Osteocytic Lacunar-canalicular System Research Articles

Expansion of the osteocytic lacunar-canalicular system involved in pharmacological action of PTH revealed by AI-driven fluorescence morphometry in female rabbits

Osteoporosis is an age-related disorder that is characterized by reduced bone mass. Its prevention and treatment are important healthcare issues for maintaining social activity in aged societies. Although bone fractures mostly occur at sites of weakened cortical bone, pathophysiological and pharmacological evaluations of bone mass have tended to be predominantly assessed in trabecular bone. To statistically characterize cortical bone remodeling, we originally established multimode fluorescence imaging and artificial intelligence (AI)-driven morphometric analyses in six-month-old female rabbits with well-defined cortical remodeling, similar to that in humans. We evaluated three distinct administration frequencies of teriparatide [TPTD; human parathyroid hormone, hPTH (1–34)]: once (1/w), twice (2/w), and seven times (7/w) a week, with the same total dose (140 μg/kg/week). Our analyses revealed significant expansions of the osteocytic lacunar-canalicular system and Haversian canals accompanied by the development of cortical porosity and endosteal naïve bone formation induced by a frequent administration regimen (7/w) of TPTD; however, once-weekly (1/w) and twice-weekly (2/w) administration of TPTD showed little effect. These findings demonstrate a clear contrast between the effects of frequent and infrequent administration of TPTD on cortical bone metabolism and suggest that osteocytic bone remodeling is involved in the pharmacological action of PTH.

Interactive effects of various loading parameters on the fluid dynamics within the lacunar-canalicular system for a single osteocyte.

The osteocyte lacunar-canalicular system (LCS) serves as a mechanotransductive core where external loading applied to the skeleton is transduced into mechanical signals (e.g., fluid shear) that can be sensed by mechanosensors (osteocytes). The fluid velocity and shear stress within the LCS are affected by various loading parameters. However, the interactive effect of distinct loading parameters on the velocity and shear stress in the LCS remains unclear. To address this issue, we developed a multiscale modeling approach, combining a poroelastic finite element (FE) model with a single osteocytic LCS unit model to calculate the flow velocity and shear stress within the LCS. Next, a sensitivity analysis was performed to investigate individual and interactive effects of strain magnitude, strain rate, number of cycles, and intervening short rests between loading cycles on the velocity and shear stress around the osteocyte. Lastly, we developed a relatively simple regression model to predict those outcomes. Our results demonstrated that the strain magnitude or rate alone were the main factors affecting the velocity and shear stress; however, the combination of these two was not directly additive, and addition of a short rest between cycles could enhance the combination of these two related factors. These results show highly interactive effects of distinct loading parameters on fluid velocity and shear stress in the LCS. Specifically, our results suggest that an enhanced fluid dynamics environment in the LCS can be achieved with a brief number of load cycles combined with short rest insertion and high strain magnitude and rate.

A Potential Participant in Type 2 Diabetes Bone Fragility: TIMP-1 at Sites of Osteocyte Lacunar-Canalicular System.

Type 2 diabetes mellitus (T2DM) is associated with an increased risk of bone fracture, but the bone mineral density (BMD) is typically normal or higher in such patients. Because the fracture risk is independent of reduced BMD, bone fragility in T2DM may be partially due to poor bone quality. The mechanisms triggering bone quality abnormalities in T2DM are complex, and include the accumulation of advanced glycation end-products, the increased inflammation, and low bone turnover. Matrix metalloproteinases (MMPs) in bone can hydrolyze the bone matrix. Tissue inhibitors of MMPs (TIMPs) can inhibit the activity of MMPs. Both MMPs and TIMPs participate in mediating bone quality. Among all types of TIMPs, TIMP-1 is mostly reportedly increased in the serum of T2DM patients. Because osteocytes can express TIMP-1, and osteocyte pericellular matrix influences bone quality partially regulated by perilacunar/canalicular remodeling, we hypothesized that TIMP-1 at sites of osteocyte lacunar-canalicular system is involved in T2DM bone fragility.

