio.
ACKNOWLEDGMENTS This study was supported by a grant of the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (101578). The authors reported no conflicts of interest related to this study.
REFERENCES 1. Pjetursson BE, Tan WC, Zwahlen M, Lang NP. A systematic review of the success of sinus floor elevation and survival of implants inserted in combination with sinus floor elevation. J Clin Periodontol 2008;35:216–240. 2. Nkenke E, Stelzle F. Clinical outcomes of sinus floor augmentation for implant placement using autogenous bone or bone substitutes: A systematic review. Clin Oral Implants Res 2009;20(suppl 4):124–133. 3. LeGeros RZ, Lin S, Rohanizadeh R, Mijares D, LeGeros JP. Biphasic calcium phosphate bioceramics: Preparation, properties and applications. J Mater Sci Mater Med 2003;14:201–209.
4. Nery EB, LeGeros RZ, Lynch KL, Lee K. Tissue response to biphasic calcium phosphate ceramic with different ratios of HA/beta TCP in periodontal osseous defects. J Periodontol 1992;63:729–735. 5. Froum SJ, Wallace SS, Cho SC, Elian N, Tarnow DP. Histomorphometric comparison of a biphasic bone ceramic to anorganic bovine bone for sinus augmentation: 6- to 8-month postsurgical assessment of vital bone formation. A pilot study. Int J Periodontics Restorative Dent 2008;28:273–281. 6. Lee JH, Jung UW, Kim CS, Choi SH, Cho KS. Histologic and clinical evaluation for maxillary sinus augmentation using macroporous biphasic calcium phosphate in human. Clin Oral Implants Res 2008;19:767–771. 7. Mangano C, Perrotti V, Shibli JA, et al. Maxillary sinus grafting with biphasic calcium phosphate ceramics: Clinical and histologic evaluation in man. Int J Oral Maxillofac Implants 2013;28:51–56. 8. Bae JH, Kim YK, Kim SG, Yun PY, Kim JS. Sinus bone graft using new alloplastic bone graft material (Osteon)-II: Clinical evaluation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e14–e20. 9. Cha JK, Park JC, Jung UW, Kim CS, Cho KS, Choi SH. Case series of maxillary sinus augmentation with biphasic calcium phosphate: A clinical and radiographic study. J Periodontal Implant Sci 2011;41:98–104. 10. Kim YK, Yun PY, Lim SC, Kim SG, Lee HJ, Ong JL. Clinical evaluations of OSTEON as a new alloplastic material in sinus bone grafting and its effect on bone healing. J Biomed Mater Res B Appl Biomater 2008;86:270–277. 11. Mangano C, Sinjari B, Shibli JA, et al. A human clinical, histological, histomorphometrical, and radiographical study on biphasic HABeta-TCP 30/70 in maxillary sinus augmentation. Clin Implant Dent Relat Res 2013 Sep 4. doi: 10.1111/cid.12145. [Epub ahead of print] 12. Hatano N, Shimizu Y, Ooya K. A clinical long-term radiographic evaluation of graft height changes after maxillary sinus floor augmentation with a 2:1 autogenous bone/xenograft mixture and simultaneous placement of dental implants. Clin Oral Implants Res 2004;15:339–345. 13. Lambert F, Leonard A, Drion P, Sourice S, Layrolle P, Rompen E. Influence of space-filling materials in subantral bone augmentation: Blood clot vs. autogenous bone chips vs. bovine hydroxyapatite. Clin Oral Implants Res 2011;22:538–545. 14. Jensen SS, Broggini N, Hjorting-Hansen E, Schenk R, Buser D. Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2006;17:237–243. 15. Altintas NY, Senel FC, Kayipmaz S, Taskesen F, Pampu AA. Comparative radiologic analyses of newly formed bone after maxillary sinus augmentation with and without bone grafting. J Oral Maxillofac Surg 2013;71:1520–1530. 16. Lundgren S, Andersson S, Gualini F, Sennerby L. Bone reformation with sinus membrane elevation: A new surgical technique for maxillary sinus floor augmentation. Clin Implant Dent Relat Res 2004;6:165–173. 17. Zijderveld SA, Zerbo IR, van den Bergh JP, Schulten EA, ten Bruggenkate CM. Maxillary sinus floor augmentation using a beta-tricalcium phosphate (Cerasorb) alone compared to autogenous bone grafts. Int J Oral Maxillofac Implants 2005;20:432–440. 18. Szabo G, Huys L, Coulthard P, et al. A prospective multicenter randomized clinical trial of autogenous bone versus beta-tricalcium phosphate graft alone for bilateral sinus elevation: Histologic and histomorphometric evaluation. Int J Oral Maxillofac Implants 2005;20:371–381. 19. von Arx T, Cochran DL, Hermann JS, Schenk RK, Higginbottom FL, Buser D. Lateral ridge augmentation and implant placement: An experimental study evaluating implant osseointegration in different augmentation materials in the canine mandible. Int J Oral Maxillofac Implants 2001;16:343–354. 20. Zijderveld SA, Schulten EA, Aartman IH, ten Bruggenkate CM. Long-term changes in graft height after maxillary sinus floor elevation with different grafting materials: Radiographic evaluation with a minimum follow-up of 4.5 years. Clin Oral Implants Res 2009;20:691–700.
