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.

Les molaires humaines ont été extraites en suivant les procédures standard pour l&#39;extraction de l&#39;École de médecine dentaire Ostrow de l&#39;Université de Californie du Sud et manipulés avec l&#39;approbation de l&#39;Institutional Review Board. 1. Préparation de la Dent dépoli à l&#39;acide Slice <ol><li> Sélectionner une troisième molaire humaine sans caries restaurées. </li><li> Retirer la partie racine de la molaire, et couper la couronne de la molaire longitudinalement en tranches de 2 mm d&#39;épaisseur en utilisant un faible débit diamant scie refroidi à l&#39;eau. Nettoyer par ultrasons ces tranches pendant 2 min, puis les rincer avec de l&#39;eau déminéralisée 3 fois. </li><li> Pour simuler des lésions érosives, graver les tranches de dents avec 30% d&#39;acide phosphorique pendant 30 secondes puis les rincer immédiatement avec de l&#39;eau déminéralisée. </li><li> Nettoyer par ultrasons des tranches gravées pendant 2 min, puis les rincer avec de l&#39;eau déminéralisée 3 fois. </li></ol> 2. Préparation d&#39;hydrogel chitosane-Amelogenin <ol><li> Preparation de la solution mère de chitosane <ol><li> Dissoudre 1% (w / v) chitosane (masse moléculaire moyenne, 75 à 85% désacétylée) dans une (v / v) d&#39;une solution à 1% d&#39;acide acétique suivi par une agitation à 80 ° CO / N. </li><li> Après refroidissement de la solution à la température ambiante, filtrer la solution de chitosane à l&#39;aide d&#39;un filtre de 0,45 um. Ajuster le pH à 5,0 par addition d&#39;une solution de NaOH. </li></ol></li><li> Préparation de calcium et de solution stock de phosphate (0,1 M) <ol><li> Peser le chlorure de calcium (CaCl 2) et préparer une solution à 0,1 M, puis mélanger à l&#39;aide d&#39;un vortex jusqu&#39;à ce que la solution est claire. Ajuster la valeur du pH à 11 par addition d&#39;une solution de NaOH. </li><li> Peser le phosphate disodique (Na 2 HPO 4) et préparer une solution à 0,1 M, puis mélanger à l&#39;aide d&#39;un vortex jusqu&#39;à ce que la solution est claire. </li></ol></li><li> Expression et purification de l&#39;amélogénine recombinant 17-18 La pleine longueur amelogenin porcin recombinant (rP172) utilisé ici a 172 acides aminés et est un analogue de la pleineLongueur porcine P173 natif, mais sans la méthionine N-terminale ainsi que d&#39;un groupe phosphate sur Ser16 18. <ol><li> Ajouter 20 g de bouillon Lysogénie (LB) Agar en poudre dans 500 ml d&#39;eau déminéralisée, l&#39;autoclave la solution à 121 ° C (réglage de liquide) pendant 20 min. Laisser refroidir la gélose à ~ 55 ° C, ajouter de l&#39;ampicilline (100 ug / ml) dans une solution d&#39;agar. </li><li> Transférer la solution de gélose des boîtes de Petri, laisser chaque plaque froide jusqu&#39;à ce qu&#39;il soit solide, puis retournez pour éviter la condensation sur la gélose. plaques de magasins dans des sacs en plastique à 4 ° C. </li><li> Streak la plaque de gélose LB avec des cellules recombinantes (E. coli-BC21) du stock de glycérol (-80 ° C). Incuber les plaques O / N à 37 ° C. </li><li> Préparer 50 ml de milieu NZCYM et autoclave les médias à 121 ° C pendant 30 min. Autoriser les médias à refroidir, ajouter 50 ul de 100 pg / ml d&#39;ampicilline à 50 ml de milieu. </li><li> Inoculer une colonie unique à partir de la plaque d&#39;agar-agar dans le milieu LB 50 ml de NZCYM supplémenté avec ampicillIncuber les cellules po O / N dans un incubateur agitateur à 37 ° C. </li><li> Préparer 1 L de médias NZCYM et autoclave les médias à 121 ° C pendant 30 min. Ajouter 1 ml de 100 mg / ml d&#39;ampicilline à 1000 ml de milieu. Prenez une densité optique (DO) de la lecture comme une vierge à l&#39;aide d&#39;un spectrophotomètre UV-Vis. NOTE: Ne pas jeter ce après la première lecture. </li><li> Inoculer 10 ml de la culture cellulaire de l&#39;étape 2.3.5 dans 1000 ml de milieu de l&#39;étape 2.3.6. Lire la densité optique à 595 nm immédiatement après l&#39;inoculation. Prendre la mesure de la densité optique pour assurer le suivi de la croissance. REMARQUE: Lire la vierge à chaque fois la DO est prise. </li><li> Induire avec 700 ul d&#39;isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 M) lorsque la croissance bactérienne atteint à environ 0,75 ~ 0,8 DO à 595 nm. </li><li> Récolter les cellules lorsque la DO atteint 1.1. Verser le contenu de la fiole dans des bouteilles en plastique 6. Et peser les bouteilles avant d&#39;utiliser la centrifugeuse. Centrifuger pendant 20 min à 8554 g à 4 ° C.Conserver le culot cellulaire dans l&#39;-20 ° CO / N. </li><li> Sortez les culots de cellules et de les combiner dans un tube de 50 ml. Ajouter 3,5 ml de guanidine HCl 4M par L de fermentation. Fois Soniquer 2-3 sur la glace, 30 secondes à chaque fois, avec des intervalles de 1 min. </li><li> Faire des dilutions 6x de volume de la cellule avec de l&#39;acide formique à 0,5% et laisser remuer dans la chambre froide pendant 2 heures. Centrifuger pendant 30 min, 8554 g à 4 ° C. Gardez le surnageant. Ajouter 20% de sulfate d&#39;ammonium saturé à l&#39;aide de la solution mère saturée et remuer dans la chambre froide, O / N. </li><li> Centrifuger pendant 30 min, 8554 g à 4 ° C et garder le culot. Reconstituer les pastilles dans 0,1% d&#39;acide trifluoroacétique (TFA), de la charge sur une colonne C4 (10 x 250 mm, 5 um), et fractionner en utilisant un gradient linéaire de A = 0,1% de TFA et B = 60% d&#39;acétonitrile dans du TFA, à une taux de 1,5 ml · min -1 débit. </li><li> Congeler la solution de protéine sur glace sèche et lyophiliser l&#39;échantillon congelé O / N. </li></ol></li><li> Préparation de amelogenin-chitosane (CS-AMEL) hydrographieel (figure 1A) <ol><li> Ajouter 200 ug de rp172 dans un tube contenant 960 pi de solution de chitosane à 1%, puis mélanger à l&#39;aide d&#39;un vortex jusqu&#39;à ce que la solution est claire. </li><li> Ajouter 25 ul de 0,1 M CaCl 2 solution suivis par 15 ul de 0,1 M de Na 2 HPO 4 solution à la solution de chitosane contenant amélogénine-, puis mélanger à l&#39;aide d&#39;un vortex pendant 5 min. </li><li> Ajuster le pH de la solution de CS-AMEL à 6,5 en ajoutant avec précaution une solution de NaOH. CS-AMEL hydrogel se forme lorsque la valeur du pH atteigne 6,5. </li></ol></li></ol> 3. Émail repousse dans Amelogenin-chitosane hydrogel <ol><li> Préparation de la solution de salive artificielle <ol><li> Dissoudre une certaine quantité de chlorure de magnésium (MgCl 2, 0,2 mM), du chlorure de calcium (CaCl 2, 1 mM), du dihydrogénophosphate de potassium (KH 2 PO 4, 4 mM), du chlorure de potassium (KCl, 16 mM) et de chlorure d&#39;ammonium ( NH 4 Cl, 4,5 mM) dans 20 mM d&#39;HEPES(4 - (2-hydroxyéthyl) pipérazine-1-éthane-sulfonique) en mémoire tampon 12. </li><li> Ajuster le pH à 7,0 avec NaOH 1 M et de stocker la solution de salive artificielle à 4 ° C. </li><li> Avant d&#39;utiliser la solution, ajouter du fluorure de sodium (NaF, 300 ppm). </li></ol></li><li> Application de CS-AMEL hydrogel <ol><li> Appliquer environ 20 ul hydrogel CS-AMEL à la tranche de la dent mordancée à l&#39;aide