Adaptive enhancement of apatite crystal orientation and Young's modulus under elevated load in rat ulnar cortical bone.

Functional adaptation refers to the active modification of bone structure according to the mechanical loads applied daily to maintain its mechanical integrity and adapt to the environment. Functional adaptation relates to bone mass, bone mineral density (BMD), and bone morphology (e.g., trabecular bone architecture). In this study, we discovered for the first time that another form of bone functional adaptation of a cortical bone involves a change in bone quality determined by the preferential orientation of apatite nano-crystallite, a key component of the bone. An in vivo rat ulnar axial loading model was adopted, to which a 3-15 N compressive load was applied, resulting in approximately 440-3200 µÉ› of compression in the bone surface. In the loaded ulnae, the degree of preferential apatite c-axis orientation along the ulnar long axis increased in a dose-dependent manner up to 13 N, whereas the increase in BMD was not dose-dependent. The Young's modulus along the same direction was enhanced as a function of the degree of apatite orientation. This finding indicates that bone has a mechanism that modifies the directionality (anisotropy) of its microstructure, strengthening itself specifically in the loaded direction. BMD, a scalar quantity, does not allow for load-direction-specific strengthening. Functional adaptation through changes in apatite orientation is an excellent strategy for bones to efficiently change their strength in response to external loading, which is mostly anisotropic.

Deterioration of apatite orientation in the cholecystokinin B receptor gene (Cckbr)-deficient mouse femurs.

INTRODUCTION: The discrepancy between bone mineral density (BMD), the gold standard for bone assessment, and bone strength is a constraint in diagnosing bone function and determining treatment strategies for several bone diseases. Gastric hypochlorhydria induced by clinically used proton pump inhibitor (PPI) therapy indicates a discordance between changes in BMD and bone strength. Here, we used Cckbr-deficient mice with gastric hypochlorhydria to examine the effect of gastric hypochlorhydria on bone mass, BMD, and preferential orientation of the apatite crystallites, which is a strong indicator of bone strength. MATERIALS AND METHODS: Cckbr-deficient mice were created, and their femurs were analyzed for BMD and preferential orientation of the apatite c-axis along the femoral long axis. RESULTS: Cckbr-deficient mouse femurs displayed a slight osteoporotic bone loss at 18 weeks of age; however, BMD was comparable to that of wild-type mice. In contrast, apatite orientation in the femur mid-shaft significantly decreased from 9 to 18 weeks. To the best of our knowledge, this is the first report demonstrating the deterioration of apatite orientation in the bones of Cckbr-deficient mice. CONCLUSION: Lesions in Cckbr-deficient mice occurred earlier in apatite orientation than in bone mass. Hence, bone apatite orientation may be a promising method for detecting hypochlorhydria-induced osteoporosis caused by PPI treatment and warrants urgent clinical applications.

Host bone microstructure for enhanced resistance to bacterial infections.

Postoperative bacterial infection is a serious complication of orthopedic surgery. Not only infections that develop in the first few weeks after surgery but also late infections that develop years after surgery are serious problems. However, the relationship between host bone and infection activation has not yet been explored. Here, we report a novel association between host bone collagen/apatite microstructure and bacterial infection. The bone-mimetic-oriented micro-organized matrix structure was obtained by prolonged controlled cell alignment using a grooved-structured biomedical titanium alloy. Surprisingly, we have discovered that highly aligned osteoblasts have a potent inhibitory effect on Escherichia coli adhesion. Additionally, the oriented collagen/apatite micro-organization of the bone matrix showed excellent antibacterial resistance against Escherichia coli. The proposed mechanism for realizing the antimicrobial activity of the micro-organized bone matrix is by the controlled secretion of the antimicrobial peptides, including ß-defensin 2 and ß-defensin 3, from the highly aligned osteoblasts. Our findings contribute to the development of anti-infective strategies for orthopedic surgeries. The recovery of the intrinsically ordered bone matrix organization provides superior antibacterial resistance after surgery.

Quantitative Threshold Determination of Auditory Brainstem Responses in Mouse Models.

