Takuya Ishimoto

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 Youngs 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 4 weeks and 12 weeks, respectively. Apparent recovery of the macroscopic shape and bio-mineralization of BAp was almost complete in the ulna defect after 4 weeks. However, an additional 2 weeks 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.

Optimization of Cr content of metastable β-type Ti–Cr alloys with changeable Young’s modulus for spinal fixation applications

Metallic implant rods used in spinal fixtures should have a Youngs modulus that is sufficiently low to prevent stress shielding for the patient and sufficiently high to suppress springback for the surgeon. Therefore, we propose a new concept: novel biomedical titanium alloys with a changeable Youngs modulus via deformation-induced ω phase transformation. In this study, the Cr content in the range of 10-14 mass% was optimized to produce deformation-induced ω phase transformation, resulting in a large increase in the Youngs modulus of binary Ti-Cr alloys. The springback and cytotoxicity of the optimized alloys were also examined. Ti-(10-12)Cr alloys exhibit an increase in Youngs modulus owing to deformation-induced ω phase transformation. In this case, such deformation-induced ω phase transformation occurs along with {332}(β) mechanical twinning, resulting in the maintenance of acceptable ductility with relatively high strength. Among the examined alloys, the lowest Youngs modulus and largest increase in Youngs modulus are obtained from the Ti-12Cr alloy. This alloy exhibits smaller springback than and comparable cytocompatibility to the biomedical Ti alloy Ti-29Nb-13Ta-4.6Zr.

Design and optimization of the oriented groove on the hip implant surface to promote bone microstructure integrity.

We proposed a novel surface modification for an artificial hip joint stem from the viewpoint of maintenance and establishment of appropriate bone function and microstructure, represented by the preferred alignment of biological apatite (BAp) and collagen (Col). Oriented grooves were introduced into the proximal medial region of the femoral stem to control the principal stress applied to the bone inside the grooves, which is a dominant factor contributing to the promotion of Col/BAp alignment. The groove angle and the stem material were optimized based on the stress inside the grooves through a finite element analysis (FEA). Only the groove oriented proximally by 60° from the normal direction of the stem surface generated the healthy maximum principal stress distribution. The magnitude of the maximum principal stress inside the groove decreased with increasing the stem Youngs modulus, while the direction of the stress did not largely changed. An in vivo implantation experiment showed that this groove was effective in inducing the new bone with preferential Col/BAp alignment along the groove depth direction which corresponded to the direction of maximum principal stress inside the groove. The anisotropic principal stress distribution and the oriented microstructure inside the groove are similar to those found in the femoral trabeculae; therefore, the creation of the oriented groove is a potent surface modification for optimizing implant design for a long-term fixation.

Biomechanical evaluation of regenerating long bone by nanoindentation

It is crucial to measure the mechanical function of regenerating bone in order to assess the mechanical performance of the regenerating portion as well as the efficiency of the regeneration methods. In this study, nanoindentation was applied to regenerating and intact rabbit ulnae to determine the material properties of hardness and elasticity; viscoelasticity was also investigated to precisely evaluate the material properties. Both intact and regenerating bones exhibited remarkable viscoelasticity manifested as a creep behavior during load hold at the maximum load, and the creep was significantly greater in the regenerating bone than the intact bone. The creep resulted in an overestimation of the hardness and Young’s modulus. Hence, during nanoindentation testing of bones, the effect of creep should be eliminated. Moreover, the regenerating bone had lower hardness and Young’s modulus than the intact bone. The nanoindentation technique proved to be a powerful approach for understanding the mechanical properties of regenerating bone.

Zirconia–hydroxyapatite composite material with micro porous structure

OBJECTIVES Titanium plates and apatite blocks are commonly used for restoring large osseous defects in dental and orthopedic surgery. However, several cases of allergies against titanium have been recently reported. Also, sintered apatite block does not possess sufficient mechanical strength. In this study, we attempted to fabricate a composite material that has mechanical properties similar to biocortical bone and high bioaffinity by compounding hydroxyapatite (HAp) with the base material zirconia (ZrO(2)), which possesses high mechanical properties and low toxicity toward living organisms. METHODS After mixing the raw material powders at several different ZrO(2)/HAp mixing ratios, the material was compressed in a metal mold (8 mm in diameter) at 5 MPa. Subsequently, it was sintered for 5 h at 1500°C to obtain the ZrO(2)/HAp composite. The mechanical property and biocompatibility of materials were investigated. Furthermore, osteoconductivity of materials was investigated by animal studies. RESULTS A composite material with a minute porous structure was successfully created using ZrO(2)/HAp powders, having different particle sizes, as the starting material. The material also showed high protein adsorption and a favorable cellular affinity. When the mixing ratio was ZrO(2)/HAp=70/30, the strength was equal to cortical bone. Furthermore, in vivo experiments confirmed its high osteoconductivity. SIGNIFICANCE The composite material had strength similar to biocortical bones with high cell and tissue affinities by compounding ZrO(2) and HAp. The ZrO(2)/HAp composite material having micro porous structure would be a promising bone restorative material.

Role of stress distribution on healing process of preferential alignment of biological apatite in long bones

Preferential alignment of biological apatite (BAp) crystallites seems closely related to the bone mechanical function due to the crystallographic anisotropy of its hexagonal structure. BAp alignment as well as bone shape and bone mineral density (BMD) is therefore one of the important factors for understanding the recovery of microstructure and mechanical properties in regenerated bones. A rabbit osteotomy model was examined to investigate the recovery process of defected bones. After the introduction of a 10 mm long segmental defect in mature rabbit ulnae, the bone defect was healed spontaneously for 4 and 20 weeks. To evaluate the original and regenerated hard tissues, BMD and BAp alignment along the longitudinal direction were analyzed using peripheral quantitative computed tomography (pQCT) and microbeam X-ray diffractometer system, respectively Four weeks after the operation, the regenerated tissue showed quite a lower degree of BAp alignment than the intact original tissue; it showed no preferential BAp alignment center in the (002) pole figure. In this stage, the regenerated new bone was hardly subjected to loading. At 20 weeks, in contrast, BMD was restored to almost original levels, and the BAp alignment of the regenerated new bone depended strongly on its position under loading conditions. The regenerated degree of BAp preferential alignment showed a high correlation (R 2 = 0.85) with the local stress component proportionally expressed by the reciprocal value of the ulnar cross-sectional area Recovery of BAp alignment of regenerated bones was finally concluded to depend on the stress component in vivo along the longitudinal direction after BMD restoration.

The influence of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide on the proliferation, differentiation and mineralization of odontoblast-like cells

A dentin primer incorporating an antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) shows strong antibacterial effects, and may provide better prognosis for direct capping of infected pulp exposed by caries removal compared with conventional adhesives. However, influences of MDPB on healing of the pulp have not yet been fully elucidated. The purpose of this study was to compare the influences of unpolymerized MDPB on proliferation, differentiation and mineralization of odontoblast-like MDPC-23 cells with those of other resin monomers, Bis-GMA, MDP, TEGDMA and HEMA. The inhibitory effects of MDPB on the proliferation of MDPC-23 were lower than those of Bis-GMA. While MDPB strongly affected the differentiation compared with the other monomers, it was less inhibitory than Bis-GMA and MDP on the mineralization ability of odontoblast-like cells. These findings indicate that MDPB has superior biocompatibility than Bis-GMA in terms of hard tissue formation by odontoblastic cells, suggesting its possible less negative influences on dentinogenesis.

Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC.

Comprehensive analyses of how tubule occlusion and advanced glycation end-products diminish strength of aged dentin.

In clinical dentistry, since fracture is a major cause of tooth loss, better understanding of mechanical properties of teeth structures is important. Dentin, the major hard tissue of teeth, has similar composition to bone. In this study, we investigated the mechanical properties of human dentin not only in terms of mineral density but also using structural and quality parameters as recently accepted in evaluating bone strength. Aged crown and root dentin (age ≥ 40) exhibited significantly lower flexural strength and toughness than young dentin (age < 40). Aged dentin, in which the dentinal tubules were occluded with calcified material, recorded the highest mineral density; but showed significantly lower flexural strength than young dentin. Dentin with strong alignment of the c-axis in hydroxyapatite exhibited high fracture strength, possibly because the aligned apatite along the collagen fibrils may reinforce the intertubular dentin. Aged dentin, showing a high advanced glycation end-products (AGEs) level in its collagen, recorded low flexural strength. We first comprehensively identified significant factors, which affected the inferior mechanical properties of aged dentin. The low mechanical strength of aged dentin is caused by the high mineral density resulting from occlusion of dentinal tubules and accumulation of AGEs in dentin collagen.