עבודה זו נתמכה על ידי קרן המחקר הלאומית סינגפור מנוהלת על ידי אחד ממרכזי המחקר שלה למצוינות, Mechanobiology המכון, סינגפור. MFAC נתמך על ידי הסוכנות למדע, טכנולוגיה ומחקר (סינגפור) והסוכנות הלאומית למחקר (צרפת) התכנית משותפת במסגרת פרויקט מספר 1122703037. BKKT נתמכת על ידי מכון Mechanobiology. אנו מודים למר דניאל HC וונג לכתב היד והגב &#39;שחר JH ניאו לסיוע בהפקת וידאו קריאת הוכחה.

1. הכנת פתרונות polyelectrolyte <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לטהר chitosan, כמפורט בet al ליאו. בקצרה, ליצור 1% (w / v) פתרון של chitosan ב 2% (V / V) מסנן חומצה אצטית וואקום באמצעות נייר סינון הכיתה 93. לנטרל את התסנין באמצעות 5M NaOH עד pH התייצב ל7. צנטריפוגה chitosan זירז ב1,200 XG במשך 10 דקות. למזוג supernatant ולהוסיף מים ללא יונים לשטוף chitosan. חזור על שלב צנטריפוגה ושטיפת פעמיים נוספות. להקפיא את chitosan זירז ב -80 ° C וLyophilize O / N להשיג הצורה המטוהרת. אחסן chitosan המטוהר בממשלה נטולת לחות. </li><li style=";text-align:right;direction:rtl"> לשקול את 1 גרם של chitosan מטוהר לתוך צלחת תרבית רקמת סטרילי. מניחים את chitosan בצלחת תרבית הרקמה הקרובה ככל האפשר למנורת UV בארון הבטיחות הביולוגית ולחשוף לאור UV במשך 15 דקות. בעזרת מלקחיים סטריליות, למקם את chitosan המעוקרים לתוך מיכל זכוכית. לפזר chitosan באמצעות מסונן 0.חומצה אצטית 15M לריכוז סופי בין 0.5% לבין 1% (w / v). </li><li style=";text-align:right;direction:rtl"> לשקול את 0.1 גרם של מלח נתרן של חומצת alginic ומתמוסס במים מזוקקים 10 מיליליטר deionized (DDI) כדי להשיג 1% (w / v) פתרון. מערבבים את מלח נתרן של חומצת alginic לפחות 2 שעות במיקסר המערבולת כדי להבטיח פירוק מלא. סנן את פתרון אלגינט דרך מסנן מזרק 0.2 מיקרומטר. אחסן את פתרון אלגינט על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> מחדש גורמים אנושיים רקומביננטי צמיחה כגון גורם כלי דם האנדותל צמיחה (VEGF) או ביתא - גורם גדילה עצבי (NGF), כפי שהומלץ על ידי יצרן. </li></ol> 2. ציור של סיבי IPC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לערבב חלבונים, גורמי צמיחה או מולקולות ביולוגיות אחרות ל10-20 aliquot μl של פתרון polyelectrolyte שיש מטען נטו דומה. מולקולות ביולוגיות עם מטען שלילי (למשל אלבומין בסרום שור [BSA]) נטו צריכה להיות מעורבות עם פתרון אלגינט. מולקולות ביולוגיות עם מטען חיובי נטו (למשל VEGF) צריכים להיות מעורבות עםפתרון chitosan. </li><li style=";text-align:right;direction:rtl"> הנח aliquots הקטן (10-20 μl) של chitosan ואלגינט על משטח שטוח ויציב שמכוסה parafilm. הטיפות של chitosan ואלגינט צריכה להיות ממוקמות בקרבה אך לא במגע אחד עם השני. </li><li style=";text-align:right;direction:rtl"> קל לטבול כל טיפ על זוג מלקחיים לטיפי chitosan ואלגינט. להביא את הטיפות של פוליאלקטרוליטים יחד על ידי צובט את המלקחיים. כאשר הטיפות באות במגע זה עם זה, לאט למשוך את המלקחיים אנכי כלפי מעלה לצייר סיבי IPC מהממשק של שתי טיפות (איור 1 א). </li><li style=";text-align:right;direction:rtl"> בזהירות במקום סוף סיבי IPC צוירו על המלקחיים על אספן, כגון פיגום פולימרים שטוח המודבק על mandrel מסתובב (ראה סעיף 3 ו -4). סובב את mandrel במהירות קבועה של 10 מ&quot;מ / שנייה כדי לאפשר היווצרות של מדים וbeadless סיבי IPC. להגדיל את המהירות של ציור סיבי IPC יהווה חרוזים, דבר שיגרום לשחרור פרץ של התאגדסיום סיבים ביוכימיים ומוקדם. 