Biological Apatite C-axis Research Articles

Structural characteristics of the bone surrounding dental implants placed into the tail-suspended mice

BackgroundThere are many unclear points regarding local structural characteristics of the bone surrounding the implant reflecting the mechanical environment.PurposeThe purpose of this study is to quantitatively evaluate bone quality surrounding implants placed into the femurs of mice in an unloading model, and to determine the influence of the mechanical environment on bone quality.MethodsTwenty 12-week-old male C57BL6/NcL mice (n = 5/group) were used as experimental animals. The mice were divided into two groups: the experimental group (n = 10) which were reared by tail suspension, and the control group (n = 10) which were reared normally. An implant was placed into the femur of a tail-suspended mouse, and after the healing period, they were sacrificed and the femur was removed. After micro-CT imaging, Villanueva osteochrome bone stain was performed. It was embedded in unsaturated polyester resin. The polymerized block was sliced passing through the center of the implant body. Next, 100-μm-thick polished specimens were prepared with water-resistant abrasive paper. In addition to histological observation, morphometric evaluation of cancellous bone was performed, and the anisotropy of collagen fibers and biological apatite (BAp) crystals was analyzed.ResultsAs a result, the femoral cortical bone thickness and new peri-implant bone mass showed low values in the tail suspension group. The uniaxial preferential orientation of BAp c-axis in the femoral long axis direction in the non-implant groups, but biaxial preferential orientation of BAp c-axis along the long axis of implant and femoral long axis direction were confirmed in new bone reconstructed by implant placement. Collagen fiber running anisotropy and orientation of BAp c-axis in the bone surrounding the implant were not significantly different due to tail suspension.ConclusionsFrom the above results, it was clarified that bone formation occurs surrounding the implant even under extremely low load conditions, and bone microstructure and bone quality adapted to the new mechanical environment are acquired.

Osteocalcin is necessary for the alignment of apatite crystallites, but not glucose metabolism, testosterone synthesis, or muscle mass

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.

Effects of long-term cigarette smoke exposure on bone metabolism, structure, and quality in a mouse model of emphysema.

Smoking is a common risk factor for both chronic obstructive pulmonary disease (COPD) and osteoporosis. In patients with COPD, severe emphysema is a risk factor for vertebral fracture; however, the effects of smoking or emphysema on bone health remain largely unknown. We report bone deterioration in a mouse model of emphysema induced by nose-only cigarette smoke (CS) exposure. Unexpectedly, short-term exposure for 4-weeks decreased bone turnover and increased bone volume in mice. However, prolonged exposure for 20- and 40-weeks reversed the effects from suppression to promotion of bone resorption. This long-term CS exposure increased osteoclast number and impaired bone growth, while it increased bone volume. Strikingly, long-term CS exposure deteriorated bone quality of the lumbar vertebrae as illustrated by disorientation of collagen fibers and the biological apatite c-axis. This animal model may provide a better understanding of the mechanisms underlying the deterioration of bone quality in pulmonary emphysema caused by smoking.

Disruption of collagen/apatite alignment impairs bone mechanical function in osteoblastic metastasis induced by prostate cancer

Prostate cancer (PCa) frequently metastasizes to the bone, generally inducing osteoblastic alterations that increase bone brittleness. Although there is growing interest in the management of the physical capability of patients with bone metastasis, the mechanism underlying the impairment of bone mechanical function remains unclear. The alignment of both collagen fibrils and biological apatite (BAp) c-axis, together with bone mineral density, is one of the strongest contributors to bone mechanical function. In this study, we analyzed the bone microstructure of the mouse femurs with and without PCa cell inoculation. Histological assessment revealed that the bone-forming pattern in the PCa-bearing bone was non-directional, resulting in a spongious structure, whereas that in the control bone was unidirectional and layer-by-layer, resulting in a compact lamellar structure. The degree of preferential alignment of collagen fibrils and BAp, which was evaluated by quantitative polarized microscopy and microbeam X-ray diffraction, respectively, were significantly lower in the PCa-bearing bone than in the control bone. Material parameters including Young's modulus and toughness, measured by the three-point bending test, were simultaneously decreased in the PCa-bearing bone. Specifically, there was a significant positive correlation between the degree of BAp c-axis orientation and Young's modulus. In conclusion, the impairment of mechanical function in the PCa-bearing bone is attributable to disruption of the anisotropic microstructure of bone in multiple phases. This is the first report demonstrating that cancer bone metastasis induces disruption of the collagen/BAp alignment in long bones, thereby impairing their mechanical function.

