Name: wet-spinning-based molding process of gelatin for tissue regeneration
Uploaded: 2019-03-07T14:15:00
Description: Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Bu çalışmada Milli Savunma Bakanlığı tarafından (MAB-105-070; desteklenmiştir MAB-106-077; MAB-107-032; Mab-107-065), Bakanlığı bilim ve Teknoloji (çoğu 107-2320-B016-016), Tri-hizmet Hastanesi'ne, ulusal savunma Tıp Merkezi, Tayvan (TSGH-C106-046; TSGH-C106-115; TSGH-C107-041) ve Cheng-Hsin General Hospital ve ulusal savunma Tıp Merkezi işbirliği (CH-NDMC-107-8).

Yağlı doku ortopedik ameliyatlar kurumsal inceleme kurulu, Tri-hizmet genel hastane tarafından sertifikalı olarak elde edilmiştir, Taipei, Tayvan, R.O.C. yordamlar hayvan konular içeren ulusal, hayvan bakımı Komitesi tarafından onaylanmış olan Savunma Tıp Merkezi, Tayvan (R.O.C).
1. işlem iplik ıslak
<ol><li>
Çözüm hazırlık
	<ol>
<li>5 g jelatin tozu %5 (w/v) çözüm konsantrasyonu elde etmek için çift distile su 100 ml geçiyoruz.</li>
		<li>Karışım...

<ol>
	<li>Bagnall, A. M., Jones, L., Duffy, S., Riemsima, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+fixation+surgery+for+acute+traumatic+spinal+cord+injury.">Spinal fixation surgery for acute traumatic spinal cord injury.</a> Cochrane Database of Systematic Reviews. 1, 004725 (2008).</li><li>Fehlings, M. G., Perrin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+timing+of+early+decompression+for+cervical+spinal+cord+injury:+update+with+a+review+of+recent+clinical+evidence.">The role and timing of early decompression for cervical spinal cord injury: update with a review of recent clinical evidence.</a> Injury. 36, 13-26 (2005).</li><li>Yang, L., Jones, N. R., Stoodley, M. A., Blumbergs, P. C., Brown, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+model+of+post-traumatic+syringomyelia+in+the+rat.">Excitotoxic model of post-traumatic syringomyelia in the rat.</a> Spine. 26, 1842-1849 (2001).</li><li>Rolls, 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=Two+faces+of+chondroitin+sulfate+proteoglycan+in+spinal+cord+repair:+a+role+in+microglia/macrophage+activation.">Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation.</a> PLoS Medicine. 5, 1262-1277 (2008).</li><li>Properzi, F., Asher, R. A., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulphate+proteoglycans+in+the+central+nervous+system:+changes+and+synthesis+after+injury.">Chondroitin sulphate proteoglycans in the central nervous system: changes and synthesis after injury.</a> Biochemical Society Transactions. 31, 335-336 (2003).</li><li>Fawcett, J. W., Asher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+scar+and+central+nervous+system+repair.">The glial scar and central nervous system repair.</a> Brain Research Bulletin. 49, 377-391 (1999).</li><li>Yang, Z., Mo, L., Duan, H., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chitosan/collagen+substrates+on+the+behavior+of+rat+neural+stem+cells.">Effects of chitosan/collagen substrates on the behavior of rat neural stem cells.</a> Science China Life Sciences. 53, 215-222 (2010).</li><li>Chawla, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fibrous Materials. , (1998).</li><li>Pickering, K. L., Aruan Efendy, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+recent+developments+in+natural+fibre+composites+and+their+mechanical+performance.">A review of recent developments in natural fibre composites and their mechanical performance.</a> Composites Part A-Applied Science and Manufacturing. 83, 98-112 (2016).</li><li>Lundgren, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+fibers+made+from+proteins.">Synthetic fibers made from proteins.</a> Advances in Protein Chemistry. 5, 305-351 (1954).</li><li>Radishevskii, M. B., Serkov, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+mechanism+in+wet+spinning+of+fibres.">Coagulation mechanism in wet spinning of fibres.</a> Fibre Chemistry. 37, 266-271 (2005).</li><li>Yannas, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+and+gelatin+in+the+solid+state.">Collagen and gelatin in the solid state.