Structural and Qualitative Bone Remodeling Around Repetitive Loaded Implants in Rabbits Shinichiro Kuroshima, DDS, PhD;* Munenori Yasutake, DDS;† Kotaro Tsuiki, DDS;† Takayoshi Nakano, PhD;‡ Takashi Sawase, DDS, PhD§

ABSTRACT Background: Bone mechanical function is regulated by bone quality and bone mineral density (BMD) that reflect bone strength. The preferential alignment of biological apatite (BAp) c-axis/collagen fibers and osteocytes is a determinant factor of bone quality. However, the effect of mechanical loading on bone quality around dental implants is unclear. Purpose: The aim of this study was to clarify the effects of mechanical loading on osseointegration, bone volume BMD, and bone quality around dental implants. Materials and Methods: Twenty anodized Ti-6Al-4V alloy implants (KYOCERA Co., Kyoto, Japan) were placed in the proximal tibial metaphysis of 10 rabbits. Twelve weeks after surgery, mechanical loading was applied along the long axis of the implant (50 N, 3 Hz, 1,800 cycles, 2 days/week) for 8 weeks. Osseointegration, bone volume, BMD, and bone quality were evaluated using light microscopy, microcomputed tomography, polarized light microscopy, and microbeam X-ray diffractometer. Results: Mechanical loading increased osseointegration, bone volume, and BMD. Bone quality around dental implant was altered with increased osteocyte numbers and the preferential alignment direction and degree of BAp c-axis/collagen fibers. Conclusions: These findings suggest that mechanical loading effectively induces bone anabolic responses around dental implants. Altered bone quality may upregulate bone strength, contributing to long-term implant stability. KEY WORDS: anisotropy, bone quality, collagen, dental implant, osseointegration, osteocytes, x-ray diffraction

INTRODUCTION

important role in long-term therapeutic success. Originally, osseointegration is defined as “a direct structural and functional connection between ordered living bone and the surface of a load-carrying implant.”1 Most clinicians believe that osseointegration could be established after implant placement irrespective of loading. However, based on the original definition of osseointegration, bone wound healing just occurs when the placed implant does not receive a loading. Data on the effects of dynamic loading on osseointegration are limited and controversial, although dental implants are repetitively subjected to dynamic loadings such as functional and/or parafunctional forces, following implantsupported prosthesis use.2 Additionally, it is unclear which loading parameters have an anabolic effect on bone around dental implants, due to difficulties in establishing load conditions that increase bone quantity in human and animal studies.3

Dental implant treatment is one of the most successful procedures for replacing missing teeth. The establishment and maintenance of osseointegration play an

*Assistant professor, Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan; †postgraduate student, Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan; ‡professor, Division of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University, Osaka, Japan; §professor, Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Corresponding Author: Professor Takashi Sawase, Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1, Sakamoto, Nagasaki, 852-8588, Japan; e-mail: [email protected] © 2015 Wiley Periodicals, Inc. DOI 10.1111/cid.12318

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Bone quality, which is independent of bone mineral density (BMD), is a critical factor in determining bone strength with BMD.4 The definition of bone quality is “the sum total of characteristics of the bone that influence the bone’s resistance to fracture.”5 The term of “bone quality” has recently provided much discussion and debate among medical doctors and researchers because the clarification of bone quality is challenging.6–8 Bone architecture, bone turnover, damage accumulation and mineralization are thought to determine bone quality.4 On the other hand, BMD, which is presented as grams of mineral per area or volume, shows the current clinical standard of bone fragility. The use of computed tomography is available for the evaluation of BMD, while assessments for bone quality have not been developed. Recently, we established a new method for evaluating bone quality; the preferential alignment direction and degree of biological apatite (BAp) c-axis are measured using a microbeam X-ray diffractometer (μXRD) system.9–11 BAp, which is the main component of hard tissues, crystallizes in an anisotropic hexagonal-based lattice compromising the a-axis and c-axis. In particular, the c-axis of BAp is parallel to the extended collagen fibers associated with mechanical function, indicating that anisotropic changes in BAp c-axis/collagen fibers regulate bone quality in response to mechanical loading.11 Osteocytes, which maintain bone homeostasis, reside in the mineralized bone matrix containing BAp and collagen fibers. Bone-embedded osteocytes control bone modeling and remodeling by regulating osteoclasts and/or osteoblasts via mechanotransduction.12–14 Furthermore, it has been recently reported that the osteocytic canalicular network may influence bone quality through mechanical loading.15 Accordingly, mechanical loading would become an important factor that controls bone quality from the viewpoint of bone architecture characterized by osteocytes and the preferential alignment direction and degree of BAp c-axis/ collagen fibers. However, the influence of mechanical loading on bone quality around dental implants has never been documented in implant dentistry. The aims of this study were: (1) to clarify the effects of mechanical repetitive loading on osseointegration, bone volume; and BMD around dental implants; and (2) to document whether mechanical repetitive loading influences bone quality, defined as the alignment of BAp c-axis/collagen fibers and osteocytes.

