The authors would like to thank Prof. Steven Nutt and Mr. Yuzheng Zhang for assistant with the Focus Ion Beam, and the Center for Electron Microscopy and Microanalysis (CEMMA) at USC for electron microscopy. Research was supported by NIH-NIDCR grants; DE-13414 and DE-020099 to J.M.O.

Die menschliche Molaren wurden nach den Standardverfahren zur Extraktion bei der Ostrow School of Dentistry der University of Southern California extrahiert und mit Zustimmung des Institutional Review Board behandelt. 1. Vorbereitung der geätzten Zahn Scheibe <ol><li> Wählen Sie einen menschlichen dritten Molaren ohne restauriert Karies. </li><li> Entfernen der Wurzelabschnitt des molaren, und schneiden die Krone des molaren Längsrichtung in Scheiben von 2 mm Dicke unter Verwendung einer wassergekühlten Niedergeschwindigkeits-Diamantsäge. Diese Scheiben reinigen mit Ultraschall für 2 Minuten und dann spülen Sie mit VE-Wasser 3-mal. </li><li> Um Erosionen zu simulieren, ätzen die Zahnscheiben mit 30% Phosphorsäure für 30 Sekunden, dann sie sofort gründlich mit VE-Wasser. </li><li> Ultraschall reinigen die Scheiben geätzt für 2 Minuten und dann spülen Sie mit VE-Wasser 3-mal. </li></ol> 2. Vorbereitung der Amelogenin-Chitosan-Hydrogel <ol><li> Präparaten von Chitosan-Stammlösung <ol><li> Man löst 1% (w / v) Chitosan (mittleres Molekulargewicht 75-85% deacetyliertes) in einer 1% (v / v) Essigsäure-Lösung, gefolgt von Rühren bei 80 ° CO / N </li><li> Nach Abkühlen der Lösung auf Raumtemperatur filtriert man das Chitosan-Lösung unter Verwendung eines 0,45 &amp;mgr; m. Einstellen des pH-Wertes auf 5,0 durch Zugabe von 1 M NaOH-Lösung. </li></ol></li><li> Herstellung von Calcium-und Phosphat-Stammlösung (0,1 M) <ol><li> Abwiegen Calciumchlorid (CaCl 2) und bereiten eine 0,1 M Lösung, dann mischen mit einem Vortex, bis die Lösung klar ist. Einstellen des pH-Werts auf 11 durch Zugabe von 1 M NaOH-Lösung. </li><li> Abwiegen Natriummonohydrogenphosphat (Na 2 HPO 4) und bereiten eine 0,1 M Lösung, dann mischen mit einem Vortex, bis die Lösung klar ist. </li></ol></li><li> Expression und Reinigung von rekombinantem Amelogenin 17-18 Das rekombinante voller Länge Schweine amelogenin (rP172) welche hier hat 172 Aminosäuren und ist ein analoges VollLängen-Nativschweine P173, aber nicht über die N-terminale Methionin sowie eine Phosphatgruppe auf Ser16 18. <ol><li> 20 g Lysogenität Brühe (LB)-Agar-Pulver in 500 ml VE-Wasser, die Lösung im Autoklaven bei 121 ° C (flüssiger Einstellung) für 20 min. Lassen Agar cool zu ~ 55 ° C, fügen Ampicillin (100 ug / ml) in Agar-Lösung. </li><li> Übertragen Sie die Agar-Lösung auf Petrischalen, lassen jede Platte abkühlen, bis es fest ist, dann drehen, um Kondensation auf dem Agar zu vermeiden. Speicher Platten in Kunststoffbeutel bei 4 ° C. </li><li> Streak die LB-Agar-Platte mit rekombinanten Zellen (E.coli-BC21) von der Glycerin-Stamm (-80 ° C). Die Platten O / N bei 37 ° C. </li><li> Vorbereitung 50 ml NZCYM-Medium und das Medium autoklaviert bei 121 º C für 30 min. Ermöglichen, dass die Medien abkühlen, 50 &amp;mgr; l 100 &amp;mgr; g / ml Ampicillin für die 50 ml Medium. </li><li> Impfen eine einzige Kolonie von der LB-Agar-Platte in den Medien mit 50ml NZCYM ampicill ergänztin. die Zellen O / N bei 37 ° C inkubieren in einem Schüttelinkubator </li><li> Herstellung von 1 l NZCYM-Medien und autoklavieren Medien bei 121 ° C für 30 min. 1 ml 100 mg / ml Ampicillin bis 1000 ml Medium. Nehmen Sie eine optische Dichte (OD) als Rohling mit einem UV-Vis-Spektrophotometer. HINWEIS: Werfen Sie diese nach der ersten Lesung. </li><li> Beimpfen von 10 ml der Zellkultur von Schritt 2.3.5 in 1000 ml Medium aus Schritt 2.3.6. Messen der optischen Dichte bei 595 nm unmittelbar nach Inokulation. Nehmen Sie die optische Dichte Lese regelmäßig zu verfolgen, das Wachstum zu halten. HINWEIS: Lesen Sie die leere jedes Mal die OD genommen wird. </li><li> Induziert mit 700 &amp;mgr; l Isopropyl-β-D-1-thiogalactopyranosid (IPTG, 1 M), wenn das bakterielle Wachstum erreicht, bis etwa 0,75 bis 0,8 OD bei 595 nm auf. </li><li> Ernten Sie die Zellen, wenn die OD erreicht 1.1. Den Inhalt des Kolbens in 6 Kunststoffflaschen. Balancieren und wiegen die Flaschen vor der Verwendung der Zentrifuge. Zentrifugieren für 20 min bei 8.554 × g bei 4 ° C.Halten des Zellpellets in der -20 ° CO / N </li><li> Nehmen Sie die Zellpellets und kombinieren sie in einem 50-ml-Tube. In 3,5 ml 4 M Guanidin-HCl pro Liter der Gärung. Ultraschallbad 2-3 mal auf Eis, 30 Sek. jedes Mal, mit 1-Minuten-Intervallen. </li><li> Machen 6x Verdünnungen des Zellvolumens mit 0,5% Ameisensäure und lassen Sie es im Kühlraum für 2 h rühren. Zentrifugieren für 30 min, 8.554 x g bei 4 ° C. Halten Sie den Überstand. In 20% Gesättigte Ammoniumsulfat mit der gesättigten Stammlösung und Aufsehen in der kalten Raum, O / N. </li><li> Zentrifugieren für 30 min, 8554 × g bei 4 ° C und halten die Pellets. Rekonstitution der Pellets in 0,1% Trifluoressigsäure (TFA), Last auf eine C4-Säule (10 x 250 mm, 5 &amp;mgr; m) und Fraktionierung unter Verwendung eines linearen Gradienten von A = 0,1% TFA und B = 60% Acetonitril in TFA, bei einer Durchflussrate von 1,5 ml · min -1. </li><li> Einfrieren der Proteinlösung auf Trockeneis und lyophilisiert die gefrorene Probe O / N. </li></ol></li><li> Vorbereitung der amelogenin-Chitosan (CS-AMEL) HYDROGel (Fig. 1A) <ol><li> In 200 ug rp172 in ein Röhrchen mit 960 ul 1% Chitosan-Lösung, dann mit einem Vortex mischen, bis die Lösung klar ist. </li><li> In 25 ul 0,1 M CaCl 2-Lösung, gefolgt von 15 ul 0,1 M Na 2 HPO 4 Lösung des amelogenin haltigen Chitosan-Lösung, dann mischen mit einem Vortex für 5 min. </li><li> Den pH-Wert des CS-AMEL Lösung auf 6,5 durch vorsichtige Zugabe von 1 M NaOH-Lösung. CS-AMEL Hydrogel zu bilden, wenn der pH-Wert 6,5 erreicht. </li></ol></li></ol> 3. Emaille Nachwachsen in Amelogenin-Chitosan-Hydrogel <ol><li> Herstellung von künstlichen Speichellösung <ol><li> Man löst eine bestimmte Menge von Magnesiumchlorid (MgCl 2, 0,2 mM), Calciumchlorid (CaCl 2, 1 mM), Kaliumdihydrogenphosphat (KH 2 PO 4, 4 mM), Kaliumchlorid (KCl, 16 mM) und Ammoniumchlorid ( NH 4 Cl, 4,5 mM) in 20 mM HEPES(4 - (2-Hydroxyethyl) piperazin-1-ethan-sulfonsäure)-Puffer 12. </li><li> PH-Wert auf 7,0 mit 1 M NaOH und speichern die künstliche Speichellösung bei 4 ° C. </li><li> Vor der Verwendung der Lösung werden Natriumfluorid (NaF, 300 ppm). </li></ol></li><li> Anwendung von CS-Hydrogel AMEL <ol><li> Gelten etwa 20 &amp;mgr; l CS-AMEL Hydrogel auf die geätzte Zahnscheibe mit einer Spritze (Abbildung 1B). </li><li> Trocknen Sie die Hydrogel-bedeckten Zahnscheibe in einem Exsikkator bei RT für 2 Stunden. </li><li> Übertragen der Zahnscheibe in ein Becherglas, das 30 ml Lösung künstlichen Speichels. Das Becherglas mit Aluminiumfolie, und dann halten Sie den Becher in einem Ofen bei 37 ° C für 7 Tage. </li><li> Entfernen Sie die Zahnscheibe und trocknen Sie sie in einem Trockenschrank bei RT. </li></ol></li></ol> 4. Charakterisierung der Schnittstelle zwischen neu gewachsenen Schicht und Emaille Die Mikrostruktur der Grenzfläche zwischen dem neu gewachsenen Schicht und Emaille Beobachdurch Rasterelektronenmikroskopie (SEM) und hohen Drehtransmissionselektronenmikroskopie (HR-TEM) ved. Die TEM-Probe wurde unter Verwendung eines fokussierten Ionenstrahls (FIB)-Technik, die für die Schritte sind wie folgt hergestellt. <ol><li> Legen Sie die Probe in ein FIB-SEM Instrument und beenden Sie alle Ausrichtungen nach der Betriebsanleitung. HINWEIS: Z Feineinstellung sollte mindestens 2 mal durchgeführt werden. </li><li> Abscheiden einer Kohlenstoffschicht (15 × 3 &amp;mgr; m) auf der Probe, um die darunterliegende Struktur (2A) zu schützen. </li><li> Mühle die Probe vorsichtig an den oberen, unteren und rechten Seite der Kohlenstoffschicht, ein dünnes Stück Probe herzustellen, und dann schneiden Sie die untere Seite dieser dünnen Stück. HINWEIS: Überprüfen Sie die Ionenstrahl-Ausrichtung vor jedem Schleifschritt (Abbildung 2B). </li><li> Weld ein Pt-Spitze auf dem dünnen Stück und schneiden Sie die linke Seite, um das Probestück (3C) zu trennen. HINWEIS: Überprüfen Sie die Ionenstrahl-Ausrichtung vor dem Schweißschritt. </li><li> Heben Sie tPT er mit der Probe langsam kippen, und montieren Sie die Lamellen Probe auf einem Lift-out-TEM-Gitter. </li><li> Nehmen Sie die Spitze aus der Probe und die Probe dünn, bis seine Dicke weniger als 100 nm (Abbildung 3D). Entfernen der Probe aus dem Gerät, um die TEM-Beobachtung durchzuführen. HINWEIS: Überprüfen Sie die Ionenstrahl-Ausrichtung vor jedem Verdünnungsprozess. </li></ol> 5. Beurteilung der Bindung und mechanische Eigenschaften der neu gewachsenen Schicht <ol><li> Die Bindungsfestigkeit wird durch Ultraschallbehandlung beurteilt. Halten Sie die reparierte Zahnscheibe in einem Ultraschallreiniger (42 kHz, 100 W) für 10 min und dann beobachten, die Schnittstelle zwischen der Schicht und repariert natürlichen Zahnschmelz mit einem Rückstreuelektronen-REM-Analyse. </li><li> Messung der Härte und der Elastizitätsmodul bei 20 Testpunkte für jedes repariert Schmelzoberfläche (n = 3) durch eine Nanoindenters mit einer Berkovich-Spitze. </li></ol> 6. Immunfluoreszenzfärbungen <ol><li> Waschen Sie die Zahnscheibe mit Tris buffer Kochsalzlösung (TBS) für 15 min. </li><li> Blockieren mit 1% Rinderserumalbumin (BSA) in TBS für 15 min. </li><li> Entfernen Sie die Flüssigkeit und Inkubation der Probe O / N 1 ° Ab (1:500 Huhn Anti-Amelogenin) in TBS (0,1% BSA, 0,3% Triton X-100) verdünnt. </li><li> Waschen Sie die Probe mit TBS für 30 min und Inkubation O / N mit 2 ° Ab (1:100 Anti-Huhn FITC) in TBS verdünnt. </li><li> Waschen Sie die Probe mit TBS für 30 min und zu verlassen, um für weitere 30 Minuten trocknen. </li><li> Beachten durch Fluoreszenz-Mikroskop. </li></ol>

