Name: characterization of leukocyte-platelet rich fibrin, a novel biomaterial
Uploaded: 2015-09-29T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

צריכים להיעשות כל נהלי דם-הציור על ידי אנשי מקצוע מוסמכים ומאושרים. שימוש בבני אדם למחקר כרוך באישור מהמועצה לביקורת המוסדית או הסמכות מתאימה אחרת. אמצעי זהירות מיוחד לגבי הסכמה מדעת והגנה על זיהוי משתתף צריך להיות אחרי. כל הניסויים המפורטים בפרוטוקול זה כרוך טיפול בדם אדם ו / או מוצרי דם וציוד מגן אישי מתאים צריכים להיות משוחק בכל העת. הפסולת צריכה להיחשב כBiohazard ומסולק בהתאם לתקנות. 1. venipuncture <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה את המטופל / משתתף ואשר ברשומות קיימות. הסבר את המחקר בפירוט ולקבל הסכמה מדעת. </li><li style=";text-align:right;direction:rtl"> יש מגש להגדיר עבור מטופל עם צינורות מסומנים על משטח יציב שטוח. </li><li style=";text-align:right;direction:rtl"> הסבר את ההליך ולהפוך את מושב המטופל בנוחות עם הזרוע נתמכת. ליידע את החולה שהוא או SHדואר ירגיש צביטה קטנה וצריך להישאר עדיין בכל ההליך. </li><li style=";text-align:right;direction:rtl"> צרף את המחט למתאם. </li><li style=";text-align:right;direction:rtl"> שטוף את הידיים וללבוש כפפות. </li><li style=";text-align:right;direction:rtl"> הכן את גומץ antecubital לvenipuncture על ידי ניקוי עם 70% אלכוהול איזופרופיל במעגלים ממרכז החוצה. תאפשר לאתר לאוויר יבש למשך 30 שניות. </li><li style=";text-align:right;direction:rtl"> זהה את הווריד המתאים על ידי מישוש. </li><li style=";text-align:right;direction:rtl"> החל חוסם עורקים 3-4 במעל האתר לנקב לוודא שזה לא חזק מדי (ועדיין מרגיש את דופק רדיאלי). </li><li style=";text-align:right;direction:rtl"> בצע venipuncture ידי הוספת השיפוע של 15-30 מעלות המחט לעור בתנועה אחת חלקה. דחוף את צינור איסוף דם דרך המחט ולאסוף 9 מיליליטר של דם מלא. </li><li style=";text-align:right;direction:rtl"> הסר את חוסם עורקים. הסר את הצינור מהמחט. </li><li style=";text-align:right;direction:rtl"> למשוך את המחט ולהחיל את כיכר הגזה באתר לנקב לפני הסרת מחט. השלך את המחט במכל Biohazard מתאים. </li><li style=";text-align:right;direction:rtl"> שאל מטופל לשמור על יחסי ציבורessure באתר לנקב. </li><li style=";text-align:right;direction:rtl"> תווית הצינורות </li><li style=";text-align:right;direction:rtl"> בדוק את האתר לנקב כדי להיות בטוח דימום הפסק. </li><li style=";text-align:right;direction:rtl"> החל תחבושת או דבק מעל כיכר הגזה, לשאול אם המטופל מרגיש בסדר (אין כאב, נפיחות או קור אור) </li><li style=";text-align:right;direction:rtl"> תודה המטופל / משתתף קודם לתחילת הזרמה. </li></ol> 2. L-PRF הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מייד לאחר איסוף דם ורידים בשפופרת הזכוכית יבשה מצופה האדומה, למקם אותו בצנטריפוגה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 400 XG במשך 12 דקות ב RT לאחר הצבת איזון נגדי מתאים. </li><li style=";text-align:right;direction:rtl"> הסר את הצינור בסוף המחזור. שים לב לשלוש שכבות: הפלזמה טסיות-העני, הפיברין טסיות דם עשיר (L-PRF) ובסיס RBC (איור 1) (PPP). </li><li style=";text-align:right;direction:rtl"> לשאוב PPP בעזרת פיפטה. פינצטה שימוש משוך בעדינות L-PRF החוצה ולשים אותו ברשת מתכת סטרילי, מחוררת. </li><li