TY - JOUR
AU - Amizuka,, Norio
AB - Abstract 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. FGF23, osteocyte, DMP1, bone remodeling, immunohistochemistry Introduction Osteocytes are the most abundant cells in bone and are in an ideal location for influencing bone turnover by communicating with osteoblasts and bone lining cells. All osteocytes lie within osteocytic lacunae, connecting to other osteocytes and to cells on the bone surface by means of thin cytoplasmic processes that pass through narrow channels referred to as osteocytic canaliculi. Osteocytes can build up functional syncytia where osteocytic processes interconnect through gap junctions [1–3], and through which embedded osteocytes and osteoblasts on the bone surface communicate, i.e. the osteocytic lacunar canalicular system(s) (OLCS) [4–6]. The three-dimensional network of OLCS has been examined in vivo [7], and our group has recently demonstrated that OLCS become progressively more regular as an individual mouse grows [8]. The most accepted theory for osteocytic function places these cells as transducers of mechanical strains that are translated into biochemical signals affecting the communication among osteocytes and between osteocytes and osteoblasts [5,9–11]. This cascade of events may ultimately regulate bone remodeling [12–15] and mineral metabolism [15–17]. Reports on the processes that lead to OLCS formation are not conclusive. It has been suggested that the OLCS is formed as a consequence of the ostecytes’ mechanosensing capabilities and that osteocytic cell bodies should ultimately align parallel to the main vector of mechanical loading [18]. The osteocyte is the main candidate for bearing the responsibility of maintaining bone's mechanical properties, since osteocytic death is eventually followed by bone fatigue and osteoclastic resorption [19]. Consistently, ablation of osteocytes in transgenic mice expressing osteocyte-specific HB-EGF, a receptor for diphtheria toxin, strongly indicated that osteocytes control mineral trafficking in bone and might as well regulate osteoclastic and osteoblastic activities on the bone surface [15]. These putative functions imply that OLCS feature a finely tuned arrangement that may be altered by physical or chemical imbalances. However, research on the dynamics of such alterations is extremely rare. Dentin matrix protein (DMP) 1 was originally identified in the rat incisor's pulp cDNA library [20]. Though initial expression studies suggested that DMP1 was odontoblast-specific [20], this protein was later found to be expressed in other mineralizing tissues [21,22]. DMP1 has been shown to be a bone matrix protein expressed in osteocytes, but not in osteoblasts, and is assumed to play a role in bone mineral homeostasis due to its high calcium ion-binding capacity [23]. Small integrin-binding ligand N-linked glycoproteins, or SIBLINGs, including DMP1, osteopontin, bone sialoprotein, MEPE/osteogenic factor 45 and dentin sialophosphoprotein, are found in a gene cluster located in the human chromosome 4q21 and in the mouse chromosome 5q21 [24–28]. Such clustering indicates a potential similarity in the role of these molecules in the processes leading to bone mineralization. Since a recent report demonstrated that DMP1 absence leads to rickets or osteomalacia in mice [17], a possible role for osteocytes as regulators of bone mineralization must be investigated. Fibroblast growth factor (FGF) 23 modulates the serum phosphate concentration, an important function required for normal skeletal development and for the preservation of bone integrity [29]. FGF23 was originally reported as a phosphaturic factor in autosomal dominant hypophosphatemic rickets [30], tumor-induced osteomalacia [31], McCune–Albright syndrome/fibrous dysplasia [32], familial tumoral calcinosis [33] and possibly in X-linked hypophosphatemic rickets [34]. Although FGF23 mRNA is found in several tissues [30,31,35], this molecule is most abundantly expressed in bone [36]. A circulating factor synthesized by osteocytes, FGF23 serves as a phosphaturic agent that inhibits 1,25(OH)2D3 production in the kidney and maintains the balance between phosphate homeostasis and skeletal mineralization [37]. A recent in vitro study demonstrated that over-expression of FGF23 suppressed matrix mineralization [38]. Thus, investigations on the biological functions of FGF23 have broadened the understanding of the systemic regulation of phosphate homeostasis, as well as of the maintenance of proper mineralization in the bone matrix [37]. In this study, we have examined the conformation of OLCS in sites of bone remodeling and attempted to establish a correlation between the immunolocalization of FGF23 and DMP1 and the regularity of the OLCS. Methods Tissue preparation Twelve-weeks-old male ICR mice (CLEA Japan, Inc., Tokyo, n = 6) were used in our experiments under Niigata University's guidelines for animal experimentation. Seven and two days before fixation, 10 mg kg−1 of calcein (Dojindo Laboratories, Kumamoto, Japan) was subcutaneously injected into mice [39]. Anesthesia was performed with diethyl ether and pentobarbital (Nembutal, Dinabot, Osaka, Japan), and perfusion was performed with 4% paraformaldehyde in a 0.1 M cacodylate buffer (pH 7.4) through the cardiac left ventricle. Femora were dissected free of soft tissue and immersed in the same fixative for additional 12 h at 4°C. After decalcification with a 5% EDTA-2Na solution for 2–3 weeks at 4°C, selected specimens were dehydrated through a graded series of ethanol and embedded into paraffin. Non-decalcified samples were embedded into methyl methacrylate (MMA) for visualization of calcein labeling under fluorescent microscopy (Eclipse E800, Nikon Instruments Inc., Tokyo, Japan). Triple staining for silver pigmentation, alkaline phosphatase and