MATERIALS AND METHODS Animal Experiments Ten adult female Japanese White rabbits weighing 4.10 1 0.22 kg (3.82 to 4.39 kg) (Biotek Co., Ltd., Saga, Japan) were obtained. Twenty anodized Ti-6Al-4V alloy dental implants (3.7 x 6.0 mm; KYOCERA Co., Kyoto, Japan) were used. Forty Ti-6Al-4V screws (KYOCERA Co.) were also used to anchor a custom-made loading device (Higuchi Co., Nagasaki, Japan). Implants were placed in both right and left of each proximal tibial metaphysis unicortically under anesthesia (35 mg/kg of ketamine and 5 mg/kg of xylazine cocktail). Two anchor screws for the loading device were also placed on each side of the implants. There was 0.05 mg/kg of buprenorphine given (intramuscular injection) immediately postoperatively, and 0.01 mg/kg of buprenorphine was also given every 12 hours for 3 days after surgery. Twelve weeks after surgery, all implants received postabutments. Randomly selected implants from each rabbit were stimulated at 50 N with a frequency of 3 Hz for 1,800 cycles, 2 days/week, for 8 successive weeks using the loading device supported by two lateral screws on each implant under anesthesia (35mg/kg of ketamine and 5 mg/kg xylazine cocktail) (n = 10, loading group) (Figure 1, A and B). Loading direction was parallel to the long axis of the implants. The remaining implants served as unloaded control (n = 10, control group). Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University, with the approval of the Ethics Committee for Animal Research. Microcomputed Tomography (microCT) Assessment Rabbits were euthanized with an overdose of pentobarbital (60 mg/kg, intravenous injection) at 8 weeks after the initiation of mechanical repetitive loading. Tibial bone blocks including the implants and screws were dissected with a diamond saw (Exakt®, Heraeus Kulzer GmbH, Hanau, Germany). Forty-eight hours after fixation in 10% formalin, microCT assessments were performed at 20-μm voxel resolution with an energy level of 90 kV (R_mCT2; Rigaku Co., Tokyo, Japan). Bone around the implant in the proximal tibial metaphysis was segmented and reconstructed using the semimanual contouring method16 with TRI/3D-Bon (Ratoc System

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Figure 1 A, Twelve weeks after implant placement, randomly selected implants for each rabbit (n = 10) received mechanical repetitive loading (50 N with a frequency of 3 Hz for 1,800 cycles, 2 days/week) for 8 weeks. The remaining implants were used as control (n = 10). B, Mechanical repetitive loading was applied to tibial implants (arrow) using a custom-made loading device supported by two anchor screws. Distance between the implant surface and the screw surface was 2 mm.