The auditory brainstem response (ABR) is a scalp recording of potentials produced by sound stimulation, and is commonly used as an indicator of auditory function. However, the ABR threshold, which is the lowest audible sound pressure, cannot be objectively determined since it is determined visually using a measurer, and this has been a problem for several decades. Although various algorithms have been developed to objectively determine ABR thresholds, they remain lacking in terms of accuracy, efficiency, and convenience. Accordingly, we proposed an improved algorithm based on the mutual covariance at adjacent sound pressure levels. An ideal ABR waveform with clearly defined waves I-V was created; moreover, using this waveform as a standard template, the experimentally obtained ABR waveform was inspected for disturbances based on mutual covariance. The ABR testing was repeated if the value was below the established cross-covariance reference value. Our proposed method allowed more efficient objective determination of ABR thresholds and a smaller burden on experimental animals.

Innovative design of bone quality-targeted intervertebral spacer: accelerated functional fusion guiding oriented collagen and apatite microstructure without autologous bone graft.

BACKGROUND CONTEXT: Although autologous bone grafting is widely considered as an ideal source for interbody fusion, it still carries a risk of nonunion. The influence of the intervertebral device should not be overlooked. Requirements for artificial spinal devices are to join the vertebrae together and recover the original function of the spine rapidly. Ordered mineralization of apatite crystals on collagen accelerates bone functionalization during the healing process. Particularly, the stable spinal function requires the ingrowth of an ordered collagen and apatite matrix which mimics the intact intervertebral microstructure. This collagen and apatite ordering is imperative for functional bone regeneration, which has not been achieved using classical autologous grafting. PURPOSE: We developed an intervertebral body device to achieve high stability between the host bone and synthesized bone by controlling the ordered collagen and apatite microstructure. STUDY DESIGN: This was an in vivo animal study. METHODS: Intervertebral spacers with a through-pore grooved surface structure, referred to as a honeycomb tree structure, were produced using metal 3D printing. These spacers were implanted into normal sheep at the L2-L3 or L4-L5 disc levels. As a control group, grafting autologous bone was embedded. The mechanical integrity of the spacer/bone interface was evaluated through push-out tests. RESULTS: The spacer with honeycomb tree structure induced anisotropic trabecular bone growth with textured collagen and apatite orientation in the through-pore and groove directions. The push-out load of the spacer was significantly higher than that of the conventional autologous graft spacer. Moreover, the load was significantly correlated with the anisotropic texture of the newly formed bone matrix. CONCLUSIONS: The developed intervertebral spacer guided the regenerated bone matrix orientation of collagen and apatite, resulting in greater strength at the spacer/host bone interface than that obtained using a conventional gold-standard autologous bone graft. CLINICAL SIGNIFICANCE: Our results provide a foundation for designing future spacers for interbody fusion in human.

Deletion of Tfam in Prx1-Cre expressing limb mesenchyme results in spontaneous bone fractures.

INTRODUCTION: Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. MATERIALS AND METHODS: Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. RESULTS: Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. CONCLUSION: Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.

A Novel Ex Vivo Bone Culture Model for Regulation of Collagen/Apatite Preferential Orientation by Mechanical Loading.

The anisotropic microstructure of bone, composed of collagen fibers and biological apatite crystallites, is an important determinant of its mechanical properties. Recent studies have revealed that the preferential orientation of collagen/apatite composites is closely related to the direction and magnitude of in vivo principal stress. However, the mechanism of alteration in the collagen/apatite microstructure to adapt to the mechanical environment remains unclear. In this study, we established a novel ex vivo bone culture system using embryonic mouse femurs, which enabled artificial control of the mechanical environment. The mineralized femur length significantly increased following cultivation; uniaxial mechanical loading promoted chondrocyte hypertrophy in the growth plates of embryonic mouse femurs. Compressive mechanical loading using the ex vivo bone culture system induced a higher anisotropic microstructure than that observed in the unloaded femur. Osteocytes in the anisotropic bone microstructure were elongated and aligned along the long axis of the femur, which corresponded to the principal loading direction. The ex vivo uniaxial mechanical loading successfully induced the formation of an oriented collagen/apatite microstructure via osteocyte mechano-sensation in a manner quite similar to the in vivo environment.