<ol>
	<li>Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-+mediated+enamel+mineralization.">Protein- mediated enamel mineralization.</a> Frontiers in Bioscience. 17, 1996-2023 (2012).</li><li>Palmer, L. C., Newcomb, C. J., Kaltz, S. R., Spoerke, E. D., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Systems+for+Hydroxyapatite+Mineralization+Inspired+By+Bone+and+Enamel.">Biomimetic Systems for Hydroxyapatite Mineralization Inspired By Bone and Enamel.</a> Chem. Rev. 108, 4754-4783 (2008).</li><li>Onuma, K., Yamagishi, K., Oyane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleation+and+growth+of+hydroxyapatite+nanocrystals+for+nondestructive+repair+of+early+caries+lesions.">Nucleation and growth of hydroxyapatite nanocrystals for nondestructive repair of early caries lesions.</a> J. Cryst. Growth. 282, 199-207 (2005).</li><li>Xie, R. Q., Feng, Z. D., Li, S. W., Xu, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDTA-Assisted+Self-Assembly+of+Fluoride-Substituted+Hydroxyapatite+Coating+on+Enamel+Substrate.">EDTA-Assisted Self-Assembly of Fluoride-Substituted Hydroxyapatite Coating on Enamel Substrate.</a> Cryst. Growth Des. 11, 5206-5214 (2011).</li><li>Li, L., 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=Bio-Inspired+Enamel+Repair+via+Glu-Directed+Assembly+of+Apatite+Nanoparticles:+an+Approach+to+Biomaterials+with+Optimal+Characteristics.">Bio-Inspired Enamel Repair via Glu-Directed Assembly of Apatite Nanoparticles: an Approach to Biomaterials with Optimal Characteristics.</a> Advanced Materials. 23, (2011).</li><li>Yin, Y. J., Yun, S., Fang, J. S., Chen, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+regeneration+of+human+tooth+enamel+under+near-physiological+conditions.">Chemical regeneration of human tooth enamel under near-physiological conditions.</a> Chem. Commun. , 5892-5894 (2009).</li><li>Fan, Y., Sun, Z., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+remineralization+of+enamel+in+the+presence+of+amelogenin+and+fluoride.">Controlled remineralization of enamel in the presence of amelogenin and fluoride.</a> Biomaterials. 30, 478-483 (2009).</li><li>Li, L., 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=Repair+of+enamel+by+using+hydroxyapatite+nanoparticles+as+the+building+blocks.">Repair of enamel by using hydroxyapatite nanoparticles as the building blocks.</a> J. Mater. Chem. 18, 4079-4084 (2008).</li><li>Chen, H., 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=Acellular+synthesis+of+a+human+enamel-like+microstructure.">Acellular synthesis of a human enamel-like microstructure.</a> Advanced Materials. 18, (2006).</li><li>Yamagishi, K., 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=A+synthetic+enamel+for+rapid+tooth+repair.">A synthetic enamel for rapid tooth repair.</a> Nature. 433, 819-819 (2005).</li><li>Bleek, K., Taubert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+polymer-controlled,+bioinspired+calcium+phosphate+mineralization+from+aqueous+solution.">New developments in polymer-controlled, bioinspired calcium phosphate mineralization from aqueous solution.</a> Acta Biomaterialia. 9, 6283-6321 (2013).</li><li>Fletcher, J., Walsh, D., Fowler, C. E., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+mats+of+PVP/ACP+nanofibres+for+remineralization+of+enamel+tooth+surfaces.">Electrospun mats of PVP/ACP nanofibres for remineralization of enamel tooth surfaces.</a> Crystengcomm. 13, 3692-3697 (2011).