</a> Journal of Macromolecular Science Part C Polymer Reviews. 7, 49-106 (1972).</li><li>Baer, E., Cassidy, J. J., Hiltner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+structure+of+collagen+composite+Systems:+lessons+from+biology.">Hierarchical structure of collagen composite Systems: lessons from biology.</a> Pure and Applied Chemistry. 6, 961-973 (2009).</li><li>Harrington, W. F., Von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+collagen+and+gelatin.">The structure of collagen and gelatin.</a> Advances in Protein Chemistry. 16, 1-138 (1961).</li><li>Veis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Macromolecular Chemistry of Gelatin. , (1994).</li><li>Freyman, T. M., Yannas, I. V., Gibson, 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=Cellular+materials+as+porous+scaffolds+for+tissue+engineering.">Cellular materials as porous scaffolds for tissue engineering.</a> Progress in Materials Science. 46, 273-282 (2001).</li><li>Michalczyk, K., Ziman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin+structure+and+predicted+function+in+cellular+cytoskeletal+organization.">Nestin structure and predicted function in cellular cytoskeletal organization.</a> Histology and Histopathology. 20, 665-671 (2005).</li></ol>

Biz jelatin tabanlı Biyomalzeme gelişimi basit bir ıslak doku rejenerasyonu için doğal polimerler çalışmada uygulanan teknik iplik kullanarak sundu. Bu eser olasılığını jelatin imalat jelatin kendisi özelliklerini optimize etmek amacı ile diğer kaynaklardan ek olmadan büyük protein kaynağı olarak gösterdi. Jelatin tabanlı Biyomalzeme gelişimi tamamen oda sıcaklığında (22-26 ° C) gerçekleştirilmiştir. Bir nazik çözüm iletişim kuralına, önemli bir adım olarak bir kesin çözüm konsant...

Doku rejenerasyonu en son gelişme tıbbi tedaviler yeni tedavi stratejileri geliştirilmesi için bir meydan okuma gösteren doku Mühendisliği uygulama içerir. Örneğin, sınırlı potansiyeli sinir sistemi yeniden oluşturma işlemi, aşağıdaki yaralanma veya hastalık, dünya çapında bir önemli sağlık sorunu teşkil etmektedir. Sinir sistemi ile ilgili patofizyolojik süreçlerin karmaşıklığı, nedeniyle geleneksel otogrefti kullanımı veya sabitleme cerrahi uygulamaları fonksiyonel sonuçlar avantajlar gösterilmiştir, ancak için güçlü hiçbir kanıt Omurilik fiksasyon ameliyat1,2etkileri. Hasarlı bölgeyi, doku kaybetti ve sonunda yoğun gliyal yara4,5şekillendirme hypertrophically indüklenen astrocytes3ile eski yerine koymak. Bu matris sinir kurtarılması6,7 işlev ve ki, bloklar böylece, büyük ölçüde engel olduğunu rejenerasyon bir engel olarak davranır. Bu nedenle, uygun doldurma boşluğu malzeme doku kaybı önlemek ve hasarlı bölgeyi bütünlüğünü muhafaza yanı sıra sinir hücreleri doğrudan değiştirme sağlayan bağ dokusu skar ilişkili oluşumunu azaltmak için bekleniyor ve devresi Axon rejenerasyon teşvik etmek.
Polimer Biyomalzeme iskele için doku yenileme terapisi, hücre veya axon davranış ve doku ilerleme doğal hücre dışı Matriks (ECM) desteği ile düzenlenmesi dayalı olarak tercih edilmiştir. Fiber biçimi genellikle onun tek boyutlu yapısı8sayesinde çeşitli malzemeler için bir yapı taşı olarak kabul edilir. Lifler genellikle eritebilir ekstrüzyon veya ıslak eğirme yöntemi tarafından elde edilebilir; Ancak, büyük boy ve maliyet ekipman ve bu yöntemler gerçekleştirmek için zorluk zor vardır. Buna ek olarak, polimer lifleri için ilgili çalışmaları çoğunluğu sentetik veya kompozit malzemeler üzerinde odaklanmıştır. Biomaterial kaynağı olarak doğal polimerler daha iyi Biyouyumluluk özellikleri insan vücudu için sunuyoruz. Yine de, doğal polimer lifleri hizalama elde etmek için sentetik polimer kaynakları9/ nispeten daha zordur. Bu nedenle, doğal bir polimer protein zengin bir kaynak olarak biomaterial lifleri dönüşüm önemli bir stratejidir — sadece biomaterial lifleri olabilir doğrudan hammadde, böylece monomerleri, gereksiz bir dönüşümü kaçınarak dan izole ama protein lifleri de iyi görünüm ve olumlu özelliklerini10var.