10 </li><li style=";text-align:right;direction:rtl"> כדי לקבוע את יעילות התאגדות, לאסוף את כל הנוזלים שנותרו נשארו על parafilm על ידי דילול עם 500 μl של פוספט 1X שנאגרו (PBS). מדוד את תוכן גורם חלבון או צמיחה בשאריות באמצעות assay BCA (לBSA), ELISA (לVEGF וNGF) או assay המתאים כדי לזהות biomolecule משולבת. </li></ol> 3. ייצור של Composite Hydrogel פיגום של pullulan-dextran (PD) פוליסכריד וסיבי IPC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בודה מסגרת pullulan ההקרבה לאוסף סיבי IPC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את פוליסכריד pullulan ולערבב עם מים מזוקקים deionized (DDI) כדי ליצור 20% (w נ /) תמיסה מימית. מערבבים את O / N פתרון pullulan כדי להבטיח אחידות. </li><li style=";text-align:right;direction:rtl"> יצוק 15 גרם של פתרון pullulan לפוליסטירן תרבית רקמה בקוטר 10 סנטימטרים צלחת (TCPS). ייבש את O פתרון pullulan / N על 37 מעלות צלזיוס. חותך את סרטי pullulan ל7מ&quot;מ x 7 מ&quot;מ מסגרות מרובעות. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנת פתרון פוליסכריד pullulan-dextran <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור 30% (w / v) פתרון של סוכרים pullulan וdextran (3: 1 יחס) במי DDI. מערבבים O / N כדי להבטיח אחידות של פתרון פוליסכריד. להוסיף לאט בסודה לשתייה לפתרון פוליסכריד כדי להשיג ריכוז סופי של 20% (w / v). מערבבים O / N כדי להבטיח אחידות של הפתרון. אחסן את פתרון פוליסכריד ב 4 מעלות צלזיוס. </li></ol></li><li style=";text-align:right;direction:rtl"> איסוף סיבי IPC במסגרת pullulan <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להדביק את מסגרת הקרבת pullulan (סעיף 3.1) באמצעות קליפ תנין. היצמד קליפ התנין ומסגרת pullulan בסוף mandrel מסתובב באמצעות דבק מצופה פלסטיק. סובב את mandrel עם המסגרת המודבקת במהירות קבועה של 10 מ&quot;מ / sec. מסגרת pullulan יכולה להיות מודבקת על mandrel מסתובב בכיוונים רצויים. </li></ol></li><li style=";text-align:right;direction:rtl"> צייר את סיבי IPC באמצעות זוג המלקחיים (סעיף 1) וATTACh הסוף נמשך של סיבי IPC על מסגרת pullulan מסתובבת. צייר את סיבי IPC במהירות קבועה. כשהגיע לסוף המסוף של סיבי IPC, לייבש את O מבנה סיבים-על-מסגרת / N ב RT. </li><li style=";text-align:right;direction:rtl"> הטבעת סיבי IPC לתוך פיגום הידרוג&#39;ל PD </li><li style=";text-align:right;direction:rtl"> לcrosslink כל גרם של פתרון pullulan-dextran, להוסיף 100 μl של 11% (w / v) נתרן trimetaphosphate תמיסה מימית ונתרן הידרוקסידי 10M 100 μl. 7 מערבבים את הפתרון ב 60 סל&quot;ד באמצעות stirplate 1 עד 2 דקות. לאחר התוספת של trimetaphosphate נתרן ונתרן הידרוקסידי, הפתרון רב-סוכר יהיה crosslink כמעט מייד. יוצקים את פתרון פוליסכריד צמיג על מבנה סיבים-על-מסגרת להטביע מלא סיבי IPC. דגירה סיבי pullulan-dextran-IPC המשולב (PD-IPC) בשעה 60 מעלות צלזיוס במשך 30 דקות כדי ליצור פיגומים כימיים crosslinked מרוכבים (איור 1). </li><li style=";text-align:right;direction:rtl"> זהירות: בצע צעד 3.3.2 במנדף ולהשתמש שוה מגן המתאימהpment כחומצה אצטית היא מאכל ודליק. </li><li style=";text-align:right;direction:rtl"> כדי לגרום להיווצרות נקבובית בפיגום PD-IPC, להטביע את כל הפיגום ב -20% (w / v) חומצה אצטית עבור 20 דקות. </li><li style=";text-align:right;direction:rtl"> הסר ריאגנטים unreacted ידי פיגומי PD-IPC כביסה ב1X PBS במשך 5 דקות בזמן שלוחץ על 100 סל&quot;ד. חזור על פעולה זו 2 פעמים. </li><li style=";text-align:right;direction:rtl"> הסר את PBS העודף ולהקפיא מייד את פיגומי PD-IPC ב -80 CO ° / N. Lyophilize הפיגומים לפחות 24 שעות לפני שימוש בכל מבחני שחרור או פעילות ביולוגית מבוקרים. </li></ol> 4. ייצור של Composite פיגום של PCL וסיבי IPC זהירות: dichloromethane היא חומר מסוכן. השתמש במנדף וציוד מגן אישי בעת טיפול dichloromethane. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יצירת מצעי PDMS טהורים ובדוגמת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור polydimethylsiloxane טהור (PDMS) מצע אלסטומרי באמצעות פיסת TCPS של ממד רצוי באמצעות תהליך ליתוגרפיה רך. צור pattePDMS rned מצעים באמצעות שיטות ליתוגרפי רכות סטנדרטיים בפולי (methacrylate מתיל) תבניות עם הטופוגרפיה הרצויה. 12 </li></ol></li><li style=";text-align:right;direction:rtl"> בודה מסגרת PCL ההקרבה לאוסף סיבי IPC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את PCL ולפזר בdichloromethane ליצור 0.9% (w / v) פתרון. לכל סנטימטר 2 אזור 1 של מצע PDMS, שחרר 500 μl של 0.9% פתרון PCL. תאפשר לכל ממס dichloromethane להתאדות באופן מלא במנדף. חזור על תהליך ליהוק PCL 0.9% כדי לעבות את הסרט לעובי הרצוי. הסר את סרט PCL המיובש מהמצע PDMS. ליצור חור במסגרת PCL באמצעות אגרופן מתאים בגודל. 2 </li></ol></li><li style=";text-align:right;direction:rtl"> איסוף סיבי IPC במסגרת PCL <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להדביק את מסגרת הקרבת PCL עם חור (מ4.2.1) בקליפ תנין. היצמד קליפ התנין על mandrel מסתובב באמצעות דבק מצופה פלסטיק. חבר את הקצה נמשך של סיבי IPC על fram PCLדואר לפני שמתחיל את הסיבוב במהירות קבועה של 10 מ&quot;מ / שנייה (סעיף 2). לאחר סיום ציור סיבי IPC, לייבש את C ° O מבנה סיבים במסגרת / N ב 4. </li></ol></li><li style=";text-align:right;direction:rtl"> הטבעת סיבים במסגרת לבנות לתוך מצע PCL דוגמת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זרוק 500 μl של 0.9% פתרון PCL על מצע PDMS ליצור בסיס PCL טהור או בדוגמת, כנדרש. יצוק מספר שכבות של 0.9% פתרון PCL להשיג פיגום עם העובי הרצוי. תאפשר לכל ממס dichloromethane להתאדות באופן מלא במנדף. </li><li style=";text-align:right;direction:rtl"> הנח את מבנה סיבים על-מסגרת (סעיף 4.3.1) על גבי בסיס PCL. להוסיף 0.9% פתרון PCL על מספר רב של פעמים בסיבים במסגרת לבנות כדי לקבל את העובי הרצוי ולהטביע באופן מלא סיבי IPC, בודה פיגום מרוכבים PCL-IPC (איור 1 ג). </li></ol></li></ol> 5. מדידה של שחרור הסוכנים ביולוגיים מComposite IPC פיגומים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח PD-IPC המורכב אופיגומי PCL-IPC ועצמאי סיבי IPC בנפרד בצלחת 24 גם. </li><li style=";text-align:right;direction:rtl"> לטבול את הפיגום ועצמאי סיבי IPC עם 500 μl של 1X PBS. דגירה הדגימות ב 37 מעלות צלזיוס. לאסוף PBS בנקודות זמן שונים (תקשורת שחרור) ולהחליף עם 500 μl של 1X PBS. </li><li style=";text-align:right;direction:rtl"> למדוד את כמות החלבון או גורם גדילה בתקשורת השחרור באמצעות assay BCA (BSA), ELISA (VEGF וNGF) או assay המתאים אחר כדי לחשב את פרופיל השחרור מצטבר לbiomolecule המשולב. </li></ol> 6. זריעה של תאים על פיגומים IPC Composite כדי לבדוק את הפעילות הביולוגית של גורמים ביולוגיים פורסם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעקר את הפיגומים מרוכבים lyophilized PD-IPC או PCL-IPC באמצעות אור UV בארון הבטיחות הביולוגי לפחות 20 דקות. </li><li style=";text-align:right;direction:rtl"> להשתמש בטכניקות תרבית תאים סטנדרטיים לקבל השעיה תא של 2 x 10 5 תאים ב200 תקשורת צמיחת μl. זרעי ההשעיה התא המרוכזת על הפיגומים המורכבים. After 20 דקות, למעלה למעלה הנפח של תקשורת צמיחה לצלול באופן מלא את הפיגומים. </li><li style=";text-align:right;direction:rtl"> למדוד תא פעילות באמצעות טכניקות סטנדרטיים כגון assay חילוף חומרים הכחולים Alamar פעילות, assay תולדת neurite PC12 או immunofluorescence. </li></ol>