</li><li>Fang, P. -. A., Conway, J. F., Margolis, H. C., Simmer, J. P., Beniash, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+self-assembly+of+amelogenin+and+the+regulation+of+biomineralization+at+the+nanoscale.">Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 14097-14102 (2011).</li><li>Yang, X., 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=How+Amelogenin+Orchestrates+the+Organization+of+Hierarchical+Elongated+Microstructures+of+Apatite.">How Amelogenin Orchestrates the Organization of Hierarchical Elongated Microstructures of Apatite.</a> Journal of Physical Chemistry B. 114, 2293-2300 (2010).</li><li>Li, H., Fujiki, Y., Sada, K., Estroff, 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=Gel+incorporation+inside+of+organic+single+crystals+grown+in+agarose+hydrogels.">Gel incorporation inside of organic single crystals grown in agarose hydrogels.</a> Crystengcomm. 13, 1060-1062 (2011).</li><li>Ruan, Q., Zhang, Y., Yang, X., Nutt, S., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+amelogenin-chitosan+matrix+promotes+assembly+of+an+enamel-like+layer+with+a+dense+interface.">An amelogenin-chitosan matrix promotes assembly of an enamel-like layer with a dense interface.</a> Acta Biomater. 9, 7289-7297 (2013).</li><li>Hu, C. C., 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=Cloning,+cDNA+sequence,+and+alternative+splicing+of+porcine+amelogenin+mRNAs.">Cloning, cDNA sequence, and alternative splicing of porcine amelogenin mRNAs.</a> Journal of Dental Research. 75, 1735-1741 (1996).</li><li>Bromley, K. M., 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=Amelogenin+Processing+by+MMP-20+Prevents+Protein+Occlusion+Inside+Calcite+Crystals.">Amelogenin Processing by MMP-20 Prevents Protein Occlusion Inside Calcite Crystals.</a> Cryst. Growth Des. 12, 4897-4905 (2012).</li><li>Fan, Y., Sun, Z., Abbott, C., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+enamel+inspired+nanocomposite+fabrication+through+amelogenin+supramolecular+assembly.">A enamel inspired nanocomposite fabrication through amelogenin supramolecular assembly.</a> Biomaterials. 28, 3034-3042 (2007).</li><li>Wright, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+etiologies+and+associated+phenotypes+of+amelogenesis+imperfecta.">The molecular etiologies and associated phenotypes of amelogenesis imperfecta.</a> Am. J. Med. Gent. A. 140 (23), 2547-2555 (2006).</li><li>Eimar, H., 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=Regulation+of+enamel+hardness+by+its+crystallographic+dimensions.">Regulation of enamel hardness by its crystallographic dimensions.</a> Acta Biomaterialia. 8, 3400-3410 (2012).</li><li>Ruan, Q., Siddiqah, N., Li, X., Nutt, S., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelogenin-chitosan+matrix+for+human+enamel+regrowth:+effects+of+viscosity+and+supersaturation+degree.">Amelogenin-chitosan matrix for human enamel regrowth: effects of viscosity and supersaturation degree.</a> Connect Tissue Res. , (2014).</li><li>Pouget, E. M., 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=The+Initial+Stages+of+Template-Controlled+CaCO3+Formation+Revealed+by+Cryo-TEM.">The Initial Stages of Template-Controlled CaCO3 Formation Revealed by Cryo-TEM.</a> Science. 323, 1455-1458 (2009).</li><li>Gebauer, D., Volkel, A., Colfen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Prenucleation+Calcium+Carbonate+Clusters.">Stable Prenucleation Calcium Carbonate Clusters.</a> Science. 322, 1819-1822 (2008).</li></ol>

