3D interrelationship between osteocyte network and forming mineral during human bone remodeling

During bone remodeling, osteoblasts are known to deposit unmineralized collagenous tissue (osteoid), which mineralizes after some time lag. Some of the osteoblasts differentiate into osteocytes, forming a cell network within the lacunocanalicular network (LCN) of bone. To get more insight into the potential role of osteocytes in the mineralization process of osteoid, sites of bone formation are three-dimensionally imaged in nine forming human osteons using focused ion beam-scanning electron microscopy (FIB-SEM). In agreement with previous observations, the mineral concentration is found to gradually increase from the central Haversian canal toward pre-existing mineralized bone. Most interestingly, a similar feature is discovered on a length scale more than 100-times smaller, whereby mineral concentration increases from the LCN, leaving around the canaliculi a zone virtually free of mineral, the size of which decreases with progressing mineralization. This suggests that the LCN controls mineral formation but not just by diffusion of mineralization precursors, which would lead to a continuous decrease of mineral concentration from the LCN. The observation is, however, compatible with the codiffusion and reaction of precursors and inhibitors from the LCN into the bone matrix

Phenotypic spectrum in osteogenesis imperfecta due to mutations in TMEM38B: unravelling a complex cellular defect.

Context: Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Objectives: Clinical and bone material phenotype description and osteoblast differentiation studies. Design and Setting: Natural history study in paediatric research centres. Patients: Eight patients with type XIV OI. Main Outcome Measures: Clinical examinations included: bone mineral density, radiographs, echocardiography and muscle biopsy. Bone biopsy samples (n=3) were analysed using histomorphometry, quantitative backscattered electron microscopy and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Results: The clinical phenotype of type XIV OI ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband L1-L4 bone density Z-score was reduced (median -3.3 [range -4.77 to +0.1; n=7]), and increased by +1.7 (1.17 to 3.0; n=3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased late and mineralization-related markers. Predominance of TRIC-B over TRIC-A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. Conclusions: OI type XIV has a bone histology, matrix mineralization and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities

Postembedding iodine staining for contrast-enhanced 3D imaging of bone tissue using focused ion beam-scanning electron microscopy

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high-resolution imaging techniques. In the case of bone, focused ion beam-scanning electron microscopy (FIB-SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin-embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB-SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization

The contribution of the pericanalicular matrix to mineral content in human osteonal bone

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The osteocyte lacunar-canalicular network (LCN) penetrates bone and houses the osteocytes and their processes. Despite its rather low volume fraction, the LCN represents an outstanding large surface that is possibly used by the osteocytes to interact with the surrounding mineralized bone matrix thereby contributing to mineral homeostasis. The aim of this study was to quantitatively describe such contributions by spatially correlating the local density of the LCN with the mineral content at the same location in micrometer-sized volume elements in human osteons. For this purpose, 65 osteons from the femur midshaft from healthy adults (n = 4) and children (n = 2) were structurally characterized with two different techniques. The 3D structure of the LCN in the osteons was imaged with confocal laser scanning microscopy after staining the bone samples with rhodamine. Subsequent image analysis provided the canalicular length density, i.e. the total length of the canaliculi per unit volume (μm/μm3). Quantitative information on the mineral content (wt%Ca) from the identical regions was obtained using quantitative backscattered electron imaging. As the LCN-porosity lowers the mineral content, a negative correlation between Ca content and network density was expected. Calculations predict a reduction of around −0.97 fmol Ca per μm of network. However, the experiment revealed for 62 out of 65 osteons a positive correlation resulting in an average additional Ca loading of +1.15 fmol per μm of canalicular network, i.e. an accumulation of mineral has occurred at dense network regions. We hypothesize that this accumulation happens in the close vicinity of canaliculi forming mineral reservoirs that can be utilized by osteocytes. Significant differences found between individuals indicate that the extent of mineral loading of the reservoir zone reflects an important parameter for mineral homeostasis.German Federal Ministry of Education and ResearchAUVA (Research Funds of the Austrian Workers Compensation Board, Austria)WGKK (Viennese sickness insurance funds, Austria)

Heterogeneity of the osteocyte lacuno-canalicular network architecture and material characteristics across different tissue types in healing bone

