Peri-Implant Crestal Bone Loss: A Putative Mechanism
Identifieur interne : 004215 ( Ncbi/Merge ); précédent : 004214; suivant : 004216Peri-Implant Crestal Bone Loss: A Putative Mechanism
Auteurs : Yuko Ujiie ; Reynaldo Todescan ; John E. DaviesSource :
- International Journal of Dentistry [ 1687-8728 ] ; 2012.
Abstract
Url:
DOI: 10.1155/2012/742439
PubMed: 23091492
PubMed Central: 3467942
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Peri-Implant Crestal Bone Loss: A Putative Mechanism</title>
<author><name sortKey="Ujiie, Yuko" sort="Ujiie, Yuko" uniqKey="Ujiie Y" first="Yuko" last="Ujiie">Yuko Ujiie</name>
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<author><name sortKey="Todescan, Reynaldo" sort="Todescan, Reynaldo" uniqKey="Todescan R" first="Reynaldo" last="Todescan">Reynaldo Todescan</name>
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<author><name sortKey="Davies, John E" sort="Davies, John E" uniqKey="Davies J" first="John E." last="Davies">John E. Davies</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Peri-Implant Crestal Bone Loss: A Putative Mechanism</title>
<author><name sortKey="Ujiie, Yuko" sort="Ujiie, Yuko" uniqKey="Ujiie Y" first="Yuko" last="Ujiie">Yuko Ujiie</name>
</author>
<author><name sortKey="Todescan, Reynaldo" sort="Todescan, Reynaldo" uniqKey="Todescan R" first="Reynaldo" last="Todescan">Reynaldo Todescan</name>
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<author><name sortKey="Davies, John E" sort="Davies, John E" uniqKey="Davies J" first="John E." last="Davies">John E. Davies</name>
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<series><title level="j">International Journal of Dentistry</title>
<idno type="ISSN">1687-8728</idno>
<idno type="eISSN">1687-8736</idno>
<imprint><date when="2012">2012</date>
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<front><div type="abstract" xml:lang="en"><p><italic>Purpose</italic>
. The immunological mechanisms of peri-implant crestal bone loss have, hitherto, not been elucidated. We hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri-implant cells, which results in osteoclast formation and, ultimately, in bone resorption. <italic>Materials and Methods</italic>
. We used RT-PCR and ELISA to assay mediators of osteoclastogenesis in rat and human macrophages (r-and hMO); bone marrow derived stromal cells (r-and hBMCs); and human gingival fibroblasts (hGF)—with or without stimulation by LPS. TRAP positive multinucleate cells were assessed for their resorptive ability. <italic>Results</italic>
. We show that IL-1<italic><italic>α</italic>
</italic>
, IL-1<italic><italic>β</italic>
</italic>
, and IL-6 were expressed by all examined cell types, and TNF-<italic><italic>α</italic>
</italic>
was upregulated in hGF. Secretion of IL-1<italic><italic>α</italic>
</italic>
and IL-1<italic><italic>β</italic>
</italic>
proteins was stimulated in hMO by LPS, and IL-6 protein secretion was highly stimulated in hBMCs and hGF. Both LPS and RANKL stimulated macrophages to form osteoclast-like TRAP positive cells, which resorbed calcium phosphate substrates. <italic>Conclusion</italic>
. Taken together, the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri-implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Dent</journal-id>
<journal-id journal-id-type="iso-abbrev">Int J Dent</journal-id>
<journal-id journal-id-type="publisher-id">IJD</journal-id>
<journal-title-group><journal-title>International Journal of Dentistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">1687-8728</issn>
<issn pub-type="epub">1687-8736</issn>
<publisher><publisher-name>Hindawi Publishing Corporation</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">23091492</article-id>
<article-id pub-id-type="pmc">3467942</article-id>
<article-id pub-id-type="doi">10.1155/2012/742439</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Peri-Implant Crestal Bone Loss: A Putative Mechanism</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Ujiie</surname>
<given-names>Yuko</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name><surname>Todescan</surname>
<given-names>Reynaldo</given-names>
</name>
</contrib>
<contrib contrib-type="author"><name><surname>Davies</surname>
