In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering
Identifieur interne : 002878 ( Pmc/Curation ); précédent : 002877; suivant : 002879In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering
Auteurs : Kitaek Lim [Corée du Sud] ; Jangho Kim [Corée du Sud] ; Hoon Seonwoo [Corée du Sud] ; Soo Hyun Park [Corée du Sud] ; Pill-Hoon Choung [Corée du Sud] ; Jong Hoon Chung [Corée du Sud]Source :
- BioMed Research International [ 2314-6133 ] ; 2013.
Abstract
Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of
time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm2 and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*
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DOI: 10.1155/2013/269724
PubMed: 24195067
PubMed Central: 3806253
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en"><italic>In Vitro</italic> Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering</title><author><name sortKey="Lim, Kitaek" sort="Lim, Kitaek" uniqKey="Lim K" first="Kitaek" last="Lim">Kitaek Lim</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation></author><author><name sortKey="Kim, Jangho" sort="Kim, Jangho" uniqKey="Kim J" first="Jangho" last="Kim">Jangho Kim</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Seonwoo, Hoon" sort="Seonwoo, Hoon" uniqKey="Seonwoo H" first="Hoon" last="Seonwoo">Hoon Seonwoo</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Park, Soo Hyun" sort="Park, Soo Hyun" uniqKey="Park S" first="Soo Hyun" last="Park">Soo Hyun Park</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Choung, Pill Hoon" sort="Choung, Pill Hoon" uniqKey="Choung P" first="Pill-Hoon" last="Choung">Pill-Hoon Choung</name><affiliation wicri:level="1"><nlm:aff id="I2">Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Tooth Bioengineering National Research Laboratory of Post BK21, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Tooth Bioengineering National Research Laboratory of Post BK21, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation></author><author><name sortKey="Chung, Jong Hoon" sort="Chung, Jong Hoon" uniqKey="Chung J" first="Jong Hoon" last="Chung">Jong Hoon Chung</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I4">Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151- 921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151- 921</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24195067</idno><idno type="pmc">3806253</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806253</idno><idno type="RBID">PMC:3806253</idno><idno type="doi">10.1155/2013/269724</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">002878</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002878</idno><idno type="wicri:Area/Pmc/Curation">002878</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">002878</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main"><italic>In Vitro</italic> Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering</title><author><name sortKey="Lim, Kitaek" sort="Lim, Kitaek" uniqKey="Lim K" first="Kitaek" last="Lim">Kitaek Lim</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation></author><author><name sortKey="Kim, Jangho" sort="Kim, Jangho" uniqKey="Kim J" first="Jangho" last="Kim">Jangho Kim</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Seonwoo, Hoon" sort="Seonwoo, Hoon" uniqKey="Seonwoo H" first="Hoon" last="Seonwoo">Hoon Seonwoo</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Park, Soo Hyun" sort="Park, Soo Hyun" uniqKey="Park S" first="Soo Hyun" last="Park">Soo Hyun Park</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation></author><author><name sortKey="Choung, Pill Hoon" sort="Choung, Pill Hoon" uniqKey="Choung P" first="Pill-Hoon" last="Choung">Pill-Hoon Choung</name><affiliation wicri:level="1"><nlm:aff id="I2">Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Tooth Bioengineering National Research Laboratory of Post BK21, School of Dentistry, Seoul National University, Seoul, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Tooth Bioengineering National Research Laboratory of Post BK21, School of Dentistry, Seoul National University, Seoul</wicri:regionArea></affiliation></author><author><name sortKey="Chung, Jong Hoon" sort="Chung, Jong Hoon" uniqKey="Chung J" first="Jong Hoon" last="Chung">Jong Hoon Chung</name><affiliation wicri:level="1"><nlm:aff id="I1">Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="I4">Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151- 921, Republic of Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151- 921</wicri:regionArea></affiliation></author></analytic><series><title level="j">BioMed Research International</title><idno type="ISSN">2314-6133</idno><idno type="eISSN">2314-6141</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of
