Papers by Author: Takuya Ishimoto

Abstract: Most bones are anisotropically loaded and seem to be adapted to the anisotropic stress or strain field by changing the anisotropy in their microstructure. Osteocyte (OCY) is believed to play an important role as a mechanosensor and regulator of modeling and/or remodeling orchestrating osteoblast and osteoclast activity to make bone suitable to resist the mechanical environment. In general, osteocytes sense magnitude of stress (strain) applied upon the bone and then work as a trigger to change bone mass to adjust bone’s mechanical function to the stress field. This structural optimization is an important aspect of the bone functional adaptation; another inevitable optimization might be achieved through the change in intrinsic material anisotropy including the preferential c-axis orientation of biological apatite (BAp) crystal. To achieve this adaptation through material anisotropy, osteocyte needs to be a mechanosensor which can detect anisotropic stress field. In the present study, osteocyte lacunae and canaliculi in the mid-diaphysis and the distal part of the rat femur were stained by a fluorescein dye for visualization and analysis. The mid-diaphysis shows greater degree of the preferential c-axis orientation of BAp crystal than the distal part in relation to the magnitude of uni-axial stress field. It was found that the osteocytes in long bone preferentially align along the bone long axis and the degree of alignment is greater in the mid-diaphysis than in the distal region, which seems to be effective for the sensation of the site-dependent specific stress field applied on the long bone.

Abstract: Bone microstructure is dominantly composed of anisotropic extracellular matrix (ECM) in which collagen fibers and epitaxially-oriented biological apatite (BAp) crystals are preferentially aligned depending on the bone anatomical position, resulting in exerting appropriate mechanical function. The regenerative bone in bony defects is however produced without the preferential alignment of collagen fibers and the c-axis of BAp crystals, and subsequently reproduced to recover toward intact alignment. Thus, it is necessary to produce the anisotropic bone-mimetic tissue for the quick recovery of original bone tissue and the related mechanical ability in the early stage of bone regeneration. Our group is focusing on the methodology for regulating the arrangement of bone cells, the following secretion of collagen and the self-assembled mineralization by oriented BAp crystallites. Cyclic stretching in vitro to bone cells, principal-stress loading in vivo on scaffolds, step formation by slip traces on Ti single crystal, surface modification by laser induced periodic surface structure (LIPSS), anisotropic collagen substrate with the different degree of orientation, etc. can dominate bone cell arrangement and lead to the construction of the oriented ECM similar to the bone tissue architecture. This suggests that stress/strain loading, surface topography and chemical anisotropy are useful to produce bone-like microstructure in order to promote the regeneration of anisotropic bone tissue and to understand the controlling parameters for anisotropic osteogenesis induction.

Abstract: Co-Cr-Mo based alloys have been widely employed as heat resistant materials and as biomaterials for implants because of their high strength and superior wear resistance. In general, the alloys exhibit a very complicated composition-dependent microstructure containing stacking faults and related mechanical properties. Thus, the essential properties must be clarified by using not only polycrystals but also single crystals. To our knowledge, single crystals and related properties have not been reported elsewhere. Thus, Co-Cr-Mo single crystals were grown and used to analyze the microstructure and the related properties. Single crystals with a composition Co-27 mass% Cr-6 mass% Mo alloy defined by ASTM F75 were grown by two single crystal apparatuses: the optical floating zone and the Bridgman methods. The single crystals with the smooth-surface shape were successfully obtained in the Bridgman method under an Ar gas atmosphere at a crystal growth rate of 5.0 or 2.5 mm/h. A portion of the crystals contain Al as Al₂O₃ precipitates from the crucible. Since the Al₂O₃ precipitate induces martensitic phase transformation from fcc (γ) phase to hcp (ε) phase, the single crystals were separated into two parts (a) containing Al₂O₃ precipitate and (b) in the absence of the clear precipitate. The microstructure was significantly altered by the martensitic phase transformation from the γ to ε phase induced by stress field or heating. In addition, variant formation of ε phase has a large influence on the mechanical functions of these Co-Cr-Mo alloys. Novel findings were preliminary obtained in the single crystals.