The International Journal of Oral & Maxillofacial Implants 71 © 2015 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.
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21. Schopper C, Ziya-Ghazvini F, Goriwoda W, et al. HA/TCP compounding of a porous CaP biomaterial improves bone formation and scaffold degradation—A long-term histological study. J Biomed Mater Res B Appl Biomater 2005;74:458–467. 22. Frenken JW, Bouwman WF, Bravenboer N, Zijderveld SA, Schulten EA, ten Bruggenkate CM. The use of Straumann bone ceramic in a maxillary sinus floor elevation procedure: A clinical, radiological, histological and histomorphometric evaluation with a 6-month healing period. Clin Oral Implants Res 2010;21:201–208. 23. Kim DM, Nevins ML, Lin Z, et al. The clinical and histologic outcome of dental implant in large ridge defect regenerated with alloplast: A randomized controlled preclinical trial. J Oral Implantol 2013;39:148–153. 24. Nevins M, Nevins ML, Schupbach P, Kim SW, Lin Z, Kim DM. A prospective, randomized controlled preclinical trial to evaluate different formulations of biphasic calcium phosphate in combination with a hydroxyapatite collagen membrane to reconstruct deficient alveolar ridges. J Oral Implantol 2013;39:133–139. 25. Yamada S, Heymann D, Bouler JM, Daculsi G. Osteoclastic resorption of biphasic calcium phosphate ceramic in vitro. J Biomed Mater Res 1997;37:346–352.
26. Yamada S, Heymann D, Bouler JM, Daculsi G. Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/ beta-tricalcium phosphate ratios. Biomaterials 1997;18:1037–1041. 27. Rice JM, Hunt JA, Gallagher JA. Quantitative evaluation of the biocompatible and osteogenic properties of a range of biphasic calcium phosphate (BCP) granules using primary cultures of human osteoblasts and monocytes. Calcif Tissue Int 2003;72:726–736. 28. Wang C, Duan Y, Markovic B, et al. Phenotypic expression of bonerelated genes in osteoblasts grown on calcium phosphate ceramics with different phase compositions. Biomaterials 2004;25:2507–2514. 29. Hong JY, Lee JS, Pang EK, Jung UW, Choi SH, Kim CK. Impact of different synthetic bone fillers on healing of extraction sockets: An experimental study in dogs. Clin Oral Implants Res 2014;25:e30–e37. 30. Stubinger S, Dard M. The rabbit as experimental model for research in implant dentistry and related tissue regeneration. J Invest Surg 2013;26:266–282. 31. Artzi Z, Weinreb M, Givol N, et al. Biomaterial resorption rate and healing site morphology of inorganic bovine bone and beta-tricalcium phosphate in the canine: A 24-month longitudinal histologic study and morphometric analysis. Int J Oral Maxillofac Implants 2004;19:357–368.