time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm<sup>2</sup> and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*<italic>P</italic> < 0.05) than that of control cells. The alkaline phosphatase (ALP) was significantly enhanced at 1 week with LIPUS treatment in osteogenic cultures as compared to control. Gene expressions showed significantly higher expression levels of CD29, CD44, COL1, and OCN in the hABMSCs under LIPUS treatment when compared to control after two weeks of treatment. The effects were partially controlled by LIPUS treatment, indicating that modulation of osteogenesis in hABMSCs was related to the specific stimulation. Furthermore, mineralized nodule
formation was markedly increased after LIPUS treatment than that seen in untreated cells. Through simple staining methods such as Alizarin red and von Kossa staining, calcium deposits generated their highest levels at about 3 weeks. These results suggest
that LIPUS could enhance the cell viability and osteogenic differentiation of hABMSCs, and could be part of effective treatment methods for clinical applications.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Buckley, Mj" uniqKey="Buckley M">MJ Buckley</name></author><author><name sortKey="Banes, Aj" uniqKey="Banes A">AJ Banes</name></author><author><name sortKey="Levin, Lg" uniqKey="Levin L">LG Levin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Binderman, I" uniqKey="Binderman I">I Binderman</name></author><author><name sortKey="Zor, U" uniqKey="Zor U">U Zor</name></author><author><name sortKey="Kaye, Am" uniqKey="Kaye A">AM Kaye</name></author><author><name sortKey="Shimshoni, Z" uniqKey="Shimshoni Z">Z Shimshoni</name></author><author><name sortKey="Harell, A" uniqKey="Harell A">A Harell</name></author><author><name sortKey="Somjen, D" uniqKey="Somjen D">D 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biomed Res Int</journal-id><journal-id journal-id-type="iso-abbrev">Biomed Res Int</journal-id><journal-id journal-id-type="publisher-id">BMRI</journal-id><journal-title-group><journal-title>BioMed Research International</journal-title></journal-title-group><issn pub-type="ppub">2314-6133</issn><issn pub-type="epub">2314-6141</issn><publisher><publisher-name>Hindawi Publishing Corporation</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24195067</article-id><article-id pub-id-type="pmc">3806253</article-id><article-id pub-id-type="doi">10.1155/2013/269724</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title><italic>In Vitro</italic> Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lim</surname><given-names>KiTaek</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Jangho</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">http://orcid.org/0000-0002-1827-9359</contrib-id><name><surname>Seonwoo</surname><given-names>Hoon</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">http://orcid.org/0000-0002-5846-4233</contrib-id><name><surname>Park</surname><given-names>Soo Hyun</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Choung</surname><given-names>Pill-Hoon</given-names></name><xref ref-type="aff" rid="I2"><sup>2</sup></xref><xref ref-type="aff" rid="I3"><sup>3</sup></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Chung</surname><given-names>Jong Hoon</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I4"><sup>4</sup></xref><xref ref-type="corresp" rid="cor2">*</xref></contrib></contrib-group><aff id="I1"><sup>1</sup>Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 151-921, Republic of Korea</aff><aff id="I2"><sup>2</sup>Department of Oral and Maxillofacial Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea</aff><aff id="I3"><sup>3</sup>Tooth Bioengineering National Research Laboratory of Post BK21, School of Dentistry, Seoul National University, Seoul, Republic of Korea</aff><aff id="I4"><sup>4</sup>Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151- 921, Republic of Korea</aff><author-notes><corresp id="cor1">*Pill-Hoon Choung: <email>choungph@snu.ac.kr</email> and </corresp><corresp id="cor2">*Jong Hoon Chung: <email>jchung@snu.ac.kr</email></corresp><fn fn-type="other"><p>Academic Editor: Aaron W. James</p></fn></author-notes><pub-date pub-type="ppub"><year>2013</year></pub-date><pub-date pub-type="epub"><day>30</day><month>9</month><year>2013</year></pub-date><volume>2013</volume><elocation-id>269724</elocation-id><history><date date-type="received"><day>4</day><month>5</month><year>2013</year></date><date date-type="rev-recd"><day>4</day><month>7</month><year>2013</year></date><date date-type="accepted"><day>9</day><month>7</month><year>2013</year></date></history><permissions><copyright-statement>Copyright © 2013 KiTaek Lim et al.</copyright-statement><copyright-year>2013</copyright-year><license xlink:href="https://creativecommons.org/licenses/by/3.0/"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of