Abstract: Cells are known to sense the topographic features of the substrate and align along the direction of the surface pattern, and this is believed to be an important aspect in the formation and regeneration of anisotropic biological tissues. In this study, a unique and anisotropic stepped pattern was produced on single crystals of α-Ti with the h.c.p. lattice by plastic deformation in compression to demonstrate the effect of the pattern on cell behavior. Because the Schmid factor for the operative slip system of prismatic (100)[110] was set to be 0.5, the slip traces with an acute angle of 45° appeared on the surface. A smooth substrate without plastic deformation was used as a control. MC3T3-E1 osteoblastic cells were cultured on the substrate for 24 h, followed by observation of the morphology and alignment of the cells by Giemsa staining. On stepped substrates, cells aligned along the slip traces, and the filopodia of the aligned cells were found to extend parallel to the slip traces. The slip traces induced by plastic deformation of a single crystal was successfully proven to be a potent substrate to control the alignment of cells.

Abstract: 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.

Abstract: Recently, more attention has been devoted to porous implants to avoid stress-shielding effects and facilitate anchor effects. In addition, our previous research revealed that uniaxially aligned pores promoted early recovery of bone tissue with high bone quality similar to that of intact bone. In this study, Ti-based implant materials with uniaxially aligned pores were fabricated using the electron beam melting (EBM) method with 2 types of grid spacing, 0.5 and 1.0 mm. Although grid spacing was varied, the constituent phase and microstructure of the products were homogenous regardless of the grid spacing. Uniaxially aligned pores were created when the grid spacing was 1.0 mm, whereas almost solid structures with random pores were formed when the grid spacing was 0.5 mm. Young’s modulus of the products with the grid spacing of 1.0 mm was 34 GPa; this value is close to that of the bone. It is concluded that the porous material with aligned pores is suitable as a bone implant to reduce stress-shielding effects and to induce bone regeneration with good bone quality.

Abstract: 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.

Abstract: To evaluate the material parameters of regenerated bone, it is important to clarify the mechanical performance of the regenerated portion. In general, the shape and size of regenerated bone tissue is heterogeneous. It is often difficult to elucidate material properties by means of conventional mechanical tests such as compressive and/or tensile tests and bending tests. The nanoindentation technique has been utilized to evaluate the material properties of small or microstructured materials because they do not necessarily require a large well-designed specimen. Thus, this technique may be useful for the evaluation of the material properties of regenerated bone tissue. In this study, this technique was applied for the assessment of the Young’s modulus and hardness of regenerated and intact long bones of a rabbit. The regenerated bone exhibited a significantly lower Young’s modulus and hardness than the intact bone. The regenerated long bone also exhibited impaired mechanical properties, which may have been caused by the difference in the nano-organization of its collagen fibers and mineral crystals (the main components of bone tissue), from that of the intact bone.

Abstract: 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.

Abstract: Amid increasing numbers of artificial joint implantation surgeries, improving the quality of life (QOL) for patients by accounting for individual variation is a primary concern. Thus, we aim to develop implants designed to optimize the interface between implant and living bone. In particular, for ensuring long-term durability and stability after implantation, we focused on inducement of appropriate alignment for biological apatite (BAp) crystallites and the related collagen (Col) fibers as a bone quality parameter. In this study, we predicted that when stress is applied to bone, the BAp/Col preferential alignment can be formed on the basis of our previous result if osteocytes, which can sense its around stress field, are in an environment that is aligned with the principal stress vector. We tested this idea by introducing grooves with the different angles on the implant surface, considering the principal stress direction. This study finally analyses the effect of stress transmission by a load at the proximal femur on the bone inside and near the grooves by using a mechanical simulation in which groove angles and positions can be changed on the implant surface. Furthermore, we carried out animal experiments using a 2-years-old beagle to examine the effect of grooves in the principal stress direction on the surface in vivo. As a result, bone formation in grooves on the implant surface strongly depends on the grooved angle to the principal stress vector and the grooved position on implants. The new bone preferentially formed inside the grooves parallel to the principal stress direction predicted by three dimensional finite element analysis (FEA) in the proximal area of beagle femur.