Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Dette arbejde blev støttet i del af Japan-samfund til fremme af videnskab (JSP'ER) licensbetaling for videnskabelig forskning (26861689 og 17 K 11813). Forfatterne vil gerne takke Mr. Nathaniel Green for korrekturlæsning.

Denne protokol er designet til at skabe menneskelige gingival væv ækvivalenter, gingival sår modeller og tandkødsbetændelse modeller. Menneskelige hud epidermale keratinocytter (HaCaT) blev venligt leveret fra Professor Norbert E. Fusenig af Deutsches Krebsforschungszentrum (Heidelberg, Tyskland)7. Menneskelige gingival fibroblaster (HGFs) blev isoleret fra gingival væv ifølge tidligere publicerede protokoller8. Informeret samtykke blev opnået på forhånd, og under...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+fibroblasts+during+inflammation+and+tissue+repair+in+models+of+organ+regeneration.">Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration.</a> Regeneration. 4 (2), 39-53 (2017).</li><li>Struillou, X., Boutigny, H., Soueidan, A., Layrolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Animal+Models+in+Periodontology:+A+Review.">Experimental Animal Models in Periodontology: A Review.</a> The Open Dentistry Journal. 4, 37-47 (2010).</li><li>Han, Y. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gingival+fibroblasts+are+critical+in+sustaining+inflammation+in+periodontal+disease.">Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.</a> J Periodontal Res. 44 (1), 21-27 (2009).</li><li>Park, E. K., Jung, H. S., Yang, H. I., Yoo, M. C., Kim, C., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+THP-1+differentiation+is+required+for+the+detection+of+responses+to+weak+stimuli.">Optimized THP-1 differentiation is required for the detection of responses to weak stimuli.</a> Inflamm Res. 56 (1), 45-50 (2007).</li><li>Sharif, O., Bolshakov, V. N., Raines, S., Newham, P., Perkins, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+LPS+induced+NF-kappaB+response+in+macrophages.">Transcriptional profiling of the LPS induced NF-kappaB response in macrophages.</a> BMC Immunol. 8, 1 (2007).</li><li>Shetty, S., Gokul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinization+and+its+disorders.">Keratinization and its disorders.</a> Oman Med J. 27 (5), 348-357 (2012).</li><li>Klinge, B., Matsson, L., Attstr&#246;m, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+initial+gingivitis+in+humans.+A+pilot+study.">Histopathology of initial gingivitis in humans. A pilot study.</a> J Clin Periodontol. 10 (4), 364-369 (1983).</li><li>Nagarakanti, S., Ramya, S., Babu, P., Arun, K. V., Sudarsan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+E-cadherin+and+cytokeratin+19+and+net+proliferative+rate+of+gingival+keratinocytes+in+oral+epithelium+in+periodontal+health+and+disease.">Differential expression of E-cadherin and cytokeratin 19 and net proliferative rate of gingival keratinocytes in oral epithelium in periodontal health and disease.</a> J Periodontol. 78 (11), 2197-2202 (2007).</li><li>Goodpaster, T., Legesse-Miller, A., Hameed, M. R., Aisner, S. C., Randolph-Habecker, J., Coller, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunohistochemical+method+for+identifying+fibroblasts+in+formalin-fixed,+paraffin-embedded+tissue.">An immunohistochemical method for identifying fibroblasts in formalin-fixed, paraffin-embedded tissue.</a> J Histochem Cytochem. 56 (4), 347-358 (2008).</li><li>Langeland, K., Rodrigues, H., Dowden, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+disease,+bacteria,+and+pulpal+histopathology.">Periodontal disease, bacteria, and pulpal histopathology.</a> Oral Surg Oral Med Oral Pathol. 37 (2), 257-270 (1974).</li><li>Holness, C. L., Simmons, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin+is+secreted+by+activated+macrophages.">Vimentin is secreted by activated macrophages.</a> Nat Cell Biol. 5 (1), 59-63 (2003).</li><li>Xiao, L., Aoshima, H., Saitoh, Y., Miwa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+squalane-dissolved+on+adipogenesis-accompanied+oxidative+stress+and+macrophage+in+a+preadipocyte-monocyte+co-culture+system.">The effect of squalane-dissolved on adipogenesis-accompanied oxidative stress and macrophage in a preadipocyte-monocyte co-culture system.</a> Biomaterials. 31 (23), 5976-5985 (2010).</li><li>Xiao, L., Aoshima, H., Saitoh, Y., Miwa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+hydroxylated+fullerene+localizes+at+the+cytoskeleton+and+inhibits+oxidative+stress+in+adipocytes+and+a+subcutaneous+adipose-tissue+equivalent.">Highly hydroxylated fullerene localizes at the cytoskeleton and inhibits oxidative stress in adipocytes and a subcutaneous adipose-tissue equivalent.</a> Free Radic Biol Med. 51 (7), 1376-1389 (2011).</li></ol>