<given-names>John E.</given-names>
</name>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
</contrib-group>
<aff id="I1">Institute of Biomaterials Biomedical Engineering and Faculty of Dentistry, University of Toronto, 164 College Street, Room 407, Toronto, ON, Canada M5S 3G9</aff>
<author-notes><corresp id="cor1">*John E. Davies: <email>davies@ecf.utoronto.ca</email>
</corresp>
<fn fn-type="other"><p>Academic Editor: Michael E. Razzoog</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><year>2012</year>
</pub-date>
<pub-date pub-type="epub"><day>2</day>
<month>10</month>
<year>2012</year>
</pub-date>
<volume>2012</volume>
<elocation-id>742439</elocation-id>
<history><date date-type="received"><day>26</day>
<month>5</month>
<year>2012</year>
</date>
<date date-type="accepted"><day>14</day>
<month>7</month>
<year>2012</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2012 Yuko Ujiie et al.</copyright-statement>
<copyright-year>2012</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><italic>Purpose</italic>
. The immunological mechanisms of peri-implant crestal bone loss have, hitherto, not been elucidated. We hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri-implant cells, which results in osteoclast formation and, ultimately, in bone resorption. <italic>Materials and Methods</italic>
. We used RT-PCR and ELISA to assay mediators of osteoclastogenesis in rat and human macrophages (r-and hMO); bone marrow derived stromal cells (r-and hBMCs); and human gingival fibroblasts (hGF)—with or without stimulation by LPS. TRAP positive multinucleate cells were assessed for their resorptive ability. <italic>Results</italic>
. We show that IL-1<italic><italic>α</italic>
</italic>
, IL-1<italic><italic>β</italic>
</italic>
, and IL-6 were expressed by all examined cell types, and TNF-<italic><italic>α</italic>
</italic>
was upregulated in hGF. Secretion of IL-1<italic><italic>α</italic>
</italic>
and IL-1<italic><italic>β</italic>
</italic>
proteins was stimulated in hMO by LPS, and IL-6 protein secretion was highly stimulated in hBMCs and hGF. Both LPS and RANKL stimulated macrophages to form osteoclast-like TRAP positive cells, which resorbed calcium phosphate substrates. <italic>Conclusion</italic>
. Taken together, the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri-implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts.</p>
</abstract>
</article-meta>
</front>
<floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Expression of pro-inflammatory and osteoclastogenic genes with several LPS stimulation in human gingival fibroblasts. IL-1<italic>β</italic>
, IL-6, TNF-<italic>α</italic>
, RANL, RANKL, and GAPDH gene expression in hGF cultures with different types of LPS stimulation: <italic>E</italic>
. <italic>coli</italic>
, <italic>P</italic>
. <italic>gingivalis</italic>
, <italic>P</italic>
. <italic>intermedia</italic>
and without LPS for 2 hours. 1 <italic>μ</italic>
g/mL of LPS was used in the experiment. mRNA was isolated at 2 hours. RT-PCR was carried out at different cycles for each gene. Annealing temperature was 55 degrees for all genes. </p>
</caption>
<graphic xlink:href="IJD2012-742439.001"></graphic>
</fig>
<fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>Expression of LPS receptors and their signaling factor's genes with several LPS stimulation in human fibroblasts. TRL 2, TRL 4, CD14, and MD-2 gene expression in hGF cultures with different types of LPS stimulation: <italic>E. coli</italic>
, <italic>P. gingivalis</italic>
, <italic>P. intermedia,</italic>
and without LPS for 2 hours. 1 <italic>μ</italic>
g/mL of LPS was used in the experiment. mRNA was isolated at 2 hours. RT-PCR was carried out at different annealing temperature that TRL2 and CD14 were 57 degrees, and TRL 4 and MD-2 were 54 degrees. PCR cycle was 30 cycles for all genes.</p>
</caption>
<graphic xlink:href="IJD2012-742439.002"></graphic>
</fig>
<fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label>
<caption><p>Expression of pro-inflammatory cytokine genes in human macrophages. IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, TNF-<italic>α</italic>