time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm<sup>2</sup> and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*<italic>P</italic> < 0.05) than that of control cells. The alkaline phosphatase (ALP) was significantly enhanced at 1 week with LIPUS treatment in osteogenic cultures as compared to control. Gene expressions showed significantly higher expression levels of CD29, CD44, COL1, and OCN in the hABMSCs under LIPUS treatment when compared to control after two weeks of treatment. The effects were partially controlled by LIPUS treatment, indicating that modulation of osteogenesis in hABMSCs was related to the specific stimulation. Furthermore, mineralized nodule
formation was markedly increased after LIPUS treatment than that seen in untreated cells. Through simple staining methods such as Alizarin red and von Kossa staining, calcium deposits generated their highest levels at about 3 weeks. These results suggest
that LIPUS could enhance the cell viability and osteogenic differentiation of hABMSCs, and could be part of effective treatment methods for clinical applications.</p></abstract></article-meta></front><floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Schematic diagram of LIPUS treatment (frequency: 1 MHz, intensity: 50 mW/cm<sup>2</sup>, exposure time: 10 min/day, and duty cycle: 20% or 50%), as compared to static culture as control. </p></caption><graphic xlink:href="BMRI2013-269724.001"></graphic></fig><fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Representative immunocytochemistry images of hABMSCs. Fluorescence images of hABMSCs showed cell nuclei (A1), actin filaments (B1), Stro-1 (C1), and merged images (D1) of the fluorescence stains (a). Fluorescence images of hABMSCs showed cell nuclei (A2), actin filaments (B2), CD146 (C2), and merged images (D2) of the fluorescence stains (b) as MSC markers.</p></caption><graphic xlink:href="BMRI2013-269724.002"></graphic></fig><fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Representative optical microscopic images of hABMSCs stimulated for 4 days in static condition (A), at 20% duty cycle (B), and at 50% duty cycle (C) under LIPUS treatment (a). Representative FE-SEM morphologies of hABMSCs stimulated for 7 days in static condition (A), at 20% duty cycle (B), and 50% duty cycle (C) under LIPUS treatment (b). FE-SEM images showed more lining up observation at stimulation groups compared to control group (arrows: cell direction). Cell metabolic viability as optical density of hABMSCs measured using WST-1 (c). Overhead brackets with asterisks indicate significant difference between groups.</p></caption><graphic xlink:href="BMRI2013-269724.003"></graphic></fig><fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>DNA concentration as percent of initial of hABMSCs measured using CyQUANT Cell Proliferation Assay Kit (D) (<italic>n</italic> = 3).</p></caption><graphic xlink:href="BMRI2013-269724.004"></graphic></fig><fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label><caption><p><italic>In vitro</italic> cell migration as representative optical microscopic images of LIPUS group compared to the static culture (a), indicating that the stimulation group exposed at 20% duty cycle LIPUS for 10 min/day was significantly different (*<italic>P</italic> < 0.05) among groups (b) (<italic>n</italic> = 3).</p></caption><graphic xlink:href="BMRI2013-269724.005"></graphic></fig><fig id="fig6" orientation="portrait" position="float"><label>Figure 6</label><caption><p>RT-PCR analysis of cell cultures between LIPUS treatment and static culture for 2 weeks. Expression of genes associated with the osteoblastic differentiation was examined using real time PCR to investigate the effect of LIPUS treatment on gene expression (a). Expression levels (b) of CD29 (A), CD44 (B), COL1 (C), and OCN (D) at 2 weeks were significantly higher in LIPUS treatment. Stimulation groups exposed during 10 min/day at 20% duty cycle (in CD44) or 50% duty cycle (in OCN) were significantly different (*<italic>P</italic> < 0.05) among groups.