Outstanding in vivo mechanical integrity of additively manufactured spinal cages with a novel "honeycomb tree structure" design via guiding bone matrix orientation.

BACKGROUND CONTEXT: Therapeutic devices for spinal disorders, such as spinal fusion cages, must be able to facilitate the maintenance and rapid recovery of spinal function. Therefore, it would be advantageous that future spinal fusion cages facilitate rapid recovery of spinal function without secondary surgery to harvest autologous bone. PURPOSE: This study investigated a novel spinal cage configuration that achieves in vivo mechanical integrity as a devise/bone complex by inducing bone that mimicked the sound trabecular bone, hierarchically and anisotropically structured trabeculae strengthened with a preferentially oriented extracellular matrix. STUDY DESIGN/SETTINGS: In vivo animal study. METHODS: A cage possessing an anisotropic through-pore with a grooved substrate, that we termed "honeycomb tree structure," was designed for guiding bone matrix orientation; it was manufactured using a laser beam powder bed fusion method through an additive manufacturing processes. The newly designed cages were implanted into sheep vertebral bodies for 8 and 16 weeks. An autologous bone was not installed in the newly designed cage. A pull-out test was performed to evaluate the mechanical integrity of the cage/bone interface. Additionally, the preferential orientation of bone matrix consisting of collagen and apatite was determined. RESULTS: The cage/host bone interface strength assessed by the maximum pull-out load for the novel cage without an autologous bone graft (3360±411 N) was significantly higher than that for the conventional cage using autologous bone (903±188 N) after only 8 weeks post-implantation. CONCLUSIONS: These results highlight the potential of this novel cage to achieve functional fusion between the cage and host bone. Our study provides insight into the design of highly functional spinal devices based on the anisotropic nature of bone. CLINICAL SIGNIFICANCE: The sheep spine is similar to the human spine in its stress condition and trabecular bone architecture and is widely recognized as a useful model for the human spine. The present design may be useful as a new spinal device for humans.

Periodontal Tissue as a Biomaterial for Hard-Tissue Regeneration following bmp-2 Gene Transfer.

The application of periodontal tissue in regenerative medicine has gained increasing interest since it has a high potential to induce hard-tissue regeneration, and is easy to handle and graft to other areas of the oral cavity or tissues. Additionally, bone morphogenetic protein-2 (BMP-2) has a high potential to induce the differentiation of mesenchymal stem cells into osteogenic cells. We previously developed a system for a gene transfer to the periodontal tissues in animal models. In this study, we aimed to reveal the potential and efficiency of periodontal tissue as a biomaterial for hard-tissue regeneration following a bmp-2 gene transfer. A non-viral expression vector carrying bmp-2 was injected into the palate of the periodontal tissues of Wistar rats, followed by electroporation. The periodontal tissues were analyzed through bone morphometric analyses, including mineral apposition rate (MAR) determination and collagen micro-arrangement, which is a bone quality parameter, before and after a gene transfer. The MAR was significantly higher 3-6 d after the gene transfer than that before the gene transfer. Collagen orientation was normally maintained even after the bmp-2 gene transfer, suggesting that the bmp-2 gene transfer has no adverse effects on bone quality. Our results suggest that periodontal tissue electroporated with bmp-2 could be a novel biomaterial candidate for hard-tissue regeneration therapy.

Ibandronate Suppresses Changes in Apatite Orientation and Young's Modulus Caused by Estrogen Deficiency in Rat Vertebrae.