Alignment of Biological Apatite c-Axis Under Functional Loading: A Preliminary Report.

To understand the bone modeling/remodeling process of functionally loaded implants by observing the alignment of the biological apatite (BAp) c-axis. Commercially available titanium implants with TiO2 grit-blasted surfaces were placed in the fourth premolar and the first molar of the beagle dog and were functionally loaded with casted crowns and were compared to the non-loaded controls. After being killed, the samples were subjected to histological observation and to a microbeam x-ray diffraction (μXRD) analysis with a transmission optical system to evaluate the preferential BAp c-axis alignment. The non-loaded group presented mostly nonoriented BAp c-axis around the implant, whereas for the loaded group, preferential alignment of BAp c-axis was observed along the loading direction of the implant suggesting that the Bap aligned itself based on the direction of the functional mechanical loading. The μXRD revealed that the c-axis orientation was perpendicular to the implant thread working against the functional loaded direction, which may contribute in designing an optimal implant macrogeometry.

Induction of Biological Apatite Orientation as a Bone Quality Parameter in Bone Regeneration Using Hydroxyapatite/Poly ɛ-Caprolactone Composite Scaffolds.

Changes in the biological apatite (BAp) c-axis orientation were investigated as a bone quality parameter in bone regeneration using hydroxyapatite/poly ɛ-caprolactone (HA/PCL) composite scaffolds. Three-dimensional (3D) HA/PCL composite scaffolds were fabricated using a layer manufacturing process in three grid sizes (200-, 600-, and 1000 μm) and grafted into the forearm ulna of New Zealand white rabbits. The cross-sectional areas of the bones regenerated from the scaffolds with 600- and 1000-μm grid sizes were significantly larger than those from the scaffold with 200-μm grid sizes, whereas bone mineral density in the regenerated regions did not differ between the three grid sizes. Moreover, the BAp c-axis orientation in the bones regenerated from the scaffolds with grid sizes of 600- and 1000 μm was not significantly different; however, both scaffolds showed enhanced BAp orientation, although the degree of BAp orientation was lower than that in intact bones. In conclusion, HA/PCL composite 3D scaffolds with 600- and 1000-μm grid sizes induced BAp c-axis orientation and showed good bone regeneration behavior in vivo.

Evaluation of crystallographic orientation of biological apatite in vertebral cortical bone in ovariectomized cynomolgus monkeys treated with minodronic acid and alendronate.

Quantitative analysis of the orientational distribution of biological apatite (BAp) crystals is proposed as a new index of bone quality. This study aimed to analyze BAp c-axis orientation in ovariectomized (OVX) monkeys treated with amino-bisphosphonates minodronic acid and alendronate as reference. Sixty female monkeys aged 9-17years were divided into five groups: one sham group and four OVX groups. The sham group and one OVX group were treated daily with vehicle for 17months. The other three groups were treated daily with minodronic acid at doses of 0.015 and 0.15mg/kg, and alendronate at 0.5mg/kg orally, respectively. The seventh lumbar vertebrae were subjected to analysis of the preferential BAp c-axis orientation in the ventral cortical bone. The BAp c-axis orientation along the craniocaudal axis was significantly increased in the OVX monkeys. The high dose of minodronic acid suppressed the OVX-induced increase in the BAp c-axis orientation, whereas alendronate showed a non-significant tendency to suppress the increase in the orientation. In analysis with other parameters, the BAp c-axis orientation was positively correlated with bone formation indices in biochemical markers and bone histomorphometry and negatively correlated with the increase in lumbar bone mineral density. On the other hand, the BAp c-axis orientation was not correlated with bone resorption indices, except for the eroded surface. These results indicate that the increase in BAp c-axis orientation was ameliorated by minodronic acid treatment in OVX monkeys, mainly by suppression of bone formation increase.