<ol>
	<li>Annabi, N., Tamayol, A., 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=25th+Anniversary+Article:+Rational+design+and+applications+of+hydrogels+in+regenerative+medicine.">25th Anniversary Article: Rational design and applications of hydrogels in regenerative medicine.</a> Adv. Mater. 26 (1), 85-124 (2014).</li><li>Teo, B. K. K., Tan, G. D. S., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synergistic+effect+of+nanotopography+and+sustained+dual+release+of+hydrophobic+and+hydrophilic+neurotrophic+factors+on+human+mesenchymal+stem+cell+neuronal+lineage+commitment.">The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.</a> Tissue Eng. Part A. 20 (15-16), 2151-2161 (2014).</li><li>Lee, K., Silva, E. A., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+delivery-based+tissue+engineering:+general+approaches+and+a+review+of+recent+developments.">Growth factor delivery-based tissue engineering: general approaches and a review of recent developments.</a> J. R. Soc. Interface. 8 (55), 153-170 (2011).</li><li>Sun, Q., Chen, R. R., Shen, Y., Mooney, D. J., Rajagopalan, S., Grossman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+vascular+endothelial+growth+factor+delivery+enhances+angiogenesis+and+perfusion+in+ischemic+hind+limb.">Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb.</a> Pharm. Res. 22 (7), 1110-1116 (2005).</li><li>Rui, J., Dadsetan, 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=Controlled+release+of+vascular+endothelial+growth+factor+using+poly-lactic-co-glycolic+acid+microspheres:+in+vitro+characterization+and+application+in+polycaprolactone+fumarate+nerve+conduits.">Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: in vitro characterization and application in polycaprolactone fumarate nerve conduits.</a> Acta Biomater. 8 (2), 511-518 (2012).</li><li>King, T. W., Patrick, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+in+vitro+characterization+of+vascular+endothelial+growth+factor+(VEGF)-loaded+poly(DL-lactic-co-glycolic+acid)/poly(ethylene+glycol)+microspheres+using+a+solid+encapsulation/single+emulsion/solvent+extraction+technique.">Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique.</a> J. Biomed. Mater. Res. 51 (3), 383-390 (2000).</li><li>Cutiongco, M. F. A., Tan, M. H., Ng, M. Y. K., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+pullulan-dextran+polysaccharide+scaffold+with+interfacial+polyelectrolyte+complexation+fibers:+A+platform+with+enhanced+cell+interaction+and+spatial+distribution.">Composite pullulan-dextran polysaccharide scaffold with interfacial polyelectrolyte complexation fibers: A platform with enhanced cell interaction and spatial distribution.</a> Acta Biomater. 10 (10), 4410-4418 (2014).</li><li>Yow, S. Z., Quek, C. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-based+fibrous+scaffold+for+spatial+organization+of+encapsulated+and+seeded+human+mesenchymal+stem+cells.">Collagen-based fibrous scaffold for spatial organization of encapsulated and seeded human mesenchymal stem cells.</a> Biomaterials. 30 (6), 1133-1142 (2009).</li><li>Yim, E. K. F., Wan, A. C. A., Le Visage, C., Liao, I. C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+human+mesenchymal+stem+cell+encapsulated+in+polyelectrolyte+complexation+fibrous+scaffold.">Proliferation and differentiation of human mesenchymal stem cell encapsulated in polyelectrolyte complexation fibrous scaffold.</a> Biomaterials. 27 (36), 6111-6122 (2006).</li><li>Liao, I. C., Wan, A. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+from+fibers+of+polyelectrolyte+complexes.">Controlled release from fibers of polyelectrolyte complexes.</a> J. Control. Release. 104 (2), 347-358 (2005).</li><li>Wan, A. C. A., Liao, I. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+fiber+formation+by+interfacial+polyelectrolyte+complexation.">Mechanism of fiber formation by interfacial polyelectrolyte complexation.</a> Macromolecules. 37 (18), 7019-7025 (2004).