d&#39;une seringue (Figure 1B). </li><li> Sécher la dent tranche recouverte d&#39;un hydrogel dans un dessiccateur à la température ambiante pendant 2 h. </li><li> Transférer la tranche de la dent dans un bêcher contenant 30 ml d&#39;une solution de salive artificielle. Recouvrir de papier d&#39;aluminium, puis garder le bécher dans un four à 37 ° C pendant 7 jours. </li><li> Retirez la tranche de la dent et le sécher dans un dessiccateur à la température ambiante. </li></ol></li></ol> 4. Caractérisation de l&#39;interface entre la couche nouvellement Cultivé et émail La microstructure de l&#39;interface entre la couche nouvellement développé et l&#39;émail a été observed par microscopie électronique à balayage (MEB) et microscopie électronique à transmission à haute révolution (HR-TEM). L&#39;échantillon de MET a été préparé en utilisant un faisceau d&#39;ions focalisé (FIB) de la technique, pour les étapes qui sont les suivantes. <ol><li> Chargez l&#39;échantillon dans un instrument FIB-SEM et terminer tous les alignements selon les instructions de fonctionnement. REMARQUE: Le réglage fin de Z doit être effectuée au moins 2 fois. </li><li> Déposer une couche de carbone (15 x 3 um) sur l&#39;échantillon afin de protéger la structure sous-jacente (figure 2A). </li><li> Mill soigneusement l&#39;échantillon sur les côtés supérieur, inférieur et droit de la couche de carbone pour préparer un petit morceau de l&#39;échantillon, et ensuite coupé la partie inférieure de cette pièce mince. REMARQUE: Vérifiez l&#39;alignement du faisceau d&#39;ions avant chaque étape de broyage (figure 2B). </li><li> Souder une pointe de Pt sur ​​la pièce mince et couper la partie gauche de séparer l&#39;échantillon pièce (figure 3C). REMARQUE: Vérifiez l&#39;alignement du faisceau d&#39;ions avant l&#39;étape de soudage. </li><li> Sortez til pt basculer avec l&#39;échantillon lentement, et monter l&#39;échantillon lamellaire sur une grille de TEM-soulèvement. </li><li> Détachez la pointe de l&#39;échantillon, et mince de l&#39;échantillon jusqu&#39;à ce que son épaisseur est inférieure à 100 nm (figure 3D). Enlever l&#39;échantillon de l&#39;instrument pour effectuer l&#39;observation TEM. REMARQUE: Vérifiez l&#39;alignement du faisceau d&#39;ions avant chaque processus d&#39;amincissement. </li></ol> 5. Évaluation des propriétés de liaison et mécaniques de la couche nouvellement Grown <ol><li> La résistance de liaison est évaluée par un traitement aux ultrasons. Gardez la tranche de dent réparée dans un nettoyeur à ultrasons (42 kHz, 100 W) pendant 10 min, puis observer l&#39;interface entre la couche réparé et l&#39;émail naturel avec un électron rétrodiffusé analyse SEM. </li><li> Mesurer la dureté et le module d&#39;élasticité à 20 points de test sur chaque surface de l&#39;émail réparé (n = 3) par un nano-indentation avec une pointe Berkovich. </li></ol> 6. Immunofluorescence <ol><li> Laver la tranche de la dent avec du Tris Buffer solution saline (TBS) pendant 15 min. </li><li> Bloquer avec 1% d&#39;albumine de sérum bovin (BSA) dans du TBS pendant 15 minutes. </li><li> Éliminer le liquide de l&#39;échantillon et incuber O / N avec 1 ° Ab (1:500 poulet anti-Amelogenin) dilués dans du TBS (0,1% de BSA, 0,3% de Triton X-100). </li><li> Laver l&#39;échantillon avec le SCT pour 30 min et incuber O / N avec 2 ° Ab (1:100 anti-poulet FITC) dilué dans du TBS. </li><li> Laver l&#39;échantillon avec le SCT pour 30 min et laisser sécher pendant 30 min. </li><li> Observez par microscope à fluorescence. </li></ol>