tartrate-resistant acid phosphatase We devised a triple-staining protocol based on a modification of Bodian's protargol-S [8,40,41] procedure. First, we performed the detection of tartrate-resistant acid phosphatase (TRAPase) [42]. In brief, dewaxed sections were incubated in a mixture of 8 mg of naphthol AS-BI phosphate (Sigma, St. Louis, MO, USA), 70 mg of red violet LB salt (Sigma) and 50 mM l(+) tartaric acid (0.76 g, Nacalai Tesque, Kyoto, Japan) diluted in 60 ml of a 0.1 M sodium acetate buffer (pH 5.0) for 20 min at 37°C. After visualizing TRAPase positivity, we proceeded to silver pigmentation, when the sections were soaked in a 1% Protargol-S solution diluted in borax-boric acid (pH 7.4, Wako Pure Chemical Industries Ltd, Osaka, Japan) for 12–48 h at 37°C. After rinsing with distilled water, the reaction was enhanced by an aqueous solution containing 0.2% hydroquinone, 0.2% citric acid and 0.7% nitric silver. After additional rinsing, the sections were reduced for 5 min with an aqueous solution of 2.5% anhydrous sodium sulfite, 0.5% potassium bromide and 0.5% amidol diaminophenol dihydrochloride. They were then treated with 1% gold chloride, and subsequently reduced by 2% oxalic acid amidol until black-stained osteocytic canaliculi could be seen. After rinsing with distilled water, sections were fixed in 5% sodium thiosulfate. Next, the sections were subsequently subjected to alkaline phosphatase (ALPase) immunodetection [43,44]. The sections were treated with 0.1% hydrogen peroxide for 15 min to inhibit endogenous peroxidase, and pre-incubated with 1% bovine serum albumin in phosphate-buffered saline (BSA–PBS) for 30 min at room temperature. Rabbit antisera against tissue-nonspecific ALPase [42,45] and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Chemicon International Inc., Temecula, CA, USA) were employed for this step. All sections were counterstained with hematoxylin and observed under a light microscope (Eclipse E800, Nikon). Images were acquired at various magnifications using a digital camera (Nikon DXM1200C, Nikon). Immunohistochemistry for DMP1 and FGF23 Dewaxed sections were treated with 0.1% hydrogen peroxide for 15 min and with 1 μg ml−1 trypsin (Wako Pure Chemical) for 30 min. After pre-incubation with 1% BSA–PBS for 30 min at room temperature, sections were incubated with a rabbit antibody against dentin matrix protein 1 (DMP1) (dilution 1:500, Takara Bio Inc., Otsu, Japan) overnight at 4°C. Incubation with HRP-conjugated goat anti-rabbit IgG (Chemicon) proceeded rinsing with PBS. Immune complexes were visualized using DAB. For the detection of FGF23, dewaxed paraffin sections were treated with rat anti-FGF23 (dilution 1:100, R&D systems, Inc., Minneapolis, MN, USA) for 2 h. The sections were subsequently incubated in ALPase-conjugated goat anti-rat IgG (Chemicon). For the visualization of the ALPase-conjugated complex, the treated sections were immersed in an aqueous solution of 8 mg of naphthol AS–BI phosphate (Sigma), 70 mg of red violet LB salt (Sigma) diluted in 60 ml of a 0.1 M Tris–HCl buffer (pH 9.0) for 20 min at 37°C. All sections were faintly counterstained with methyl green. In order to immunolocalize DMP1 and FGF23 in the same section, we performed double staining of DMP1 and FGF23. Dewaxed sections were subjected to immunostaining for FGF23 as described above, but using fast blue BR salt (Sigma) instead of red violet LB salt. The same sections were subsequently employed for DMP1 detection as described. These sections were not counterstained with methyl green or hematoxylin to facilitate the recognition of the blue (FGF23) reactivity. Bone histomorphometry for mineral appositional rate and mineralization surface/bone surface Histomorphometric parameters in calcein-labeled specimens were examined under fluorescence microscopy and measured as described previously [39]. The cortical bone divided into three regions, namely the metaphyseal region (from below the inferior border of the growth plate up to 2.0 mm distally), the middle region (comprehending the next 2.0 mm distal from the area above) and the diaphyseal region (a 2.0-mm segment distal from the middle region). Mineral appositional rate (MAR, μm/day) and mineralized surface/bone surface (MS/BS, %) were calculated for assessing the speed of mineral deposition and bone maturation, respectively. All values are presented as means ± standard deviation. Differences among groups were assessed by an unpaired Student's t-test, and considered statistically significant when P < 0.05. Quantification of ALP- and TRAP-positive cells Metaphyseal trabeculae were divided in primary and secondary regions. ALPase- or TRAPase-positive cells were counted with the aid of the ImagePro Plus 6.2 software (Media Cybernetics, Silver Spring, MD, USA) and values were expressed using bone surface as the referent. All values are presented as means ± standard deviation. Differences among groups were assessed by an unpaired Student's t-test, and considered statistically significant when P < 0.05. Quantification of the percentage of DMP1- or FGF23-positive osteocytes Metaphyseal trabeculae were divided in primary and secondary regions. For the quantification of the percentage of DMP1- and FGF23-positive osteocytes in the cortical bone, we divided the cortical bone into periosteal and endosteal halves with a line paralleling the longitudinal axis. The number of DMP1- or FGF23-positive osteocytes was divided by the total number of osteocytes in the respective region. Primary and secondary trabeculae values, as well as those from the endosteal and periosteal regions of the cortical bone were compared and statistically analyzed. Results Triple staining for silver impregnation, ALPase and TRAPase In the femoral primary metaphyseal trabeculae, intensely ALPase-positive osteoblasts and several TRAPase-positive