Preferential Orientation of Collagen/Biological Apatite in Growing Rat Ulna under an Artificial Loading Condition

Mechanical loading plays a key role in altering macroscopic and microscopic bone structure through functional adaptation; however, the anisotropic micro-organization of collagen and biological apatite (BAp) in adaptively created bone tissue is not well understood in spite of its importance in the mechanical function of bone. In this study, we artificially applied axial compressive loading (15N at 2Hz) to a growing rat ulna 10min/day for 3 weeks to induce new bone under increased mechanical stimulus. Artificial loading induced marked increases in the structural properties of the loaded ulna; the cortical bone area of the mid-shaft was 43.3% larger in the loaded ulna than the contralateral control ulna. The newly formed bone was located mainly on the medial periosteal surface of the ulnar mid-shaft, which experienced the highest compressive strain. The present study firstly clarified that new bone induced by an artificial load showed preferential orientation of collagen and BAp c-axis along the ulnar long axis, which is similar to the pre-existing bone, although the degree of orientation and bone mineral density (BMD) were still impaired after loading for 3 weeks. This anisotropic organization of collagen and BAp crystals corresponded to that of osteocytes, implying that osteocytes are involved in the formation of anisotropic bone micro-organization which is important aspect of bone mechanical function regarding material properties. [doi:10.2320/matertrans.ME201314]

Degree of biological apatite c-axis orientation rather than bone mineral density controls mechanical function in bone regenerated using recombinant bone morphogenetic protein-2

The aim of the present study was to assess the bone regeneration process in defects introduced into rabbit long bones, which were regenerated with controlled release of recombinant bone morphogenetic protein-2 (rBMP-2). The orientation of the biological apatite (BAp) c-axis and bone mineral density (BMD) were compared as predictors of bone mechanical function. A 20-mm-long defect was introduced in rabbit ulnas, and 17 µg of rBMP-2 was controlled-released into the defect using a biodegradable gelatin hydrogel as the carrier. In the bone regeneration process, two characteristic phases may have been governed by different factors. First, new bone formation actively occurred, filling the bone defect with newly formed bone tissue and increasing the BMD. This process was regulated by the strong osteoinductive capacity of rBMP-2. Second, after filling of the defect and moderate BMD restoration, preferential BAp c-axis orientation began to increase, coincident with initiation of remodeling. In addition, the BAp c-axis orientation, rather than BMD, was strongly correlated with Young's modulus, an important index of bone mechanical function, particularly in the later stage of bone regeneration. Thus, preferential BAp c-axis orientation is a strong determinant and predictor of the mechanical function of tissue-engineered bone. Therefore, analysis of BAp preferential c-axis orientation in addition to measurement of BMD is crucial in assessment of bone mechanical function.

Biological apatite (BAp) crystallographic orientation and texture as a new index for assessing the microstructure and function of bone regenerated by tissue engineering

Recently, there have been remarkable advances in medical techniques for regenerating bone defects. To determine the degree of bone regeneration, it is essential to develop a new method that can analyze microstructure and related mechanical function. Here, quantitative analysis of the orientation distribution of biological apatite (BAp) crystallites by a microbeam X-ray diffractometer system is proposed as a new index of bone quality for the evaluation of regenerated bone microstructure. Preferential alignment of the BAp c-axis in the rabbit ulna and skull bone, regenerated by controlled release of basic fibroblast growth factor (bFGF) was investigated. The BAp c-axis orientation was evaluated by the relative intensity between the (002) and (310) diffraction peaks, or the three-dimensional texture for the (002) peak. It was found that new bone in the defects was initially produced without preferential alignment of the BAp c-axis, and subsequently reproduced to recover towards the original alignment. In other words, the BAp density recovered prior to the BAp orientation. Perfect recovery of BAp alignment was not achieved in the ulna and skull defects after 4weeks and 12weeks, respectively. Apparent recovery of the macroscopic shape and bio-mineralization of BAp was almost complete in the ulna defect after 4weeks. However, an additional 2weeks was required for complete repair of BAp orientation. It is finally concluded that orientation distribution of BAp crystallites offers an effective means of evaluating the degree of microstructural regeneration, and also the related mechanical function, in regenerated hard tissues.