</p></caption><graphic xlink:href="BMRI2013-269724.006"></graphic></fig><fig id="fig7" orientation="portrait" position="float"><label>Figure 7</label><caption><p>ALP activity cultured in different types of hABMSCs stimulated with LIPUS for 1 week. LIPUS groups exposed during 10 min/day at 20% or 50% duty cycle were significantly different (*<italic>P</italic> < 0.05) among groups (<italic>n</italic> = 3).</p></caption><graphic xlink:href="BMRI2013-269724.007"></graphic></fig><fig id="fig8" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Representative images of hABMSCs after Alizarin red and von Kossa staining treatment in static condition (a), at 20% duty cycle (b), and at 50% duty cycle under LIPUS treatment (c) at 2-3 weeks after the addition of osteogenic differentiation media. Representative optical microscopic images of hABMSCs after Alizarin red staining treatment in static condition (A), at 20% duty cycle under LIPUS (B), and at 50% duty cycle under LIPUS (C) at 2-3 weeks were indicated (d). Mineralization images of von Kossa staining are shown in <xref ref-type="fig" rid="fig8">Figure 8(e)</xref>. The short-pulsed duty cycle group was interestingly increased compared to the longer duty cycle group. Representative images were shown in Figures <xref ref-type="fig" rid="fig8">8(d)</xref> and <xref ref-type="fig" rid="fig8">8(e)</xref>. <xref ref-type="fig" rid="fig8"> Figure 8(f)</xref> shows the optical density value of a mineralized nodule (absorbance of 562 nm) measured after destaining treatment.</p></caption><graphic xlink:href="BMRI2013-269724.008"></graphic></fig><fig id="fig9" orientation="portrait" position="float"><label>Figure 9</label><caption><p>(a) Representative confocal laser microscopy images of hABMSCs cultured for 7 days in static conditions (A) or LIPUS induction at 20% duty cycle (B) or 50% duty cycle (C) after the addition of differentiation media: cell nuclei (blue), actin filaments (red), vinculin (green), and merged images of the fluorescence stains. Confocal laser microscopy images showed more intense observation in the LIPUS group compared to the control group. (b) Representative confocal laser microscopy images of hABMSCs cultured for 7 days in static conditions (A) or LIPUS induction at 20% duty cycle (B), or 50% duty cycle (C) after the addition of differentiation media: cell nuclei (blue), actin filaments (red), osteocalcin (green), and merged images of the fluorescence stains. Confocal laser microscopy images showed more intense observation in the LIPUS group compared to the control group. (c) Brightness level of vinculin (A) and osteocalcin (B) in florescence cell image treated by LIPUS induction.</p></caption><graphic xlink:href="BMRI2013-269724.009"></graphic></fig><table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label><caption><p>Primers used for RT-PCR.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Gene</th><th align="center" rowspan="1" colspan="1">Accession number</th><th align="center" rowspan="1" colspan="1">Primer sequence</th><th align="center" rowspan="1" colspan="1">Predicted size (bp)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">CD29</td><td align="center" rowspan="1" colspan="1">NM_002211</td><td align="center" rowspan="1" colspan="1">5′-AATGAAGGGCGTGTTGGTAG-3′ <break></break>5′-CGTTGCTGGCTTCACAAGTA-3′</td><td align="center" rowspan="1" colspan="1">337</td></tr><tr><td align="left" rowspan="1" colspan="1">CD44</td><td align="center" rowspan="1" colspan="1">X55938</td><td align="center" rowspan="1" colspan="1">5′-ACCGACCTTCCCACTTCACAG-3′ <break></break>5′-GCACTACACCCCAATCTTCAT-3′</td><td align="center" rowspan="1" colspan="1">168–200</td></tr><tr><td align="left" rowspan="1" colspan="1">Col-I</td><td align="center" rowspan="1" colspan="1">NM_000088</td><td align="center" rowspan="1" colspan="1">5′-CTGGCAAAGAAGGCGGCAAA-3′ <break></break>5′-CTCACCACGATCACCCACTCT-3′</td><td align="center" rowspan="1" colspan="1">503</td></tr><tr><td align="left" rowspan="1" colspan="1">OCN</td><td align="center" rowspan="1" colspan="1">X53698.1</td><td align="center" rowspan="1" colspan="1">5′-CATGAGAGCCCTCACACTC-3′ <break></break>5′-AGAGCGACACCCTAGACCG-3′</td><td align="center" rowspan="1" colspan="1">315</td></tr><tr><td align="left" rowspan="1" colspan="1">GAPDH</td><td align="center" rowspan="1" colspan="1">AF017079</td><td align="center" rowspan="1" colspan="1">5′-GGGCATGAACCATGAGAAGT-3′ <break></break>5′-CCCCAGCATCAAAGGTAGAA-3′</td><td align="center" rowspan="1" colspan="1">497</td></tr></tbody></table></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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