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.

يجب أن تتم جميع الإجراءات الدم الرسم من قبل المتخصصين مرخصة ومعتمدة. استخدام الإنسان للبحث ينطوي على موافقة مجلس المراجعة المؤسسية أو سلطة مناسبة أخرى. احتياطات خاصة فيما يتعلق الموافقة المستنيرة وحماية الهوية مشارك يتعين أن يتبع. جميع التجارب الواردة في هذا البروتوكول تنطوي على معالجة الدم البشري و / أو منتجات الدم ومعدات الوقاية الشخصية المناسبة تحتاج إلى أن ترتديه في جميع الأوقات. ينبغي النظر في النفايات كما اقية والتخلص منها وفقا للوائح. 1. بزل الوريد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التعرف على المريض / مشارك وتأكيد مع السجلات الموجودة. شرح الدراسة في التفاصيل والحصول على الموافقة المسبقة. </li><li style=";text-align:right;direction:rtl"> هل لديك علبة إعداد للمريض مع أنابيب ملحوظ على سطح مستو وثابت. </li><li style=";text-align:right;direction:rtl"> شرح الإجراءات وجعل المقعد المريض بشكل مريح مع الذراع معتمد. إعلام المريض انه او SHوه يشعرون قرصة صغيرة ويجب أن تبقى لا تزال في جميع أنحاء الداخلي. </li><li style=";text-align:right;direction:rtl"> إرفاق الإبرة إلى محول. </li><li style=";text-align:right;direction:rtl"> غسل اليدين وارتداء القفازات. </li><li style=";text-align:right;direction:rtl"> تحضير الحفرة المرفقية لبزل الوريد عن طريق تنظيف مع 70٪ ايزوبروبيل في دوائر متحدة المركز من مركز إلى الخارج. يسمح الموقع لتجف في الهواء لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> تحديد الوريد المناسب بالجس. </li><li style=";text-align:right;direction:rtl"> تطبيق وقف النزف 3-4 في أعلى الموقع ثقب التأكد من أنها ليست ضيقة جدا (في حين لا يزال جس النبض الكعبري). </li><li style=";text-align:right;direction:rtl"> أداء بزل الوريد عن طريق إدخال شطبة من الإبرة 15-30 درجة إلى الجلد في الحركة السلسة واحد. دفع أنبوب جمع الدم من خلال إبرة وجمع 9 مل من الدم الكامل. </li><li style=";text-align:right;direction:rtl"> إزالة وقف النزف. إزالة أنبوب من الإبرة. </li><li style=";text-align:right;direction:rtl"> سحب الإبرة وتطبيق ساحة الشاش في موقع ثقب قبل إزالة الإبرة. التخلص الإبرة في وعاء واقية مناسبة. </li><li style=";text-align:right;direction:rtl"> نسأل المريض للحفاظ على العلاقات العامةessure في موقع ثقب. </li><li style=";text-align:right;direction:rtl"> تسمية أنابيب </li><li style=";text-align:right;direction:rtl"> التحقق من موقع ثقب للتأكد من توقف النزيف. </li><li style=";text-align:right;direction:rtl"> تطبيق ضمادة لاصقة أو الشريط على الساحة الشاش، أسأل ما إذا كان المريض يشعر بخير (لا أشعر بأي ألم، تورم أو الدوار الخفيف) </li><li style=";text-align:right;direction:rtl"> شكرا المريض / مشارك قبل تصريفها. </li></ol> 2. L-PRF إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مباشرة بعد جمع الدم الوريدي في الحمراء تصدرت أنبوب زجاجي جاف، وضعه في أجهزة الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في 400 x ج لمدة 12 دقيقة في RT بعد وضع توازن مناسب مضادة. </li><li style=";text-align:right;direction:rtl"> إزالة أنبوب في نهاية الدورة. لاحظ ثلاث طبقات: الصفائح الدموية الفقراء البلازما (PPP)،-الصفائح الدموية الغنية الفيبرين (L-PRF) وقاعدة RBC (الشكل 1). </li><li style=";text-align:right;direction:rtl"> نضح PPP باستخدام ماصة. باستخدام الملقط سحب بلطف L-PRF من ووضعه في العقيمة، مثقب شبكة معدنية. </li><li