</li><li>Yim, E. K. F., Reano, R., Pang, S., Yee, A., Chen, C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Sun, H., Xu, F., Guo, D., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+NGF-microsphere+conduits+for+regeneration+of+defective+nerves.">Preparation and evaluation of NGF-microsphere conduits for regeneration of defective nerves.</a> Neurol. Res. 34 (5), 491-497 (2012).</li><li>Sim&#243;n-Yarza, T., Formiga, F. R., Tamayo, E., Pelacho, B., Prosper, F., Blanco-Prieto, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated-PLGA+microparticles+containing+VEGF+for+long+term+drug+delivery.">PEGylated-PLGA microparticles containing VEGF for long term drug delivery.</a> Int. J. Pharm. 440 (1), 13-18 (2013).</li><li>Patel, Z. S., Ueda, H., Yamamoto, M., Tabata, Y., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+release+of+vascular+endothelial+growth+factor+from+gelatin+microparticles+and+biodegradable+composite+scaffolds.">In vitro and in vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds.</a> Pharm. Res. 25 (10), 2370-2378 (2008).</li><li>Sun, H., Xu, F., Guo, D., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+effects+of+various+additives+and+polymers+on+nerve+growth+factor+microspheres.">In vitro evaluation of the effects of various additives and polymers on nerve growth factor microspheres.</a> Drug Dev. Int. Pharm. 40 (4), 452-457 (2014).</li><li>Lee, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+drug+release+from+hydrogel+matrices.">Kinetics of drug release from hydrogel matrices.</a> J. Control. Release. 2, 277-288 (1985).</li><li>Cutiongco, M. F. A., Choo, R. K. T., Shen, N. J. X., Chua, B. M. X., Sju, E., Choo, A. W. L., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+scaffold+of+poly(vinyl+alcohol)+and+interfacial+polyelectrolyte+complexation+fibers+for+controlled+biomolecule+delivery.">Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.</a> Front. Bioeng. Biotechnol. 3 (3), 1-12 (2015).</li><li>Yow, S. Z., Lim, T. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Electroactive+Polypyrrole-Collagen+Fibrous+Scaffold+for+Tissue+Engineering.">A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering.</a> Polymers. 3 (1), 527-544 (2011).</li><li>Leong, M. F., Toh, J. K. 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=Patterned+prevascularised+tissue+constructs+by+assembly+of+polyelectrolyte+hydrogel+fibres.">Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres.</a> Nat. Commun. 4, 2353 (2013).</li><li>Di Benedetto, F., Biasco, A., Pisignano, D., Cingolani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+polyacrylamide+hydrogels+by+soft+lithography.">Patterning polyacrylamide hydrogels by soft lithography.</a> Nanotechnology. 16 (5), S165 (2005).</li><li>Revzin, A., Russell, R. J., 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=Fabrication+of+poly(ethylene+glycol)+hydrogel+microstructures+using+photolithography.">Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography.</a> Langmuir. 17 (18), 5440-5447 (2001).</li><li>Yim, E. K. F., Liao, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+compatibility+of+interfacial+polyelectrolyte+complexation+fibrous+scaffold:+evaluation+of+blood+compatibility+and+biocompatibility.">Tissue compatibility of interfacial polyelectrolyte complexation fibrous scaffold: evaluation of blood compatibility and biocompatibility.</a> Tissue Eng. Part A. 13 (2), 423-433 (2007).</li><li>Chaouat, M., Le Visage, C., Autissier, A., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+a+small-diameter+polysaccharide-based+arterial+graft+in+rats.">The evaluation of a small-diameter polysaccharide-based arterial graft in rats.</a> Biomaterials. 27, 5546-5553 (2006).</li><li>Eshraghi, S., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+microstructural+properties+of+polycaprolactone+scaffolds+with+one-dimensional,+two+dimensional,+and+three-dimensional+orthogonally+oriented+porous+architectures+produced+by+selective+laser+sintering.">Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering.</a> Acta Biomater. 6, 2467-2476 (2010).</li></ol>