, IL-6, and GAPDH gene expression in hMO cultures with and without LPS stimulation for 24 hours. 1 <italic>μ</italic>
g/mL of LPS was used in the experiment. mRNA was isolated at 24 hours. RT-PCR was carried out at 30 cycles for all genes.</p>
</caption>
<graphic xlink:href="IJD2012-742439.003"></graphic>
</fig>
<fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label>
<caption><p>Expression of pro-inflammatory cytokine genes in mouse macrophages (RAW 264.7). Gene expression of IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, TNF-<italic>α</italic>
, IL-6, and GAPDH in mMO cultures in the absence (0 <italic>μ</italic>
g/mL) and presence (1 <italic>μ</italic>
g/mL) of LPS. RNA was isolated at 12, 24, 36, 48, and 60 hours. RT-PCR was carried out at 30 cycles for all genes in this experiment. </p>
</caption>
<graphic xlink:href="IJD2012-742439.004"></graphic>
</fig>
<fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label>
<caption><p>Expression of pro-inflammatory cytokine genes in human bone marrow cells. IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, TNF-<italic>α</italic>
, IL-6, and GAPDH gene expression in hBMCs cultures with or without LPS stimulation. LPS concentrations were 0 <italic>μ</italic>
g/mL, 0.1 <italic>μ</italic>
g/mL or 1 <italic>μ</italic>
g/mL. RNA was isolated at 24, 48, and 96 hours. RT-PCR was carried out at 30 cycles for GAPDH, IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, and TNF-<italic>α</italic>
, and 25 cycles for IL-6.</p>
</caption>
<graphic xlink:href="IJD2012-742439.005"></graphic>
</fig>
<fig id="fig6" orientation="portrait" position="float"><label>Figure 6</label>
<caption><p>Expression of pro-inflammatory cytokine genes in rat stromal bone marrow cells. Gene expression for IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, TNF-<italic>α</italic>
, IL-6 and GAPDH in rSBMCs cultures in the presence and absence of LPS. The LPS concentrations used were 0.1 <italic>μ</italic>
g/mL and 1 <italic>μ</italic>
g/mL. RNA was isolated at 6, 24, 48 and 96 hours. RT-PCR was carried out at 30 cycles for GAPDH, IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, and TNF-<italic>α</italic>
, and 25 cycles for IL-6.</p>
</caption>
<graphic xlink:href="IJD2012-742439.006"></graphic>
</fig>
<fig id="fig7" orientation="portrait" position="float"><label>Figure 7</label>
<caption><p>Expression of pro-inflammatory cytokine genes in human gingival fibroblasts. Gene expression for IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, TNF-<italic>α</italic>
, IL-6, and GAPDH genes in hGF cultures with and without LPS stimulation. LPS concentrations were 0 <italic>μ</italic>
g/mL, 0.1 <italic>μ</italic>
g/mL or 1 <italic>μ</italic>
g/mL. RNA was isolated at 24, 48, and 96 hours. RT-PCR was carried out at 30 cycles for GAPDH, 35 cycles for IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, and TNF-<italic>α</italic>
, and 25 cycles for IL-6. </p>
</caption>
<graphic xlink:href="IJD2012-742439.007"></graphic>
</fig>
<fig id="fig8" orientation="portrait" position="float"><label>Figure 8</label>
<caption><p>(a) Secretion of IL-6 protein in human bone marrow cells IL-6 secretion in hBMCs cultures with and without LPS stimulation for 24, 48, and 96 hours. LPS concentrations were 0 <italic>μ</italic>
g/mL, 0.1 <italic>μ</italic>
g/mL or 1 <italic>μ</italic>
g/mL. The amount of IL-6 in cell-free culture supernatants was analyzed by ELISA. (b) secretion of IL-6 protein in human gingival fibroblasts Secretion of IL-6 in hGFs with or without LPS stimulation for 24, 48, and 96 hours. LPS concentrations were 0 <italic>μ</italic>
g/mL, 0.1 <italic>μ</italic>
g/mL or 1 <italic>μ</italic>
g/mL. The amount of IL-6 in cell-free culture supernatants was analyzed by ELISA.</p>
</caption>
<graphic xlink:href="IJD2012-742439.008"></graphic>
</fig>
<fig id="fig9" orientation="portrait" position="float"><label>Figure 9</label>
<caption><p>TRAP positive reactions were showed as level of absorbance in each group. The data were calculated by triplicate samples in each group. One day after mMO cells were seeded into 96 wells they were stimulated with <italic>E. coli</italic>
, <italic>P. gingivalis</italic>
, <italic>P. intermedia</italic>