style=";text-align:right;direction:rtl"> باستخدام مشرط جراحي، كشط الجزء الأكبر من طبقة RBC ترك بعناية برتقاليذ معطف سليمة. </li><li style=";text-align:right;direction:rtl"> ضغط بلطف الجلطة L-PRF (باستخدام لوحة معدنية معقمة، الوزن التقريبي 225 غ) لمدة 30 ثانية. سوف يكون ضغط الصفائح الدموية البلازما الفقيرة. </li><li style=";text-align:right;direction:rtl"> إزالة لوحة ورفع بلطف الغشاء L-PRF. غشاء L-PRF جاهزا للاستخدام في التجارب 12. </li></ol> 3. أحادي المحور اختبار الشد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان الأغشية L-PRF (ن = 6) على ورق الترشيح لسهولة التعامل وكمة إلى &quot;عظام الكلب&quot; باستخدام يموت حسب الطلب المعدنية (2.75 ملم واسعة عند أضيق نقطة مع طول قياس 7.5 ملم). </li><li style=";text-align:right;direction:rtl"> قياس سماكة من كل عينة في ثلاث نقاط واتخاذ المتوسط. </li><li style=";text-align:right;direction:rtl"> المشاركة بعناية غشاء L-PRF في مركز السيطرة الفك من نظام الاختبار ذو محورين. </li><li style=";text-align:right;direction:rtl"> المسيل للدموع بعناية دعامة الورق المدون لفضح غشاء L-PRF. </li><li style=";text-align:right;direction:rtl"> برنامج الصك حتى أن رئيس متحرك يعمل بمعدل ثابت (10.0 مم / دقيقة) وتبدأ التجربة عندما L-PRF لا تزال رطبة. </li><li style=";text-align:right;direction:rtl"> تسجيل معامل المرونة والطاقة لكسر، والتوتر في استراحة من البرامج المصاحبة للاختبار نظام أحادي المحور. وتحسب هذه القيم تلقائيا ودون حاجة للمدخلات التي يحددها المستخدم. الرجاء انظر الشكل 3. </li></ol> 4. خياطة الاحتفاظ القوة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان الأغشية L-PRF (ن = 3) على ورقة مرشح لسهولة المناولة ومقطعة إلى عينات مستطيلة قياس (10 ملم × 25 ملم) باستخدام مشرط جراحي. </li><li style=";text-align:right;direction:rtl"> قياس سماكة من كل عينة (متوسط ​​3). </li><li style=";text-align:right;direction:rtl"> جعل ثقب صغير في وسط العينة باستخدام الفولاذ المقاوم للصدأ سلك رباط التقويمي (220 ميكرون في القطر). </li><li style=";text-align:right;direction:rtl"> تمرير سلك رباط من خلال الثقب لتشكيل حلقة واصلاحها لآلة اختبار الشد. ضع حافة الغشاء L-PRF إلى انخفاض قبضة الفك 13. </li><li style=";text-align:right;direction:rtl"> برنامج الصك حتى أن رئيس متحرك يعمل بمعدل ثابت (10 مم / دقيقة) وبدايةالتجربة. </li><li style=";text-align:right;direction:rtl"> تسجيل معامل المرونة والطاقة لكسر، والتوتر في استراحة من البرامج المصاحبة للاختبار نظام أحادي المحور. وتحسب هذه القيم تلقائيا ودون حاجة للمدخلات التي يحددها المستخدم. الرجاء انظر الشكل 3. </li></ol> 5. فحص الصرفي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد العينات L-PRF للفحص SEM باستخدام 10 ملم خزعة الجلد لكمة ووضعها في لوحة 24-جيدا. </li><li style=";text-align:right;direction:rtl"> غسل العينات مع PBS وإصلاح مع 2.5٪ غلوتارالدهيد (في PBS) لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> يذوى العينات عن طريق الغمر في زيادة بالتسلسل تركيزات الإيثانول (50٪، 70٪، 80٪، 90٪ و 100٪) لمدة 5 دقائق لكل منهما. </li><li style=";text-align:right;direction:rtl"> علاج مع 0.5 مل من HMDS٪ 100 (Hexamethyldisilazane) لمدة 5 دقائق. تهوية O / N لإزالة الزائدة HMDS 14. </li><li style=";text-align:right;direction:rtl"> جبل العينات على بذرة باستخدام الشريط على الوجهين، تفل معطف البلاتين لمدة 70 ثانية، وتدرس في المجهر الإلكتروني الماسح