Collagen osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization
Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. Th...
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| description | Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology. |
| doi_str_mv | 10.1371/journal.pone.0057344 |
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A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0057344</identifier><identifier>PMID: 23460841</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Acids ; Adhesion tests ; Alkaline phosphatase ; Animals ; Apatite ; Apatites - metabolism ; Biocompatibility ; Biology ; Biomedical materials ; Bone (long) ; Bone and Bones - metabolism ; Bone and Bones - ultrastructure ; Bone density ; Bone Matrix - metabolism ; Bone Matrix - ultrastructure ; Bone morphogenetic protein 2 ; Calcification ; Calcification, Physiologic - genetics ; Calcium ; Calcium ions ; Cell culture ; Cells, Cultured ; Chemical Sciences ; Collagen ; Collagen (type I) ; Collagen - metabolism ; Contaminants ; Dentin ; Deposition ; Diffraction patterns ; Electron microscopy ; Experiments ; Extracellular matrix ; Gene expression ; Gene Expression Regulation ; Histology ; Human health and pathology ; Humans ; Hydroxyapatite ; Kinetics ; Life Sciences ; Long bone ; Male ; Materials Science ; Middle Aged ; Mimicry ; Mineralization ; Minerals - metabolism ; Models, Biological ; NMR ; Nuclear magnetic resonance ; Osteoblasts ; Osteoblasts - cytology ; Osteoblasts - metabolism ; Osteoblasts - ultrastructure ; Osteoid ; Phosphatase ; Proteins ; Rats ; Rhumatology and musculoskeletal system ; Scaffolds ; Studies ; Surgical implants ; Viability</subject><ispartof>PloS one, 2013-02, Vol.8 (2), p.e57344-e57344</ispartof><rights>2013 Silvent et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>2013 Silvent et al 2013 Silvent et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c560t-2c3be971eb07161480e18e792e8f5857161c2040012ffc9ff1602d9f06af25e13</citedby><cites>FETCH-LOGICAL-c560t-2c3be971eb07161480e18e792e8f5857161c2040012ffc9ff1602d9f06af25e13</cites><orcidid>0000-0001-9312-7278 ; 0000-0002-4094-4909 ; 0000-0002-9031-872X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583827/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583827/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23865,27923,27924,53790,53792,79371,79372</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23460841$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.sorbonne-universite.fr/hal-01142641$$DView record in HAL$$Hfree_for_read</backlink></links><search><contributor>Zeugolis, Dimitrios</contributor><creatorcontrib>Silvent, Jérémie</creatorcontrib><creatorcontrib>Nassif, Nadine</creatorcontrib><creatorcontrib>Helary, Christophe</creatorcontrib><creatorcontrib>Azaïs, Thierry</creatorcontrib><creatorcontrib>Sire, Jean-Yves</creatorcontrib><creatorcontrib>Guille, Marie Madeleine Giraud</creatorcontrib><title>Collagen osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.</description><subject>Acids</subject><subject>Adhesion tests</subject><subject>Alkaline phosphatase</subject><subject>Animals</subject><subject>Apatite</subject><subject>Apatites - metabolism</subject><subject>Biocompatibility</subject><subject>Biology</subject><subject>Biomedical materials</subject><subject>Bone (long)</subject><subject>Bone and Bones - metabolism</subject><subject>Bone and Bones - ultrastructure</subject><subject>Bone density</subject><subject>Bone Matrix - metabolism</subject><subject>Bone Matrix - ultrastructure</subject><subject>Bone morphogenetic protein 2</subject><subject>Calcification</subject><subject>Calcification, Physiologic - genetics</subject><subject>Calcium</subject><subject>Calcium ions</subject><subject>Cell culture</subject><subject>Cells, Cultured</subject><subject>Chemical Sciences</subject><subject>Collagen</subject><subject>Collagen (type I)</subject><subject>Collagen - metabolism</subject><subject>Contaminants</subject><subject>Dentin</subject><subject>Deposition</subject><subject>Diffraction