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Histological studies have revealed that some proteins, such as BMP2, BMPR1A, and Connexin 43, are expressed in and around sites of ectopic ossification. However, the relationship between the expressed proteins considered to be associated with the ossification and mineral formation in vivo is not clear. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1)\u2010mutant spinal hyperostotic TWY mice and ICR mice as controls were euthanized after calcein labeling, and undecalcified frozen sections were obtained from the middle thoracic spine. Intervertebral disc areas were examined histologically and by measuring calcein\u2010labeled areas and areas showing immunoreactivity for BMP2, BMPR1A, and Connexin 43. Calcein\u2010labeled areas, indicating mineralization in the ectopic mineralization sites, were significantly larger in the mutant mice than in controls. The expression of Connexin 43 was elevated in the annulus fibrosus. Increases in the calcein\u2010labeled areas was not correlated with increases in the areas showing immunoreactivity for Connexin 43 in the annulus fibrosus. There was no statistical correlation between enhanced immunohistochemical expression and elevated calcein\u2010labeled areas. By applying the morphometrical analysis method using undecalcified frozen sections to ENPP1\u2010mutant mice, quantitative evaluation of the mineralization and proteins expressed in the surrounding area in the same animal became possible.<\/jats:p>","DOI":"10.1002\/jemt.23140","type":"journal-article","created":{"date-parts":[[2018,10,8]],"date-time":"2018-10-08T07:24:34Z","timestamp":1538983474000},"page":"1318-1324","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Histomorphometry of ectopic mineralization using undecalcified frozen bone sections"],"prefix":"10.1002","volume":"81","author":[{"given":"Ryuji","family":"Fujihara","sequence":"first","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"},{"name":"Department of Orthopaedic Surgery Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoichi","family":"Chiba","sequence":"additional","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toshitaka","family":"Nakagawa","sequence":"additional","affiliation":[{"name":"Division of Research Instrument and Equipment, Life Science Research Center Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryuta","family":"Murakami","sequence":"additional","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koichi","family":"Matsumoto","sequence":"additional","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Machi","family":"Kawauchi","sequence":"additional","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takayuki","family":"Fujii","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryuichi","family":"Shimono","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tetsuji","family":"Yamamoto","sequence":"additional","affiliation":[{"name":"Department of Orthopaedic Surgery Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6592-3573","authenticated-orcid":false,"given":"Masaki","family":"Ueno","sequence":"additional","affiliation":[{"name":"Department of Pathology and Host Defense Kagawa University  Kagawa Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2018,10,8]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00223-002-2074-6"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1155\/2013\/125492"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00157958"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00441-015-2277-6"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/jbmr.1805"},{"key":"e_1_2_6_7_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/srep04868","article-title":"In vivo neuronal action potential recordings via three\u2010dimensional microscale needle\u2010electrode arrays","volume":"4","author":"Fujishiro A.","year":"2014","journal-title":"Scientific Reports"},{"issue":"3","key":"e_1_2_6_8_1","first-page":"199","article-title":"Characteristics and mechanism of the ossification of posterior longitudinal ligament in the tip\u2010toe walking Yoshimura (twy) mouse","volume":"40","author":"Furusawa N.","year":"1996","journal-title":"European Journal of Histochemistry"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0101000"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00776-004-0823-2"},{"key":"e_1_2_6_11_1","first-page":"4","article-title":"Computer\u2010automated static, dynamic and cellular bone histomorphometry","volume":"05","author":"Hong S.\u2010H.","year":"2012","journal-title":"Journal of Tissue Science & Engineering"},{"key":"e_1_2_6_12_1","first-page":"157","article-title":"A new breed of mouse showing multiple osteochondral lesions\u2010\u2010twy mouse","volume":"21","author":"Hosoda Y.","year":"1981","journal-title":"Ryumachi. 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