style=";text-align:right;direction:rtl"> באמצעות אזמל כירורגי, לגרד את חלק הארי של שכבת RBC משאיר חובב בזהירותמעיל y ללא פגע. </li><li style=";text-align:right;direction:rtl"> בעדינות לדחוס את קריש L-PRF (באמצעות לוחית מתכת סטרילי, גרם משקל משוער 225) למשך 30 שניות. פלזמה עניה טסיות דם תהיה סחטה. </li><li style=";text-align:right;direction:rtl"> הסר את הצלחת ועדינות להרים את קרום L-PRF. קרום L-PRF מוכן לשימוש בניסויים 12. </li></ol> 3. uniaxial מתיחה בדיקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קרומי מקום L-PRF (n = 6) על נייר סינון כדי להקל על טיפול ואגרוף ל&quot; עצמות כלב &quot;באמצעות מחוייט מתכת מת (2.75 מ&quot;מ רחב בנקודה הצרה שלהם באורך של 7.5 מ&quot;מ מד). </li><li style=";text-align:right;direction:rtl"> למדוד את העובי של כל דגימה בשלוש נקודות ולקחת את הממוצע. </li><li style=";text-align:right;direction:rtl"> לעסוק בזהירות את קרום L-PRF במרכז אוחז הלסת של מערכת בדיקות uniaxial. </li><li style=";text-align:right;direction:rtl"> לקרוע בזהירות את תמיכת נייר פילר לחשוף את קרום L-PRF. </li><li style=";text-align:right;direction:rtl"> תכנית המכשיר כך שראש מטלטלין פועל בקצב קבוע (10.0 מ&quot;מ / דקה) ולהתחיל את הניסוי כאשר L-PRF הוא עדיין רטוב. </li><li style=";text-align:right;direction:rtl"> רשום את מודולוס האלסטיות, אנרגיה לשבור, ומתח בהפסקה מהתוכנה המלווה את מערכת בדיקת uniaxial. ערכים אלו מחושבים באופן אוטומטי ולא קלט משתמש מוגדר נדרש. אנא ראה איור 3. </li></ol> כוח 4. תפר שייר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קרומי מקום L-PRF (n = 3) על נייר סינון כדי להקל על טיפול וחותך לדגימות מלבניות מדידה (10 מ&quot;מ x 25 מ&quot;מ) באמצעות אזמל כירורגי. </li><li style=";text-align:right;direction:rtl"> למדוד את העובי של כל דגימה (ממוצע של 3). </li><li style=";text-align:right;direction:rtl"> הפוך חריר במרכז המדגם באמצעות חוט הנירוסטה אורתודונטי מייתר (220 מיקרומטר בקוטר). </li><li style=";text-align:right;direction:rtl"> להעביר את חוט הקשירה דרך חריר כדי ליצור לולאה ולתקן אותה למכונת בדיקת מתיחה. הנח את הקצה של קרום L-PRF לאחיזת הלסת התחתונה 13. </li><li style=";text-align:right;direction:rtl"> לתכנת את המכשיר כך שראש מטלטלין פועל בקצב קבוע (10 מ&quot;מ / דקה) והתחלההניסוי. </li><li style=";text-align:right;direction:rtl"> רשום את מודולוס האלסטיות, אנרגיה לשבור, ומתח בהפסקה מהתוכנה המלווה את מערכת בדיקת uniaxial. ערכים אלו מחושבים באופן אוטומטי ולא קלט משתמש מוגדר נדרש. אנא ראה איור 3. </li></ol> 5. מורפולוגי בחינה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את דגימות L-PRF לבדיקת SEM באמצעות אגרוף ביופסית עורי 10 מ&quot;מ ומניח אותם בצלחת 24 גם. </li><li style=";text-align:right;direction:rtl"> שטוף את הדגימות עם PBS ולתקן עם glutaraldehyde 2.5% (PBS) במשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> מייבש את הדגימות על ידי טבילה ברצף הגדלת ריכוזים של אתנול (50%, 70%, 80%, 90% ו -100%) במשך 5 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> פנק עם 0.5 מיליליטר של 100% HMDS (Hexamethyldisilazane) במשך 5 דקות. לאוורר O / N כדי להסיר את עודפי 14 HMDS. </li><li style=";text-align:right;direction:rtl"> דגימות הר על ספחים באמצעות קלטת דו צדדית, גמגום-מעיל