Engineering, Tokyo, Japan). Region of interest (ROI) was defined as the region surrounded between 50 μm and 550 μm away from the implant surface to avoid the metal-induced artifacts,17 and from the implant neck to the inferior border of newly formed bone extending downward from original cortex. Extracortical bone above the implant neck was not included in the ROI (Figure 2A). Bone volume fraction (BVF [%] = bone volume in ROI/tissue volume in ROI), the number of trabecular bone (Tb.N), the thickness of trabecular bone (Tb.Th), the distance between trabecular bones (Tb.Sp), and BMD were semi-automatically measured following the guideline for assessment of bone microstructure using microCT.18 Histology Tibial bone blocks were embedded in methyl methacrylate resin (methyl methacrylate polymer and monomer, Wako Pure Chemical Industries, Ltd., Osaka, Japan) following dehydration in increasing grades of ethanol. Longitudinal central resin-embedded specimens including the implant were made for each tibial bone block using Exakt. These specimens were ground to a final thickness of approximately 10 μm. Toluidine blue staining was performed for the detection of: (1) bone area fraction (BAF); (2) bone implant contact (BIC); (3) bone thickness; (4) bone formation; (5) extracortical bone formation; and (6) osteocyte numbers. Each area of interest (AOI) was determined as follows: (1) BAF (%) = bone area surrounded between 0 μm and 500 μm away from

the implant surface, and from the implant neck to the inferior border of newly formed bone extending downward from original cortex (mm2)/tissue area containing bone marrow in AOI (mm2) × 100 (Figure 2B); (2) BIC (%) = length of bone in contact from the implant neck to the inferior border of newly formed bone extending downward from original cortex (mm)/length of implant from the implant neck to the inferior border of newly formed bone extending downward from original cortex (mm) × 100 (Figure 2C); (3) bone thickness = length of bone from the implant neck to the inferior border of newly formed bone extending downward from original cortex at 500 μm, 750 μm, 1,000 μm, and 1,250 μm away from the implant surface (Figure 2D); (4) bone formation = blue-stained area surrounded between 500 μm and 750 μm, 750 μm and 1,000 μm, and 1,000 μm and 1,250 μm away from the implant surface, and from the implant neck to the inferior border of newly formed bone extending downward from original cortex (Figure 2E); (5) extracortical bone formation = bone area stained with blue above the implant neck; and (6) osteocyte numbers = the number of osteocyte in each AOI (500 μm × 500 μm) around the implant neck and the newly formed bone extending downward from the original cortex, respectively (Figure 2F). Stained sections were photomicrographed using microscopy (BZ-9000, Keyence, Osaka, Japan) and were histomorphometrically analyzed using a BZ Analyzer (Keyence). The measurements were conducted on each side of the implant, and a mean value was calculated.

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Figure 2 A, ROI for microCT analysis was the surrounded region between 50 μm and 550 μm away from the implant surface, and from the implant neck to the inferior border of newly formed bone extending downward from original cortex. B, The red area was used for BAF assessment (two-headed arrow; 500 μm, and from the implant neck to the inferior border of newly formed bone extending downward from original cortex) (dotted line indicates the top of the implant). C, Red line indicates the length of the implant from the implant neck to the inferior border of newly formed bone extending downward from original cortex for assessment of BIC (dotted line indicates the top of the implant). D, Length of two-headed arrows from the implant neck to the inferior border of newly formed bone extending downward from original cortex in 500 μm, 750 μm, 1,000 μm, and 1,250 μm away from the implant surface was used for detection of bone thickness (dotted line indicates the top of the implant). E, Each colored area between 500 μm and 750 μm, 750 μm and 1,000 μm, and 1,000 μm and 1,250 μm was used to evaluate bone formation away from the implant surface (dotted line indicates the top of the implant). F, Assessment of osteocyte numbers and collagen alignment was performed in hard tissues around the implant neck (red area; AOI: 500 × 500 μm) and around the newly formed bone area extending downward from the original cortex (blue area; AOI: 500 × 500 μm). G, Assessment of BAp c-axis alignment was also performed in bone around the implant neck (red circle; AOI: 100-μm diameter with center of the AOI for osteocyte and collagen assessment) and around newly formed bone area extending downward from the original cortex (blue circle; AOI: 100-μm diameter with center of the AOI for osteocyte and collagen assessment). Stained sections were photomicrographed using microscopy. AOI = area of interest; BAF = bone area fraction; BAp = biological apatite; BIC = bone implant contact; ROI = region of interest.

