עבודה זו נתמכה באופן חלקי מקופות הפעלה ממרכז הרפואי של אוניברסיטת נברסקה ומכון הלאומי לבריאות (מספר מענק 1R15 AR063901-01).

1. הכנת הפתרון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן פתרון של פולי (ε-caprolactone) (PCL) (M w = 80,000 g / mol) בריכוז של כ 100 מ&quot;ג / מיליליטר. לפזר PCL בתערובת של dichloromethane (DCM) וN, N-dimethlyformamide (DMF) ביחס של 4: 1 (V / V) בריכוז של 10% (w / v). </li><li style=";text-align:right;direction:rtl"> מניחים את הפתרון בצינור זכוכית 20 מיליליטר לערבוב. מניחים שפופרת זכוכית לתוך שואב קולי במשך 30 דקות, או עד שהפתרון הוא שקוף. </li></ol> 2. מכשירי הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מוסיף את פתרון PCL הכין לתוך מזרק 5 מיליליטר עם מחט קהה 21 מד המצורף. </li><li style=";text-align:right;direction:rtl"> מניחים משאבת מזרק בעמדת electrospinning אנכית, על פי איור 1. </li><li style=";text-align:right;direction:rtl"> השתמש אספן הפלדה אל-חלד עם פער חלל פתוח של 2 x סנטימטר 5 סנטימטרים למצע סיבים מיושרים. הנח את האספן 12 סנטימטר מקצה המחט. </li><li style=";text-align:right;direction:rtl"> חבר את הזרם ישר (DC) אספקת חשמל מתח הגבוה למחט וקרקע האספן. ודא שהאספן מעוגן בנפרד ללא קשר לציוד מעבדה האחר. </li></ol> 3. Electrospinning <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> משאבת Set מזרק ל1.50 מיליליטר / שעה, עד שהטיפין יוצרות בקצה המחט מוחלפות מייד כשהוסר. ואז, להגדיר את קצב הזרימה עד 0.50 מיליליטר / שעה. </li><li style=";text-align:right;direction:rtl"> הפעל מפעיל מתח kV 12. </li><li style=";text-align:right;direction:rtl"> Electrospin עד הסיבים מיושרים uniaxially לכסות לחלוטין את הפער באספן. </li><li style=";text-align:right;direction:rtl"> החל דבק לקצוות של צלחת זכוכית קטנה ולהעביר את הסיבים מאספן הפער לצלחת הזכוכית. מניחים את צלחת הזכוכית בחלק העליון של פיסת נייר אלומיניום שיש באותו גודל כמו צלחת הזכוכית ומעוגנת. </li><li style=";text-align:right;direction:rtl"> מקם את אספן המזרק השני על פי איור 3. </li><li style=";text-align:right;direction:rtl"> צרף את מסכת הפלסטיק למשאבה השנייה מזרק וזה 2 מ&quot;מ מעל עמדת האספן. </li><li style=";text-align:right;direction:rtl"> להוסיף Coumarin 6 ב 1% (w / w) לפתרון-אם נאן PCLoencapsulation הוא desired- ולערבב עד הפתרון הוא שקוף. </li><li style=";text-align:right;direction:rtl"> טען את PCL או פתרון PCL / Coumarin 6 למחט 5 מיליליטר עם מחט 21 מד בוטה. הגדר