LPS for 3 days. 1 <italic>μ</italic>
g/mL of LPS was added into the cell culture medium individually. As positive control, we used 300 ng/mL GST-RANKL which was treated the same way with LPS groups. Negative control was not including RANKL and LPS. </p>
</caption>
<graphic xlink:href="IJD2012-742439.009"></graphic>
</fig>
<fig id="fig10" orientation="portrait" position="float"><label>Figure 10</label>
<caption><p>(a) and (b) Cell morphology of mouse macrophages in the presence of LPS Cells images were taken by phase contrast microscope (original magnification, ×10). mMO (RAW 264.7) were cultured for 7days in the absence of LPS (a) and in the presence of 1 <italic>μ</italic>
g/mL LPS (b). (c) to (g) Osteoclast-like cells in RAW264.7 cultures induced by LPS and RANKL mMO (RAW 264.7) were incubated with 1 <italic>μ</italic>
g/mL of LPS or 200 ng/mL of RANKL for 7 days. TRAP staining images were taken by phase contrast microscope (c, e and h). Pit formation activity of osteoclasts was observed by SEM (d, f and g). (c and d) mMO seeded on CaP substrate (control), (e and f) treated with LPS and (h and g) treated with RANKL.</p>
</caption>
<graphic xlink:href="IJD2012-742439.010"></graphic>
</fig>
<table-wrap-group id="tab1" orientation="portrait" position="float"><label>Table 1</label>
<caption><p>Specific primers for the reverse transcriptase polymerase chain reaction (RT-PCR) and the predicted sizes of the PCR products (bp).</p>
</caption>
<table-wrap id="tab1a" orientation="portrait" position="anchor"><caption><p>(a) Specific primers for mouse</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Specificity</th>
<th align="center" rowspan="1" colspan="1">Primer</th>
<th align="center" rowspan="1" colspan="1">Detection</th>
<th align="center" rowspan="1" colspan="1">Product size (bp)</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="2" colspan="1">IL-1alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TGCCACCAAAGAACAAAGTCG</td>
<td align="center" rowspan="2" colspan="1">599</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CCCACTGAGGTAGGAAAGATGTAGC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-1beta</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TCTTTGAAGTTGACGGACCCC</td>
<td align="center" rowspan="2" colspan="1">269</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CCAGCAGGTTATCATCATCATCCC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">TNF-alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">ACCGTCAGCCGATTTGCTATC</td>
<td align="center" rowspan="2" colspan="1">288</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TCAGAGTAAAGGGGTCAGAGTGGG</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-6</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">GAGCCCACCAAGAACGATAG</td>
<td align="center" rowspan="2" colspan="1">229</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TCCACGATTTCCCAGAGAAC</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab1b" orientation="portrait" position="anchor"><caption><p>(b) Specific primers for rat</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Specificity</th>
<th align="center" rowspan="1" colspan="1">Primer</th>
<th align="center" rowspan="1" colspan="1">Detection</th>
<th align="center" rowspan="1" colspan="1">Product size (bp)</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="2" colspan="1">IL-1alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">AGGTTCCACGTTTCCTCCTT</td>
<td align="center" rowspan="2" colspan="1">233</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">GCCTCCAGGTCATCTTCAGT</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-1beta</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">CAACAAAAATGCCTCGTGC</td>
<td align="center" rowspan="2" colspan="1">395</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">AAGTCAACTATGTCCCGAC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">TNF-alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TACTGAACTTCGGGGTGATCG</td>
<td align="center" rowspan="2" colspan="1">292</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CCTTGTCCCTTGAAGAGAACC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-6</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TGTGCAATGGCAATTCTGAT</td>