The authors have nothing to disclose.

סיבי IPC נוצרים על ידי האינטראקציה של שני פוליאלקטרוליטים טעונים הפוך. התהליך מנצל את הפקת המורכבת מהממשק של פוליאלקטרוליטים, הקלת תהליך הרכבה עצמית להיווצרות סיבים יציבה. מנגנון היווצרות סיבי IPC מבטיח כי כל biomolecule הוסיפה לpolyelectrolyte טעון דומה יכול להיות משולב בתהליך comp...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pullulan&#160;</td>
			<td>Hayashibara Inc Okayama Japan</td>
			<td></td>
			<td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma Aldrich</td>
			<td>D1037</td>
			<td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Trimetaphosphate</td>
			<td>Sigma Aldrich</td>
			<td>T5508</td>
			<td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>448877</td>
			<td>MW 190-310 kDa. Acetylation degree of 75 to 85%. Purification of chitosan is required to create stable IPC fibers.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td></td>
			<td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&#160;</td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt from brown algae, low viscosity</td>
			<td>Sigma Aldrich</td>
			<td>A2158</td>
			<td>Dissolve in water overnight. Filter through sterile 0.2&#181;m syringe filter before use. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td></td>
			<td>Dissolve in sterile PBS and filter using 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>This kit was used to measure BSA release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Human Recombinant Vascular Endothelial Growth Factor</td>
			<td>R&#38;D systems</td>
			<td>293-VE</td>
			<td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt From Porcine</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&#160;</td>
		</tr>
		<tr>
			<td>ScanVac Coolsafe Lyophilizer</td>
			<td>Labogene</td>
			<td>7.001.200.060</td>
			<td>This is a non-programmable freeze dryer that operates at -105 to -110 &#176;C, This can be replaced by other standard lab lyophilizers.</td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL)</td>
			<td>Sigma Aldrich</td>
			<td>181609</td>
			<td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>V800151</td>
			<td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td>
		</tr>
		<tr>
			<td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td>
			<td>R&#38;D systems</td>
			<td>256-GF</td>
			<td>Reconstituted in sterile DI water to a final concentration of 100 &#181;g/ml. Aliquot and store in -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells (hMSC)</td>
			<td>Cambrex</td>
			<td></td>
			<td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td>
		</tr>
		<tr>
			<td>Rat PC12 Pheochromocytoma Cells&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td>
		</tr>
		<tr>
			<td>Grade 93 filter paper</td>
			<td>Whatman</td>
			<td>Z699675</td>
			<td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.</td>
		</tr>
		<tr>
			<td>Swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>To be used during the purification of chitosan using 1200 x g speed.&#160;</td>
		</tr>
		<tr>
			<td>Motor with mandrel rotating at constant speed</td>
			<td>Rhymebus</td>
			<td>RM5E</td>
			<td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>FirstBase</td>
			<td></td>
			<td>Sterilize through filtration (0.2 &#181;m filter) and autoclave.&#160;</td>
		</tr>
		<tr>
			<td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td>
			<td>Greiner</td>
			<td></td>
			<td>The TCPS dish is used for casting of pullulan frame.&#160;</td>
		</tr>
		<tr>
			<td>Human VEGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DVE00</td>
			<td>The ELISA kit is used for detection of VEGF in the release medium.</td>
		</tr>
		<tr>
			<td>Human NGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY256</td>
			<td>&#160;The ELISA kit is used for detection of NGF in the release medium.</td>
		</tr>
		<tr>
			<td>Plastic Coated Adhesive Tape</td>
			<td>Bel-Art</td>
			<td>9040336</td>
			<td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td>
		</tr>
	</tbody>
</table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