פלטינה במשך 70 שניות ולבחון במיקרוסקופ אלקטרונים סורק הפועלות באקסלמתח eration של 20 קילו וולט (או הגדרה מתאימה). </li></ol> 6. Genipin Crosslinking של L-PRF, טריפסין רגישות וNinhydrin Assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להכין L-PRF צולבים genipin, לשטוף ממברנות עם PBS ולספוג ב 4 מיליליטר של 1% פתרון genipin (באתנול 70%) במשך 48 שעות. לשטוף עם PBS לפני ניסויים כדי להסיר genipin העודף 15, 16. </li><li style=";text-align:right;direction:rtl"> להעריך את היציבות של crosslinking genipin של L-PRF על ידי התנגדותה לשפלה על ידי טריפסין. קרום מקום L-PRF (n = 3) וgenipin L-PRF crosslinked ב500 μl של טריפסין 0.01% וטופחו על 37 מעלות צלזיוס במשך 3 ימים עם שינוי יומי של טריפסין. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול דגימות ביום 1 לפני אנזים חשיפה וביום 3. ההבדל בהתחלה וסיום במשקל מייצג השפלה האנזימטית 17. </li></ol></li><li style=";text-align:right;direction:rtl"> לכמת את כמות cross-linking בgenipin טופל L-PRF (G-PRF) על ידי ninhydrin assay. ראשית להכין הכמתמ&quot;כ הסטנדרטיעקומת דואר באמצעות גליצין (1 מ&quot;מ 0.031 מ&quot;מ) כדי לקבוע את הקשר בין ריכוז חומצת אמינו החופשי (FAA) וספיגה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגימות החום PRF עם 1 מיליליטר של 2% (w / v) ninhydrin במשך 15 דקות ב 100 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> אפשר הפתרון להתקרר לRT ולהוסיף 1.5 מיליליטר של 50% אתנול. </li><li style=";text-align:right;direction:rtl"> לנתח את הספיגה ב 570 ננומטר באמצעות ספקטרופוטומטר מתאים. </li><li style=";text-align:right;direction:rtl"> לקבוע אחוז cross-linking השתמש בנוסחא הבאה 15 </li></ol></li></ol><img alt="משוואת 1" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. Assay התפשטות התאים MTS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול MC3T3 (עכבר preosteoblasts calvarial) בשינוי -alpha בינוני חיוני מינימאלי (αMEM) בT-75 צלוחיות עד monolayer ומחוברות 80% מתקבלת. </li><li style=";text-align:right;direction:rtl"> הכן קרום טרי, סטרילי L-PRF (לפתוח את צינור L-PRF בתוך מכסה המנוע תרבית תאים) ולהעביר את הקרום על צלחת תרבית תאים חדשה. </li><li style=";text-align:right;direction:rtl"> תקשורת לשאוב מהבקבוק ולשטוף את monolayer עם PBS, להוסיף 5 מיליליטר של 0.05% טריפסין ומניח את הבקבוק בC חממת 37 o במשך 5 דקות, פיפטה תכולת הבקבוק לתוך צינור צנטריפוגות ו צנטריפוגות ב 400 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> למזוג supernatant, לטפוח בעדינות את הצינור כדי לשבור את התא גלולה מחדש להשעות עם 4 מיליליטר של ממ α הטרי, לוותר תערובת של השעיה תא (50 μl) וtrypan כחול (50 μl) לhemocytometer ולספור את מספר תאים </li><li style=";text-align:right;direction:rtl"> זרעי 4x10 5 תאים בתוך טבעות שיבוט זכוכית 10 מ&quot;מ מונחות על גבי קרומי L-PRF כדי לשמר את התאים בתוך ממברנות (ניתן להסיר טבעות אחרי 24 שעות). </li><li style=";text-align:right;direction:rtl"> ביום 4, לשטוף בונה עם שלוש פעמים PBS במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף 1 מיליליטר של תקשורת חופשית בסרום ומגיב MTS 200 μl לכל היטב דגירה עבור שעה 2 על 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> למדוד הספיגה בין 200 aliquots μl ב 490 ננומטר. </li></ol>