العاملة في لالتسارعeration الجهد 20 كيلو فولت (أو الإعداد المناسب). </li></ol> 6. Genipin يشابك من L-PRF، التربسين حساسية والنينهيدرين الفحص <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لإعداد genipin عبر ربط L-PRF، وشطف الأغشية مع برنامج تلفزيوني ونقع في 4 مل من 1٪ محلول genipin (في 70٪ من الإيثانول) لمدة 48 ساعة. شطف مع برنامج تلفزيوني قبل التجارب لإزالة الزائدة genipin 15، 16. </li><li style=";text-align:right;direction:rtl"> تقييم استقرار يشابك genipin من L-PRF التي كتبها مقاومته للتحلل بواسطة التربسين. المكان L-PRF غشاء (ن = 3) وgenipin crosslinked L-PRF في 500 ميكرولتر من 0.01٪ التربسين وحضنت عند 37 درجة مئوية لمدة 3 أيام مع التغيير اليومي من التربسين. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وزن العينات في يوم 1 قبل انزيم التعرض وفي اليوم 3. الفرق في البداية والنهاية الوزن يمثل تدهور الأنزيمية 17. </li></ol></li><li style=";text-align:right;direction:rtl"> قياس كمية من عبر ربط في genipin تعامل L-PRF (G-PRF) من خلال النينهيدرين الفحص. أولا إعداد المنحنيات القياسيةالبريد باستخدام الجلايسين (1 ملم 0،031 ملم) منحنى لتأسيس العلاقة بين تركيز الأمينية الحرة حمض (FAA) والامتصاصية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات الحرارة PRF مع 1 مل من 2٪ (ث / ت) النينهيدرين لمدة 15 دقيقة في 100 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> السماح للحل لتبريد لRT وإضافة 1.5 مل من 50٪ من الإيثانول. </li><li style=";text-align:right;direction:rtl"> تحليل الامتصاصية في 570 نانومتر باستخدام مقياس الطيف الضوئي مناسبة. </li><li style=";text-align:right;direction:rtl"> تحديد النسبة المئوية عبر ربط باستخدام صيغة أقل من 15 </li></ol></li></ol><img alt="المعادلة 1" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS تكاثر الخلايا الفحص <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو MC3T3 (الماوس preosteoblasts قبي) في الحد الأدنى الضروري المتوسطة -alpha تعديل (αMEM) في T-75 قوارير حتى يتم الحصول على متكدسة أحادي الطبقة 80٪. </li><li style=";text-align:right;direction:rtl"> إعداد جديدة، معقم L-PRF غشاء (فتح أنبوب L-PRF داخل الخلية هود الثقافة) ونقل الغشاء على طبق ثقافة الخلايا الجديدة. </li><li style=";text-align:right;direction:rtl"> وسائل الإعلام نضح من القارورة وشطف أحادي الطبقة مع برنامج تلفزيوني، إضافة 5 مل من 0.05٪ التربسين ووضع القارورة في C الحاضنة 37 درجة لمدة 5 دقائق، ماصة محتويات القارورة في أنبوب الطرد المركزي وأجهزة الطرد المركزي في 400 x ج لمدة 5 دقيقة. </li><li style=";text-align:right;direction:rtl"> صب طاف، بلطف الاستفادة من أنبوب لكسر بيليه الخلايا وإعادة تعليق مع 4 مل من MEM α جديدة، الاستغناء عن خليط من تعليق خلية (50 ميكرولتر) والأزرق التريبان (50 ميكرولتر) في عدادة الكريات وحساب عدد الخلايا </li><li style=";text-align:right;direction:rtl"> البذور 4x10 5 الخلايا في غضون 10 مم حلقات الزجاج الاستنساخ وضعت على رأس الأغشية L-PRF الإبقاء على الخلايا داخل الأغشية (حلقات يمكن إزالتها بعد 24 ساعة). </li><li style=";text-align:right;direction:rtl"> في يوم 4، وشطف يبني مع PBS ثلاث مرات لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من وسائل الإعلام الحرة المصل و 200 ميكرولتر MTS الكاشف إلى كل بئر واحتضان لمدة 2 ساعة على 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> قياس الامتصاصية من 200 مكل في 490 نانومتر. </li></ol>

