Cellular function of osteocytes in normal and αklotho-deficient mice

During the last decade, osteocyte-derived factors i.e., sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) that reduces serum phosphate concentration by mediating FGF receptor 1c/αklotho in the kidney, have been highlighted for osteocytes’ fine-turned regulation on bone remodeling and phosphate homeostasis. Osteocytes are interconnected through gap junctions between their cytoplasmic processes, and thereby, build upon the functional syncytia, referred to as the osteocytic lacunar-canalicular system (OLCS). Osteocytes appear to communicate surrounding osteocytes and osteoblasts by means of two possible pathways of molecular transport throughout the OLCS : One is a passageway of their cytoplasmic processes, and the other is a pericellular space in the osteocytic canaliculi. The regularly-oriented OLCS in mature compact bone appears to efficiently serve for molecular transport, mechanosensing and targeted bone remodeling that would erase microdamages in bone. In a disrupted signaling state of FGF23/αklotho, serum concentration of phosphate would be markedly-elevated. Despite highly-elevated serum phosphate, αklotho -deficient mice revealed defective mineralization in bone matrix. OLCS in αklotho -deficient mice were irregularly-distributed and the connectivity of cytoplasmic processes of osteocytes was very poor, so that osteocytes did not seem to form functional syncytia. Therefore, osteocytes’function cooperated with other bone cells, rather than serum concentration of calcium/phosphate, and this seems to play a central role in maintaining bone mineralization. In this review, the biological function of the regularly-arranged OLCS in a normal state will be introduced, as well as dysfunctional osteocytes in αklotho-deficient state, using animal models

Effects of drug discontinuation after short-term daily alendronate administration on osteoblasts and osteocytes in mice

In order to determine whether osteoclastic bone resorption is restarted after withdrawn of bisphosphonates, we conducted histological examinations on murine osteoclasts, osteoblasts and osteocytes after discontinuation of a daily regimen of alendronate (ALN) with a dosage of 1 mg/kg/day for 10 days. After drug discontinuation, metaphyseal trabecular number and bone volume remained unaltered for the first 4 days. Osteoclast number did not increase, while the number of apoptotic osteoclasts was elevated. On the other hand, tissue non-specific alkaline phosphatase-immunoreactive area was markedly reduced after ALN discontinuation. In addition, osteocytes showed an atrophic profile with empty lacunar areas during and after ALN treatment. Interestingly, as early as 36 h after a single ALN injection, osteocytes show signs of atrophy despite the presence of active osteoblasts. Structured illumination microscopy system showed shortening of osteocytic cytoplasmic processes after drug cessation, suggesting a possible morphological and functional disconnection between osteocytes and osteoblasts. Taken together, it appears that osteoclastic bone resorption is not resumed after ALN discontinuation; also, osteoblasts and osteocytes hardly seem to recover once they are inactivated and atrophied by ALN. In summary, it seems that one must pay more attention to the responses of osteoblasts and osteocytes, rather focusing on the resuming of osteoclastic bone resorption after the ALN discontinuation

Hertwig's epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars

This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle

Histological Assessment of Endochondral Ossification and Bone Mineralization

Finely tuned cartilage mineralization, endochondral ossification, and normal bone formation are necessary for normal bone growth. Hypertrophic chondrocytes in the epiphyseal cartilage secrete matrix vesicles, which are small extracellular vesicles initiating mineralization, into the intercolumnar septa but not the transverse partitions of the cartilage columns. Bone-specific blood vessels invade the unmineralized transverse septum, exposing the mineralized cartilage cores. Many osteoblast precursors migrate to the cartilage cores, where they synthesize abundant bone matrices, and mineralize them in a process of matrix vesicle-mediated bone mineralization. Matrix vesicle-mediated mineralization concentrates calcium (Ca) and inorganic phosphates (Pi), which are converted into hydroxyapatite crystals. These crystals grow radially and are eventually get out of the vesicles to form spherical mineralized nodules, leading to collagen mineralization. The influx of Ca and Pi into the matrix vesicle is regulated by several enzymes and transporters such as TNAP, ENPP1, PiT1, PHOSPHO1, annexins, and others. Such matrix vesicle-mediated mineralization is regulated by osteoblastic activities, synchronizing the synthesis of organic bone material. However, osteocytes reportedly regulate peripheral mineralization, e.g., osteocytic osteolysis. The interplay between cartilage mineralization and vascular invasion during endochondral ossification, as well as that of osteoblasts and osteocytes for normal mineralization, appears to be crucial for normal bone growth

Medial vascular calcification: a new concept challenging the classical paradigm of dystrophic calcification