<td align="center" rowspan="2" colspan="1">156</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">GGAACTCCAGAAGACCAGAGC</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab1c" orientation="portrait" position="anchor"><caption><p>(c) Specific primers for human</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Specificity</th>
<th align="center" rowspan="1" colspan="1">Primer</th>
<th align="center" rowspan="1" colspan="1">Detection</th>
<th align="center" rowspan="1" colspan="1">Product size (bp)</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="2" colspan="1">IL-1alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TTCATTGGCGTTTGAGTCAG</td>
<td align="center" rowspan="2" colspan="1">163</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">GGAGTGGGCCATAGCTTACA</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-1beta</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">GCATCCAGCTACGAATCTCC</td>
<td align="center" rowspan="2" colspan="1">193</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TCGTTATCCCATGTGTCGAA</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">TNF-alpha</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">ACAAGCCTGTAGCCCATGTT</td>
<td align="center" rowspan="2" colspan="1">265</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TTGATGGCAGAGAGGAGGTT</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">IL-6</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">GCTATGAACTCCTTCTCCACAAGC</td>
<td align="center" rowspan="2" colspan="1">264</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TTCTGCCAGTGCCTCTTTGC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">RANK</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">ATGCGGTTTGCAGTTCTTCT</td>
<td align="center" rowspan="2" colspan="1">384</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CGTAGGGACCACCTCCTACA</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">RANKL</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">AGAGCGCAGATGGATCCTAA</td>
<td align="center" rowspan="2" colspan="1">180</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TTCCTTTTGCACAGCTCCTT</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">TRL 2</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TTAGCAACAGTGACCTACAGAG</td>
<td align="center" rowspan="2" colspan="1">503</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CAAATCAGTATCTCGCAGTTCC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">TRL 4</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">TGGATACGTTTCCTTATAAG</td>
<td align="center" rowspan="2" colspan="1">507</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">GAAATGGAGGCACCCCTTC</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">CD 14</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">CAACTTCTCCGAACCTCAGC</td>
<td align="center" rowspan="2" colspan="1">271</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TAGGTCCTCGAGCGTCAGTT</td>
</tr>
<tr><td align="left" colspan="4" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="2" colspan="1">MD-2</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">GCACATTTTCTACATTCC</td>
<td align="center" rowspan="2" colspan="1">157</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">CACAGTCTCTCCCTTCAG</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab1d" orientation="portrait" position="anchor"><caption><p>(d) Specific primers for House keeping gene</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Specificity</th>
<th align="center" rowspan="1" colspan="1">Primer</th>
<th align="center" rowspan="1" colspan="1">Detection</th>
<th align="center" rowspan="1" colspan="1">Product size (bp)</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="2" colspan="1">GAPDH</td>
<td align="center" rowspan="1" colspan="1">Sense</td>
<td align="center" rowspan="1" colspan="1">ACCACAGTCCATGCCATCAC</td>
<td align="center" rowspan="2" colspan="1">452</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Antisense</td>
<td align="center" rowspan="1" colspan="1">TCCACCACCCTGTTGCTGTA</td>
</tr>
</tbody>
</table>
</table-wrap>
</table-wrap-group>
<table-wrap id="tab2" orientation="portrait" position="float"><label>Table 2</label>