משאבה אנכית עד 0.50 מיליליטר / שעה ואופקית למשוך בשעה 9 מיליליטר / שעה או במהירות משוערת של 1 מ&quot;מ / דקה. </li><li style=";text-align:right;direction:rtl"> Electrospin עד המסכה עבר כמעט לחלוטין את האספן, אבל עם אזור הקצה עדיין מתחת למסכה. </li></ol> 4. סיבי אפיון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגימות מקום עם קלטת מוליך דו-צדדית להרבעה מתכתית ומעיל עם פלטינה במשך 40 שניות באמצעות coater גמגום בגיל 40 mA. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבחון סיבים באמצעות מיקרוסקופ אלקטרונים סורק (SEM) על פי המחקרים הקודמים שלנו 17,18. </li><li style=";text-align:right;direction:rtl"> לאסוף תמונות במתח מאיץ של 15 קילו וולט. </li></ol></li><li style=";text-align:right;direction:rtl"> לבצע ניתוח מהיר התמרה (FFT) על מדגם סיבים נפרד למדוד יישור סיבים. המידע מפורט על יישור סיבי מדידה על ידי FFT יכול להתייחס to מחקרים קודמים 19,20. </li></ol> 5. בתאי גזע זריעה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעקר את דגימות סיבים על ידי שריה בתמיסת אתנול 70% עבור שעה 2. לאחר מכן לשטוף את הסיבים עם מים מזוקקים כדי להסיר כל מזהמים. </li><li style=";text-align:right;direction:rtl"> להשיג ADSCs אנושי ותאי תרבות בבקבוק 2 25 סנטימטר על 37 מעלות צלזיוס באווירה של 95% CO אוויר / 5% 2. לשנות את מדיום תרבית תאים בכל יום אחר 10. </li><li style=";text-align:right;direction:rtl"> Trypsinize התאים ולספור את מספר התאים. באופן ספציפי, להסיר את כל מדיום התרבות מהבקבוק תרבית תאים ולשטוף את התאים עם ופר פוספט (PBS) פעמיים. לאחר מכן להוסיף l 1 מ &#39;של פתרון 0.25% טריפסין EDTA (טרום חם באמבט מים עד 37 ° C) כדי לכסות את monolayer תא דגירה הבקבוק בתרבית תאי החממה במשך 2-3 דקות. הוסף בינוני תרבות 4 מיליליטר ולשטוף את כל התאים מפני השטח על ידי pipetting הבינוני בכל רחבי השטח. ה צנטריפוגה ההשעיה התא ומחדש להתפזרתא גלולה דואר במדיום התרבות. ספירת תאים באמצעות hemacytometer. זרע סביב 1 × 10 4 תאים לפיגום nanofiber הניח בצלחת -culture 35 מ&quot;מ ודגירה של 3 ו -7 ימים. </li><li style=";text-align:right;direction:rtl"> תאי כתם באמצעות diacetate והעמסת (5 מ&quot;ג / מיליליטר באצטון) (FDA) ואז תמונה הדגימות באמצעות מיקרוסקופ פלואורסצנטי. באופן ספציפי, להוסיף של פתרון ה- FDA 100 μl למנת התרבות ודגירה למשך 30 דקות. לשטוף את התאים עם PBS שלוש פעמים לפני ההדמיה ניאון. לנתח כיוון תא על ידי תכנית מעבדה-מחצלת מותאמת אישית המבוססת על המחקרים הקודמים שלנו 10. </li></ol>