Klotho deficient （kl/kl） mice are well known to　develop hyperphosphatemia and resultant Möncheberg’s vascular sclerosis, which consists of elongated or　fragmented elastic lamellae and abundant collagen fibrils inside the vessels. Instead of normal vascular smooth muscle cells （VSMCs）, the tunica media of the kl/kl aorta has cells rich with abundant endoplasmic reticulum and Golgi apparatus, somewhat resembling osteoblasts. There were many matrix vesicle-like structures and calcifying nodules in the vicinity of these osteoblast-like cells in kl/kl aorta. The calcifying nodules seem to trigger calcification in the elastic lamellae, without promoting it in the collagen fibrils inside the kl/kl aorta. Also, mineral deposition was observed within the intravascular amorphous organic component, suggesting dystrophic calcification. Thus, two possible pathways for vascular calcification exist: one mediated by matrix vesicle-like structures, and another taking place after the deposition of calcium phosphates in the amorphous organic component. Compared to the latter, which consists of the classical view of intravascular calcification, the former appears to mimic osteoblastic mineralization in bone, and could be the result of trans-differentiation of VSMCs into osteoblastic cells. In this work, we will review our current findings on the process of medial vascular calcification found in kl/kl mice

Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats

To elucidate the effect of evocalcet, a new oral calcimimetic to bone of secondary hyperparathyroidism (SHPT) with chronic kidney disease (CKD), the rats were 5/6 nephrectomized and fed on a high-phosphate diet. The treated rats were then divided into vehicle groups and evocalcet administered groups. The rats in the vehicle groups exhibited increased levels of serum PTH and inorganic phosphate (Pi) levels, high bone turnover, and severe cortical porosity, mimicking SHPT (CKD-SHPT rats). The cortical bone of the CKD-SHPT rats showed broad demineralization around the osteocytes, suppression of Phex/small integrin-binding ligand N-linked glycoprotein-mediated mineralization in the periphery of the osteocytic lacunae, and increased levels of osteocytic cell death, all of which were considered as the first steps of cortical porosity. In contrast, evocalcet ameliorated the increased serum PTH levels, the enlarged osteocytic lacunae, and the cortical porosity of the CKD-SHPT rats. Osteocytes of CKD-SHPT rats strongly expressed PTH receptor and Pit1/Pit2, which sense extracellular Pi, indicating that PTH and Pi affected these osteocytes. Cell death of cultured osteocytes increased in a Pi concentration-dependent manner, and PTH administration rapidly elevated Pit1 expression and enhanced osteocytic death, indicating the possibility that the highly concentrated serum PTH and Pi cause severe perilacunar osteolysis and osteocytic cell death. It is likely therefore that evocalcet not only decreases serum PTH but also reduces the exacerbation combined with PTH and Pi to the demineralization of osteocytic lacunae and osteocytic cell death, thereby protecting cortical porosity in CKD-SHPT rats

Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice

In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca2+-binding bone matrix proteins - osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) - were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca2+-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix. (C) 2013 Elsevier Inc. All rights reserved

Intermittent PTH Administration Increases Bone-Specific Blood Vessels and Surrounding Stromal Cells in Murine Long Bones

To verify whether PTH acts on bone-specific blood vessels and on cells surrounding these blood vessels, 6-week-old male mice were subjected to vehicle (control group) or hPTH [1-34] (20 mu g/kg/day, PTH group) injections for 2 weeks. Femoral metaphyses were used for histochemical and immunohistochemical studies. In control metaphyses, endomucin-positive blood vessels were abundant, but alpha SMA-reactive blood vessels were scarce. In the PTH-administered mice, the lumen of endomucin-positive blood vessels was markedly enlarged. Moreover, many alpha SMA-positive cells were evident near the blood vessels, and seemed to derive from those vessels. These alpha SMA-positive cells neighboring the blood vessels showed features of mesenchymal stromal cells, such as immunopositivity for c-kit and tissue nonspecific alkaline phosphatase (TNALP). Thus, PTH administration increased the population of perivascular/stromal cells positive for alpha SMA and c-kit, which were likely committed to the osteoblastic lineage. To understand the cellular events that led to increased numbers and size of bone-specific blood vessels, we performed immunohistochemical studies for PTH/PTHrP receptor and VEGF. After PTH administration, PTH/PTHrP receptor, VEGF and its receptor flk-1 were consistently identified in both osteoblasts and blood vessels (endothelial cells and surrounding perivascular cells). Our findings suggest that exogenous PTH increases the number and size of bone-specific blood vessels while fostering perivascular/stromal cells positive for alpha SMA/TNALP/c-kit