The authors have nothing to disclose.

Während der Mineralgehalt des Schmelz Hoch macht es das härteste mineralisiertem Gewebe im menschlichen Körper, ist dieses biokeramischen anfällig Entmineralisierung Prozesse, die oft auftreten, wie Karies oder Erosion. Genmutationen können auch dazu führen, dünnen oder weichen Zahnschmelz, was zu einer Reihe von Erbkrankheiten der Schmelzfehlbildungen genannt Amelogenesis imperfecta 20. Oral Gesundheitsprodukten Fluorid oder CPP-ACP enthalten, sind auf dem Markt seit mehreren Jahren (dh,<...

Dental enamel is the hard mineralized surface of human teeth. It is composed of numerous needle-like apatite crystals, which are bundled in organized, parallel prisms to ensure the unique mechanical strength and biological protection that enamel provides1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss1-2, which often occurs as dental caries or erosion. Commercially available products such as fluoride containing varnishes, tooth pastes and mouthwashes are effective in re-mineralizing enamel but none of them have the potential to promote the formation of organized apatite crystals. Clinically, the conventional treatment for enamel repair involves a filling procedure with artificial materials such as amalgam, ceramics, or composite resin 3. However, these materials usually do not interface well with the natural tissue surrounding the lesion, because their structures, components and properties are different from the natural enamel. As a result, secondary caries frequently develops overtime at the interface between the tooth and foreign materials. Therefore, in situ regrowth of enamel with a dense interface is an attractive target for the fields of materials science and stomatology. One particularly promising way to achieve this purpose is biomimetic synthesis of enamel-like material on the enamel surface. Recently, numerous in vitro attempts have been made to prepare enamel-like materials using biomimetic systems that contain nano-apatites or different organic additives 4-12. However, developing the optimal biomimetic strategy to promote remineralization crystals to achieve a perfect dense interface is still a challenge. 
During enamel mineralization, the oriented growth and elongation of apatite crystals is regulated by an amelogenin-rich matrix 1,13-14. To mimic the organic matrix in developing enamel, herein we describe a detailed protocol for fabricating an amelogenin-chitosan (CS-AMEL) hydrogel for in situ enamel regrowth on an acid-etched enamel surface used as a model for erosive lesions. As &#8220;the most versatile growth media&#8221; for crystals 15, hydrogel matrices have an advantage over a solution system since clinically they are easier to handle. Moreover, CS-AMEL is biocompatible, biodegradable, and has unique antimicrobial and adhesion properties that compare favorably with other biomimetic systems for dental applications 16. Importantly, the in situ mineralization of apatite crystals on the enamel surface provides a dense interface between the repaired layer and the natural enamel, which can potentially improve the durability of restorations and prevent the formation of new caries at the margin of the restoration.

<table border="0" cellpadding="0" cellspacing="0" width="1219">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Name</td>
			<td style="width:209px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:516px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Material/ Reagent</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:331px;">Human Third Molar&#160;</td>
			<td style="width:209px;">Ostrow School of Dentistry of the University of Southern California&#160;</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">The human molars were extracted following the standard procedures for extraction at the Ostrow School of Dentistry of the University of Southern California and handled with the approval of the Institutional Review Board.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Recombinant Pocine Amelogenin</td>
			<td style="width:209px;">Expression and purification&#160; in lab</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">rP172, full-length&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chitosan&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">448877</td>
			<td style="width:516px;">medium molecular weight, 75-85% deacetylated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Phosphoric Acid&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA033266</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetic Acid Glacial</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">A036289</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Hydroxide</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9292</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Calcium Chloride&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">223506</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dibasic Sodium Phosphate Anhydrous</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0316</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">BL21-CodonPlus (DE3)-RP&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">230255</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Sulfate</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH8001</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trifluoroacetic Acid</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAL06374</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetonitrile</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH1103</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Magnesium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0244</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Dihydrogen Phosphate&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9268</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0258</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAA15000</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAA14777</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Fluoride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA11561</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tris-Buffered Saline</td>
			<td style="width:209px;">Bio-Rad</td>
			<td style="width:164px;">170-6435</td>
			<td style="width:516px;">10&#215; TBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Serum Albumin</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">12659</td>
			<td style="width:516px;">CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Triton X-100</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">TX1568-1</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chicken Anti-Amelogenin</td>
			<td style="width:209px;">N/A</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">A gift from Prof. Malcolm Snead, University of Southern California</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Anti-Chicken IgY-FITC</td>
			<td style="width:209px;">Santa Cruz Biotechnology</td>
			<td style="width:164px;">Sc-2700</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:331px;">Equipments</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:331px;">High Performance Liquid Chromatography System</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">Varian Prostar 210</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">C4 column</td>
			<td style="width:209px;">Phenomenx&#160;</td>
			<td style="width:164px;">Jupiter 5&#956; 300A</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Scanning Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JSM-7001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FIB-SEM&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JIB-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Transmission Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL</td>
			<td style="width:164px;">JEM-2100F</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital Low Speed Diamond Saw</td>
			<td style="width:209px;">MTI Corporation</td>
			<td style="width:164px;">SYJ-150</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescence Microscopy</td>
			<td style="width:209px;">Leica</td>
			<td style="width:164px;">DMI3000 B</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultrasonic Cleaner&#160;</td>
			<td style="width:209px;">Branson&#160;</td>
			<td style="width:164px;">2510</td>
			<td style="width:516px;">42 kHz, 100 W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nano-indenter&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">MTS XP</td>
			<td style="width:516px;"></td>
		</tr>
	</tbody>
</table>