osteoclasts covered the bone surface (Fig. 1a). Round-shaped osteocytes embedded in the primary trabeculae extended their cytoplasmic processes in multiple directions. However, the inner cartilaginous cores of the primary trabeculae abruptly interrupted the connections among osteocytic processes. In contrast, in the remodeled secondary trabeculae, OLCS were regularly arranged, osteocytes showed flattened cells bodies whose longitudinal axis was parallel to the bone surface and osteocytic canaliculi ran perpendicular to the bone surfaces (Fig. 1b). In addition, the number of ALP-positive cells per bone surface (Fig. 1c, 132.0 ± 26.2 versus 84.4 ± 11.7) as well as the number of TRAP-positive cells per bone surface (Fig. 1d, 65.6 ± 11.6 versus 31.5 ± 9.0) was significantly higher in the primary trabeculae compared to the secondary ones. Fig. 1 Open in new tabDownload slide Triple staining for silver impregnation, ALPase and TRAPase. (a) Primary metaphyseal trabeculae displaying intensely ALPase-positive osteoblasts (ob, brown) and several TRAPase-positive osteoclasts (oc, red) cover the bone surfaces. The cytoplasmic processes of ovoid osteocytes (ocy, black) spread in multiple directions, but the connectivity among these processes is interrupted by the presence of inner cartilaginous cores (cc) in the primary trabeculae. (b) In the secondary trabeculae, OLCS are regularly arranged and osteocytes (ocy) are flattened and distributed parallel to the bone surface. Osteocytic canaliculi run perpendicular to the bone surface. (c) The number of ALPase-positive cells per bone surface is significantly higher in the primary trabeculae. (d) Likewise, the number of TRAPase-positive cells per bone surface was significantly higher in the primary trabeculae. Numerical values are given in the ‘Methods’ section. Bar, a and b: 20 μm. Fig. 1 Open in new tabDownload slide Triple staining for silver impregnation, ALPase and TRAPase. (a) Primary metaphyseal trabeculae displaying intensely ALPase-positive osteoblasts (ob, brown) and several TRAPase-positive osteoclasts (oc, red) cover the bone surfaces. The cytoplasmic processes of ovoid osteocytes (ocy, black) spread in multiple directions, but the connectivity among these processes is interrupted by the presence of inner cartilaginous cores (cc) in the primary trabeculae. (b) In the secondary trabeculae, OLCS are regularly arranged and osteocytes (ocy) are flattened and distributed parallel to the bone surface. Osteocytic canaliculi run perpendicular to the bone surface. (c) The number of ALPase-positive cells per bone surface is significantly higher in the primary trabeculae. (d) Likewise, the number of TRAPase-positive cells per bone surface was significantly higher in the primary trabeculae. Numerical values are given in the ‘Methods’ section. Bar, a and b: 20 μm. Distribution of DMP1 and FGF23 in the metaphyseal trabeculae DMP1 immunolocalization was seen throughout the femoral epiphysis and metaphysis (Fig. 2a). DMP1 immunoreactivity was seen in osteocytic lacunae and canaliculi in primary and secondary metaphyseal trabeculae (Fig. 2b and c). Statistical analysis did not show significant differences between the percentages of DMP1-positive osteocytes between the primary and secondary trabeculae (Table 1). Thus, the localization of DMP1 did not seem to be associated with the regularity of the OLCS. Fig. 2 Open in new tabDownload slide Distribution of DMP1 immunopositivity in the metaphyseal trabeculae. (a) DMP1 immunopositivity is seen throughout the femoral epiphysis (epi) and metaphysis (meta). DMP1 positivity is found in the osteocytic lacunae and canaliculi of both the primary (b) and secondary (c) metaphyseal trabeculae. Note the similar intensity of immunoreaction between the primary and secondary trabeculae. GP: growth plate, ocy: osteocytes. Bar, a: 100 μm; b and c: 40 μm. Fig. 2 Open in new tabDownload slide Distribution of DMP1 immunopositivity in the metaphyseal trabeculae. (a) DMP1 immunopositivity is seen throughout the femoral epiphysis (epi) and metaphysis (meta). DMP1 positivity is found in the osteocytic lacunae and canaliculi of both the primary (b) and secondary (c) metaphyseal trabeculae. Note the similar intensity of immunoreaction between the primary and secondary trabeculae. GP: growth plate, ocy: osteocytes. Bar, a: 100 μm; b and c: 40 μm. Table 1. Statistical analysis of the percentage of DMP1-positive and FGF23-positive osteocytes in different regions of the bone Metaphysis Cortical bone Primary trabeculae Secondary trabeculae Endosteal region Periosteal region DMP1 64.96 ± 3.01 59.49 ± 1.22 50.92 ± 4.22 70.24 ± 5.12** FGF23 10.34 ± 1.52 43.97 ± 0.63* 24.55 ± 2.66 21.16 ± 2.56 Metaphysis Cortical bone Primary trabeculae Secondary trabeculae Endosteal region Periosteal region DMP1 64.96 ± 3.01 59.49 ± 1.22 50.92 ± 4.22 70.24 ± 5.12** FGF23 10.34 ± 1.52 43.97 ± 0.63* 24.55 ± 2.66 21.16 ± 2.56 The percentage of DMP1- and FGF23-positive cells were examined in primary trabeculae, secondary trabeculae and the endosteal and periosteal region of the cortical bone. Statistical analysis was performed by Student's t-test. All data were expressed as mean + standard deviation (SD). *P < 0.005, **P < 0.05. Open in new tab Table 1. Statistical analysis of the percentage of DMP1-positive and FGF23-positive osteocytes in different regions of the bone Metaphysis Cortical bone Primary trabeculae Secondary trabeculae Endosteal region Periosteal region DMP1 64.96 ± 3.01 59.49 ± 1.22 50.92 ± 4.22 70.24 ± 5.12** FGF23 10.34 ± 1.52 43.97 ± 0.63* 24.55 ± 2.66 21.16 ± 2.56 Metaphysis Cortical bone Primary trabeculae Secondary trabeculae Endosteal region Periosteal region DMP1 64.96 ± 3.01 59.49 ± 1.22 50.92 ± 4.22 70.24 ± 5.12** FGF23 10.34 ± 1.52 43.97 ± 0.63* 24.55 ± 2.66 21.16 ± 2.56 The percentage of DMP1- and FGF23-positive