<ol>
	<li>Xie, J., Li, X., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+electrospun+nanofibers+to+work+for+biomedical+research.">Putting electrospun nanofibers to work for biomedical research.</a> Macromol. Rapid Commun. 29 (22), 1775-1792 (2008).</li><li>Genin, G. 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=Functional+grading+of+mineral+and+collagen+in+the+attachment+of+tendon+to+bone.">Functional grading of mineral and collagen in the attachment of tendon to bone.</a> Biophys. J. 97 (4), 976-985 (2009).</li><li>Thomopoulos, S., Marquez, J. P., Weinberger, B., Birman, V., Genin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+orientation+at+the+tendon+to+bone+insertion+and+its+influence+on+stress+concentrations.">Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations.</a> J. Biomech. 39 (10), 1842-1851 (2006).</li><li>Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+biomechanical,+structural,+and+compositional+properties+along+the+tendon+to+bone+insertion+site.">Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site.</a> J. Orthop. Res. 21 (3), 413-419 (2003).</li><li>Thomopoulos, S., Genin, G. M., Galatz, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+morphogenesis+of+the+tendon-to-bone+insertion+-+What+development+can+teach+us+about+healing.">The development and morphogenesis of the tendon-to-bone insertion - What development can teach us about healing.</a> Musculoskelet Neuronal Interact. 10 (1), 35-45 (2010).</li><li>Li, X., Xie, J., Lipner, J., Yuan, X., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+scaffolds+with+gradations+in+mineral+content+for+mimicking+the+tendon-to-bone+insertion+site.">Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site.</a> Nano Lett. 9 (7), 2763-2768 (2009).</li><li>Kunzler, T. P., Huwiler, C., Drobek, T., V&#246;r&#246;s, J., Spencer, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+study+of+osteoblast+response+to+nanotopography+by+means+of+nanoparticle-density+gradients.">Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients.</a> Biomaterials. 28 (33), 5000-5006 (2007).</li><li>Huwiler, C., Kunzler, T. P., Textor, M., V&#246;r&#246;s, J., Spencer, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalizable+nanomorphology+gradients+via+colloidal+self-assembly.">Functionalizable nanomorphology gradients via colloidal self-assembly.</a> Langmuir. 23 (11), 5929-5935 (2007).</li><li>Xie, 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='Aligned-to-random'+nanofiber+scaffolds+for+mimicking+the+structure+of+the+tendon-to-bone+insertion+site.">'Aligned-to-random' nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.</a> Nanoscale. 2 (6), 923-926 (2010).</li><li>Xie, J., Ma, B., Michael, P. L., Shuler, F. D. <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+nanofiber+scaffolds+with+gradations+in+fiber+organization+and+their+potential+applications.">Fabrication of nanofiber scaffolds with gradations in fiber organization and their potential applications.</a> Macromol. Biosci. 12 (10), 1336-1341 (2012).</li><li>James, R., Kumbar, S. G., Laurencin, C. T., Balian, G., Chhabra, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+tissue+engineering:+adipose-derived+stem+cell+and+GDF-5+mediated+regeneration+using+electrospun+matrix+systems.">Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.</a> Biomed. Mater. 6 (2), 025011 (2011).</li><li>Bodle, J. C., Hanson, A. D., Loboa, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+in+functional+bone+tissue+engineering:+lessons+from+bone+mechanobiology.">Adipose-derived stem cells in functional bone tissue engineering: lessons from bone mechanobiology.</a> Tissue Eng. Part B Rev. 17 (3), 195-211 (2011).</li><li>Lee, J. H., Rhie, J. W., Oh, D. Y., Ahn, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+human+adipose+tissue-derived+stromal+cells+(hASCs)+in+a+porous+three-dimensional+scaffold.">Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold.</a> Biochem. Biophys. Res. Commun. 370 (3), 456-460 (2008).</li><li>Tapp, H., Hanley, E. N., Patt, J. C., Gruber, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+characterization+and+current+application+in+orthopaedic+tissue+repair.">Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair.</a> Exp. Biol. Med. 234 (1), 1-9 (2009).</li><li>Gimble, J. M., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+adult+stem+cells:+isolation,+characterization,+and+differentiation+potential.">Adipose-derived adult stem cells: isolation, characterization, and differentiation potential.</a> Cytotherapy. 5 (5), 362-369 (2003).</li><li>Zuk, P. 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=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Xie, 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=The+differentiation+of+embryonic+stem+cells+seeded+on+electrospun+nanofibers+into+neural+lineages.">The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages.</a> Biomaterials. 30 (3), 354-362 (2009).</li><li>Xie, J., MacEwan, M. R., Li, X., Sakiyama-Elbert, S. E., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+on+nanofiber+scaffolds+with+different+orders,+structures,+and+surface+properties.">Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties.</a> ACS Nano. 3 (5), 1151-1159 (2009).</li><li>Ayres, 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=Modulation+of+anisotropy+in+electrospun+tissue+engineering+scaffolds:+analysis+of+fiber+alignment+by+the+fast+Fourier+transform.">Modulation of anisotropy in electrospun tissue engineering scaffolds: analysis of fiber alignment by the fast Fourier transform.</a> Biomaterials. 27 (32), 5524-5534 (2006).</li><li>Ayres, 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=Measuring+fiber+alignment+in+electrospun+scaffolds:+a+user&#8217;s+guide+to+the+2D+fast+Fourier+transform+approach.">Measuring fiber alignment in electrospun scaffolds: a user&#8217;s guide to the 2D fast Fourier transform approach.</a> J. Biomater. Sci. Poly. Ed. 19 (5), 603-621 (2008).</li></ol>