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Part II: platelet-related biologic features.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e45-e50 (2006).</li><li>Choukroun, 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=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+V:+histologic+evaluations+of+PRF+effects+on+bone+allograft+maturation+in+sinus+lift.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), 299-303 (2006).</li><li>Dohan Ehrenfest, D. M., Bielecki, T., 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=Do+the+Fibrin+Architecture+and+Leukocyte+Content+Influence+the+Growth+Factor+Release+of+Platelet+Concentrates?+An+Evidence-based+Answer+Comparing+a+Pure+Platelet-Rich+Plasma+(P-PRP)+Gel+and+a+Leukocyte-+and+Platelet-Rich+Fibrin+(L-PRF).">Do the Fibrin Architecture and Leukocyte Content Influence the Growth Factor Release of Platelet Concentrates? An Evidence-based Answer Comparing a Pure Platelet-Rich Plasma (P-PRP) Gel and a Leukocyte- and Platelet-Rich Fibrin (L-PRF).</a> Curr Pharma Biotechno. 13 (7), 1145-1152 (2012).</li><li>Dohan Ehrenfest, D. M., Rasmusson, L., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+platelet+concentrates:+from+pure+platelet-rich+plasma+(P-PRP)+to+leucocyte-+and+platelet-rich+fibrin+(L-PRF).">Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).</a> Trends Biotechno. 27 (3), 158-167 (2009).</li><li>Mine, Y., 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=Suture+Retention+Strength+of+Expanded+Polytetrafluoroethylene+(ePTFE)+Graft.">Suture Retention Strength of Expanded Polytetrafluoroethylene (ePTFE) Graft.</a> Acta Med Okayam. 64 (2), 121-128 (2010).</li><li>Braet, F., De Zanger, R., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drying+cells+for+SEM+,+AFM+and+TEM+by+hexamethyldisilazane:+a+study+on+hepatic+endothelial+cells.">Drying cells for SEM , AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.</a> J Micros. 186 (1), 84-87 (1997).</li><li>Yuan, Y., 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+effect+of+cross-linking+of+chitosan+microspheres+with+genipin+on+protein+release.">The effect of cross-linking of chitosan microspheres with genipin on protein release.</a> Carbohydr Poly. 68 (3), 561-567 (2007).</li><li>Sell, S. 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=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., 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=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. The Role of Platelet Gel in Regenerative Medicine.</a> Advances In Regenerative Medicine. , 319-349 (2011).</li><li>Dohan Ehrenfest, ., Lemo, D. M., Jimbo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+a+relevant+animal+model+for+testing+the+in+vivo+effects+of+Choukroun&#8217;s+platelet-rich+fibrin+(PRF):+Rabbit+tricks+and+traps.">Selecting a relevant animal model for testing the in vivo effects of Choukroun&#8217;s platelet-rich fibrin (PRF): Rabbit tricks and traps.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110 (4), 413-416 (2010).</li><li>Guilak, F., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+Ten+more+years+of+progress.">Functional tissue engineering: Ten more years of progress.</a> J Biomec. 47 (9), 1931-1932 (2014).</li><li>Guilak, F., Butler, D. L., Goldstein, S. A., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+mechanobiology+in+functional+tissue+engineering.">Biomechanics and mechanobiology in functional tissue engineering.</a> J Biomec. 47 (9), 1933-1940 (2014).</li><li>Collet, J. P., Shuman, H., Ledger, R. E., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+an+individual+fibrin+fiber+in+a+clot.">The elasticity of an individual fibrin fiber in a clot.</a> Proc Natl Acad Sci. 102 (26), 9133-9137 (2005).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+fibers+have+extraordinary+extensibility+and+elasticity.">Fibrin fibers have extraordinary extensibility and elasticity.</a> Science. 313 (5787), 634 (2006).</li><li>American National Standard. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8:+Test+methods+for+vascular+prostheses.">Chapter 8: Test methods for vascular prostheses.</a> Association for the Advancement of Medica lnstrumentation Guidance document: Cardiovascular Implants - Tubular vascular prostheses. , 33-34 (2001).</li></ol>