Denne protokol er baseret på metoder til at skabe gingival væv ækvivalenter og subkutane fedtvæv ækvivalenter beskrevet af tidligere rapporter8,21,22. Selv om dette er en enkel og nem metode, kræver nogle trin særlig opmærksomhed. For eksempel, bør kollagen blandingen holdes på is indtil brugen for at undgå gel dannelse i løsningen. Når du tilføjer kollagen blandingen ind i kultur-Indsæt, sikre løsningen blev inj...

Parodontale sygdomme er den hyppigste årsag til tandtab hos voksne. Tandkødsbetændelse og paradentose er de mest almindelige parodontale sygdomme. Begge præsentere biofilm-medieret akut eller kronisk inflammatoriske forandringer i gingiva. Tandkødsbetændelse er karakteriseret ved akut betændelse, hvorimod parodontitis normalt præsenterer som kronisk betændelse. På niveauet histologiske udløser bakteriel komponenter aktivering af immunceller, såsom makrofager, lymfocytter, plasma celler og mastceller1,2. Disse immunceller, især makrofager, interagere med lokale celler (herunder gingival epitel celler, fibroblaster, endotelceller og osteoblaster) resulterer i inflammatoriske læsioner i parodontale væv3,4. Eksperimentelle modeller af parodontale sygdomme har været etableret i forskellige typer af dyr, som rotter, hamstere, kaniner, fritter, hjørnetænder og primater. Patofysiologi af dyremodeller er dog forskellige fra mennesker, hvilket gør det vanskeligt at analysere cellulære og molekylære mekanismer og vurdere nye lægemidler parodontale sygdomme5. Co dyrkning af paradentose bakterier og éncellelag menneskelige mundtlige epitelceller er blevet brugt til at efterforske mekanismen af parodontale infektioner6. Ikke desto mindre mangler éncellelag kulturer af mundtlige celler den tredimensionale (3D) cellulære arkitektur af intakt væv; Derfor kan de efterligner in vitro- situation.
Her, er 3D inflammatoriske humant væv ækvivalenter af gingiva (iGTE) etableret til at repræsentere parodontale sygdomme i vitro. Denne 3D model af parodontale sygdomme indtager en mellemposition mellem éncellelag cellekulturer og dyremodeller. Tre typer af humane celler, herunder HaCaT keratinocytter, gingival fibroblaster og THP-1 makrofager, er co dyrket på kollagen gel og stimuleret af inflammatoriske initiatorer til at bygge iGTE. IGTE simulerer nøje i vivo -betingelserne for Celledifferentiering, celle-celle interaktion og makrofag aktivering i gingiva. Denne model har mange mulige anvendelser for stof screening og afprøvning af nye farmakologiske tilgange i parodontale sygdomme og analysere cellulære og molekylære mekanismer i sårheling, inflammation og væv revitalisering.

<table>
	<tbody>
		<tr>
			<td>Collagen type I-A</td>
			<td>Nitta Gelatin Inc</td>
			<td></td>
			<td>For making dermis of GTE</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>Thermo Fisher Scientific</td>
			<td>11900073</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Cell Culture Insert (for 24-well plate), pore size 3.0 &#956;m</td>
			<td>Corning, Inc.</td>
			<td>353096</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31600034</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Tissue puncher</td>
			<td>Shibata system service co., LTD</td>
			<td>SP-703</td>
			<td>For punching holes in GTE</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>31800022</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Hoechst 33342 (NucBlue Live Cell stain)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td>For labeling nuclei</td>
		</tr>
		<tr>
			<td>Fluorescence mount medium</td>
			<td>Dako</td>
			<td></td>
			<td>For mounting samples after immunostaining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 8+18 antibody [5D3]</td>
			<td>abcam</td>
			<td>ab17139</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Scaning electron microscope</td>
			<td>Hitachi, Ltd.</td>
			<td>HITACHI S-4000</td>
			<td>For observing samples&#39; surface topography and composition</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscopy</td>
			<td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td>
			<td>LSM 700</td>
			<td>For observing samples&#39; immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 19 antibody</td>
			<td>abcam</td>
			<td>ab52625</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Anti-vimentin antibody</td>
			<td>abcam</td>
			<td>ab92547</td>
			<td>For identifying fibroblasts and activated macrophages</td>
		</tr>
		<tr>
			<td>Anti-TE-7 antibody</td>
			<td>Millipore</td>
			<td>CBL271</td>
			<td>For identifying fibroblasts in the dermis</td>
		</tr>
		<tr>
			<td>Anti-CD68 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB2700244</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Human CD14 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB3832-SP</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11012</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>For observing the sample&#39;s immunofluorescence staining</td>
		</tr>
		<tr>
			<td>THP-1 cells</td>
			<td>Riken BRC cell bank</td>
			<td>RCB1189</td>
			<td>For making iGTE</td>
		</tr>
		<tr>
			<td>PMA(Phorbol 12-myristate 13-acetate)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For differentiatting THP-1 cells</td>
		</tr>
	</tbody>
</table>

three-dimensional inflammatory human tissue equivalents of gingiva

Parodontale sygdomme (såsom gingivitis og parodontitis) er de førende årsager til tandtab hos voksne. Betændelse i gingiva er den grundlæggende Patofysiologi af parodontale sygdomme. Nuværende eksperimentelle modeller af parodontale sygdomme har været etableret i forskellige typer af dyr. Patofysiologi af dyremodeller er dog forskellige fra mennesker, hvilket gør det vanskeligt at analysere cellulære og molekylære mekanismer og vurdere nye lægemidler til parodontale sygdomme. Vi præsenterer her, en detaljeret protokol til rekonstruere menneskelige inflammatorisk væv ækvivalenter af gingiva (iGTE) i vitro. Vi bygger først humant væv ækvivalenter af gingiva (GTE) ved at bruge to typer af humane celler, herunder menneskelige gingival fibroblaster (HGF) og menneskelige hud epidermale keratinocytter (HaCaT), tre-dimensionelle betingelser. Vi skaber en såret model ved hjælp af en væv puncher til punch et hul i GTE. Næste, menneskelige THP-1 monocytter blandet med kollagen gel er sprøjtet ind i hullet i GTE. Ved adimistration af 10 ng/mL phorbol 12-myristate 13-acetat (PMA) for 72 h, THP-1 celler differentieret til makrofager at form inflammatoriske foci i GTE (iGTE) (IGTE også kan være stumilated med 2 µg/mL af lipopolysaccharides (LPS) for 48 h at indlede betændelse ). IGTE er den første i vitro model af inflammatoriske gingiva ved hjælp af menneskelige celler med en tre-dimensionel arkitektur. IGTE afspejler store patologiske forandringer (immunocytes aktiviteter, intracellulære interaktioner blandt fibryoblasts, epitelceller, monocytter og makrofager) i parodontale sygdomme. GTE, sårede GTE og iGTE kan bruges som alsidige værktøjer til at studere sårheling, vævsregeneration, betændelse, celle-celle interaktion og skærmen potentielle lægemidler til parodontale sygdomme.

HaCaT celler vises typisk keratinocytter morfologi under fasekontrast mikroskopiske observation (figur 2A). Scanner elektron mikroskopisk (SEM) billeder viste, at HaCaT celle overflader var dækket af mange microvilli. Intercellulære forbindelser mellem HaCaT celler blev formidlet af membran processer (figur 2B). HaCaT celler udtrykt gingival epitel markør K8/1814, hvilket indikerer at HaCaT celler er eg...

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Målet med protokollen er at opbygge en inflammatorisk menneskelige gingiva model in vitro. Dette væv model dyrker Co tre typer af humane celler, HaCaT keratinocytter, gingival fibroblaster og THP-1 makrofager, tre-dimensionelle betingelser. Denne model kan anvendes til at undersøge parodontale sygdomme, såsom gingivitis og parodontitis.

Tre-dimensionelle inflammatoriske humant væv ækvivalenter af Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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