The authors have nothing to disclose.

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Fu...

Nanofibers הוא כלי פופולרי להנדסת רקמות בגלל היכולת שלהם לחקות את המטריצה ​​תאית במבנה שלה וגודלה היחסי 1. עם זאת, חלק מממשקים האם של הרקמה, כגון אתר הכנסת גיד לעצם, מכילים סיבי קולגן, אשר מפגינים מבנה ארגוני משתנה שמגדיל ביישור לגיד ופוחת באתר העצם 2-5. אז, לשחזור רקמות יעילות יש צורך לפברק פיגום שיכול ביעילות לחקות שיפוע מבני זו. מחקר בעבר, חל נערך על שינויים הדרגתיים בהרכב סיבים, במיוחד, תכולת מינרלים 6. עם זאת, יצירה מחדש של המרכיב המבני של רקמות חיבור נשארה נחקרה במידה רבה. מחקר מוקדם יותר בדק הדרגתיים מורפולוגיים על ידי לימוד ההשפעה של צפיפות חלקיקי סיליקה משטח על ההתפשטות של osteoblasts calvarial החולדה ומצא Inverמערכת יחסים בין se צפיפות חלקיקי סיליקה ותא התפשטות 7. אבל השינויים מורפולוגיים שתיווך שגשוג תאים בעבודה קודמת היו בעיקר קשורים למשטח חספוס חסר היכולת במחק שינויים ארגוניים סיבים 7,8. אחד מחקרים אחרונים ניסו לפברק פיגום שחיקה אוריינטציות קולגן סיבים הייחודיות באמצעות אספן חדשני לelectrospinning 9. בעוד מחקר זה הצליח לייצר פיגום עם סיבים שני מיושרים ואקראיים, היא לא הצליחה לחקות את השינויים הדרגתיים שהוצגו ברקמות המקומיות. כמו כן, בייצור רכיבים נפרדים, עם שינוי מיידי ממיושר לכיוון אקראי, המאפיינים ביו-המכאניים של פיגום זה ירד באופן משמעותי. אין עבודה קודמת הצליחה לייצר פיגומי nanofiber ישימים עם דרגות רציפות באורינטציות סיבים ממיושרים ואקראי. המחקר שנערך לאחרונה שלנו הראה בילוי מוצלח של פיגומי nanofiberעם דרגות בארגון סיבים שעלולים לחקות את הארגון האם של קולגן בגיד לעצם ההכנסה 10. עבודה זו מבקשת להציג את הפרוטוקולים המשמשים לייצור פיגומי nanofiber עם מבנה דומה מאוד לזו של ארגון סיבים בממשק רקמת גיד לעצם הילידים. מבני nanofiber Gradient יש פוטנציאל מרחיק לכת יישומים על פני מגוון רחב של תחומים. אנחנו מתמקדים ביישומים להנדסת רקמות של אתר הכנסת גיד לעצם על ידי שילוב של הפיגומים שלנו עם תאי שמקורם שומן גזע (ADSCs) שכבר מנוצלים לשחזור רקמות על מצעים שונים 11-14. בנוסף, ADSCs דומה מאוד באופיים לתאי גזע של מח עצם במונחים של multipotency והמשאבים שלהם הוא בשפע שניתן לקצור באמצעות 15,16 הליך שאיבת שומן פשוט. זריעת תאים אלה לפיגומי nanofiber מדורגים משפרת עוד יותר את tisלתבוע יישומים הנדסיים כך שהוא מאפשר להפצה מבוקרת של התאים שיכולים להתמיין לרקמות שעלולים להיות שונות. בנוסף לזריעת תאי גזע, יכול להיות במארז nanofibers עם מולקולות איתות לרגולציה של תגובה תאית. צימוד nanoencapsulation עם השיפוע הארגוני של פיגומים אלה מאפשר לחקר התנהגות סלולרית או עיצובי שתל אפשריים וציפויים. Encapsulation של מולקולות חלבון פונקציונליות כמו עצם המוךפו&quot;גנטי 2 (BMP2), אשר הוכח לגרום להתמיינות תא עצם 15,16, עוד יכול לשפר את יישומי הנדסת רקמות של פיגומים אלה 10.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone</td>
			<td>Sigma-Aldrich</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethlyformamide</td>
			<td>Fisher Chemical</td>
			<td>D-119-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Chemical</td>
			<td>AC61093-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 6</td>
			<td>Sigma-Aldrich</td>
			<td>546283</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipose Derived Stem Cells</td>
			<td>Cellular engineering Technologies</td>
			<td>HMSC.AD-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>26140-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Modified Eagle&#39;s Medium</td>
			<td>Invitrogen</td>
			<td>a10490-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>s25120a</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>VWR international, LLC</td>
			<td>101412-842</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td>Single syringe</td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Branson</td>
			<td>1510</td>
			<td></td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Gamma High Voltage Research</td>
			<td>ES30</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>FEI</td>
			<td>Nova 2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospun nanofiber scaffolds with gradations in fiber organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