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A
utogenous bone has long been considered an ideal material for bone regeneration because of
1Instructor,
Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 2Researcher, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 3Clinical Assistant Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 4 Associate Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. 5Professor, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea. Correspondence to: Prof Seong-Ho Choi, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, South Korea. Fax: +82-2-3920398. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.
its osteogenic potential, but the amount of tissue that can be harvested from intraoral donor sites is limited, and the procedure is associated with problematic morbidity. In a review on sinus augmentation, Pjetursson et al1 reported a low annual failure rate of rough-surfaced implants placed in bone substitute–augmented sinuses (1.13%). Moreover, because there is no clear evidence that autogenous bone should be chosen over a bone substitute for sinus augmentation,2 various bone substitutes have been used for that procedure. One of the bone substitutes, biphasic calcium phosphate (BCP), was demonstrated to be effective in sinus augmentation. BCP is composed of slowly resorbing hydroxyapatite (HA) and fast-resorbing β-tricalcium phosphate (β-TCP). HA stabilizes the augmented space, and β-TCP provides the space for de novo bone formation, so the HA:β-TCP ratio is an important factor determining its reactivity.3,4 However, the optimum HA:β-TCP ratio has yet to be determined; moreover, it is possible that the optimum HA:β-TCP ratio is site-specific according to the defect type. The most commonly documented HA:β-TCP ratio in BCP for sinus augmentation is 60:40, the resulting outcomes for which are The International Journal of Oral & Maxillofacial Implants 65
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Lim et al
a
b
Fig 1 Clinical photographs of the surgery. (a) Two osteotomies were prepared, and the sinus membranes were carefully elevated. (b) The sinuses were then grafted with BCP with an HA: β-TCP ratio of 70:30 (TCP30) on one side and an HA:β-TCP ratio of 30:70 (TCP70) on the contralateral side.
reportedly highly predictable5–7; however, this in itself is not strong evidence that this is the optimum ratio. Some studies have also demonstrated that other ratios, such as 70:30 and 30:70, can be used successfully for sinus augmentation.8–11 Space maintenance is essential for bone regeneration. A higher HA ratio may be advantageous for volume stability, given that the maxillary sinus is constantly exposed to air pressure and that repneumatization has been reported.12,13 Conversely, a higher HA ratio may result in a reduction in the space available for de novo bone formation, because HA is highly stable and either remains unresorbed or is slowly resorbed over a long time.13 In the long term, the increased amount of vital new bone may strengthen the predictability of sinus augmentation in terms of higher bone-to-implant contact and better load distribution. It was demonstrated in an animal study that β-TCP has similar characteristics to autogenous bone14; accordingly, a higher β-TCP ratio may lead to the production of a greater amount of vital bone. Moreover, the maxillary sinus is reported to have good healing potential,15,16 which may increase the degree of β-TCP resorption. Thus, it can be postulated that for sinus augmentation, the relative amount of HA in BCP can be minimized without disturbing volume stability, and conversely, the relative amount of β-TCP can be maximized to enable greater new bone formation. The aforementioned situation prompted the present study to compare the volume stability and bone-forming capacity between BCP with a higher ratio of β-TCP (HA:β-TCP ratio of 30:70) and a lower ratio of β-TCP (HA:β-TCP ratio of 70:30) in the rabbit sinus model.
MATERIALS AND METHODS Eight adult New Zealand white rabbits (2.5 to 3.5 kg) were used in this study. The protocol for this research was approved by the Institutional Animal Care and Use
Fig 2 Color-coded reconstructed micro-CT image of the grafted sinuses. Blue color, TCP70; red color, TCP30.
Committee, Yonsei Medical Center. In each rabbit, each bilateral sinus window was assigned to one of two experimental groups according to the composition of the grafted BCP (ie, the HA:β-TCP ratio): (1) TCP30 (Osteon I, Genoss; HA:β-TCP = 70:30, porosity = 77%, pore size = 300 to 500 mm) or (2) for the contralateral sinus, TCP70 (Osteon II, Genoss; HA:β-TCP = 30:70, porosity = 70%, pore size = 250 mm).
Surgical Procedure
The animals were anesthetized using an intramuscular injection of ketamine hydrochloride (Ketalar, Yuhan) and xylazine (Rompun, Bayer Korea). The surgical area was shaved, disinfected, and then anesthetized locally with 2% lidocaine (lidocaine HCl, Huons). A skin incision was made along the midsagittal line on the nasal bone, and a full-thickness flap was reflected sufficiently to reveal the lateral antral wall. A trephine bur (outer diameter, 6 mm) was carefully used to create windows under saline irrigation. The bony window was detached, and the sinus membrane was gently elevated. Each sinus was grafted with 0.2 mL of one of the two BCP formulations (ie, Osteon I into one sinus and Osteon II into the contralateral sinus; Fig 1). After grafting, the flaps were sutured with 4-0 Monosyn (B-Braun Aesculap). The animals were sacrificed after either 2 weeks (n = 4) or 8 weeks (n = 4) of healing.
