Analysis and Imaging of Osteocytes

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological ...

University of Missouri-Kansas City

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development.

Differential regulation of dentin matrix protein 1 expression during odontogenesis.

The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells, may have multiple functions, including acting as a mechanosensor in bone (re)modeling. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes and, when deleted in mice, results in a hypomineralized bone phenotype. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P(i)) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.

Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo.

Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism.

The biological function of DMP-1 in osteocyte maturation is mediated by its 57-kDa C-terminal fragment.

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG-SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno-canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)-tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP-fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.

Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo.

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder primarily due to mutations in the type I collagen genes (COL1A1 and COL1A2), leading to compromised biomechanical integrity in type I collagen-containing tissues such as bone. Bone is inherently mechanosensitive and thus responds and adapts to external stimuli, such as muscle mass and contractile strength, to alter its mass and shape. Myostatin, a member of the TGF-β superfamily, signals through activin receptor type IIB to negatively regulate muscle fiber growth. Because of the positive impact of myostatin deficiency on bone mass, we utilized a soluble activin receptor type IIB-mFc (sActRIIB-mFc) fusion protein in two molecularly distinct OI mouse models (G610C and oim) and evaluated their bone properties. Wild-type (WT), +/G610C, and oim/oim mice were treated from 2 to 4 months of age with either vehicle (Tris-buffered saline) or sActRIIB-mFc (10 mg/kg). Femurs of sActRIIB-mFc-treated mice exhibited increased trabecular bone volume regardless of genotype, whereas the cortical bone microarchitecture and biomechanical strength were only improved in WT and +/G610C mice. Dynamic histomorphometric analyses suggest the improved cortical bone geometry and biomechanical integrity reflect an anabolic effect due to increased mineral apposition and bone formation rates, whereas static histomorphometric analyses supported sActRIIB-mFc treatment also having an anti-catabolic impact with decreased osteoclast number per bone surface on trabecular bone regardless of sex and genotype. Together, our data suggest that sActRIIB-mFc may provide a new therapeutic direction to improve both bone and muscle properties in OI. © 2018 American Society for Bone and Mineral Research.

Skeletal Response to Soluble Activin Receptor Type IIB in Mouse Models of Osteogenesis Imperfecta.

Bone formation, remodeling and repair are dynamic processes, involving cell migration, ECM assembly, osteocyte embedding, and bone resorption. Using live-cell imaging, we previously showed that osteoblast assembly of the ECM proteins fibronectin and collagen is highly dynamic and is integrated with cell motility. Additionally, osteoblast-to-osteocyte transition involved arrest of cell motility, followed by dendrite extension and retraction that may regulate positioning of embedding osteocytes. To further understand how osteocytes differentiate and embed in collagen, mice were generated that co-expressed GFP-tagged collagen with a Dmp1-Cre-inducible tdTomato reporter targeted to preosteocytes/osteocytes. Dual live-cell imaging of collagen and osteocyte dynamics in mineralizing primary calvarial cell cultures showed that Dmp1-Cre/tdTomato turned on in early bone nodule forming regions, demarcated by foci of concentrated GFP-collagen bundles that appeared structurally distinct from the surrounding collagen. Dmp1-Cre/tdTomato-positive cells were post-mitotic and were continuously induced throughout the 2 week timecourse, whereas the majority of collagen was assembled by day 7. GFP-collagen fibrils showed global (tissue-level) motions, suggesting coordinated cell layer movement, and local fibril motions mediated by cell-generated forces. Condensation of collagen fibril networks occurred within bone nodules prior to mineralization. Intravital imaging confirmed a similar structural appearance of GFP-collagen in calvarial bone, with analogous global motions of mineralizing areas adjacent to sutures. In early (unmineralized) calvarial cell cultures, Dmp1-Cre/tdTomato-positive cells were motile (mean velocity 4.8 μm/h), moving freely in and around the forming bone nodule, with a small number of these cells embedded in collagen, constraining their motion. In mineralizing cultures, the average velocity of Dmp1-Cre/tdTomato-positive cells was significantly reduced (0.7 μm/h), with many immobilized in the mineralizing nodule. Three apparent mechanisms for embedding of Dmp1-Cre/tdTomato-positive cells were observed. In some cases, a previously motile Dmp1-Cre/tdTomato-positive cell became immobilized in collagen fibril networks that were newly assembled around the cell, thereby entrapping it. In other cases, a motile Dmp1-Cre/tdTomato-positive cell moved into an already formed "collagen lacuna," arrested its motility and became embedded. Alternatively, some cells switched on tdTomato expression  within a lacuna. These data provide new insight into the dynamic process of bone collagen assembly and suggest multiple mechanisms for osteocyte entrapment in collagen matrix.

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization.

Mouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Yixia Xie

Department of Oral and Craniofacial Sciences,
School of Dentistry,
Department of Oral and Craniofacial Sciences, School of Dentistry

University of Missouri-Kansas City

Analysis and Imaging of Osteocytes

Yixia Xie

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University of Missouri-Kansas City

Analysis and Imaging of Osteocytes

Department of Oral and Craniofacial Sciences,
School of Dentistry,
Department of Oral and Craniofacial Sciences, School of Dentistry