patterns</subject><subject>Electron microscopy</subject><subject>Experiments</subject><subject>Extracellular matrix</subject><subject>Gene expression</subject><subject>Gene Expression Regulation</subject><subject>Histology</subject><subject>Human health and pathology</subject><subject>Humans</subject><subject>Hydroxyapatite</subject><subject>Kinetics</subject><subject>Life Sciences</subject><subject>Long bone</subject><subject>Male</subject><subject>Materials Science</subject><subject>Middle Aged</subject><subject>Mimicry</subject><subject>Mineralization</subject><subject>Minerals - metabolism</subject><subject>Models, Biological</subject><subject>NMR</subject><subject>Nuclear magnetic resonance</subject><subject>Osteoblasts</subject><subject>Osteoblasts - cytology</subject><subject>Osteoblasts - metabolism</subject><subject>Osteoblasts - ultrastructure</subject><subject>Osteoid</subject><subject>Phosphatase</subject><subject>Proteins</subject><subject>Rats</subject><subject>Rhumatology and musculoskeletal system</subject><subject>Scaffolds</subject><subject>Studies</subject><subject>Surgical 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osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization</title><author>Silvent, Jérémie ; Nassif, Nadine ; Helary, Christophe ; Azaïs, Thierry ; Sire, Jean-Yves ; Guille, Marie Madeleine Giraud</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c560t-2c3be971eb07161480e18e792e8f5857161c2040012ffc9ff1602d9f06af25e13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Acids</topic><topic>Adhesion tests</topic><topic>Alkaline phosphatase</topic><topic>Animals</topic><topic>Apatite</topic><topic>Apatites - metabolism</topic><topic>Biocompatibility</topic><topic>Biology</topic><topic>Biomedical materials</topic><topic>Bone (long)</topic><topic>Bone and Bones - metabolism</topic><topic>Bone and Bones - ultrastructure</topic><topic>Bone density</topic><topic>Bone Matrix - 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Dimitrios</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Collagen osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-02-27</date><risdate>2013</risdate><volume>8</volume><issue>2</issue><spage>e57344</spage><epage>e57344</epage><pages>e57344-e57344</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23460841</pmid><doi>10.1371/journal.pone.0057344</doi><orcidid>https://orcid.org/0000-0001-9312-7278</orcidid><orcidid>https://orcid.org/0000-0002-4094-4909</orcidid><orcidid>https://orcid.org/0000-0002-9031-872X</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2013-02, Vol.8 (2), p.e57344-e57344 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1330883068 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Acids Adhesion tests Alkaline phosphatase Animals Apatite Apatites - metabolism Biocompatibility Biology Biomedical materials Bone (long) Bone and Bones - metabolism Bone and Bones - ultrastructure Bone density Bone Matrix - metabolism Bone Matrix - ultrastructure Bone morphogenetic protein 2 Calcification Calcification, Physiologic - genetics Calcium Calcium ions Cell culture Cells, Cultured Chemical Sciences Collagen Collagen (type I) Collagen - metabolism Contaminants Dentin Deposition Diffraction patterns Electron microscopy Experiments Extracellular matrix Gene expression Gene Expression Regulation Histology Human health and pathology Humans Hydroxyapatite Kinetics Life Sciences Long bone Male Materials Science Middle Aged Mimicry Mineralization Minerals - metabolism Models, Biological NMR Nuclear magnetic resonance Osteoblasts Osteoblasts - cytology Osteoblasts - metabolism Osteoblasts - ultrastructure Osteoid Phosphatase Proteins Rats Rhumatology and musculoskeletal system Scaffolds Studies Surgical implants Viability |
| title | Collagen osteoid-like model allows kinetic gene expression studies of non-collagenous proteins in relation with mineral development to understand bone biomineralization |
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