יש המחברים אין לחשוף ולאשר שאין התנגשויות ידועות של עניין הקשורים בפרסום זה.

תרכיזי טסיות אוטולוגית מבטיחים בתחום של רפואת רגנרטיבית 18 בגלל השפע של גורמי גדילה. עם זאת, הכנות אלה לעתים קרובות חסרי מבנה מוגדר שעושה מניפולציה כירורגית קשה מאוד. פעמים רבות, ההשעיות וג&#39;לים אינן נשמרים בצורה יעילה באתר של משלוח, וכתוצאה מכך תוצאות בלתי צפו?...

The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4. 
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used. 
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.

<table><tbody><tr><td>Needle 19G</td><td>BD</td><td>305186</td><td></td></tr><tr><td>Needle Disposal Container</td><td>Fisherbrand</td><td>14-827-122</td><td></td></tr><tr><td>Red-Topped Glass Collection Tube</td><td>BD</td><td>8020129</td><td></td></tr><tr><td>Gauze Pads</td><td>Tyco</td><td>5750</td><td></td></tr><tr><td>Bandage</td><td>Johnson &amp;amp; Johnson</td><td>5005989</td><td></td></tr><tr><td>Surshield</td><td>Terumo</td><td>SV*S19BL</td><td>Safety winged infusion set</td></tr><tr><td>Blood Collection Assembly</td><td>BD</td><td>303380</td><td></td></tr><tr><td>Tourniquets</td><td>BD</td><td>367203</td><td></td></tr><tr><td>Brand Luer Adapter</td><td>Vacutainer</td><td>L42179</td><td></td></tr><tr><td>Intra-Spin System&amp;nbsp;</td><td>Intra-Lock International</td><td>ISS110</td><td>Centrifuge and Xpression L-PRF FabricationKit&amp;nbsp;</td></tr><tr><td>Pipettes (Serological &amp;amp; Micro)</td><td>Corning</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Exelint</td><td>29552</td><td></td></tr><tr><td>MTS Bionix 200</td><td>MTS Systems Corporation</td><td></td><td>Material testing systems</td></tr><tr><td>MTS Test Works 4</td><td>MTS Systems Corporation</td><td></td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Whatman</td><td>1004 070</td><td></td></tr><tr><td>SS Orthodontic ligature wire</td><td>Patterson Dental</td><td>628-4228</td><td></td></tr><tr><td>200 Proof Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Aldrich</td><td>440191</td><td></td></tr><tr><td>Aluminium Mounting Stubs</td><td>Ted Pella</td><td>16324</td><td></td></tr><tr><td>Double Sided Carbon Tape</td><td>PELCO Tabs</td><td>16084-1</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>JEOL</td><td>LV 5610</td><td></td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH30042.01</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo Fisher Scientific Inc</td><td>51026282</td><td></td></tr><tr><td>Antibiotic-Antimicotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Genipin</td><td>Wako</td><td>078-03021</td><td></td></tr><tr><td>Cell Culture Media</td><td>Gibco</td><td>12000-022</td><td>Minimum Essential Medium-Alpha</td></tr><tr><td>MTS Reagent</td><td>Promega</td><td>G1118</td><td></td></tr><tr><td>PMS Reagent</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>Spectrophotometer</td><td>BioTek</td><td></td><td>Epoch Spectrophotometer</td></tr><tr><td>10mm Glass Cloning Rings</td><td>Corning</td><td>3166-10</td><td></td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>DPBS</td><td>Corning</td><td>55-031-PB</td><td></td></tr><tr><td>Ninhydrin 98%</td><td>Aldrich</td><td>454044</td><td></td></tr><tr><td>24 Well Plate</td><td>Corning</td><td>3987</td><td></td></tr><tr><td>Biopsy Punch</td><td>Acu Punch</td><td>P1025</td><td></td></tr><tr><td>Digital Micrometer</td><td>Pittsburgh</td><td>68305</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>12 Well Plate</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>96 Well Plate</td><td>Corning</td><td>3596</td><td></td></tr></tbody></table>