Microcomputed Tomography Analysis
Block sections including the grafted site and the surrounding tissues were harvested and fixed in 10% buffered formalin solution for 10 days. Microcomputed tomography (micro-CT) (SkyScan 1072, SkyScan) images of these sections were obtained at a resolution of 35 mm (achieved using settings of 100 kV and 100 mA). The images were stored in Digital Imaging and Communications in Medicine (DICOM) format and reconstructed in three dimensions using commercially available software (On-Demand3D, CyberMed). The augmented space was color-coded, and the total volume of augmented space was calculated (Fig 2).
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Histologic and Histomorphometric Analysis
After obtaining the micro-CT images, the blocks were decalcified in 5% formic acid for 14 days and embedded in paraffin. The sections were serially sliced at a thickness of 5 mm through the middle of the windows in the antral area, and the two central sections were chosen for histologic analysis. The selected sections were stained with hematoxylin-eosin and Masson’s trichrome. Histologic observation and histomorphometric analysis were performed using a stereomicroscope (MZFL III, Leica), a light microscope (DM-LB, Leica), and an automated image-analysis system (Image-Pro Plus, Media Cybernetics). The following parameters were measured for histomorphometric analysis: the total augmented area (TA), the area of newly formed bone (NB), the area of residual material (RM), the area of fibrovascular tissue (FV), and the NB-RM contact ratio. The NB-RM contact ratio was calculated after linear measurement of the perimeter of each residual particle and the contact length between the bone and the particles. The percentages of NB (%NB), RM (%RM), and FV (%FV) were measured and compared at each healing time point (ie, 2 and 8 weeks).
Statistical Analysis
Commercially available statistical software (SPSS 15.0, SPSS) was used for the statistical analysis. The histomorphometric and micro-CT data are presented as mean ± SD values. Paired t tests were used to evaluate the differences between the TCP30 and TCP70 groups at each healing time point. The Student t test was used to compare the parameters at 2 and 8 weeks in each group. The level of statistical significance was set at P < .05. In one animal, the sinus assigned to the TCP70 8-week group was excluded from paired t test analysis because one of its sinuses was severely inflamed, possibly as a result of membrane perforation.
RESULTS Clinical Results
Minor membrane perforation occurred in one sinus in the TCP70 8-week group, as described earlier. The membrane elevation was carefully extended so as to protect the perforated area, and grafting was performed. No special treatment was conducted to repair the perforation. None of the other experimental animals suffered any adverse event.
Micro-CT Analysis
The augmented sinus had an inverse dome-like shape that was elongated in the sagittal direction. The gross morphology of the augmented sinuses was similar in all animals irrespective of the group or healing time. A
Table 1 Total Augmented Volume (Mean ± SD)* TCP30 (mm3)
TCP70 (mm3)
2 wk
212.50 ± 36.28
210.02 ± 21.65
8 wk
188.12 ± 17.54
201.61 ± 56.76
Healing period
*There was no statistically significant difference between the groups at each time point, or between the time points within each group.
well-defined osteotomy was observed at 2 weeks, the appearance of which was maintained at 8 weeks. The BCP particles were uniformly distributed within the augmented space. The volume of total augmentation was calculated from micro-CT images. The total augmented volumes in the TCP30 group were 115.83 ± 11.99 mm3 and 94.35 ± 20.74 mm3 at 2 and 8 weeks, respectively; the corresponding values in the TCP70 group were 114.35 ± 4.08 mm3 and 92.03 ± 36.12 mm3. There was no statistical significance between the groups at each healing time point. There appeared to be a reduction in volume over time, but the difference was not statistically significant. A relatively high standard deviation was noted in both groups at 8 weeks (Table 1).