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The authors have nothing to disclose.

Bien que la teneur en minéraux de l&#39;émail est élevé rend plus difficile le tissu minéralisé dans le corps humain, ce biocéramique est sensible aux processus de déminéralisation, qui se produisent souvent que la carie dentaire ou de l&#39;érosion. Les mutations génétiques peuvent aussi provoquer émail fines et souples menant à une série de maladies héréditaires de malformation de l&#39;émail appelé Amélogenèse imparfaite 20. Produits de soins de santé bucco-dentaire contenant...

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.

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			<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>
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			<td height="21" style="height:21px;width:331px;">Material/ Reagent</td>
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			<td></td>
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			<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.

L&#39;efficacité du protocole décrit ici est mise en évidence par microscopie électronique à balayage (MEB), la diffraction d&#39;électrons de la zone sélectionnée (DSA) et la diffraction des rayons X (XRD) des analyses. Après la réparation par amélogénine-chitosan (CS-AMEL) hydrogel pendant 7 jours, une couche d&#39;émail en forme avec une épaisseur de 15 pm a été formée sur la surface de l&#39;émail mordancé. La couche nouvellement développée a été faite de matrices hautement ordonnées de crist...

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

Dans cet article, on décrit un protocole de fabrication d&#39;un hydrogel de chitosane-amélogénine à la reconstruction de l&#39;émail superficiel. Organisé croissance in situ de cristaux d&#39;apatite dans l&#39;hydrogel formé dans une interface émail-restauration dense, ce qui permettra d&#39;améliorer l&#39;efficacité et la durabilité des restaurations.

Le développement de l&#39;Amelogenin-chitosane Hydrogel pour<em&gt; In Vitro</em&gt; Émail repousse avec une interface dense

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 vues.  University of Southern California.  Dans cet article, on décrit un protocole de fabrication d'un hydrogel de chitosane-amélogénine à la reconstruction de l'émail superficiel. Organisé croissance in situ de cristaux d'apatite dans l'hydrogel formé dans une interface émail-restauration dense, ce qui permettra d'améliorer l'efficacité et la durabilité des restaurations. 

Vidéo: 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.

L&#39;encapsulation des cardiomyocytes dans un hydrogel de fibrine pour l&#39;ingénierie du tissu cardiaque

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

Recherche

Bio-ingénierie

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.

La culture de cellules progénitrices neurales humaines dans un hydrogel en 3 dimensions Peptide auto-assemblage

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.

Combler l&#39;interface bio-électronique avec 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.

Synthèse d&#39;une protéine artificielle induite par une intéine 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 ...

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 nanoparticules récolte de plasma ou l&#39;urine pour la détection des protéines de faible abondance

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).

Irritation des yeux Test (IET) pour l&#39;identification des dangers des produits chimiques irritants oculaires utilisant épithélial (RHCE) Tissue modèle reconstruit humain Cornea-like

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.

Modèle in vitro de physiologiques et pathologiques Blood Flow avec application aux enquêtes de cellules vasculaires Rénovation

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 ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Développement d&#39;un<em&gt; In vitro</em&gt; Plateforme Ocular pour tester Lentilles de contact

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

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.

Amélioré l’Hydrogel 3D des Cultures de cellules gliales primaires pour In Vitro la modélisation du neuro-inflammation

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 Alginate/gélatine Hydrogel 3D In Vitro systèmes modèles induisent la Formation de sphéroïde de cellule

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