<caption><p>The samples for ELISA analysis were cell-free culture supernatants and protein lysate. Both the cell-free culture supernatant samples and the protein lysate samples of hBMCs and hGFs were obtained from three different donors and analyzed twice. The cell-free culture supernatant samples of hMO were obtained from one donor. The LPS without samples were analyzed twice and the LPS with samples were analyzed four times. The protein lysate samples were not prepared from hMO.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">The samples for ELISA</th>
<th align="center" colspan="2" rowspan="1">Human Macrophages</th>
<th align="center" colspan="2" rowspan="1">HBMCs</th>
<th align="center" colspan="2" rowspan="1">HGFs</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="3" colspan="1">Cell-free culture supernatant</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 1</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 1</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">Analyzed twice</td>
<td align="center" rowspan="1" colspan="1">Analyzed four times</td>
<td align="center" rowspan="1" colspan="1">Analyzed twice</td>
<td align="center" rowspan="1" colspan="1">Analyzed twice</td>
<td align="center" rowspan="1" colspan="1">Analyzed twice</td>
<td align="center" rowspan="1" colspan="1">Analyzed twice</td>
</tr>
<tr><td align="left" colspan="7" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="3" colspan="1">Protein lysate</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
<td align="center" rowspan="1" colspan="1">LPS −</td>
<td align="center" rowspan="1" colspan="1">LPS +</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">×</td>
<td align="center" rowspan="1" colspan="1">×</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
<td align="center" rowspan="1" colspan="1"><italic>n</italic>
= 3</td>
</tr>
<tr><td align="center" rowspan="1" colspan="1">×</td>
<td align="center" rowspan="1" colspan="1">×</td>
<td align="center" rowspan="1" colspan="1">Analyzed once</td>
<td align="center" rowspan="1" colspan="1">Analyzed once</td>
<td align="center" rowspan="1" colspan="1">Analyzed once</td>
<td align="center" rowspan="1" colspan="1">Analyzed once</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap-group id="tab3" orientation="portrait" position="float"><label>Table 3</label>
<caption><p>ELISA results for hMO (a), hBMCs (b) and hGFs (c). For hMO, protein levels were assessed from the cell-free culture supernatants. hBMC and hGF protein levels were quantified from the cell lysate. </p>
</caption>
<table-wrap id="tab3a" orientation="portrait" position="anchor"><caption><p>(a) Secretion of IL-1<italic>α</italic>
and IL-1<italic>β</italic>
by human macrophages after LPS stimulation</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><break></break>
</th>
<th align="center" colspan="2" rowspan="1">IL-1<italic>α</italic>
</th>
<th align="center" colspan="2" rowspan="1">IL-1<italic>β</italic>
</th>
</tr>
<tr><th align="left" rowspan="1" colspan="1">Stimulation period</th>
<th align="center" rowspan="1" colspan="1">LPS 0</th>
<th align="center" rowspan="1" colspan="1">LPS 1 <italic>μ</italic>
g/mL</th>
<th align="center" rowspan="1" colspan="1">LPS 0</th>
<th align="center" rowspan="1" colspan="1">LPS 1 <italic>μ</italic>
g/mL</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">24 hrs</td>
<td align="center" rowspan="1" colspan="1">0 pg/mL</td>
<td align="center" rowspan="1" colspan="1">470.80 ± 79.69 pg/mL</td>
<td align="center" rowspan="1" colspan="1">32 pg/mL</td>
<td align="center" rowspan="1" colspan="1">400 pg/mL</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab3b" orientation="portrait" position="anchor"><caption><p>(b) Secretion of IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, and TNF-by human bone marrow cells after LPS stimulation</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><break></break>
</th>
<th align="center" colspan="3" rowspan="1">IL-1<italic>α</italic>
(pg/mL)</th>
<th align="center" colspan="3" rowspan="1">IL-1<italic>β</italic>
(pg/mL)</th>
<th align="center" colspan="3" rowspan="1">TNF-<italic>α</italic>
(pg/mL)</th>
</tr>
<tr><th align="left" rowspan="1" colspan="1"> </th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