במאמר זה, אנו מבקשים ליצור פיגומים מורכבים עם סיבי IPC לשחרור המושהה של מולקולות ביולוגיות שונות. מאפיינים של מולקולות ביולוגיות המשמשות במחקר זה נמצאים בטבלה 1. סיבי IPC היו מוטבעים הראשונים להידרוג&#39;ל PD הידרופילי ליצור פיגום מרוכבים PD-IPC (איור 1). מול...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

פיגומים מורכבים של סיבי Interfacial polyelectrolyte לשחרור מבוקר באופן זמני של מולקולות ביולוגיות

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

composite-scaffolds-interfacial-polyelectrolyte-fibers-for-temporally

Research

JoVE Journal

Bioengineering

8.3K Views.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Video: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

הכנת 3D הפיברין פיגומים יישומים סלולריים גזע תרבות

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

ללא קשר, לייבל ללא ניטור של תאים תאי מטריקס באמצעות ספקטרוסקופיית ראמאן

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

פיגומים דיווח עצמי לתרבית תאים 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

הכנה של ממברנות אור תגובה על ידי הארכת משטח משולבת ותהליך Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

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

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

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

דור של עור בעובי מלא תלת ממדי Equivalent ואוטומטי פציעה

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

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

מוני Microgels לטין היברידי שדה-Responsive המגנטי לשחרור סמים מבוקר

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

קומפלקס polyelectrolyte למשלוח גורם הגדילה osteogenic דומיין כריכה הפרין

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal לב פיגומים: מטריצות Novel ללימודי הרגנרציה

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

הכנת סיבי חלבון תאי מטריקס עבור ספקטרוסקופיה ברילואן

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

מגנטי תרמי רגיש פולי (<em&gt; N</em&gt; -isopropylacrylamide) מבוססי microgels עבור מגנטית מופעל מבוקר שחרור

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...