Liquid-phase ASEM imaging of cellular and structural details in cartilage\xa0and bone formed during endochondral ossification: Keap1-deficient osteomalacia

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1−/− femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells

BackgroundDegenerative changes in the skeleton play an important role in ageing. As the foremost sensors and orchestrators of bone remodelling, osteocytes contribute significantly to the health of the skeleton. Embedded in a mineralized bone matrix, the osteocyte network and the surrounding lacunar canaliculae work together as a functional syncytium—the osteocytic lacunar-canalicular system (OLCS). However, changes in the OLCS during ageing and related mechanisms cannot be fully understood by using traditional histological analysis.MethodsTo link the phenotypes of aged osteocytes and their functional changes during ageing, we analysed the changes in the gene expression profiles of bone cells and the proteomic profiles of OLCS exosomes derived from aged and young cortical bone.ResultsGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) suggested that a decline in cell energy metabolism and an increased level of the proinflammatory state are major characteristics of bone ageing. Moreover, some DEGs were key regulators of bone mechanical sensation and bone remodelling, which are indicative of reduced bone-specific function with age. Further, the identified proteins in OLCS exosomes showed potential changes in the secretory function bone. Compared with young controls, the decreased functional proteins in aged OLCS exosomes were enriched mainly in GO terms that included regulating bone development and remodelling, cell-matrix adhesion, and cell clearance and homeostasis. Notably, several functions of exosomal proteins of the aged group revealed potential new roles, such as regulating innate and adaptive immunity, wound healing, and angiogenesis and eliminating oxidative stress.ConclusionThe information obtained from bone cells and OLCS exosomes will help us discover new features of bone ageing.

1. Morphological Implication on Cellular Response to Mechanical Stress in Bone.

In bone, there are 3 distinct cell types: an osteoblast, a bone forming cell; an osteocyte embedded in bone matrix as a consequence of being differentiated from an osteoblast; and an osteoclast, a multinucleated giant cell responsible for bone resorption. Bone is always remodeled by replacing old bone with new bone (bone remodeling), by which bone can maintain its stiffness and flexibility. However, in an osteoporotic state, the disrupted balance between bone resorption and formation results in not only markedly reduced bone mass, but also in disorganized geometry of trabecules, which can often give rise to a bone fracture. Osteocytes located in their lacunae insert their fine cytoplasmic processes into narrow passageways referred to as osteocytic canaliculi. Neighboring osteocytes connect to each other by means of a gap junction in their cytoplasmic processes. Therefore, osteocytes and their lacunae/canaliculi appear to form functional syncytium called osteocytic lacunar canalicular system (OLCS). The geometrical distribution of OLCS is poorly arranged in immature bone, while it appears well-arranged distribution in mature bone (cortical bone), in which molecular transports and sensing mechanical stress seems to be efficient, and therefore, may be able to respond to mechanical stress. In this seminar, I will introduce our recent findings on the morphology and function of OLCS which may respond to mechanical stress.

Bone and Stem Cells. Cellular network in bone micro-environment - histological and ultrastructural aspects -

Bone micro-environment appears to reflect bone turnover, i.e., frequency of bone remodeling. There are many bone-synthesizing mature osteoblasts, bone-resorbing osteoclasts, and a thick cell layer of preosteoblasts overlying mature osteoblasts in the region which shows active bone remodeling. Bone lining cells, - flattened, resting form of osteoblasts cover the quiescent bone surface, in which, however, osteocyte-lacunar canalicular system tend to be geometrically well-arranged. Thus, bone micro-environment seems to be regulated by preosteoblasts, bone marrow stromal cells and vascular endothelial cells, as well as osteoblasts and osteoclasts. But, precious biological function of preosteoblasts and bone marrow stromal cells are still under the investigation, e.g., due to many phenotypes of preosteoblasts. In this review, we will introduce histological and ultrastructural aspects on cellular involvement in bone micro-environment.

Updates on rickets and osteomalacia: biological function of osteocyte and FGF23/klotho system

Osteocytes are known to synthesize FGF23 which would bind to FGFR1c/klotho complex in proximal renal tubules in kidney, thereby, reducing serum concentration of Pi and the activity of 1α-hydroxylase. Meanwhile, recent studies suggest the possibility that osteocytes might induce osteolysis of lacuna walls. Compact, cortical bone develops well-organized distribution of osteocyte-lacunar canalicular system (OLCS) , which appears to be efficient for osteocytic function. There seems some relation between the geometrical regularity of OLCS and osteocytic regulation of systemic and local mineral balance.