Histologic Observations
At 2 Weeks. The augmented area was convex in cross section at 2 weeks in both the TCP30 and TCP70 groups. The bone material was well maintained under the elevated sinus membrane in all animals. The window areas were still not healed with bone at this time point; instead, there was a fibrous tissue bridge between opposing window edges. Ingrowth of NB was observed along the surface of the sinus walls near the sinus windows. Very little bonelike tissue was observed in the central area in both groups. New bone had formed adjacent to the sinus membrane and in contact with the graft particles in both groups. The border between the particles and the NB was rougher in the TCP70 group than in the TCP30 group, and a larger number of multinucleated giant cells were observed near the sinus membrane and at the center of the augmented area in the TCP70 group (Figs 3 and 4). At 8 Weeks. The augmented areas in all groups were well maintained at 8 weeks, and the quantity of NB in the augmented areas was increased compared to that at 2 weeks. Although the windows were not still bridged with NB, a substantial amount of new bone had formed between the edges of the window in both the TCP30 and TCP70 groups. One sample in the TCP70 group presented with loss of sinus membrane integrity due to unhealed membrane perforation and significant inflammation throughout almost the entire cavity. The two groups differed in terms of RM. While The International Journal of Oral & Maxillofacial Implants 67
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Lim et al
×100
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Fig 4 Histologic observations in the center and sinus membrane areas in the TCP70 group at 2 weeks. Black arrows indicate osteoclasts. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
×200
RM
RM
NB
RM RM RM
Fig 3 Histologic observations in the center and sinus membrane areas in the TCP30 group at 2 weeks. NB = new bone; RM = residual material; black arrows indicate osteoclasts. (Left) Masson’s trichrome stain, original magnification ×100; (right) hematoxylin-eosin stain, original magnification ×200.
RM
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Fig 5 Histologic observation of the center and sinus membrane areas in the TCP30 group at 8 weeks. Yellow arrows indicate bone-forming fronts; stars, reversal line. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
Fig 6 Histologic observation of the center and sinus membrane areas in the TCP70 at 8 weeks. Black arrows indicate osteoclasts; yellow stars, reversal line. (Left) Masson’s trichrome, original magnification ×100; (right) hematoxylin-eosin, original magnification ×200.
in the TCP30 group the RM maintained its structure well, with slowly resorbing characteristics at its surface, the RM in the TCP70 group was relatively reduced and partitioned. In both groups, substantial new bone formation was observed in both the central and sinus membrane areas. The NB was in close contact with BCP particles in both groups, and reversal lines were observed. Numerous osteocytes were observed in the NB. The NB in the TCP30 group was bridged along the surface of the BCP particles, forming a linear bony outline partially surrounding the particles. The NB in the TCP70 group exhibited not only a linear pattern but also a sprouting pattern, producing small islands, and tended to be embedded in soft tissue more than in the TCP30 group. The borders of the NB against particles were rougher in TCP70 than in TCP30. In the TCP70
group, the number of multinucleated giant cells in the center and near the sinus membrane was reduced, but still higher compared to the TCP30 group (Figs 5 and 6).
Histometric Analysis
The histometric findings are summarized in Tables 2 and 3 and Fig 7. Although both the TA and the area of RM appeared to have decreased over the observation period in both groups, the differences were not statistically significant. There was a statistically significant increase in the area of NB in both groups over the observation period. Neither TA nor the area of NB differed significantly between the TCP30 and TCP70 groups at both 2 and 8 weeks. The area of RM was significantly smaller in the TCP70 group than in the TCP30 group at both healing
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Table 2 Total Augmented Area, Area of Newly Formed Bone, and Area of Residual Material TA (mm2) Healing time
Area of NB (mm2) TCP30
Area of RM (mm2)
TCP30
TCP70
TCP70
TCP30
TCP70
2 wk
16.32 ± 4.03
14.46 ± 1.55
0.91 ± 0.26
0.89 ± 0.28
6.46 ± 0.72
4.40 ± 0.32*
8 wk
14.43 ± 2.81
13.06 ± 3.71
3.11 ± 1.28†
3.06 ± 0.89†
5.67 ± 0.85
2.66 ± 0.58*
*Statistically significant difference compared to corresponding TCP30 group. †Statistically significant difference compared to corresponding 2-week group.
Table 3 NB-RM Contact Ratio (%) TCP30
TCP70
2 wk
9.63 ± 3.23
4.44 ± 2.78
8 wk
39.33 ± 9.43
39.49 ± 6.12
There was no statistically significant difference between the groups at each time point, or between the time points within each group.