השימוש בפרוטוקול זה, מחצלת סיבים עם שיפוע ארגוני הוקמה. איור 3 מציג את התמונות שצולמו SEM במקומות שונים על פיגום nanofiber. איכותי, שניתן לקבוע שיש התקדמות מהסיבים מיושרים uniaxially ב0 מ&quot;מ (איור 3 א) למגוון סיבים אקראי בשעה 6 מ&quot;מ (איור 3D). FFT נותן ערך ?...

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

כאן, אנו מציגים פרוטוקול לפברק פיגומי nanofiber electrospun עם ארגון מדורג של סיבים ולחקור את היישומים שלהם בויסות מורפולוגיה תא / אורינטציה. הדרגתיים בכל קשורים לתכונות פיסיקליות וכימיות של פיגומי nanofiber מציע מגוון רחב של יישומים בתחום ביו-רפואיים.

Electrospun nanofiber פיגומים עם דרגות בארגון סיבים

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Research

JoVE Journal

Bioengineering

9.7K Views.  University of Nebraska Medical Center.  כאן, אנו מציגים פרוטוקול לפברק פיגומי nanofiber electrospun עם ארגון מדורג של סיבים ולחקור את היישומים שלהם בויסות מורפולוגיה תא / אורינטציה. הדרגתיים בכל קשורים לתכונות פיסיקליות וכימיות של פיגומי nanofiber מציע מגוון רחב של יישומים בתחום ביו-רפואיים. 

Video: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS אלסטומרי פיגומים להנדסת רקמות העורקים

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction עיבוד Fibres Electrospun עבור הנדסת רקמות

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun סיבי פיגומים של פולי (גליצרול-dodecanedioate) להנדסה עצבית רקמות מתאי גזע עובריים בעכבר

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Repopulation תאי אפיתל והכנה של מכרסמים תאיים מטריקס פיגומים לפיתוח רקמות כליה

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

סינתזה של nanofiber קראטין מבוסס על הנדסה ביו רפואית

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

קליל ידידותי לסביבת כביש לפברק פולי (החומצה לקטית) פיגומים עם גודל מדורג נקבובי

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

להמיס Electrospinning הכתיבה של פיגומים Poly(ε-caprolactone) תלת מימדי עם מורפולוגיות לשליטה עבור יישומי הנדסת רקמות

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

ייצור ואפיון של סיבים Griffithsin-השתנה פיגומים לצורך מניעת מחלות המועברות

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Culturing בתרבית של תאים בפפטיד תלת מימדי פיגומים

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

ייצור של מטריקס הסחוס, נגזר Decellularized פיגומים

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...