development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Die Wirksamkeit der hier beschriebenen Protokoll wird durch Rasterelektronenmikroskopie (SEM), Bereichsbeugung (SAED) und Röntgenbeugung (XRD)-Analyse bewiesen. Nach der Reparatur von Amelogenin-Chitosan (CS-AMEL) Hydrogel für 7 Tage wurde ein emailartigen Schicht mit einer Dicke von 15 um auf der geätzten Zahnschmelzoberfläche ist. Die neu gewachsene Schicht von hochgeordneten Anordnungen von Kristallen mit einem Durchmesser von ~ 50 nm, wächst vorzugsweise entlang der c-Achse, senkrecht zur Oberfläche (Pfeile in...

Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In diesem Artikel beschreiben wir ein Protokoll zur Herstellung eines amelogenin-Chitosan-Hydrogel für oberflächliche Zahnschmelz Wiederaufbau. In-situ-Wachstum von Apatitkristalle in dem Hydrogel Organisiert bildeten einen dichten Schmelz-Wiederherstellung-Schnittstelle, die die Wirksamkeit und Haltbarkeit von Restaurationen zu verbessern.

Entwicklung von Amelogenin-Chitosan-Hydrogel für<em&gt; In-vitro-</em&gt; Emaille Nachwachsen mit einem dichten Schnittstelle

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

development-amelogenin-chitosan-hydrogel-for-vitro-enamel-regrowth

Research

JoVE Journal

Bioengineering

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

14.9K Aufrufe.  University of Southern California.  In diesem Artikel beschreiben wir ein Protokoll zur Herstellung eines amelogenin-Chitosan-Hydrogel für oberflächliche Zahnschmelz Wiederaufbau. In-situ-Wachstum von Apatitkristalle in dem Hydrogel Organisiert bildeten einen dichten Schmelz-Wiederherstellung-Schnittstelle, die die Wirksamkeit und Haltbarkeit von Restaurationen zu verbessern. 

Serie: Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Verkapselung von Kardiomyozyten in einer Fibrin-Hydrogel für Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Forschung

Biotechnik

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Kultivierung humaner neuronaler Vorläuferzellen in einer 3-dimensionalen Self-Montage Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Bridging the Bio-elektronische Schnittstelle mit Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Synthese einer Intein-vermittelten Protein-Hydrogel Künstliche

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Hydrogel-Nanopartikel Ernte von Plasma oder Urin zum Nachweis geringer Menge Proteine

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Test auf Augenreizung (EIT) zur Kennzeichnung der Gefahr von Augen Reizt Chemicals mit rekonstruierter menschlicher Hornhaut-ähnlichen Epithelial (RhCE) Gewebemodell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

In-vitro-Modell von physiologischen und pathologischen Blutfluss mit Anwendung auf Untersuchungen der Gefäßzell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

3D Hydrogel Kulturen von primären Gliazellen für In-vitro- Modellierung von Neuroinflammation verbessert

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable Alginat/Gelatine Hydrogel 3D In-vitro- Modellsysteme induzieren Sphäroid Zellbildung

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Herstellung einer Niere Cortex extrazelluläre Matrix abgeleitet Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...