Regeneration of Bone Mass and Bone Quality around Implant with Grooves for Aligning Bone Cells in Rabbit Hindlimb Bones

It was reported that one-dimensionally elongated pores in implants promote the production of new bone tissue possessing both high bone density and the preferential alignment of biological apatite (BAp) c-axis/collagen as a bone quality parameter. This finding indicates that the anisotropic orientation and/or migration of osteoblasts guided by the grooved-pore surface affected the establishment of the anisotropic microstructure of bone tissue. In this study, a grooved polytetrafluoroethylene (Teflon) implant, which may have a role in regulating osteoblast arrangement, was prepared to investigate the relationship between cell behavior and bone microstructure. A cylindrical Teflon implant with 8 grooves on its side was prepared. The width and depth of the groove cross-section were 0.5 and 0.75 mm, respectively. Each implant was inserted in a drill-hole defect created on a rabbit femur such that the groove direction was parallel or perpendicular to the long bone axis in which the BAp c-axis aligns one-dimensionally. The Young’s modulus of Teflon is approximately 0.5 GPa, much lower than that of bone; therefore, the effects of applied stress can be eliminated in this model. The oriented new bone was preferentially produced along the grooved surface. The alignment direction of the BAp c-axis was almost parallel to the grooved surface even near the surface vertically aligned to the long bone axis. The geometry of the implant surface can control the organization of BAp alignment through the arrangement of osteoblasts to orient and subsequently to migrate along the surface direction; hence, implant geometry, particularly the groove, is considered an important factor controlling the BAp orientation of regenerated bone tissues.

Quantity and Quality of Regenerated Bone in Grooves Aligned at Different Angles from the Implant Surface

The quantity and quality of regenerated bone strongly depends on the direction and amplitude of in vivo principal stress; therefore, in vivo stress distribution near bone implants should be optimized on the basis of the morphology of the interface between an implant and bone tissue. In this study, grooves were created on the implant surface in order to improve the surface morphology of the implant for optimizing in vivo stress distribution near the implant. The preferential alignment of the biological apatite (BAp) c-axis, which is a parameter of bone quality and controls the mechanical function of bones, is closely related to stress distribution; therefore, the direction of principal stress should be matched with the direction of the groove on the implant surface. Hip implants were prepared with grooves aligned at different angles from the surface; the grooves were located on the stem portion. These implants were inserted in a beagle femur to investigate the dependency of the quantity and quality of newly formed bone in the grooves on the groove angle. The degree of preferential alignment of the BAp c-axis of the regenerated bone in the grooves strongly depends on the angle of the groove to the principal stress vector that was estimated previously to an animal experiment. The regenerated bone forms anisotropic BAp orientation in response to the principal stress in the grooves; therefore, the direction of the grooves has to be designed on the basis of the stress distribution near the implant.

Evaluation and Control of Crystallographic Alignment of Biological Apatite Crystallites in Bones

Our group focused on the preferential degree of biological apatite (BAp) c-axis, an important bone quality parameter based on the microstructural anisotropy in intact, pathological, and regenerated bones. The preferential degree of the BAp c-axis strongly depends on the bone position, in vivo stress distribution, bone growth, degree of pathology and regeneration, activity of bone cells, gene defect, etc. We attempted to challenge clarification of the BAp preferential alignment formation mechanism and control the degree of BAp orientation by using an anisotropic biomaterial design to develop suitable distribution of the BAp c-axis orientation.

Change in Biological Apatite Orientation in Beagle Mandible

The orientation of biological apatite (BAp) is one of the bone quality parameters dominating bone mechanical function. In the mandible, the preferential orientation of the BAp c-axis changes depending on alteration of the in vivo stress condition induced by a change in the biting stress. In this study, to clarify the functional adaptation of the preferential BAp orientation and bone mineral density (BMD), all beagle mandibular molars on one side were extracted to remove the biting stress, leading to changes in both BAp orientation and BMD. The BMD exhibited discontinuous distribution around the first molar, mainly responsible for mastication, on the normal side. However, the distribution was continuous along the mesiodistal axis of the edentulous side. The preferential BAp orientation was analyzed in mandibular cross-sections at the first molar root region. Molar extraction led to a change in the BAp orientation: immediately under the root region on the lingual sides, two-dimensional preferential alignment in the mesiodistal and biting directions of the normal side changed to one-dimensional alignment along the mesiodistal axis of the edentulous side. One-dimensional alignment was also observed on the buccal sides irrespective of molar extraction. These findings clarify the close relationship between in vivo biting stress and the preferential BAp orientation, and will be useful clinically for diagnosis, implant placement, and so on.