cells were examined in primary trabeculae, secondary trabeculae and the endosteal and periosteal region of the cortical bone. Statistical analysis was performed by Student's t-test. All data were expressed as mean + standard deviation (SD). *P < 0.005, **P < 0.05. Open in new tab The distribution of FGF23 in primary and secondary trabeculae was examined for the comparison with DMP1 localization (Fig. 3). Unlike the findings for DMP1, osteocytes in the epiphysis revealed the intense immunoreactivity for FGF23, while metaphyseal osteocytes displayed markedly reduced immunoreactivity (Fig. 3a). Interestingly, osteocytes show little FGF23 in the metaphyseal primary trabeculae, where OLCS were irregularly distributed (Fig. 3b). In contrast, osteocytes exhibited relatively intense FGF23 immunoreactivity (Fig. 3c) in the region of the secondary trabeculae, where OLCS are regularly arranged. Consistently, statistical analysis revealed a significant difference between the percentages of FGF23-positive osteocytes between primary and secondary trabeculae (Table 1). Fig. 3 Open in new tabDownload slide Distribution of FGF23 immunoreactivity in the metaphyseal trabeculae. (a) While FGF23-positive osteocytes (black dots) are ubiquitous in the epiphysis (epi), hardly any positivity is seen in the metaphysic (meta). (b) When observing at a higher magnification, osteocytes (ocy) do not show FGF23 positivity in the metaphyseal primary trabeculae, where the OLCS is irregularly distributed. (c) In the region of the secondary trabeculae featuring a regular OLCS, osteocytes (ocy) exhibit relatively intense FGF23 immunoreactivity. Bar, a: 100 μm; b and c: 40 μm. Fig. 3 Open in new tabDownload slide Distribution of FGF23 immunoreactivity in the metaphyseal trabeculae. (a) While FGF23-positive osteocytes (black dots) are ubiquitous in the epiphysis (epi), hardly any positivity is seen in the metaphysic (meta). (b) When observing at a higher magnification, osteocytes (ocy) do not show FGF23 positivity in the metaphyseal primary trabeculae, where the OLCS is irregularly distributed. (c) In the region of the secondary trabeculae featuring a regular OLCS, osteocytes (ocy) exhibit relatively intense FGF23 immunoreactivity. Bar, a: 100 μm; b and c: 40 μm. Immunolocalization of FGF23 correlates with the regularity of the OLCS Using bone histomorphometry, MAR (μm/day) and MS/BS (%) were quantified at the endosteal surface of the cortical bone, where continuous bone deposition occurs. Histologically, a wider distance between the two calcein labels was seen in the metaphyseal region, whereas narrow bands were detected in the diaphyseal region (Fig. 4a). Consistently, MAR and MS/BS values showed significant differences between the metaphysis and diaphysis (Table 2). In the metaphyseal area of the cortical bone, cuboidal osteoblasts with intense ALPase positivity and ovoid osteocytes with irregularly distributed canaliculi were identified (Fig. 4b and c). When observing the middle area between the metaphysis and diaphysis on the other hand, osteocytes were elongated and seemed to be more regularly distributed (Fig. 4d and e). In the diaphyseal region, osteocytes were flattened and extended their cytoplasmic processes perpendicular to the bone surface (Fig. 4f and g). It seems likely that when the speed of bone deposition is high, the OLCS are irregular. Fig. 4 Open in new tabDownload slide Immunolocalization of FGF23 correlates with the regularity of the OLCS. (a) The distance between the two calcein labels (white lines) gradually narrows toward the diaphyseal region. (b and c [a higher magnification of b]) In the metaphyseal cortical bone, intensely ALPase-positive (black) cuboidal osteoblasts and ovoid osteocytes (ocy) with irregularly spread canaliculi are discernible. (d and e [a higher magnification of d]) In the middle region of the cortical bone, osteocytes become slender and are more regularly distributed. (f and g [a higher magnification of f]) In the diaphyseal region, osteocytes are flattened and extend their cytoplasmic processes perpendicular to the bone surface. Additionally, the layer of ALPase-positive osteoblasts becomes less pronounced toward the diaphysis. BM: bone marrow. Bar, a: 100 μm; b, d and f: 50 μm; c, e and g: 10 μm. Fig. 4 Open in new tabDownload slide Immunolocalization of FGF23 correlates with the regularity of the OLCS. (a) The distance between the two calcein labels (white lines) gradually narrows toward the diaphyseal region. (b and c [a higher magnification of b]) In the metaphyseal cortical bone, intensely ALPase-positive (black) cuboidal osteoblasts and ovoid osteocytes (ocy) with irregularly spread canaliculi are discernible. (d and e [a higher magnification of d]) In the middle region of the cortical bone, osteocytes become slender and are more regularly distributed. (f and g [a higher magnification of f]) In the diaphyseal region, osteocytes are flattened and extend their cytoplasmic processes perpendicular to the bone surface. Additionally, the layer of ALPase-positive osteoblasts becomes less pronounced toward the diaphysis. BM: bone marrow. Bar, a: 100 μm; b, d and f: 50 μm; c, e and g: 10 μm. Table 2 Statistical analysis of the mineral appositional rate and mineralization surface/bone surface at the endosteal surface of the cortical bone Metaphysis Middle region Diaphysis MAR 1.26 ± 0.37a 0.84 ± 0.12a 0.43 ± 0.08 MS/BS 34.46 ± 9.86b 41.42 ± 5.25b 60.71 ± 14.24 Metaphysis Middle region Diaphysis MAR 1.26 ± 0.37a 0.84 ± 0.12a 0.43 ± 0.08 MS/BS 34.46 ± 9.86b 41.42 ± 5.25b 60.71 ± 14.24 MAR (μm/day) and MS/BS (%) were examined in three different areas of the cortical bone (see the ‘Methods’ section for references). Statistical analysis was performed by Student's t-test. All data were expressed as mean + standard deviation (SD). aP < 0.01 versus diaphysis, bP < 