Sclerostin is differently immunolocalized in metaphyseal trabecules and cortical bones of mouse tibiae

Sclerostin, an osteocyte-derived molecule, has been reported to serve as a negative regulator of osteoblastic activity as well as bone remodeling. However, there is no report that verified the regional difference for sclerostin synthesis, and in this study we have investigated immunolocalization of sclerostin by comparing dentin matrix protein (DMP) 1, an osteocyte-derived factor broadly expressed in tibial metaphyses and cortical bone. In metaphyseal primary trabecules, a site of bone modeling, strong DMP1-reactivity was observed in osteocytic lacunar-canalicular system (OLCS), while faint staining for sclerostin was visible only in a few osteocytes. In secondary trabecules, in which bone remodeling begins, some osteocytes showed intense sclerostin-immunopositivity, though there were many DMP1-positive osteocytes. In cortical bone, there were more osteocytes reactive for sclerostin, when compared with those in the secondary trabecules. Silver impregnation verified that immature, primary trabecules contained randomly-oriented OLCS, while mature, cortical bone showed geometrically well-arrangement of OLCS. Taken together, though DMP1 is broadly synthesized in bone, sclerostin appears to be abundantly synthesized in regular OLCS of cortical bone, but less produced in irregular OLCS as seen in primary trabecules, indicating the regional difference for sclerostin synthesis.

Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats

This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor α and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system. In conclusion, estrogen deficiency appears to inhibit osteocyte-derived sclerostin synthesis in the mesial region of the interradicular septum, in a process that seems to be mediated by accelerated bone remodeling rather than by direct effects on osteocytes.

A Morphological Analysis on the Osteocytic Lacunar Canalicular System in Bone Surrounding Dental Implants

Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone.

Immunolocalization of DMP1 and sclerostin in the epiphyseal trabecule and diaphyseal cortical bone of osteoprotegerin deficient mice

In order to define the osteocytic function in accelerated bone remodeling, we examined the distribution of the osteocytic lacunar-canalicular system (OLCS) and osteocyte-secreting molecules--dentin matrix protein (DMP) 1 and sclerostin--in the epiphyses and cortical bones of osteoprotegerin deficient (OPG(-/-)) mice. Silver impregnation visualized a well-arranged OLCS in the wild-type epiphyses and cortical bone, whereas OPG(-/-) mice had an irregular OLCS in the epiphyses, but well-arranged canaliculi in the cortical bone. DMP1-positive osteocytes were evenly distributed throughout the wild-type epiphyses and cortical bone, as well as the OPG(-/-) cortical bone. However, OPG(-/-) epiphyses revealed weak DMP1-immunoreactivity. Thus, osteocytes appear to synthesize more DMP1 as the OLCS becomes regular. In contrast, sclerostin-immunoreactivity was significantly diminished in the OPG(-/-) epiphyses and cortical bone. In OPG(-/-) epiphyses and cortical bone, triple staining demonstrated few sclerostin-positive osteocytes in the periphery of a thick cell layer of alkaline phosphatase-positive osteoblasts and many tartrate resistant acid phosphatase-positive osteoclasts. Summarizing, the regular distribution of OLCS may affect DMP1 synthesis, while the cellular activities of osteoclasts and osteoblasts rather than the regularity of OLCS may ultimately influence sclerostin synthesis.

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

Modeling Fluorescence Recovery After Photobleaching in Loaded Bone: Potential Applications in Measuring Fluid and Solute Transport in the Osteocytic Lacunar-Canalicular System

Solute transport through the bone lacunar-canalicular system is essential for osteocyte viability and function, and it can be measured using fluorescence recovery after photobleaching (FRAP). The mathematical model developed here aims to analyze solute transport during FRAP in mechanically loaded bone. Combining both whole bone-level poroelasticity and cellular-level solute transport, we found that load-induced solute transport during FRAP is characterized by an exponential recovery rate, which is determined by the dimensionless Strouhal (St) number that characterizes the oscillation effects over the mean flows, and that significant transport occurs only for St values below a threshold, when the solute stroke displacement exceeds the distance between the source and sink (the canalicular length). This threshold mechanism explains the general flow behaviors such as increasing transport with increasing magnitude and decreasing frequency. Mechanical loading is predicted to enhance transport of all tracers relative to diffusion, with the greatest enhancement for medium-sized tracers and less enhancement for small and large tracers. This study provides guidelines for future FRAP experiments, based on which the model can be used to quantify bone permeability, solute-matrix interaction, and flow velocities. These studies should provide insights into bone adaptation and metabolism, and help to treat various bone diseases and conditions.

A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining

Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoen's silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.