Mechanical Loading Altered Bone Quality

Bone Quality Assessment: Osteocyte Numbers and Alignment of BAp c-Axis and Collagen Fibers In this study, the assessment of bone architecture, determined by osteocyte numbers19 and the preferential alignment of biological apatite (BAp) c-axis and collagen fibers,11 was performed for the evaluation of bone quality. Osteocyte numbers in osteocytic lacunae were counted because osteocytes are detectable in resin-embedded samples with toluidine blue staining using BZ-9000 with higher magnification.20,21 The area around the implant neck, where the original cortical bone would be located, was used to investigate osteocyte numbers and alignment of collagen fibers (AOI; 500 μm × 500 μm). Newly formed bone area extending downward from the original cortex was also used for analyses (AOI; 500 μm × 500 μm) (Figure 2F). Polarized light microscopy (Optiphot-2 Pol, Nikon Corporation, Tokyo, Japan) was used with a polarizer and analyzer in the cross position for assessing the alignment of collagen fibers. Three serial photographs, thought to contain the brightest one for each AOI, were obtained. The brightest photomicrographed image of each AOI was objectively selected from the images using NIH image J (version 1.47; National Institutes of Health, Bethesda, MD). Alignment of collagen fibers was calculated as the relative angle to the long axis of the implant. μXRD with a transmission optical system (R-Axis BQ, Rigaku, Tokyo, Japan) was also used to evaluate the preferential BAp c-axis alignment (Figure 2G) as previously reported.22 Briefly, Mo-Kα radiation was generated at 50 kV and 90 mA. The incident beam was collimated into a 100-μm circular spot using a double-pinhole metal collimator, and was projected vertically onto the specimen to analyze the 2D distribution of BAp c-axis alignment along the surface of the thin specimen. The diffracted beam was collected for 3,600 seconds. From the Debye ring obtained, the diffracted intensities from (002) and (310) planes of BAp were integrated along the azimuthal angle (β) at angle steps of 1°. Finally, the degree of BAp c-axis alignment was calculated as the intensity ratio of (002)/(310) for each β, resulting in 2D BAp c-axis alignment along the plane vertical to the incident X-ray beam. The 2D distribution of BAp alignment was expressed as a radar diagram.

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and with Levene’s test for equality of variances. For parametric data, paired t-test was performed. For nonparametric data, Wilcoxon signed-rank test was used. All statistical analyses were conducted with SYSTAT 12 (Systat Software, Chicago, IL, USA). An α-level of 0.05 was used for statistical significance. Results are presented as median (range). RESULTS Mechanical Repetitive Loading Enhanced Osseointegration and Bone Formation Around Dental Implant All implants were histologically integrated without inflammation (Figure 3A). More bone formation (BAF) and BIC were observed in the loading groups compared with controls (Figure 3, B and C). To clarify the influence of mechanical repetitive loading on hard tissue away from dental implants, bone thickness and bone formation between 500 μm and 1,250 μm away from the implant surface were also measured. Mechanical repetitive loading increased bone thickness regardless of distance from the implant (Figure 3D), while mechanical repetitive loading had a smaller effect on bone formation with increasing distance from the implant (Figure 3E). Interestingly, more extracortical bone formation around the abutment and above the implant neck was observed in the loading group (Figure 3F). Mechanical Repetitive Loading Promoted Bone Anabolic Response Around Dental Implant Bone volume (BVF) excluding extracortical bone mass around dental implants was increased in the loading group compared with control (Figure 4, A and B). Numbers and thickness of trabeculae (Tb.N and Tb.Th, respectively) were significantly higher in the loading group than the control group (Figure 4, D and E). The distance between trabecular bones (Tb.Sp) was smaller in the loading group (Figure 4E). Moreover, BMD in the loading group was increased compared with controls at 8 weeks after initiation of mechanical repetitive loading (Figure 4F). These results indicate that mechanical repetitive loading significantly improved bone anabolic action around dental implant.

Statistics

Mechanical Repetitive Loading Altered Bone Quality Around the Implant Neck

All analyses in this study were blindly conducted. Data were analyzed with the Shapiro–Wilk test for normality

Firstly, we assessed bone quality around the implant neck. In this study, bone quality was assessed by

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Figure 3 Mechanical repetitive loading enhanced bone formation around dental implants; histomorphometric analyses. A, Representative longitudinal images of toluidine blue-stained sections including dental implant in rabbit tibiae. Bar = 1 mm. B, Bone area fraction (BAF) in the loading group was significantly higher than control. C, Bone implant contact (BIC) in the loading group was also significantly higher than control. D, Bone thickness between 500 and 1,250 μm away from implant surface was measured. Each bone thickness was increased in the loading group versus control. A trend of more bone thickness in the loading group was observed at 1,000 μm away from the implant surface. E, Bone formation between 500 and 1,250 μm away from implant surface was measured. Bone formation between 500 and 750 μm away from implant surface in the loading group was increased compared with control. No statistically significant differences between 750 and 1,250 μm in either group were noted. F, Extracortical bone formation was also measured. Mechanical repetitive loading significantly enhanced extracortical bone formation compared with control. n 3 6 per group; *p < .05, **p < .01, ***p < .001.

Mechanical Loading Altered Bone Quality

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measuring osteocyte numbers, the preferential alignment of BAp c-axis, and collagen orientation. Osteocyte numbers in the loading group were higher compared with the control group (Figure 5, A and B). In the control group, the preferential BAp c-axis alignment was along the implant axis. The direction of collagen alignment was also similar to that of BAp c-axis alignment (Figure 5, C–F). Surprisingly, mechanical repetitive loading considerably altered the alignment angle of the BAp c-axis and collagen fibers. The alignment direction of BAp c-axis and collagen fibers along the implant axis rather changed perpendicular direction to the implant axis. Also, the preferential BAp c-axis alignment was approximately parallel to the preferential collagen alignment in response to mechanical loading (Figure 5, C–F). The degree of BAp c-axis alignment was not reduced irrespective of mechanical loading (Figure 5G).

Mechanical Repetitive Loading Partially Altered Bone Quality of Newly Formed Bone Extending Downward From Original Cortex Next, bone quality of newly formed bone extending downward from the original cortex was investigated with the same analyses as bone quality of the implant neck. Osteocyte numbers did not change regardless of mechanical loading (Figure 6, A and B). The preferential alignment of collagen fibers along the implant axis in controls changed with mechanical loading (Figure 6, C and D). Preferential alignment of the BAp c-axis in the loading group was not altered, while mechanical repetitive loading decreased the degree of BAp c-axis alignment compared with control (Figure 6, F and G). DISCUSSION This study demonstrated for the first time that mechanical repetitive loading along the long axis of an implant

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Figure 5 Effects of mechanical repetitive loading on bone quality around the dental implant neck. A, Representative longitudinal images of toluidine blue-stained sections. Osteocytes were observed in both groups. Bar = 100 μm. B, Mechanical repetitive loading significantly increased osteocyte numbers compared with control. C, Representative longitudinal images using polarized microscopy. Bright areas in each group show the direction of preferential collagen alignment (two-headed arrows). Bar = 100 μm. D, Angle of preferential collagen alignment relative to long axis of implants in the loading group was significantly larger than control. E, Representative 2D radar diagrams for BAp alignment defined by integrated intensity ratio of (002)/(310) diffraction intensities using μXRD system. F, Mechanical repetitive loading significantly increased the angle of BAp alignment compared with control. G, No statistically significant differences were noted for the degree of BAp alignment regardless of mechanical loading. n 3 6 per group; *p < .05. μXRD = microbeam X-ray diffractometer; BAp = biological apatite.

alters bone quality with increased osteocyte numbers and adapted preferential alignment of BAp c-axis/ collagen fibers. Furthermore, this study also demonstrated that mechanical repetitive loading upregulates osseointegration, bone quantity (bone volume), and BMD around dental implants.