100%
FV RM NB
80%
60%
40%
40%
20%
20% TCP30
TCP70
FV RM NB
80%
60%
0% a
100%
0% b
TCP30
TCP70
Fig 7 Percentages of NB, RM, and fibrovascular tissue (FV) in the augmented area at (a) 2 and (b) 8 weeks after surgery in the TCP30 and TCP70 groups.
time points. Although %NB did not differ significantly between both groups at each healing time, 2 and 8 weeks, %RM was greater at 8 weeks than at 2 weeks. The NB-RM contact ratio was found to have increased significantly in both groups over time. There was no statistically significant difference in this ratio between the TCP30 and TCP70 groups at either 2 or 8 weeks.
DISCUSSION The balance between the slowly resorbing HA and fast-resorbing β-TCP in BCP is thought to determine the amount of new bone formation at graft sites. The stability of HA acts to maintain the augmented space, while β-TCP promotes bone formation within that space. Some authors have labeled β-TCP a temporary bone replacement.17 The maxillary sinus has strong potential for bone regeneration, and this potential can be maximized with the use of active biomaterials. It was thus hypothesized that increasing the β-TCP ratio would not harm the volume stability while increasing
the amount of vital bone. In the present study, increasing the β-TCP ratio to 70% (ie, TCP70) did not impede the volume stability at the graft site; however, it also did not increase the amount of NB compared to TCP30. It has been reported that β-TCP has similar characteristics to autogenous bone in terms of its new bone formation and resorption properties. Jensen et al14 demonstrated that β-TCP grafting with an expanded polytetrafluoroethylene membrane results in substantial new bone formation at 8 weeks in the mandibles of minipigs, which is not statistically different to that achieved with autogenous bone grafts (57% vs 55%), and that the β-TCP and autogenous bone particles had almost completely resorbed by 4 weeks. In sinus augmentation, β-TCP also yielded comparable bone formation to autogenous bone. Szabo et al18 demonstrated that the percentage of NB in sinuses grafted with β-TCP was comparable to that in sinuses grafted with autogenous bone (36% vs 38%). However, the reported performance of β-TCP appears to be somewhat contradictory. Von Arx et al19 reported that the amount of NB obtained with pure phased TCP and barrier membrane was inconsistent The International Journal of Oral & Maxillofacial Implants 69
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in a lateral-ridge augmentation model, which may indicate that the degradation of TCP may be too fast to support bone formation. Similar inconsistencies were also reported in sinus grafting. Zijderveld et al17 reported a low percentage of NB in lateral-core biopsy samples in β-TCP-grafted sinuses (17% ± 5%) compared to autogenous bone–grafted sinuses (41% ± 10%). In another study, Zijderveld et al20 demonstrated a significant resorption of β-TCP 5 years after sinus augmentation. The graft height reduced 57.8% to 62.2% in comparison to the originally augmented amount. Rapid replacement or resorption of biomaterials may render them unsuitable for sinus grafting or where long-term scaffolding is required in the graft. The findings for HA in BCP reciprocates the inconsistent outcomes of β-TCP, suggesting that BCP with a low HA ratio may provide both volume stability and sufficient bone formation. In a long-term study on costal defects in sheep, the resorption and bone formation achieved with an HA:β-TCP ratio of 30:70 did not differ significantly from that obtained with a ratio of 50:50; however, the 30:70 ratio resulted in better integration in the bone-remodeling process, implying that a higher β-TCP ratio in BCP may accelerate bone formation.21 In a study evaluating the efficacy of an HA:β-TCP ratio of 30:70 in the human maxillary sinus, the NB, RM, and marrow spaces were found to be comparable to those of a study using a ratio of 60:40.11,22 Indeed, the 30:70 ratio was found to perform well even in lateral ridge augmentation; the BCP particles were well integrated with the NB, and the dental implants exhibited a high bone-implant contact ratio with the regenerated bone.23,24 However, the amount of NB in the present study did not differ significantly between the TCP70 and TCP30 groups at each healing time interval. This finding may be explained by the osteoconductive mechanism of BCP and the cellular response to the HA:β-TCP ratio. Dissolution of BCP apatite crystals in the superficial layer is known to facilitate reprecipitation of an apatite layer, which leads to bone formation on the surface of the BCP particles.25 Furthermore, physiochemical dissolution during the resorption of calcium phosphate releases calcium ions into the grafted area, and the resulting elevated level of calcium ions may act as a reservoir that promotes bone formation away from the grafted particles.26 Conversely, the content of TCP has been reported to be inversely correlated with the degree of viable cell adhesion. Higher concentrations of TCP decrease the attachment of osteoblasts and monocytes to the BCP particles,27,28 which is in agreement with the smaller bone islands observed in the present TCP70 group. Furthermore, an increased number of multinucleated giant cells were observed adjacent to the sinus membrane in the TCP70 group at