<ol>
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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>

الكتاب ليس لديهم ما تكشف وتؤكد أنه لا توجد صراعات معروف في المصالح المرتبطة بهذا المنشور.

مركزات الصفائح الدموية ذاتي واعدة في مجال الطب التجديدي 18 بسبب وفرة من عوامل النمو. ومع ذلك، هذه الاستعدادات غالبا ما تفتقر إلى بنية المعرفة التي تجعل من التلاعب جراحية صعبة للغاية. وفي كثير من الأحيان، لا يتم الاحتفاظ المعلقات والمواد الهلامية على نحو فعال في ...

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 border="0" cellpadding="0" cellspacing="0">
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			<td>Needle 19G</td>
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			<td>Fisherbrand</td>
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			<td>BD</td>
			<td>8020129</td>
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			<td>Tyco</td>
			<td>5750</td>
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			<td>Johnson &#38; Johnson</td>
			<td>5005989</td>
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			<td>Surshield</td>
			<td>Terumo</td>
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			<td>BD</td>
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			<td>Intra-Lock International</td>
			<td>ISS110</td>
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			<td>Pipettes (Serological &#38; Micro)</td>
			<td>Corning</td>
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			<td>MTS Systems Corporation</td>
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			<td>MTS Systems Corporation</td>
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			<td>Koptec</td>
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			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Aldrich</td>
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			<td>PELCO Tabs</td>
			<td>16084-1</td>
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			<td>Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>LV 5610</td>
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			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
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		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>51026282</td>
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			<td>Gibco</td>
			<td>15240-062</td>
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			<td>Genipin</td>
			<td>Wako</td>
			<td>078-03021</td>
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		<tr>
			<td>Cell Culture Media</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>Minimum Essential Medium-Alpha</td>
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		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G1118</td>
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		<tr>
			<td>PMS Reagent</td>
			<td>Sigma</td>
			<td>P9625</td>
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			<td>Spectrophotometer</td>
			<td>BioTek</td>
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			<td>10mm Glass Cloning Rings</td>
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			<td>Corning</td>
			<td>430641</td>
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			<td>Corning</td>
			<td>55-031-PB</td>
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			<td>Ninhydrin 98%</td>
			<td>Aldrich</td>
			<td>454044</td>
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			<td>24 Well Plate</td>
			<td>Corning</td>
			<td>3987</td>
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		<tr>
			<td>Biopsy Punch</td>
			<td>Acu Punch</td>
			<td>P1025</td>
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			<td>Digital Micrometer</td>
			<td>Pittsburgh</td>
			<td>68305</td>
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			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
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			<td>12 Well Plate</td>
			<td>Corning</td>
			<td>3336</td>
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		<tr>
			<td>96 Well Plate</td>
			<td>Corning</td>
			<td>3596</td>
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</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.

مسح الإلكترون صورة المجهر من جلطة L-PRF في أقسام مختلفة (أعلى والمتوسطة والقاع) ويتضح طبقة في الشكل (2). كما يمكن أن يرى، ويتكون الجزء العلوي في الغالب من شبكة الليفين مع عدم وجود الخلايا. غير المخصب وطبقة وسطى مع الصفائح الدموية مع دليل على تفعيل وتحبب. ...

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.

توصيف خلايا الدم البيضاء، الصفائح الدموية الغنية الليفين، رواية بيولوجية

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

12.8K 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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

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

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

تغليف الخلايا العضلية في هيدروجيل الفايبرين للهندسة الأنسجة القلبية

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

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

نظام الحرير ثقافة السينما ل في المختبر تحليل وتصميم مادة بيولوجية

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

مراقبة وقياس حجم فيبرين جل ضغط الخلايا الليفية بوساطة

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

البناء وتوصيف من طية تفاعلات الاحيائية الرواية الصوتية

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

إعداد وتوصيف رواية هدل-محاكاة الجسيمات النانوية لتغليف عامل النمو العصبي

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

هندسة السطح من جزيرات البنكرياس مع نانوكواتينج ستاربيج هيبارينيزيد

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

تصنيع وتوصيف أشباح الأنسجة البصرية التي تتضمن حداتها

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

ميكروديسيكشن شرائح الأنسجة الكلوية الأولية، وإدماج مع تكنولوجيا بناء رواية خالية من السقالة

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

نموذج الجنين الزرد بالتصور فيفو و المرتبطة "تحليل إينترافيتال لمادة بيولوجية" المكوّرات العنقودية الذهبية العدوى

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.