Various tissue types, including fibrous connective tissue, bone marrow, cartilage, woven and lamellar bone coexist in healing bone. Similar to all bone tissue type, healing bone contains a lacuno-canalicular network (LCN) housing osteocytes that are known to orchestrate bone remodeling in healthy bone by sensing mechanical strains and translating them into biochemical signals. The structure of the LCN is also hypothesized to influence mineralization processes. Hence, the aim of the present study was to visualize and correlate spatial variations in the LCN topology with mineral characteristics, within and at the interfaces of the different tissue types that comprise healing bone. We applied a correlative multi-method approach to visualize the LCN architecture and quantify mineral particle size and orientation within healing femoral bone in a mouse osteotomy model (26 weeks old C57BL/6 mice). This approach revealed structural differences across several length scales during endochondral ossification within the following regions: calcified cartilage, bony callus, cortical bone and the transition zone between the cortical region and callus that developed during 21 days after the osteotomy. In this transition zone, we observed a continuous convergence of mineral characteristics and osteocyte lacunae shape as well as discontinuities in the lacunae volume and LCN connectivity. The bony callus exhibits a 34% higher lacunae number density with 40% larger lacunar volume compared to cortical bone. The presented correlations between LCN architecture and mineral characteristics improves our understanding of how bone develops during healing and may indicate a contribution of osteocytes to bone (re)modeling

Materials in particulate form for tissue engineering. 2 Applications in bone

Materials in particulate form have been the subjects of intensive research in view of their use as drug delivery systems. While within this application there are still issues to be addressed, these systems are now being regarded as having a great potential for tissue engineering applications. Bone repair is a very demanding task, due to the specific characteristics of skeletal tissues, and the design of scaffolds for bone tissue engineering presents several difficulties. Materials in particulate form are now seen as a means of achieving higher control over parameters such as porosity, pore size, surface area and the mechanical properties of the scaffold. These materials also have the potential to incorporate biologically active molecules for release and to serve as carriers for cells. It is believed that the combination of these features would create a more efficient approach towards regeneration. This review focuses on the application ofmaterials in particulate formfor bone tissue engineering. A brief overview of bone biology and the healing process is also provided in order to place the application in its broader context. An original compilation of molecules with a documented role in bone tissue biology is listed, as they have the potential to be used in bone tissue engineering strategies. To sum up this review, examples of works addressing the above aspects are presented

Quantitative backscattered electron imaging of bone using a thermionic or a field emission electron source

Quantitative backscattered electron imaging is an established method to map mineral content distributions in bone and to determine the bone mineralization density distribution (BMDD). The method we applied was initially validated for a scanning electron microscope (SEM) equipped with a tungsten hairpin cathode (thermionic electron emission) under strongly defined settings of SEM parameters. For several reasons, it would be interesting to migrate the technique to a SEM with a field emission electron source (FE-SEM), which, however, would require to work with different SEM parameter settings as have been validated for DSM 962. The FE-SEM has a much better spatial resolution based on an electron source size in the order of several 100 nanometers, corresponding to an about 105 to 106 times smaller source area compared to thermionic sources. In the present work, we compare BMDD between these two types of instruments in order to further validate the methodology. We show that a transition to higher pixel resolution (1.76, 0.88, and 0.57 μm) results in shifts of the BMDD peak and BMDD width to higher values. Further the inter-device reproducibility of the mean calcium content shows a difference of up to 1 wt% Ca, while the technical variance of each device can be reduced to ±0.17 wt% Ca. Bearing in mind that shifts in calcium levels due to diseases, e.g., high turnover osteoporosis, are often in the range of 1 wt% Ca, both the bone samples of the patients as well as the control samples have to be measured on the same SEM device. Therefore, we also constructed new reference BMDD curves for adults to be used for FE-SEM data comparison

Combination of Nanoindentation and Quantitative Backscattered Electron Imaging Revealed Altered Bone Material Properties Associated with Femoral Neck Fragility

Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74–92 years) and five nonfractured controls (75–88 years). The typical calcium content (CaPeak) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (CaInd) at the indent positions (obtained from qBEI). CaPeak (−2.2%, P = 0.002), CaInd (−1.8%, P = 0.048), E (−5.6%, P = 0.040), and H (−6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, CaPeak as well as CaInd were also lower (−2.6%, P = 0.006, and –3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rar alpha, and rar beta) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgf beta 2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rar beta, meis2, hoxb5, tgf beta 2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgf beta 2, and id2 spatial distribution; to increase rar beta, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.The authors would like to thank Ana Lima for slide sectioning and Rita Lopes for contributing to the initiation of this project. This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio