Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Questo lavoro è stato supportato in parte dalla Japan Society per la promozione della scienza (JSPS) sovvenzione per la ricerca scientifica (26861689 e 17 K 11813). Gli autori vorrei ringraziare Mr. Nathaniel Green per la correzione di bozze.

Questo protocollo è progettato per creare equivalenti di tessuto gengivale umano, ferita gengivale modelli e modelli di gengivite. Pelle umana cheratinociti (HaCaT) sono state gentilmente concesse da professore Norbert E. Fusenig di Deutsches Krebsforschungszentrum (Heidelberg, Germania)7. Fibroblasti gengivali umani (HGFs) sono stati isolati dai tessuti gengivali secondo i protocolli precedentemente pubblicati8. Il consenso informato è stato ottenuto in anticipo, e lo studio è stato approvato secondo le linee guida fissano dal comitato etico, la Nippon Dental University School of Life Dentistry presso Tokyo (numero di autorizzazione: NDU-T2012-35). Procedura di protocollo 1 – 3 deve essere eseguita in una cappa di cultura cellulare.
1. preparazione del tessuto umano 3D equivalenti di gengiva (GTE) (Figura 1A)
<ol><li>Mix del collagene tipo I-A gel con 10% concentrato MEM-alfa e buffer di ricostruzione di 10% (2,2 g di NaHCO3 e 4,47 HEPES in 100 mL di 0.05 N NaOH) in una provetta sterile da 15 mL di pipettaggio. Mantenere la soluzione mista collagene su ghiaccio fino a quando non utilizzato.</li>
	<li>Inserire inserti cultura 24 pozzetti (poro dimensione 3.0 µm) in una piastra a 24 pozzetti.</li>
	<li>
Rimuovere HGFs dalla superficie di cultura utilizzando 0,25% soluzione di tripsina-EDTA. Sospendere le cellule in 10 mL di medium di coltura A (MEM-alfa + 20% FBS + 1% GlutaMAX). Contare le celle di un contatore di cellule Bürker-Türk.
	<ol>
<li>Estrarre la quantità desiderata di celle (1-5 x105 cellule/mL), quindi centrifugare per 4 min a 190 x g.</li>
		<li>Sospendere le cellule nella soluzione mista del collagene. Mantenere la miscela su ghiaccio fino al passaggio successivo.</li>
	</ol>
</li>
	<li>Aggiungere 0,5 mL della miscela delle cellule-collagene (0,5-2,5 x 105 cellule/pozzetto) nell'inserto cultura senza produrre eventuali bolle. Incubare la miscela per 10-30 min in atmosfera umidificata con 5% CO2 a 37 ° C per far coagulare la miscela in un gel. Nota: Aggiungere il composto di collagene al centro dell'inserto cultura per evitare disparità formazione di gel di collagene.</li>
	<li>Aggiungere 1 mL di terreno di coltura nel pozzo e 0,5 mL nell'inserto. Incubare la piastra di coltura per 24 ore o durante la notte in atmosfera umidificata con 5% CO2 a 37 ° C.</li>
	<li>Rimuovere le cellule HaCaT dalla superficie di cultura utilizzando 0,25% soluzione di tripsina-EDTA. Sospendere le cellule in 10 mL di medium B (DMEM 10% FBS + 1% GlutaMAX) e contare le celle. Estratto la quantità necessaria di celle (1 – 5 x 105 cellule/mL) con una pipetta, centrifugare per 4 min x 190 g. sospendere le cellule in media B.</li>
	<li>Rimuovere il supporto dagli inserti di cultura, che contengono la miscela di HGF-collagene, dall'aspirazione. Aggiungere 0,5 mL della sospensione di cellule HaCaT (0,5-2,5 x 105 cellule/pozzetto) nella parte superiore la miscela di HGF-collagene. Incubare le colture per 24 ore o durante la notte. Nota: A questo punto del tempo, il mezzo nella cultura ben dovrebbe essere A medio, mentre il mezzo nell'inserto cultura dovrebbe essere medio B.</li>
	<li>Sostituire il terreno di coltura con il mezzo di KSR (co-coltura) (MEM-alfa + 15% sostituzione di KnockOut del siero + 1% GlutaMAX) + 5% FBS. Aggiungere 1 mL di terreno di coltura nella cultura ben e 0,5 mL nell'inserto cultura. Incubare le colture per 24 – 48 h.</li>
	<li>Sostituire il terreno di coltura con KSR medium + 1% FBS. Aggiungere 1 mL nella cultura ben e 0,5 mL nell'inserto cultura. Incubare le colture per 24 ore o durante la notte.</li>
	<li>Sostituire il terreno di coltura nella cultura bene con 0,7 – 1 mL di terreno di KSR. Rimuovere completamente il mezzo dall'inserto di cultura (la superficie di cultura dovrebbe essere esposto all'aria). Incubare le colture per 1 – 2 settimane. Modificare il mezzo nella cultura ben due volte alla settimana. Nota: Non lasciare che l'innalzamento del livello medio sopra la superficie di cultura (lo strato superiore è composto da cheratinociti). Tenere sempre asciutta la superficie di cultura.</li>
</ol>2. preparazione del GTE feriti (Figura 1B)
<ol><li>Preparare un perforatore di tessuto del diametro 1 – 3 mm. Sterilizzare con autoclave a 121 ° C per 20 min.</li>
	<li>Spingere il puncher tessuto perpendicolarmente in GTE circa 300 µm profonda e fare un buco nel tessuto per simulare una ferita.</li>
	<li>A questo punto, è possibile utilizzare il GTE feriti per indagare la rigenerazione di tessuto e di guarigione della ferita. In alternativa, Difficoltà GTE utilizzando soluzione tampone neutro di formalina al 10% per eseguire l'analisi istologica.</li>
</ol>3. preparazione del GTE infiammatorie (iGTE) (Figura 1B)
<ol><li>Coltivare le cellule THP-1 secondo la precedente relazione9.</li>
	<li>Mix del collagene tipo I-A gel con 10% concentrato RPMI 1640, buffer di ricostruzione del 10% (2,2 g di NaHCO3 e 4,47 HEPES in 100 mL di 0.05 N NaOH) e 10 ng/mL PMA. Mantenere la soluzione mista collagene su ghiaccio fino all'utilizzo.</li>
	<li>Conteggio 1 – 2 x 106 THP-1 cellule e centrifugare per 4 min x 190 g. sospendere le cellule in 1 mL della soluzione mista di collagene. Mantenere la miscela su ghiaccio fino al passaggio successivo.</li>
	<li>Aggiungere 10 – 30 µ l THP-1-collagene miscela nell'area ferito del GTE. Sostituire il terreno di coltura a 0,7 – 1 mL di terreno di KSR contenente 10 ng/mL di PMA.</li>
	<li>Incubare la miscela in atmosfera umidificata con 5% CO2 a 37 ° C per 72 h.</li>
	<li>Utilizzare il modello per lo studio di gengivite o parodontite (ad esempio, aggiungere potenziale farmaco antinfiammatorio a iGTE per vedere i suoi effetti sul rilascio di citochina10). In alternativa, omettere 72h PMA-trattamento, aggiungere 2 µ g/mL di LPS nella cultura e incubare il tessuto in atmosfera umidificata con 5% CO2 a 37 ° C per 48 h11,12.</li>
</ol>4. fissazione e Immunostaining di montare tutta del GTE e iGTE culture
<ol><li>
Fissazione delle culture.
	<ol>
<li>Rimuovere il terreno di coltura dalla piastra a 24 pozzetti. Lavare i tessuti 2 volte con tamponato fosfato salino (PBS).</li>
		<li>Aggiungere 1 mL di soluzione di tampone neutro di formalina al 10% per l'inserto e 1 mL alla cultura bene. Lasciare la piastra a 24 pozzetti in frigorifero a 4 ° C durante la notte.</li>
		<li>Rimuovere la soluzione di tampone neutro di formalina 10% il giorno seguente.</li>
		<li>Per l'intero Monte immunocolorazione, lavare i tessuti 3 - 4 volte con PBS dopo la fissazione.</li>
		<li>Per ematossilina ed eosina (H & E) e immunostaining regolari, procedere con disidratazione, inclusione in paraffina e sezionamento. Nota: Per l'intero Monte immunocolorazione, questo passaggio può essere omesso.</li>
	</ol>
</li>
	<li>
Tutta Monte immunostaining Nota: Questo protocollo è stato modificato da quella di riferimento 13.<ol>
<li>Lavare i tessuti 3 x in PBS 0.5 – 1% Triton, 10 – 30 min ogni volta.</li>
		<li>Incubare i tessuti due volte per 30 min in tampone bloccante (PBS 1% Triton + 10% BSA), a temperatura ambiente.</li>
		<li>Lavare i tessuti 2 × in tampone bloccante, 5 – 10 minuti ogni volta.</li>
		<li>Aggiungere 50 µ l di soluzione di anticorpo primario alla diluizione/concentrazione necessaria agli inserti. Utilizzare una sottile pellicola di plastica per avvolgere gli inserti per evitare disidratazione del tessuto.</li>
		<li>Incubare il tessuto per 1 o 2 giorni a 4 ° C.</li>
		<li>Lavare i tessuti 3 volte, per 30 min in PBS 1% Triton + 10% BSA. Lavare nuovamente 3 volte, per 5 min in PBS 1% Triton.</li>
		<li>Aggiungere 50 µ l di anticorpo secondario in tampone bloccante (PBS 1% Triton + 10% BSA) e 5 µ l di soluzione di Hoechst 33342 (macchia di NucBlue Live Cell) per ogni inserimento. Utilizzare una sottile pellicola di plastica per avvolgere gli inserti per evitare disidratazione del tessuto.</li>
		<li>Incubare per 1-2 giorni a 4 ° C. Avvolgere la piastra a 24 pozzetti in un tovagliolo di carta bagnato e poi metterlo in un pacchetto di plastica da utilizzare durante l'incubazione.</li>
		<li>Lavare i tessuti 3 volte, per 10 min in triton 1% PBS</li>
		<li>Usare un ago affilato per tagliare la membrana dell'inserto cultura per ottenere il tessuto. Posizionare la membrana in una diapositiva. Nota: Il tessuto dovrebbe essere sulla membrana.</li>
		<li>Aggiungere 2 – 3 gocce di mezzo di montaggio di fluorescenza. Coprire il tessuto con una copertura in vetro e conservare a 4 ° C al buio fino all'analisi.</li>
		<li>Mostra tessuti con un laser confocale microscopia (LSM).</li>
	</ol>
</li>
</ol>

<ol>
	<li>Cekici, A., Kantarci, A., Hasturk, H., Van Dyke, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+immune+pathways+in+the+pathogenesis+of+periodontal+disease.">Inflammatory and immune pathways in the pathogenesis of periodontal disease.</a> Periodontology 2000. 64 (1), 57-80 (2014).</li><li>Hasturk, H., Kantarci, A., Van Dyke, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Inflammatory+Diseases+and+Systemic+Inflammation:+Role+of+the+Macrophage.">Oral Inflammatory Diseases and Systemic Inflammation: Role of the Macrophage.</a> Frontiers in Immunology. 3, 118 (2012).</li><li>Koh, T. J., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+wound+healing:+The+role+of+the+macrophage.">Inflammation and wound healing: The role of the macrophage.</a> Expert reviews in molecular medicine. 13, e23 (2011).</li><li>Mescher, A. 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. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Periodontal+Bacteria+and+Human+Oral+Epithelial+Cells:+Fusobacterium+nucleatum+Adheres+to+and+Invades+Epithelial+Cells.">Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells.</a> Infection and Immunity. 68 (6), 3140-3146 (2000).</li><li>Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Xiao, L., 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=Hydrogen-rich+water+achieves+cytoprotection+from+oxidative+stress+injury+in+human+gingival+fibroblasts+in+culture+or+3D-tissue+equivalents,+and+wound-healing+promotion,+together+with+ROS-scavenging+and+relief+from+glutathione+diminishment.">Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging and relief from glutathione diminishment.</a> Hum Cell. 30 (2), 72-87 (2017).</li><li>Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>Ara, T., Kurata, K., Hirai, K., Uchihashi, T., Uematsu, T., Imamura, Y., Furusawa, K., Kurihara, S., Wang, P. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+CD68,+a+human+macrophage+marker+related+to+lysosomal+glycoproteins.">Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.</a> Blood. 81 (6), 1607-1613 (1993).</li><li>Mor-Vaknin, N., Punturieri, A., Sitwala, K., Markovitz, D. 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>

Gli autori non dichiarano alcun conflitto di interessi.

Questo protocollo è basato su metodi di creazione di tessuto gengivale equivalenti ed equivalenti di tessuto adiposo sottocutanei descritti da precedenti rapporti8,21,22. Anche se si tratta di un metodo semplice e facile, alcuni passaggi richiedono particolare attenzione. Ad esempio, la miscela di collagene dovrebbe essere tenuta su ghiaccio fino all'utilizzo per evitare la formazione di un gel nella soluzione. Quando si aggiun...

Le malattie parodontali sono la principale causa di perdita dei denti negli adulti. Gengivite e parodontite sono le più comuni malattie parodontali. Entrambi presenti alterazioni infiammatorie acute o croniche biofilm-mediata nella gengiva. Gengivite è caratterizzata da infiammazione acuta, considerando che la parodontite si presenta solitamente come l'infiammazione cronica. A livello istologico, componenti batteriche innescano l'attivazione di cellule immunitarie, come i macrofagi, linfociti, plasmacellule e mastociti1,2. Queste cellule immuni, soprattutto macrofagi, interagiscono con le cellule locali (tra cui cellule epiteliali gingival, fibroblasti, cellule endoteliali e osteoblasti) con conseguente lesioni infiammatorie nel tessuto peridentale3,4. Modelli sperimentali di malattie parodontali sono state stabilite in vari tipi di animali, come ratti, criceti, conigli, furetti, canini e primati. Tuttavia, la fisiopatologia dei modelli animali è diversa da quella degli esseri umani, rendendo difficile per analizzare i meccanismi cellulari e molecolari e valutare nuovi farmaci di malattie parodontali5. Co-coltura di batteri parodontali e cellule epiteliali orali umane monostrato è stato utilizzato per studiare il meccanismo di infezioni parodontali6. Tuttavia, colture monostrato di cellule orali mancano l'architettura cellulare di tridimensionale (3D) di tessuto intatto; Pertanto, essi non può simulare la situazione in vitro .
Qui, gli equivalenti 3D infiammatoria del tessuto umano della gengiva (iGTE) sono istituiti per rappresentare malattie parodontali in vitro. Questo modello 3D delle malattie parodontali occupa una posizione intermedia tra colture delle cellule dello strato monomolecolare e modelli animali. Tre tipi di cellule umane, incluse HaCaT cheratinociti e fibroblasti gengivali macrofagi THP-1, sono co-coltivati su gel di collagene e stimolati dagli iniziatori infiammatori di costruire iGTE. IGTE simula molto attentamente le condizioni in vivo del differenziamento cellulare, l'interazione cellula-cellula e l'attivazione del macrofago nel gingiva. Questo modello ha molte possibili applicazioni per droga di screening e test di nuovi approcci farmacologici in malattie parodontali, nonché per l'analisi dei meccanismi cellulari e molecolari nella guarigione della ferita, infiammazione e rigenerazione dei tessuti.

<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 &amp;mu;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&#039; 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&#039; 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&amp;amp;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&#039;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

Le malattie parodontali (come gengivite e parodontite) sono le principali cause di perdita dei denti negli adulti. L'infiammazione nella gengiva è fondamentale fisiopatologia delle malattie parodontali. Attuali modelli sperimentali di malattie parodontali sono stati stabiliti in vari tipi di animali. Tuttavia, la fisiopatologia dei modelli animali è diversa da quella degli esseri umani, rendendo difficile per analizzare i meccanismi cellulari e molecolari e valutare nuovi farmaci per malattie parodontali. Qui, presentiamo un protocollo dettagliato per la ricostruzione di equivalenti di tessuto infiammatorio umano della gengiva (iGTE) in vitro. Costruiamo prima equivalenti di tessuto umano della gengiva (GTE) utilizzando due tipi di cellule umane, tra cui fibroblasti gengivali umani (HGF) e cheratinociti epidermici della pelle umana (HaCaT), in condizioni tridimensionali. Creiamo un modello di ferita utilizzando un perforatore di tessuto per fare un buco nel GTE. Prossimi, umani monociti THP-1 mescolati con gel di collagene sono iniettati nel foro il GTE. Da adimistration di 10 ng/mL phorbol 12-myristate 13-acetate (PMA) per 72 h, cellule THP-1 differenziano nei macrofagi di focolai infiammatori forma in GTE (iGTE) (IGTE inoltre può essere stumilated con 2 µ g/mL di lipopolisaccaridi (LPS) per 48 h avviare l'infiammazione ). IGTE è il primo modello in vitro di gengiva infiammatoria utilizzando cellule umane con un'architettura tridimensionale. IGTE riflette le modifiche patologiche principali (activition immunociti, interazioni intracellulari tra fibryoblasts, cellule epiteliali, monociti e macrofagi) a malattie parodontali. GTE, GTE feriti e iGTE utilizzabile come versatile di strumenti per studiare la guarigione della ferita, rigenerazione tissutale, infiammazione, interazione cellula-cellula e potenziali farmaci schermo per malattie parodontali.

Le cellule HaCaT visualizzato morfologia tipica del keratinocyte sotto osservazione al microscopio di contrasto di fase (Figura 2A). Immagini di scanner elettrone microscopiche (SEM) ha mostrato che le superfici delle cellule HaCaT erano coperti da molti microvilli. Intercellulare connessioni tra le cellule HaCaT sono state mediate da processi a membrana (Figura 2B). Le cellule HaCaT espresso epitelio gingival marcatore K8/18<sup...

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

L'obiettivo del protocollo è quello di costruire un modello di infiammatorio gengiva umana in vitro. Questo modello di tessuto Co-coltiva tre tipi di cellule umane, keratinocytes di HaCaT, fibroblasti gengivali e macrofagi THP-1, in condizioni tridimensionali. Questo modello può essere applicato a indagare le malattie parodontali, quali gengivite e parodontite.

Equivalenti di tessuto umano infiammatorio tridimensionale del Gingiva

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

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Research

JoVE Journal

Developmental Biology

9.7K Views.  The Nippon Dental University. L'obiettivo del protocollo è quello di costruire un modello di infiammatorio gengiva umana in vitro. Questo modello di tessuto Co-coltiva tre tipi di cellule umane, keratinocytes di HaCaT, fibroblasti gengivali e macrofagi THP-1, in condizioni tridimensionali. Questo modello può essere applicato a indagare le malattie parodontali, quali gengivite e parodontite.

Video: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Ricerca

Bioingegneria

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolamento, elaborazione ed analisi dei murini gengivali

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Immunologia e infezioni

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produzione, caratterizzazione e possibilità di utilizzo di un 3D tissutale umano esofageo mucose Modello

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Un nuovo approccio al monitoraggio dell'innesto neovascolarizzazione nella gengiva umana

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Medicina

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Sistemi modello di tessuto organotipico per lo studio delle interazioni ospite-patogeno in vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Creazione di un modello murino di infiammazione della polpa utilizzando un nuovo bavaglio orale 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Autore in primo piano: Migliorare la ricerca sulla polpa dentale con modelli murini migliorati

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Equivalenti di tessuto umano infiammatorio tridimensionale del Gingiva

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Ingegneria dei tessuti di un Umano 3D