In this study, jaw bone was not used for the evaluation of bone architecture around dental implants because the application of a custom-made loading device to intraoral region was technically difficult. Previous report indicates that the tibial bone volume in female New Zealand white rabbits is similar to that in maxillae.23

Mechanical Loading Altered Bone Quality

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Figure 6 Effects of mechanical repetitive loading on bone quality of the newly formed bone extending downward from the original cortex. A, Representative longitudinal images of toluidine blue-stained sections. Bar = 100 μm. B, No statistically significant differences between both groups were noted for osteocyte numbers. C, Representative longitudinal images using polarized microscopy. Bright areas in each group show the direction of preferential collagen alignment (two-headed arrows). Bar = 100 μm. D, Angle of preferential collagen alignment relative to long axis of the implant in the loading group was significantly lower than control. E, Representative 2D radar diagrams for BAp alignment using μXRD system. F, No statistically significant differences were noted for the angle of BAp alignment regardless of mechanical loading. G, mechanical repetitive loading significantly decreased the degree of BAp alignment compared with control. n 3 6 per group; *p < .05, **p < .01. μXRD = microbeam X-ray diffractometer; BAp = biological apatite.

Additionally, the natural chewing frequency of New Zealand white rabbits is approximately 3.5 Hz.24,25 Another report also shows that the number of chewing frequency of rabbits is between 3.3 Hz and 4.0 Hz.26 Hence, rabbit tibia was used instead of jaw bone with the loading frequency of 3.0 Hz in the present study. According to the mechanostat theory, a strain amplitude between 1,500 μstrain (upper boundary of

minimum effective strain) and 3,000 μstrain (upper boundary of pathologic minimum effective strain) could promote bone formation.27 An anabolic action in bone especially occurs via dynamic loading rather than static loading.28,29 In this study, dynamic loading was adopted. The force amplitude and frequency were 50 N and 3 Hz, respectively. These parameters were equivalent to 1,661 1 123.65 μstrain/second (strain rate) using

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cadaver samples (data not shown), indicating that the combination of force amplitude and frequency in this study would promote osteogenesis. Indeed, mechanical repetitive loading significantly enhanced bone mass around dental implants compared with nonloaded condition. The repetitive load condition also increased extracortical bone formation and bone thickness, regardless of the distance from the implant surface, revealing that osteogenic response was strikingly induced in this study. Mechanical loading for the early healing response around dental implants is believed to be more effective in bone formation.30,31 However, the net effect of mechanical repetitive loading on bone tissues would be masked by wound healing processes. Bone remodeling around dental implant following wound healing after implant placement may not actually present “osseointegration” because the original definition of “osseointegration” is “a direct structural and functional connection between ordered living bone and the surface of a load-carrying implant.”1 Moreover, the bone response 8 weeks after surgical intervention in rabbit tibiae is significantly weaker compared with that in rabbit maxillae.23 In rabbit tibiae, 8 to 12 weeks are required to reestablish normal bone architecture after surgical trauma.32,33 Thus, in the current study, mechanical loading was applied 12 weeks after implant placement with complete wound healing to clarify the net effect of mechanical loading on bone tissue, although the healing period of 12 weeks in rabbits is comparable with that of 8 months in humans.34 Based on the original definition of “osseointegration,” our finding shows that mechanical repetitive loading significantly enhanced osseointegration. On the other hand, the effects of load cycle number on bone are unclear. The in vivo study demonstrated that bone mineral contents were not changed by any additional increases in the number of load cycles from 36 to 1,800 cycles/day with the same load amplitude and frequency,35 while the in vitro study showed that the proliferation rate of human osteoblasts was mostly increased at 1,800 cycles/day.36 Hence, the determination of load cycle number remains controversial, although 1,800 cycles/day was effective in the present study. Most conventional studies in implant dentistry have focused on the evaluation of bone-to-implant contact and bone formation around dental implants regardless of mechanical loading. However, recent medical reports

