Dmp1 is an acidic phosphoprotein that is specifically expressed in osteocytes. During the secretory process, the full-length, precursor Dmp1 is cleaved into N- and C-terminal fragments. C-terminal Dmp1 is phosphorylated, becoming a highly negatively charged domain that may assist in bone mineralization by recruiting calcium ions and influencing subsequent mineral deposition. It has been recently reported that the Golgi-localized protein kinase Fam20C phosphorylates Dmp1 in vitro. To investigate this phosphorylation in situ, we determined the locations of phosphorylated Dmp1 and Fam20C in rat bones using immunohistochemistry. During osteocytogenesis, osteoblastic, osteoid, and young osteocytes (but not old osteocytes) express Dmp1 mRNA and contain Dmp1 protein in the Golgi apparatus. These Dmp1-producing cells were distributed across the surface layer of cortical bone. Using immunofluorescence, we found that N- and C-terminal Dmp1 fragments were predominantly distributed along the lacunar walls and canaliculi of mineralized bone, respectively, but were not present in the osteoid matrix. We also found that Fam20C and its substrate, C-terminal Dmp1, colocalized in the Golgi of osteoblastic, osteoid, and young osteocytes. Furthermore, phosphorylated C-terminal Dmp1 was present in the Golgi of young osteocytes. Double-labeling immunoelectron microscopy revealed that phosphorylated C-terminal Dmp1 localized to the canalicular wall in mineralized bone. These findings suggest that C-terminal Dmp1 is phosphorylated within osteocytes and then secreted into the pericanalicular matrix of mineralized bone. Phosphorylated, negatively charged C-terminal Dmp1 in the pericanalicular matrix may play an important role in bone mineralization by recruiting calcium ions.
Cleavage of the antigenic telopeptide region from type I collagen yields atelocollagen, and this is widely used as a scaffold for bone regeneration combined with cells, growth factors, etc. However, neither the biological effect of atelocollagen alone or its contribution to bone regeneration has been well studied. We evaluated the chronological histological changes during bone regeneration following implantation of non-crosslinked atelocollagen (Koken Co., Ltd.) in rat calvarial defects. One week after implantation, osteogenic cells positive for runt-related transcription factor 2 (Runx2) and osteoclasts positive for tartrate-resistant acid phosphatase (TRAP) were present in the atelocollagen implant in the absence of bone formation. The number of Runx2-positive osteogenic cells and Osterix-positive osteoblasts increased 2 weeks after implantation, and bone matrix proteins (osteopontin, OPN; osteocalcin, OC; dentin matrix protein 1, DMP1) were distributed in newly formed bone in a way comparable to normal bone. Some resorption cavities containing osteoclasts were also present. By 3 weeks after implantation, most of the implanted atelocollagen was replaced by new bone containing many resorption cavities, and OPN, OC, and DMP1 were deposited in the residual collagenous matrix. After 4 weeks, nearly all of the atelocollagen implant was replaced with new bone including hematopoietic marrow. Immunohistochemistry for the telopeptide region of type I collagen (TeloCOL1) during these processes demonstrated that the TeloCOL1-negative atelocollagen implant was replaced by TeloCOL1-positive collagenous matrix and new bone, indicating that new bone was mostly composed of endogenous type I collagen. These findings suggest that the atelocollagen itself can support bone regeneration by promoting osteoblast differentiation and type I collagen production.
IntroductionRecently, the applications of liquid‐based cytology (LBC) have been extended to oral cytology, and in 2015, diagnostic guidelines having roots in the Bethesda System for oral exfoliative cytology were published by the Japanese Society of Clinical Cytology (JSCC): Classification of cytology for oral mucosal disease (JSCC, 2015). Here we aimed to evaluate the applicability of LBC in the oral mucosa in accordance with the novel diagnostic guidelines.MethodsTwo preparation techniques (conventional exfoliative cytology [CEC] with LBC and LBC alone) were used in this study. Intraepithelial lesions of the oral mucosa histologically diagnosed by biopsy or resection materials were selected as samples and multiple intraepithelial lesions in a single patient were counted. Deep‐seated lesions under the oral mucosa were excluded. The performance of cytological diagnosis was evaluated in each technique by comparing cytological diagnoses with histological diagnoses in accordance with the diagnostic guidelines.ResultsThe sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of cytological diagnosis in CEC with LBC were 61%, 73%, 86%, 41%, and 64% (P ≤ .023), respectively. For LBC alone, these values were 55%, 79%, 92%, 29%, and 60% (P ≤ .024), respectively. The rates of inadequate samples were 0.83% for CEC with LBC and 1.2% for LBC alone against whole samples.ConclusionLBC showed good specificity, positive predictive value, and low rate of inadequate specimen, so it was suitable for oral cytology. The data used for this study reflect a large contribution in daily medical examination.
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