</tr>
<tr><th align="left" rowspan="1" colspan="1">Stimulation period</th>
<th align="center" rowspan="1" colspan="1"> 0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
<th align="center" rowspan="1" colspan="1">0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
<th align="center" rowspan="1" colspan="1">0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">24 hrs</td>
<td align="center" rowspan="1" colspan="1"> 0</td>
<td align="center" rowspan="1" colspan="1">5.49 ± 1.21</td>
<td align="center" rowspan="1" colspan="1">5.11 ± 2.03</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">9.17 ± 4.57</td>
<td align="center" rowspan="1" colspan="1">16.15 ± 1.78</td>
<td align="center" rowspan="1" colspan="1">19.32 ± 10.03</td>
<td align="center" rowspan="1" colspan="1">52.95 ± 5.26</td>
<td align="center" rowspan="1" colspan="1">54.55 ± 3.25</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">48 hrs</td>
<td align="center" rowspan="1" colspan="1"> 0</td>
<td align="center" rowspan="1" colspan="1">0.34 ± 0.46</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">15.59 ± 1.05</td>
<td align="center" rowspan="1" colspan="1">22.27 ± 2.12</td>
<td align="center" rowspan="1" colspan="1">51.48 ± 2.33</td>
<td align="center" rowspan="1" colspan="1">40.00 ± 4.82</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">96 hrs</td>
<td align="center" rowspan="1" colspan="1"> 0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">14.89 ± 2.44</td>
<td align="center" rowspan="1" colspan="1">42.39 ± 8.85</td>
<td align="center" rowspan="1" colspan="1">40.11 ± 11.06</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab3c" orientation="portrait" position="anchor"><caption><p>(c) Secretion of IL-1<italic>α</italic>
, IL-1<italic>β</italic>
, and TNF-by human gingival fibroblasts after LPS stimulation</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><break></break>
</th>
<th align="center" colspan="3" rowspan="1">IL-1<italic>α</italic>
(pg/mL)</th>
<th align="center" colspan="3" rowspan="1">IL-1<italic>β</italic>
(pg/mL)</th>
<th align="center" colspan="3" rowspan="1">TNF-<italic>α</italic>
(pg/mL)</th>
</tr>
<tr><th align="left" rowspan="1" colspan="1"> </th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
<th align="center" colspan="3" rowspan="1">LPS (<italic>μ</italic>
g/mL)</th>
</tr>
<tr><th align="left" rowspan="1" colspan="1">Stimulation period</th>
<th align="center" rowspan="1" colspan="1">0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
<th align="center" rowspan="1" colspan="1">0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
<th align="center" rowspan="1" colspan="1">0</th>
<th align="center" rowspan="1" colspan="1">0.1</th>
<th align="center" rowspan="1" colspan="1">1</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">24 hrs</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">23.30 ± 3.72</td>
<td align="center" rowspan="1" colspan="1">28.30 ± 1.14</td>
<td align="center" rowspan="1" colspan="1">26.93 ± 2.18</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">48 hrs</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">22.27 ± 2.05</td>
<td align="center" rowspan="1" colspan="1">17.84 ± 8.17</td>
<td align="center" rowspan="1" colspan="1">21.36 ± 3.20</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">96 hrs</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">0</td>
<td align="center" rowspan="1" colspan="1">20.00 ± 4.16</td>
<td align="center" rowspan="1" colspan="1">19.31 ± 6.89</td>
<td align="center" rowspan="1" colspan="1">22.84 ± 1.76</td>
</tr>
</tbody>
</table>
</table-wrap>
</table-wrap-group>
</floats-group>
</pmc>
<affiliations><list></list>
<tree><noCountry><name sortKey="Davies, John E" sort="Davies, John E" uniqKey="Davies J" first="John E." last="Davies">John E. Davies</name>
<name sortKey="Todescan, Reynaldo" sort="Todescan, Reynaldo" uniqKey="Todescan R" first="Reynaldo" last="Todescan">Reynaldo Todescan</name>
<name sortKey="Ujiie, Yuko" sort="Ujiie, Yuko" uniqKey="Ujiie Y" first="Yuko" last="Ujiie">Yuko Ujiie</name>
</noCountry>
</tree>
</affiliations>
</record>
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