and basic researches have emphasized that both evaluation of BMD and bone quality are ultimately required to completely account for bone strength as related to bone mechanical function.37,38 BMD and bone quality account for approximately 70% and 30% of bone strength, respectively.4,39 Moreover, bone architecture, which reflects osteocyte numbers and the preferential alignment direction and degree of BAp c-axis/collagen fibers, is one of the critical factors in determining bone quality.11,19 It has been recently reported that intermittent administration of parathyroid hormone that increases bone strength accelerates new bone formation around dental implants in rat,40,41 suggesting that upregulated bone strength may improve clinical outcome in implant therapy. Hence, we evaluated not only BMD but also bone quality around dental implants to adequately account for bone strength. Mechanical repetitive loading markedly increased BMD with increased bone mass, Tb.N, and Tb.Th, and with decreased Tb.Sp, revealing that approximately 70% of bone strength around dental implants were enhanced. Furthermore, bone quality was also assessed to account for approximately 30% of bone strength. First, bone quality around the implant neck was evaluated because the stress concentration mainly occurs in the neck of osseointegrated implants.42 More osteocyte numbers around the implant neck were noted with loading. Recent report indicates that mechanotransduction via osteocytes plays an important role in adapting bone architecture in response to mechanical loading.43 Decreased osteocyte numbers after parathyroidectomy in human patients influence bone metabolism and bone quality.19 Reduced osteocyte numbers in humans enhance microdamage accumulation that is another determinant factor of bone quality.44 Therefore, our finding shows that increased osteocyte numbers may improve bone quality around the implant neck. On the other hand, mechanical repetitive loading significantly altered the anisotropy of BAp c-axis/collagen fibers in the current study. Additionally, the altered direction of BAp c-axis alignment was approximately parallel to that of collagen alignment. The alignment of BAp c-axis is mostly identical to the direction of the extension of the collagen fibers with loading.45,46 The degree of BAp c-axis alignment contributes to the mechanical function of bone.47 Thus, the altered alignment of BAp c-axis/ collagen fibers indicates that bone quality around the implant neck may be improved. Our findings also

Mechanical Loading Altered Bone Quality

suggest that osteocytic mechanosensing may regulate the anisotropy of BAp c-axis/collagen alignment in response to mechanical repetitive loading although osteocyte-related molecular mechanisms were not demonstrated in this study. On the other hand, the effects of mechanical repetitive loading on the bone quality of newly formed bone extending downward from the original cortical bone were limited. Only collagen alignment was altered by mechanical loading. The degree of BAp c-axis alignment in the loading group was significantly reduced. The reason is uncertain, but load cycle number, load duration, and stress distribution in this area may reduce the responsiveness of bone tissues. However, a recent report showed that the functional BAp c-axis orientation occurs long after new bone formation in rabbit ulna.47 This suggests that bone architecture of newly formed bone extending downward from the original cortex did not fully alter under the present experimental conditions. CONCLUSIONS Within the limitation of the present study because of the application of one load condition, this study showed that: (1) mechanical repetitive loading along the long axis of dental implants was effective for upregulating osseointegration, bone quantity (bone volume), and BMD around dental implant; and 2) mechanical repetitive loading significantly altered bone quality around dental implants. These new insights suggest the importance of evaluating bone quality in implant dentistry. Mechanical repetitive loading via dental implants may increase bone strength, and may contribute to long-term implant stability. ACKNOWLEDGMENTS The authors thank Dr. Ikuya Watanabe for assistance with analyzing the strain rate. This work was supported by a Grant-in Aid for Science Research (B) from the Japan Society for the Promotion of Science (#22390368). All authors have no conflicts of interest. REFERENCES 1. Mavrogenis AF, Dimitriou R, Parvizi J, Babis GC. Biology of implant osseointegration. J Musculoskelet Neuronal Interact 2009; 9:61–71. 2. Chambrone L, Chambrone LA, Lima LA. Effects of occlusal overload on peri-implant tissue health: a systematic review of animal-model studies. J Periodontol 2010; 81:1367–1378.

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Structural and Qualitative Bone Remodeling Around Repetitive Loaded Implants in Rabbits.

Bone mechanical function is regulated by bone quality and bone mineral density (BMD) that reflect bone strength. The preferential alignment of biologi...
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