Bone material quality is important for evaluating the mechanical integrity of diseased and/or medically treated bones. However, compared to the knowledge accumulated regarding changes in bone mass, our understanding of the quality of bone material is lacking. In this study, we clarified the changes in bone material quality mainly characterized by the preferential orientation of the apatite c-axis associated with estrogen deficiency-induced osteoporosis, and their prevention using ibandronate (IBN), a nitrogen-containing bisphosphonate. IBN effectively prevented bone loss and degradation of whole bone strength in a dose-dependent manner. The estrogen-deficient condition abnormally increased the degree of apatite orientation along the craniocaudal axis in which principal stress is applied; IBN at higher doses played a role in maintaining the normal orientation of apatite but not at lower doses. The bone size-independent Young's modulus along the craniocaudal axis of the anterior cortical shell of the vertebra showed a significant and positive correlation with apatite orientation; therefore, the craniocaudal Young's modulus abnormally increased under estrogen-deficient conditions, despite a significant decrease in volumetric bone mineral density. However, the abnormal increase in craniocaudal Young's modulus did not compensate for the degradation of whole bone mechanical properties due to the bone loss. In conclusion, it was clarified that changes in the material quality, which are hidden in bone mass evaluation, occur with estrogen deficiency-induced osteoporosis and IBN treatment. Here, IBN was shown to be a beneficial drug that suppresses abnormal changes in bone mechanical integrity caused by estrogen deficiency at both the whole bone and material levels.

Combination treatment with ibandronate and eldecalcitol prevents osteoporotic bone loss and deterioration of bone quality characterized by nano-arrangement of the collagen/apatite in an ovariectomized aged rat model.

Combination therapy with bisphosphonates and vitamin D3 analogs has been frequently used for the treatment of osteoporosis. However, its effects on bone anisotropies, such as orientations of collagen and apatite at the nanometer-scale, which is a promising bone quality index, and its trabecular architecture at the micrometer scale, are not well understood despite its important mechanical properties and its role in fracture risk. In the present study, we analyzed the effects of ibandronate (IBN), eldecalcitol (ELD), and their combination on the collagen/apatite orientation and trabecular architectural anisotropy using an estrogen-deficiency-induced osteoporotic rat model. Estrogen deficiency caused by ovariectomy (OVX) excessively increased the degree of collagen/apatite orientation or trabecular architectural anisotropy along the craniocaudal axis in the lumbar vertebra compared to that of the sham-operated group. The craniocaudal axis corresponds to the direction of principal stress in the spine. The excessive material anisotropy in the craniocaudal axis contributed to the enhanced Young's modulus, which may compensate for the reduced mechanical resistance by bone loss to some extent. The solo administration of IBN and ELD prevented the reduction of bone fraction (BV/TV) determined by µ-CT, and combination therapy showed the highest efficacy in BV/TV gain. Furthermore, the solo administration and combination treatment significantly decreased the degree of collagen/apatite orientation to the sham level. Based on the results of bone mass and collagen/apatite orientation, combination treatment is an effective strategy. This is the first report to demonstrate the efficacy of IBN, ELD, and combination treatment with IBN and ELD relative to the bone micro-architectural anisotropy characterized by collagen/apatite orientation.

Bone fragility via degradation of bone quality featured by collagen/apatite micro-arrangement in human rheumatic arthritis.

Although increased bone fragility is a well-recognized consequence in patients with rheumatoid arthritis (RA), the essential cause of degenerate bone strength remains unknown. This study aimed to determine factors contributing to bone dysfunction in RA by focusing on the bone matrix micro-arrangement, based on the preferential orientation of collagen and the related apatite c-axis as a bone quality index. The classical understanding of RA is limited to its severe pathological conditions associated with inflammation-induced bone loss. This study examined periarticular proximal tibiae from RA patients as compared with osteoarthritis (OA) patients as controls. Bone tissue material strength was disrupted in the RA group compared with the control. Collagen/apatite micro-arrangement and vBMD were significantly lower in the RA group, and the rate of decrease in apatite c-axis orientation (-45%) was larger than that in vBMD (-22%). Multiple regression analysis showed that the degree of apatite c-axis orientation (ß = 0.52, p = 1.9 × 10-2) significantly contributed to RA-induced bone material impairment as well as vBMD (ß = 0.46, p = 3.8 × 10-2). To the best of our knowledge, this is the first report to demonstrate that RA reduces bone material strength by deteriorating the micro-arrangement of collagen/apatite bone matrix, leading to decreased fracture resistance. Our findings represent the significance of bone quality-based analysis for precise evaluation and subsequent therapy of the integrity and soundness of the bone in patients with RA.

Superior Alignment of Human iPSC-Osteoblasts Associated with Focal Adhesion Formation Stimulated by Oriented Collagen Scaffold.