Changes of Bone Quality and Quantity in rhM-CSF-Treated Osteopetrotic (op/op) Mice

Measurements of bone mineral density (BMD) cannot accurately predict the risk of bone fracture in some clinical cases; however, BMD is a useful index for assessing the bone condition. Recently, various parameters related to bone strength have been investigated. Among them, we have focused on the preferential orientation of biological apatite (BAp) crystallites analyzed by microbeam-X-ray diffraction, a powerful tool for analyzing BAp crystallites in bones. BAp, a dominant component of bone, is an anisotropic ionic crystal with a hexagonal lattice. In this study, we investigated the mechanism underlying BAp orientation during endochondral or membranous ossification by administering macrophage colony-stimulating factor (M-CSF) to osteopetrotic (op/op) mice lacking M-CSF. op/op mice were treated with intraperitoneal injections of 5 μg recombinant human M-CSF (rhM-CSF); the first injection was administered on the 14th day after birth. In the treated op/op mice, the bone marrow cavities expanded significantly, and this expansion was accompanied by an increase in the number of osteoclasts. Moreover, the degree of BAp orientation along the longitudinal axis was higher in the treated group than in the untreated group. These results suggest that M-CSF is one of the important parameters controlling the preferential alignment of the BAp c-axis.

Analysis of Biological Apatite Orientation in Rat Mandibles

Recently, significant progress has been made in medical techniques for regenerating bone. However, bone evaluation techniques generally assess bone quantity as opposed to bone quality. The use of c-axis crystallite orientation of biological apatite (BAp) as a bone quality index has recently generated great interest. BAp demonstrates strong crystallographic anisotropy, and preferential alignment of BAp in each bone varies depending on the shape and stress conditions in vivo. In the mandible, complicated bone shape and stress conditions in vivo might be associated with both bone quantity and quality. In this study, we aimed to elucidate changes in the bone microstructure in the mandible using crystallographic orientation of BAp as a bone quality index. Using Crj : CD (SD) IGS female rats, we observed changes in the dentulous mandible during bone growth. Measuring points on the mandible were determined based on its positional relationship with the teeth. For analysis of bone quantity, the area and bone mineral density of cortical bone were evaluated using peripheral quantitative computed tomography (pQCT), while the orientation of the BAp c-axis, as analyzed by a micro-beam X-ray diffraction system, was used to assess bone quality. The results of both bone quantity and quality assessments indicated that changes during bone growth varied depending on the presence of teeth. We concluded that the microstructure (especially the texture) of BAp crystallite changes in correlation with variations in stress distribution in vivo resulting from changes in chewing conditions designed to optimize the dynamic chewing function.

Orientation of Biological Apatite in Rat Calvaria Analyzed by Microbeam X-Ray Diffractometer

A microbeam X-ray diffractometer is a powerful tool to analyze oriented biological apatite (BAp) crystallites in bones since BAp orientation is one of the dominant controlling factors for bone mechanical function. The formation of BAp orientation seems to be partly affected by the bone formation process, including membranous or intracartilaginous ossification, the direction and the rate of bone growth, the mineral apposition rate, etc. However, the detailed process and the mechanisms of the organization of BAp orientation during the bone formation process are still not understood. In this study, we focused on a calvarial bone as a flat bone to establish a procedure to analyze BAp orientation in calvarial bone and examined the variation in BAp orientation with age and position in growing rats. Microbeam X-ray diffraction analysis was performed on the extracted calvaria of 5- to 10-week-old Wister rats. The transmission optical system was selected to analyze the orientation of the BAp c-axis along the bone surface. An incident molybdenum (Mo)-K X-ray, which was collimated into a 300-m diameter, was vertically radiated on the calvaria surface, and the diffraction pattern was registered on an imaging plate. Diffraction peak intensities from the (002) and (310) planes of the hexagonal BAp were detected, and then an intensity ratio of (002)/(310) was calculated to evaluate the degree of BAp orientation. BAp orientation in a calvarial bone was successfully analyzed, and the two-dimensional distribution of the BAp c-axis along the calvarial bone surface was identified. A parietal bone, which is a part of the calvarial bone, showed a unique two-dimensional distribution of the BAp c-axis. The distribution remarkably changed depending on the position on a parietal bone and age. The anisotropy in the preferred BAp orientation was very significant at a region that showed high growth rate. Even though the bone formation process seems to affect BAp orientation in the parietal bone, further investigation is needed to understand the mechanism for the development of BAp texture, which is closely related to bone mechanical function.