2 weeks. The rapid dissolution of TCP in BCP may provoke an inflammatory reaction in the initial phase, thus delaying wound healing, even though multinucleated osteoclasts are related to bone formation via their coupling with osteoblasts. Likewise, Hong et al29 observed an increased number of multinucleated osteoclasts in β-TCP– and BCP-grafted (HA:β-TCP ratio of 60:40) extraction sockets, and a pronounced inflammatory reaction at the initial phase. The rabbit sinus model is generally considered to be appropriate for evaluating bone regeneration due to its similarity to the human sinus. However, the healing conditions differ between these two species. The bony area in the rabbit sinus, where the osteotomy is made, is not the sinus floor, but the roof. Accordingly, the graft is ultimately located on the membrane.30 The difference in the final graft position between the rabbit and human sinuses may negatively contribute to new bone formation with TCP70, which means that the ventilation of the sinus and subsequent micromotion of the membrane may jeopardize the bone-forming activity in the graft. In particular, the lower HA content in TCP70 may result in insufficient stability and increase the susceptibility to micromotion relative to BCP with a higher HA content. Statistically significant new bone formation was observed in both BCP groups over time. Lambert et al13 evaluated the percentage of new bone in the augmented area with BCP in a rabbit sinus model. At 1 week, 5 weeks, and 6 months of healing, the percentage of new bone increased significantly, being 0.3% ± 0.1%, 12.9% ± 2.2%, and 21.5% ± 3.8%, respectively. In the present study, %NB at 8 weeks was comparable to that obtained by Lambert et al at 6 months (24% and 23% in the TCP30 and TCP70 groups, respectively). Significant resorption of graft material over time was observed in the TCP70 group, in agreement with earlier animal studies using β-TCP.14,19,31 The stability of the augmented volume is an important factor for the long-term survival of dental implants. In the present study, the sinuses augmented with TCP70 were also well maintained in terms of volume and area throughout the observation period. This might indicate that the augmentation can be maintained with a low proportion of HA in BCP. However, it should be noted that the resorption increased the FV component. Furthermore, it remains to be established whether the increased FV can be stable over the long term, and whether the tissue has the potential to become new bone. One of the parameters of interest after grafting procedures is the NB-RM contact ratio. A high contact ratio infers a stable network of bone and biomaterial, which can be interpreted as strong osteoconductivity. In the present study, an NB-RM contact ratio of almost 40% was calculated for both groups, with no statistically
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significant difference between them, indicating that the integration potency with new bone tissue of the BCPs with two different HA:β-TCP ratios is very similar. Lambert et al13 found that the bone–graft particles contact ratio was lower in BCP-grafted sinuses than in bovine HA–grafted sinuses; those authors speculated that the increase in particles and FV in the BCP could be a buffer against air pressure in the sinus. Jensen et al14 stated that the ideal biomaterial for maxillary sinus grafting is not necessarily an ideal material for other types of bony deficiency, which in turn means that different types of defects may have different biomaterial requirements.4 In the pursuit of preferable biomaterials for sinus grafting, the authors of the present study evaluated BCPs with different compositions. The volume stability and osteoconductive capacity of the BCP with an HA:β-TCP ratio of 30:70 was comparable to that with an HA:β-TCP ratio of 70:30. Additional studies involving greater numbers of experimental animals and extended healing periods are required to confirm these findings.
CONCLUSION The BCP with a high β-TCP ratio did not increase the amount of NB in the rabbit sinus at 8 weeks compared to that with a low β-TCP ratio. However, the high β-TCP ratio did not impair volume stability. Consequently, within the limitations of this study, it can be concluded that BCP with an HA:β-TCP ratio of 30:70 can be successfully used for sinus augmentation. Additional studies are required to confirm the stability and the destiny of the increased amount of FV observed in association with the BCP with the high β-TCP ratio.
ACKNOWLEDGMENTS This study was supported by a grant of the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (101578). The authors reported no conflicts of interest related to this study.
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