Human-induced pluripotent stem cells (hiPSCs) can be applied in patient-specific cell therapy to regenerate lost tissue or organ function. Anisotropic control of the structural organization in the newly generated bone matrix is pivotal for functional reconstruction during bone tissue regeneration. Recently, we revealed that hiPSC-derived osteoblasts (hiPSC-Obs) exhibit preferential alignment and organize in highly ordered bone matrices along a bone-mimetic collagen scaffold, indicating their critical role in regulating the unidirectional cellular arrangement, as well as the structural organization of regenerated bone tissue. However, it remains unclear how hiPSCs exhibit the cell properties required for oriented tissue construction. The present study aimed to characterize the properties of hiPSCs-Obs and those of their focal adhesions (FAs), which mediate the structural relationship between cells and the matrix. Our in vitro anisotropic cell culture system revealed the superior adhesion behavior of hiPSC-Obs, which exhibited accelerated cell proliferation and better cell alignment along the collagen axis compared to normal human osteoblasts. Notably, the oriented collagen scaffold stimulated FA formation along the scaffold collagen orientation. This is the first report of the superior cell adhesion behavior of hiPSC-Obs associated with the promotion of FA assembly along an anisotropic scaffold. These findings suggest a promising role for hiPSCs in enabling anisotropic bone microstructural regeneration.

Type I Angiotensin II Receptor Blockade Reduces Uremia-Induced Deterioration of Bone Material Properties.

Chronic kidney disease (CKD) is associated with a high incidence of fractures. However, the pathophysiology of this disease is not fully understood, and limited therapeutic interventions are available. This study aimed to determine the impact of type 1 angiotensin II receptor blockade (AT-1RB) on preventing CKD-related fragility fractures and elucidate its pharmacological mechanisms. AT-1RB use was associated with a lower risk of hospitalization due to fractures in 3276 patients undergoing maintenance hemodialysis. In nephrectomized rats, administration of olmesartan suppressed osteocyte apoptosis, skeletal pentosidine accumulation, and apatite disorientation, and partially inhibited the progression of the bone elastic mechanical properties, while the bone mass was unchanged. Olmesartan suppressed angiotensin II-dependent oxidation stress and apoptosis in primary cultured osteocytes in vitro. In conclusion, angiotensin II-dependent intraskeletal oxidation stress deteriorated the bone elastic mechanical properties by promoting osteocyte apoptosis and pentosidine accumulation. Thus, AT-1RB contributes to the underlying pathogenesis of abnormal bone quality in the setting of CKD, possibly by oxidative stress. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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.

Osteoporosis Changes Collagen/Apatite Orientation and Young's Modulus in Vertebral Cortical Bone of Rat.

This study revealed the distinguished changes of preferential orientation of collagen and apatite and Young's modulus in two different types of osteoporotic bones compared with the normal bone. Little is known about the bone material properties of osteoporotic bones; therefore, we aimed to assess material properties in osteoporotic bones. 66 female Sprague-Dawley rats were used. We analyzed the volumetric bone mineral density, collagen/apatite orientation, and Young's modulus of fifth lumbar vertebral cortex for osteoporotic rats caused by ovariectomy (OVX), administration of low calcium and phosphate content (LCaP) diet, and their combination (OVX + LCaP), as well as sham-operated control. Osteocyte conditions were assessed by hematoxylin and eosin and immunohistochemical (matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)) staining. All osteoporotic animals showed bone loss compared with the sham-operated control. OVX improved craniocaudal Young's modulus by enhancing collagen/apatite orientation along the craniocaudal axis, likely in response to the elevated stress due to osteoporotic bone loss. Conversely, LCaP-fed animals showed either significant bone loss or degraded collagen/apatite orientation and Young's modulus. Osteocytes in LCaP and OVX + LCaP groups showed atypical appearance and MEPE- and DMP1-negative phenotype, whereas those in the OVX group showed similarity with osteocytes in the control group. This suggests that osteocytes are possibly involved in the osteoporotic changes in collagen/apatite orientation and Young's modulus. This study is the first to demonstrate that osteoporosis changes collagen/apatite orientation and Young's modulus in an opposite manner depending on the cause of osteoporosis in spite of common bone loss.

Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018.