Adaptation of BAp crystal orientation to stress distribution in rat mandible during bone growth

Biological apatite (BAp) c-axis orientation strongly depends on stress distribution in vivo and tends to align along the principal stress direction in bones. Dentulous mandible is subjected to a complicated stress condition in vivo during chewing but few studies have been carried out on the BAp c-axis orientation; so the adaptation of BAp crystal orientation to stress distribution was examined in rat dentulous mandible during bone growth and mastication. Female SD rats 4 to 14 weeks old were prepared, and the bone mineral density (BMD) and BAp crystal orientation were analyzed in a cross-section of mandible across the first molar focusing on two positions: separated from and just under the tooth root on the same cross-section perpendicular to the mesiodistal axis. The degree of BAp orientation was analyzed by a microbeam X-ray diffractometer using Cu-Kα radiation equipped with a detector of curved one-dimensional PSPC and two-dimensional PSPC in the reflection and transmission optics, respectively. BMD quickly increased during bone growth up to 14 weeks, although it was independent of the position from the tooth root. In contrast, BAp crystal orientation strongly depended on the age and the position from the tooth root, even in the same cross-section and direction, especially along the mesiodistal and the biting axes. With increased biting stress during bone growth, the degree of BAp orientation increased along the mesiodistal axis in a position separated from the tooth root more than that near the tooth root. In contrast, BAp preferential alignment clearly appeared along the biting axis near the tooth root. We conclude that BAp orientation rather than BMD sensitively adapts to local stress distribution, especially from the chewing stress in vivo in the mandible.

透過型 X 線回折法による生体アパタイト結晶配向性の新評価法

Two-dimensional quantitative analysis of point focus X-ray beam diffraction was performed using a transmission optics system to examine the biological apatite (BAp) orientation in the femurs of an osteopetrotic (op/op) mouse and a normal mouse. The Mo Kα (wavelength: 0.07107 nm) was used as characteristic X-ray radiation as a substitute for conventional Cu Kα (wavelength: 0.15418 nm) radiation. At first a theoretical calculation concerning with X-ray absorption and peak resolution was performed, and subsequently X-ray diffraction measurements were carried out to confirm the usefulness of the transmission X-ray method by Mo Kα radiation. The distribution of the preferential orientation of the BAp c-axis was finally measured and calculated as an integrated intensity ratio of (002)/(310) in a plane roughly containing the bone longitudinal axis. The result resembled analysis from a conventional reflection X-ray diffraction method by Cu Kα radiation but this transmission optics system was more convenient for the screening of BAp orientation in bones without sectioning. The distribution of the preferential alignment of the BAp c-axis was measured as a function of the longitudinal axis in the femurs of a 12-week mutant op/op mouse and a littermate control mouse. The preferential alignment of the BAp c-axis along the longitudinal direction is much lower in the op/op mouse than in the control mouse in all analytical positions. The transmission optics system using Mo Kα radiation in this study provides a fuss-free method for analyzing BAp orientation as a bone quality parameter.

J0401-4-2 家兎長管骨再生過程における再生骨の力学機能評価(生体材料およびその表面改質材(4))

Bone mechanical function is given as a complex of the material and structural parameters of bone. We previously reported that the material parameter of regenerated bone can be dominantly explained by two indices, the density and the preferred orientation degree of biological apatite (BAp). Moreover, the shape of the regenerated portion remarkably changes during bone healing, which may lead to a dynamic change in the whole bone mechanical function. In this study, material and structural parameters of regenerated bone are analyzed separately. A defect was prepared on rabbit ulna and naturally regenerated, and a three-point bending test was conducted at the center of the regenerated portion. The dominant parameter which controls the whole bone mechanical function shifts from a structural to material parameter during bone healing. Furthermore, it was statistically demonstrated that the improvement in the material parameter is strongly governed by recovery of the orientation degree of the BAp c-axis along longitudinal direction of the rabbit ulna.