0.05 versus diaphysis. Open in new tab Table 2 Statistical analysis of the mineral appositional rate and mineralization surface/bone surface at the endosteal surface of the cortical bone Metaphysis Middle region Diaphysis MAR 1.26 ± 0.37a 0.84 ± 0.12a 0.43 ± 0.08 MS/BS 34.46 ± 9.86b 41.42 ± 5.25b 60.71 ± 14.24 Metaphysis Middle region Diaphysis MAR 1.26 ± 0.37a 0.84 ± 0.12a 0.43 ± 0.08 MS/BS 34.46 ± 9.86b 41.42 ± 5.25b 60.71 ± 14.24 MAR (μm/day) and MS/BS (%) were examined in three different areas of the cortical bone (see the ‘Methods’ section for references). Statistical analysis was performed by Student's t-test. All data were expressed as mean + standard deviation (SD). aP < 0.01 versus diaphysis, bP < 0.05 versus diaphysis. Open in new tab The immunolocalization for FGF23 and DMP1 was then examined on the individual regions of the cortical bone (Fig. 5). FGF23 was hardly seen in the metaphyseal region of cortical bones (Fig. 5a and d), while there were several DMP1-positive osteocytes (Fig. 5g). When the middle area was assessed, however, the cortical bone started to display FGF23-immunoreactive osteocytes (Fig. 5b and e). In its diaphyseal region, the cortical bone exhibited osteocytes with intense FGF23 immunoreactivity (Fig. 5c and e). Such gradation could not be seen in the distribution of DMP1 (compare Fig. 5g–i). Interestingly, FGF23 and DMP1 were not immunolocalized evenly in osteocytes; some osteocytes showed an intense immunoreactivity for FGF23 or DMP1, but other exhibited only weak staining. Fig. 5 Open in new tabDownload slide Immunolocalization of FGF23 and DMP1 on the various regions of the cortical bone. (a and d [a higher magnification of a]) FGF23 immunopositivity is hardly seen in the metaphyseal region of the cortical bone, where there were several DMP1-positive osteocytes (ocy) (g). (b and e [a higher magnification of b]) In the middle cortical bone, some FGF23-immunoreactive osteocytes can be identified. (c and f [a higher magnification of c]) In the diaphysis, the cortical bone exhibits intensely FGF23-immunoreactive osteocytes. On the other hand, the pattern of distribution of DMP1 positivity is essentially similar regardless of the regions of the cortical bone (compare g–i). Bar, a–c: 50 μm; d–f: 20 μm; g–i: 30 μm. Fig. 5 Open in new tabDownload slide Immunolocalization of FGF23 and DMP1 on the various regions of the cortical bone. (a and d [a higher magnification of a]) FGF23 immunopositivity is hardly seen in the metaphyseal region of the cortical bone, where there were several DMP1-positive osteocytes (ocy) (g). (b and e [a higher magnification of b]) In the middle cortical bone, some FGF23-immunoreactive osteocytes can be identified. (c and f [a higher magnification of c]) In the diaphysis, the cortical bone exhibits intensely FGF23-immunoreactive osteocytes. On the other hand, the pattern of distribution of DMP1 positivity is essentially similar regardless of the regions of the cortical bone (compare g–i). Bar, a–c: 50 μm; d–f: 20 μm; g–i: 30 μm. It is well known that, in the extremity of long bones, the periosteal area of the cortical bone permits osteoclastic bone resorption, while the endosteal area is exclusively subjected to bone formation. Triple staining of ALPase, TRAPase and silver impregnation, as well as the double detection for FGF23 and DMP1 in the periosteal and endosteal regions of the cortical bone were conducted to further support our assumption (Fig. 6). The endosteal bone surface was covered with flattened bone lining cells, and a regularly arranged OLCS could be identified (Fig. 6a). However, the periosteal region featured an irregular distribution of the OLCS, with TRAPase-positive osteoclasts populating its surface (Fig. 6b). The periosteal region displayed intense DMP1 immunoreactivity in osteocytes, whereas FGF23-reactive osteocytes seemed to dominate the endosteal region (Fig. 6c). This tendency of immunolocalization of FGF23 and DMP1 was confirmed by statistical analysis (Table 1), and was also seen in the secondary trabeculae where the OLCS were regularly arranged (Fig. 6d). Fig. 6 Open in new tabDownload slide Triple staining of ALPase, TRAPase and silver impregnation and double detection for FGF23 and DMP1 in the periosteal and endosteal regions of the cortical bone. (a) The periosteal region of the cortical bone shows irregular OLCS and some TRAPase-positive osteoclasts (red) on its bone surface. (b) The endosteal area, where the bone surface is covered by flattened ALPase-positive bone lining cells (brown), displays regularly arranged OLCS. (c) The periosteal region displays mainly DMP1 reactivity (brown, arrows) in osteocytes, whereas FGF23 positivity (blue, arrowheads) seems more significant than that of DMP1 in the endosteal region. (d) A similar pattern of FGF23 (blue, arrowheads) and DMP1 (brown, arrows) immunolocalization is discernible in the secondary trabeculae, where the OLCS are regularly arranged. BM: bone marrow. Bar, a and b: 20 μm; c and d: 50 μm. Fig. 6 Open in new tabDownload slide Triple staining of ALPase, TRAPase and silver impregnation and double detection for FGF23 and DMP1 in the periosteal and endosteal regions of the cortical bone. (a) The periosteal region of the cortical bone shows irregular OLCS and some TRAPase-positive osteoclasts (red) on its bone surface. (b) The endosteal area, where the bone surface is covered by flattened ALPase-positive bone lining cells (brown), displays regularly arranged OLCS. (c) The periosteal region displays mainly DMP1 reactivity (brown, arrows) in osteocytes, whereas FGF23 positivity (blue, arrowheads) seems more significant than that of DMP1 in the endosteal region. (d) A similar pattern of FGF23 (blue, arrowheads) and DMP1 (brown, arrows) immunolocalization is discernible in the secondary trabeculae, where the OLCS are regularly arranged. BM: bone marrow. Bar, a and b: 20 μm; c and d: 50 μm. Discussion To our knowledge, this study is the first report to successfully demonstrate immunohistochemistry and enzyme histochemistry combined with silver impregnation (modified Bodian method) [8,40,41] in a single section, unveiling the distribution of ALPase, TRAPase and osteocytic canaliculi, and also the first to demonstrate a correlation between DMP1 and FGF23 localization and the regularity of the OLCS. The establishment of a regular distribution of the OLCS appears to relate with the maturation of the bone matrix. The employment of triple staining envisioned the relationships of the OLCS and the localization of osteoblasts and osteoclasts. In triple staining and bone histomorphomertry of Fig. 1, the primary trabeculae showed an irregular distribution of OLCS with many osteoblasts and osteoclasts, while the secondary trabeculae demonstrated a regular distribution of OLCS with fewer osteoblasts and osteoclasts. Such fast bone formation carried out by numerous osteoblasts, while capable of promoting the embedding of osteocytes, does not allow for a regular osteocytic distribution. Alternatively, OLCS were regularly distributed in the secondary trabeculae and in the endosteal area of the cortical bone, strongly suggesting that bone remodeling affects the arrangement of OLCS. We postulated that the speed of bone deposition would be the most important factor influencing the regularity of OLCS, as evidenced by our data from bone histomorphometry and silver impregnation. As shown in Fig. 1, osteocytes were randomly embedded in the primary trabeculae, where the bone is formed essentially by bone modeling. These osteocytes extended their cytoplasmic processes in multiple directions, but the connectivity between processes was abruptly interrupted by the presence of an internal cartilage core. On the other hand, the connectivity of neighboring osteocytes seems to be improved in secondary trabeculae and in the cortical bone. Therefore, osteocytes in rapidly produced bone, e.g. primary trabeculae, may form small functional groups, whereas osteocytes gradually embedded into the bone matrix, e.g. the secondary trabeculae and the cortical bone, may be able to form a regular OLCS and a broader functional syncytium [4,6]. Our findings indicate that regularly arranged OLCS appear to be a result of physiological bone remodeling, especially when the speed of bone formation is low. It may also be that a regular OLCS reflects enhanced bone maturation. Is osteocytic function affected by the geometrical conformation of the OLCS? In a system where well-arranged canaliculi are present and the connectivity of osteocytic processes is maintained, higher efficiency in the transport of small molecules and better mechanosensing are more likely to happen [4,9–11]. Interestingly, FGF23 positivity was abundant in bone regions featuring regular OLCS. Assuming that the regularly arranged OLCS is a matured, fully functional syncytium, it may be that FGF23 is produced by osteocytes in such organized systems, which would then be a part of the bone–renal axis that is so central to proper mineral metabolism [37]. Unlike FGF23, however, DMP1 was seen throughout the epiphysis, metaphysis and diaphysis; therefore, it appears to work as a local regulator of mineralization, and may be controlled by the osteocytic microenvironment. It is interesting that DMP1-positive osteocytes did not express FGF23, from which one may infer that these molecules’ expression is mutually exclusive. DMP1-null mice revealed defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization [17]. Likewise, families suffering from autosomal recessive hypophosphatemic rickets were shown to carry mutations in DMP1 genes, and manifested forms of rickets and osteomalacia that feature elevated FGF23 despite normocalciuria [17]. In contrast to DMP1-deficient mice, we have recently reported abundant DMP1 in mice homozygous for the klotho gene deletion [46]. Hyperphosphatemia in klotho-deficient mice may be in part due to the inhibition of FGF23 by DMP1 overproduction. It appears that DMP1 negatively affects FGF23 expression, imbalancing a phosphaturic factor that may in turn compromise mineral metabolism [17,37,47]. Thus, FGF23 synthesis appears to be controlled mainly by the regularity of the OLCS, but may as well be negatively modulated by DMP1. Controlling of FGF23 levels may become an important tool for diagnosing and treating conditions such as hypophosphatemia and hyperphosphatemia [37]. However, in pathological bone states like osteoporosis and osteomalacia, the production of FGF23 may be affected together with possible kidney involvement. Indeed, in osteoporotic patients, osteocytic population and connectivity were shown to be attenuated, while osteomalatic bone revealed highly condensed osteocytes with a reduced connectivity [48]. While the regularity of the OLCS may result from bone metabolic diseases, it can also worsen the pathological state by affecting phosphate homeostasis. Taken together, further investigation on the molecular and cellular mechanism of the FGF23 production by mediating the regularity of OLCS is necessary. Conclusion We have demonstrated a single-section triple-staining procedure for localizing ALPase-positive osteoblasts, TRAPase-positive osteoclasts and the OLCS. The arrangement of osteocytes and its lacunar canaliculi might depend on bone remodeling, and especially on the speed of bone formation. Synthesis of FGF23 appears to be controlled mainly by OLCS arrangement, but may also be partially suppressed by DMP1 expression. This work was partially supported by grants from the Suzuken Memorial Foundation (N.A.), and from the Japanese Society for the Promotion of Science (N.A.). References 1 Doty S B . Morphological evidence of gap junctions between bone cells , Calcif. Tissue Int. , 1981 , vol. 33 5 (pg. 509 - 512 ) Google Scholar Crossref Search ADS PubMed WorldCat 2 Shapiro F . Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear stacked linear, curvilinear, oval, and annular junctions , Calcif. Tissue Int , 1997 , vol. 61 (pg. 285 - 293 ) Google Scholar Crossref Search ADS PubMed WorldCat 3 Donahue H J . Gap junctions and biophysical regulation of bone cell differentiation , Bone , 2000 , vol. 26 (pg. 417 - 422 ) Google Scholar Crossref Search ADS PubMed WorldCat 4 Aarden E M , Burger E H , Nijweide P J . Function of osteocytes in bone , J. Cell. Biochem. , 1994 , vol. 55 (pg. 287 - 299 ) Google Scholar Crossref Search ADS PubMed WorldCat 5 Burger E H , Klein-Nulend J . Mechanotransduction in bone role of the lacuno-canalicular network , FASEB J. , 1999 , vol. 13 (pg. 101 - 112 ) WorldCat 6 Knothe Tate M L , Adamson J R , Tami A E , Bauer T W . The osteocyte , Int. J. Biochem. Cell Biol. , 2004 , vol. 36 1 (pg. 1 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat 7 Kamioka H , Honjo T , Takano-Yamamoto T . A three dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy , Bone , 2001 , vol. 28 (pg. 145 - 149 ) Google Scholar Crossref Search ADS PubMed WorldCat 8 Hirose S , Li M , Kojima T , de Freitas P H , Ubaidus S , Oda K , Saito C , Amizuka N . A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining , J. Bone Miner. Metab. , 2007 , vol. 25 6 (pg. 374 - 382 ) Google Scholar Crossref Search ADS PubMed WorldCat 9 Klein-Nulend J , Van Der Plas A , Semeins C M , Ajubi N E , Frangos J A , Nijweide P J , Burger E H . Sensitivity of osteocytes to biomechanical stress in vitro , FASEB J. , 1995 , vol. 9 5 (pg. 441 - 445 ) Google Scholar PubMed WorldCat 10 Weinbaum S , Cowin S C , Zeng Y . A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses , J. Biomech. , 1994 , vol. 27 3 (pg. 339 - 360 ) Google Scholar Crossref Search ADS PubMed WorldCat 11 Burger E H , Klein-Nulend J , Van Der Plas A , Nijweide P J . Function of osteocytes in bone—their role in mechanotransduction , J. Nutr. , 1995 , vol. 125 Suppl 7 (pg. 2020S - 2023S ) Google Scholar PubMed WorldCat 12 Kusu N , Laurikkala J , Imanishi M , Usui H , Konishi M , Miyake A , Thesleff I , Itoh N . Sclerostin is a novel secreted osteoclast-derived bone morphogenetic protein antagonist with unique ligand specificity , J. Biol. Chem. , 2003 , vol. 278 26 (pg. 24113 - 24117 ) Google Scholar Crossref Search ADS PubMed WorldCat 13 Poole K E , van Bezooijen R L , Loveridge N , Hamersma H , Papapoulos S E , Löwik C W , Reeve J . Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation , FASEB J. , 2005 , vol. 19 13 (pg. 1842 - 1844 ) Google Scholar PubMed WorldCat 14 Silvestrini G , Ballanti P , Leopizzi M , Sebastiani M , Berni S , Di Vito M , Bonucci E . Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone , J. Mol Histol. , 2007 , vol. 38 4 (pg. 261 - 269 ) Google Scholar Crossref Search ADS PubMed WorldCat 15 Tatsumi S , Ishii K , Amizuka N , Li M , Kobayashi T , Kohno K , Ito M , Takeshita S , Ikeda K . Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction , Cell Metab. , 2007 , vol. 5 6 (pg. 464 - 475 ) Google Scholar Crossref Search ADS PubMed WorldCat 16 Bélanger L F . Osteocytic osteolysis , Calcif. Tissue Res. , 1969 , vol. 4 1 (pg. 1 - 12 ) Google Scholar Crossref Search ADS PubMed WorldCat 17 Feng J Q , Ward L M , Liu S , Lu Y , Xie Y , Yuan B , Yu X , Rauch F , Davis S I , Zhang S , Rios H , Drezner M K , Quarles L D , Bonewald L F , White K E . Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism , Nat. Genet , 2006 , vol. 38 11 (pg. 1230 - 1231 ) Google Scholar Crossref Search ADS PubMed WorldCat 18 Vatsa A , Breuls R G , Semeins C M , Salmon P L , Smit T H , Klein-Nulend J . Osteocyte morphology in fibula and calvaria—is there a role for mechanosensing? , Bone , 2008 , vol. 43 3 (pg. 452 - 458 ) Google Scholar Crossref Search ADS PubMed WorldCat 19 Verborgt O , Gibson G J , Schaffler M B . Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo , J. Bone Miner. Res. , 2000 , vol. 15 1 (pg. 60 - 67 ) Google Scholar Crossref Search ADS PubMed WorldCat 20 George A , Gui J , Jenkins N A , Gilbert D J , Copeland N G , Veis A . In situ localization and chromosomal mapping of the AG1 (Dmp1) gene , J. Histochem. Cytochem. , 1994 , vol. 42 (pg. 1527 - 1531 ) Google Scholar Crossref Search ADS PubMed WorldCat 21 D’Souza R N , Cavender A , Sunavala G , Alvarez J , Ohshima T , Kulkarni A B , MacDougall M . Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo , J. Bone Miner. Res. , 1997 , vol. 12 (pg. 2040 - 2049 ) Google Scholar Crossref Search ADS PubMed WorldCat 22 MacDougall M , Gu T T , Luan X , Simmons D , Chen J . Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues , J. Bone Miner. Res. , 1998 , vol. 13 (pg. 422 - 431 ) Google Scholar Crossref Search ADS PubMed WorldCat 23 Toyosawa S , Shintani S , Fujiwara T , Ooshima T , Sato A , Ijuhin N , Komori T . Dentin matrix protein 1 is predominantly expressedin chicken and rat osteocytes but not in osteoblasts , J. Bone Miner. Res. , 2001 , vol. 16 (pg. 2017 - 2026 ) Google Scholar Crossref Search ADS PubMed WorldCat 24 Aplin H M , Hirst K L , Crosby A H , Dixon M J . Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21 , Genomics , 1995 , vol. 30 (pg. 347 - 349 ) Google Scholar Crossref Search ADS PubMed WorldCat 25 MacDougall M , DuPont B R , Simmons D , Leach R J . Assignment of DMP1 to human chromosome 4 band q21 by in situ hybridization , Cytogenet. Cell Genet. , 1996 , vol. 74 pg. 189 Google Scholar Crossref Search ADS PubMed WorldCat 26 Hirst K L , Ibaraki-O’Connor K , Young M F , Dixon M J . Cloning and expression analysis of the bovine dentin matrix acidic phosphoprotein gene , J. Dent. Res. , 1997 , vol. 76 (pg. 754 - 760 ) Google Scholar Crossref Search ADS PubMed WorldCat 27 Fisher L W , Torchia D A , Fohr B , Young M F , Fedarko N S . Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin , Biochem. Biophys. Res. Commun. , 2001 , vol. 280 (pg. 460 - 465 ) Google Scholar Crossref Search ADS PubMed WorldCat 28 MacDougall M , Simmons D , Gu T T , Dong J . MEPE/OF45 a new dentin/bone matrix protein and candidate gene for dentin disease mapping to chromosome 4q21 , Connect. Tissue Res. , 2002 , vol. 43 (pg. 320 - 330 ) Google Scholar Crossref Search ADS PubMed WorldCat 29 Quarles L D . Evidence for a bone–kidney axis regulating phosphate homeostasis , J. Clin. Invest. , 2003 , vol. 112 (pg. 642 - 646 ) Google Scholar Crossref Search ADS PubMed WorldCat 30 The ADHR Consortium . Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23 , Nat. Genet. , 2000 , vol. 26 (pg. 345 - 348 ) Crossref Search ADS PubMed WorldCat 31 Shimada T , Mizutani S , Muto T , Yoneya T , Hino R , Takeda S , Takeuchi Y , Fujita T , Fukumoto S , Yamashita T . Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia , Proc. Natl. Acad. Sci. USA , 2001 , vol. 98 11 (pg. 5945 - 5946 ) Google Scholar Crossref Search ADS WorldCat 32 Riminucci M , Collins M T , Fedarko N S , Cherman N , Corsi A , White K E , Waguespack S , Gupta A , Hannon T , Econs M J , Bianco P , Gehron Robey P . FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting , J. Clin. Invest. , 2003 , vol. 112 5 (pg. 642 - 646 ) Google Scholar Crossref Search ADS PubMed WorldCat 33 Benet-Pagès A , Orlik P , Strom T M , Lorenz-Depiereux B . An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia , Hum. Mol. Genet. , 2005 , vol. 14 3 (pg. 385 - 390 ) Google Scholar Crossref Search ADS PubMed WorldCat 34 Jonsson K B , Zahradnik R , Larsson T , White K E , Sugimoto T , Imanishi Y , Yamamoto T , Hampson G , Koshiyama H , Ljunggren O , Oba K , Yang I M , Miyauchi A , Econs M J , Lavigne J , Jüppner H . Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia , N. Engl. J. Med. , 2003 , vol. 348 17 (pg. 1656 - 1663 ) Google Scholar Crossref Search ADS PubMed WorldCat 35 Liu S , Guo R , Simpson L G , Xiao Z S , Burnham C E , Quarles L D . Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX , J. Biol. Chem. , 2003 , vol. 278 39 (pg. 37419 - 37426 ) Google Scholar Crossref Search ADS PubMed WorldCat 36 Weber T J , Liu S , Indridason O S , Quarles L D . Serum FGF23 levels in normal and disordered phosphorus homeostasis , J. Bone Miner. Res. , 2003 , vol. 18 7 (pg. 1227 - 1234 ) Google Scholar Crossref Search ADS PubMed WorldCat 37 Liu S , Gupta A , Quarles L D . Emerging role of fibroblast growth factor 23 in a bone-kidney axis regulating systemic phosphate homeostasis and extracellular matrix mineralization , Curr. Opin. Nephrol. Hypertens. , 2007 , vol. 16 4 (pg. 329 - 335 ) Google Scholar Crossref Search ADS PubMed WorldCat 38 Wang H , Yoshiko Y , Yamamoto R , Minamizaki T , Kozai K , Tanne K , Aubin J E , Maeda N . Overexpression of fibroblast growth factor 23 suppresses osteoblast differentiation and matrix mineralization in vitro , J. Bone Miner. Res. , 2008 , vol. 23 6 (pg. 939 - 948 ) Google Scholar Crossref Search ADS PubMed WorldCat 39 Amizuka N , Li M , Hara K , Kobayashi M , Freitas de P H L , Ubaidus S , Oda K , Akiyama Y . Warfarin administration disrupts the assembly of mineralized nodules in osteoid , J. Electron Microsc. , 2008 , vol. 58 2 (pg. 55 - 65 ) Google Scholar Crossref Search ADS WorldCat 40 Bodian D . A new method for staining nerve fibers and nerve endings in mounted paraffin section , Anat. Rec. , 1936 , vol. 65 (pg. 89 - 97 ) Google Scholar Crossref Search ADS WorldCat 41 Bodian D . The staining of paraffin sections of nerve tissue with activated protagol. The role of fixatives , Anat. Rec. , 1937 , vol. 69 (pg. 153 - 162 ) Google Scholar Crossref Search ADS WorldCat 42 Amizuka N , Shimomura J , Li M , Seki S , Oda K , Henderson J E , Mizuno A , Ozawa H , Maeda T . Defective bone remodeling in osteoprotegerin deficient mice , J. Electron Microsc , 2003 , vol. 52 6 (pg. 503 - 513 ) Google Scholar Crossref Search ADS WorldCat 43 Amizuka N , Li M , Kobayashi M , Hara K , Akahane S , Takeuchi K , Freitas P H , Ozawa H , Maeda T , Akiyama Y . Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency , Histol. Histopathol. , 2008 , vol. 23 11 (pg. 1353 - 1366 ) Google Scholar PubMed WorldCat 44 Amizuka N , Kwan M Y , Goltzman D , Ozawa H , White J H . Vitamin D3 differentially regulates parathyroid hormone/parathyroid hormone-related peptide receptor expression in bone and cartilage , J. Clin. Invest. , 1999 , vol. 103 (pg. 373 - 381 ) Google Scholar Crossref Search ADS PubMed WorldCat 45 Oda K , Amaya Y , Fukushi-Irie M , Kinameri Y , Ohsuye K , Kubota I , Fujimura S , Kobayashi J . A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase , J. Biochem. , 1999 , vol. 126 (pg. 694 - 699 ) Google Scholar Crossref Search ADS PubMed WorldCat 46 Suzuki H , Amizuka N , Oda K , Noda M , Ohshima H , Maeda T . Involvement of the klotho protein in dentin formation and mineralization , Anat. Rec. , 2008 , vol. 291 2 (pg. 183 - 190 ) Google Scholar Crossref Search ADS WorldCat 47 Bonewald L F . Osteocytes: a proposed multifunctional bone cell , J. Musculoskelet. Neuronal. Interact. , 2002 , vol. 2 3 (pg. 239 - 241 ) Google Scholar PubMed WorldCat 48 Knothe Tate M L , Tami A , Bauer T W , Knothe U . Micropathoanatomy of osteoporosis—indications for a cellular basis of bone disease , Adv. Osteoporotic Fracture Manage. , 2002 , vol. 2 1 (pg. 9 - 14 ) WorldCat © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org Oxford University Press
TI - FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfp032
DA - 2009-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/fgf23-is-mainly-synthesized-by-osteocytes-in-the-regularly-distributed-XXgD4nnCu0
SP - 381
EP - 392
VL - 58
IS - 6
DP - DeepDyve
ER -