characterization of leukocyte-platelet rich fibrin, a novel biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

תמונת מיקרוסקופ האלקטרונים הסורקת של קריש L-PRF בחלקים שונים (למעלה, אמצעית ותחתון) שכבה מתוארת באיור 2. כפי שניתן לראות, החלק העליון מורכבת בעיקר של רשת הפיברין ללא תאים. השכבה האמצעית מועשרת בטסיות דם עם ראיות של ההפעלה וdegranulation. יש השכבה התחתונה תערובת של כדו?...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

אפיון של יקוציט-טסיות העשיר הפיברין, רומן ביולוגי

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.9K Views.  School of Dentistry, Virginia Commonwealth University. Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. 

Video: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

הנדסת Hydrogel Bilayered לשלוט בידול ASC

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP כגישה חדשה למניעת זיהום: הכנה ו<em&gt; במבחנה</em&gt; מאפיינים מיקרוביאלית של PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Microfluidic זרימה צ&#39;יימברס שימוש מחדש דם למודל המוסטאסיס ואת טסיות עירוי<em&gt; במבחנה</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

כימיה רטובה פפטיד קיבוע על polytetrafluoroethylene עבור Cell-הדבקה משופרת

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

מודל לשכת תזרים Microfluidic עבור עירוי טסיות המוסטאסיס מודד הפקדת טסיות והיווצרות הפיברין בזמן אמת

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

מבחני מבוסס זרימה שכבתית לחקור ליקוציט גיוס על תאים בתרבית וסקולרית, טסיות חסיד

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

מי כלוריד-induced Ferric מודלים פקקת Murine

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Medicine

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Live-cell הדמיה של degranulation טסיות והפרשה תחת זרימה

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Biology

<meta charset="utf8"></head><body>מקטע כלי דם מכני סטרומה בפיברין הידרוג'ל לרפואה רגנרטיבית

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

<meta charset="utf8"></head><body>מחבר זרקור: חקר הפוטנציאל של מקטע כלי דם סטרומה שמקורו בשומן לריפוי פצעים

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

השילוב של מקטע כלי דם סטרומה מבודד מכנית והידרוג'ל פיברין: פרוטוקול עיבוד

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

אפיון של יקוציט-טסיות העשיר הפיברין, רומן ביולוגי

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Chapters in this video

הנדסת Hydrogel Bilayered לשלוט בידול ASC

PRP כגישה חדשה למניעת זיהום: הכנה ו

Microfluidic זרימה צ'יימברס שימוש מחדש דם למודל המוסטאסיס ואת טסיות עירוי

כימיה רטובה פפטיד קיבוע על polytetrafluoroethylene עבור Cell-הדבקה משופרת

מודל לשכת תזרים Microfluidic עבור עירוי טסיות המוסטאסיס מודד הפקדת טסיות והיווצרות הפיברין בזמן אמת

מבחני מבוסס זרימה שכבתית לחקור ליקוציט גיוס על תאים בתרבית וסקולרית, טסיות חסיד

מי כלוריד-induced Ferric מודלים פקקת Murine

Live-cell הדמיה של degranulation טסיות והפרשה תחת זרימה

השילוב של מקטע כלי דם סטרומה מבודד מכנית והידרוג'ל פיברין: פרוטוקול עיבוד

השילוב של מקטע כלי דם סטרומה מבודד מכנית והידרוג'ל פיברין: פרוטוקול עיבוד