Bu bağlamda, biz doğal polimer lifleri için doku Mühendisliği laboratuvar ölçekte uygulanacak ıslak eğirme, temel kavramı ile üretim için bir ucuz işleme yöntemi açıklanmaktadır. Islak iplik ekstrüzyon ve uygun polimer nonsolvent içine bir polimer çözüm koagülasyon tarafından gerçekleştirilir. Uygun, yapışkan çözümünü koagülasyon orta katkılı polimer molekülleri erimeye neden olur. Faz geçiş yoluyla filamentler sonra onların çözünürlük kaybetmek ve bir katı polimer faz11şeklinde çöktürülmüş. Bu konsepte atıfta, biz o zaman jelatin geliştirme tüp şekle doku rejenerasyonu uygulama için uygun olarak kabul edilir bir kalıplama işlemi tarafından genişletilmiş. Buna ek olarak, özünde, biz de herhangi bir şekil jelatin liflerinden malzemenin gelişebilir (örneğin, jelatin conduit yerleştirilmiştir birkaç jelatin lifleri), diğeri için istenen uygulamalar.
Jelatin, bir biyolojik doğal polimer denatüre ve hidrolize kolajen herhangi bir semicrystalline, amorf veya üçlü sarmal devlet kollajen12de dahil olmak üzere oluşturulur. Bu kolajen olduğunu tüm bağ dokusu, sinir büyüme indükler ana ECM protein yapısına benzeyen omurgalı ve omurgasız13,14, temel yapısal protein bilindiği ve, aynı anda, omurilik yaralanmaları sırasında salgılanan glikozaminoglikan büyük miktarda yerini alır. Bu nedenle, bir kaynak olarak jelatin kullanımı tıbbi herhangi bir araç için mükemmel bir seçim olurdu. Ucuz bir kaynak olmasının yanı sıra, jelatin de biyolojik olarak parçalanabilen ve cytocompatible ve klinik olarak kanıtlanmış bir geçici dolgu15kusur olmak. , Tüp forma geliştirilen tüp bebek ve içinde vivo testleri burada açıklanan göstermek jelatin bir mükemmel Biyouyumluluk ve gelecekteki doku mühendisliği uygulamaları için uygunluk var. Kültürlü insan yağ kök hücreleri ile jelatin tüpleri pozitif testin bir sinir hücre marker olarak boyama kullanarak hücre farklılaşması nöral progenitör hücre içine geliştirmek. Ayrıca, jelatin boşluk, bu çalışmada, kurulan yöntemi tarafından üretilen gibi dolgu olarak yönetilebilir ve güvenli olması ve büyük yarar şu anda geliştirme doku için Bağdaşık doğal polimerler gelişmekte olan doku mühendisleri için bekleniyor rejenerasyon stratejileri.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Solution preparation:</td>
		</tr>
		<tr>
			<td>Gelatin type B (porcine)</td>
			<td>Ferak</td>
			<td>Art. -Nr. 10733</td>
			<td>500 g vial</td>
		</tr>
		<tr>
			<td>Wet spinning process:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Model M312</td>
			<td>Minipuls*3</td>
		</tr>
		<tr>
			<td>Plastic tube connector</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>1 box</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Sterican</td>
			<td>5A06258541</td>
			<td>26Gx1/2&#34;(0.45 x 12mm)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00010</td>
			<td>2.5 L vial</td>
		</tr>
		<tr>
			<td>Polycaprolactone CAPA 6500</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Scharlau</td>
			<td>CL03421000</td>
			<td>1 L vial</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (Introcan Certo)</td>
			<td>B. Braun</td>
			<td>1B03258241</td>
			<td>24Gx3/4&#34;(0.7 x 19mm)</td>
		</tr>
		<tr>
			<td colspan="4">Morphology of the gelatin tube:</td>
		</tr>
		<tr>
			<td>Ion sputter coater machine&#160;</td>
			<td>Hitachi</td>
			<td>e1010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Scanning electron microscopy</td>
			<td>Hitachi</td>
			<td>S-3000N</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cultivation of cells on the gelatin tube:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>488625</td>
			<td>100 mL vial</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>923119</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium&#160;</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>TPP</td>
			<td>90025</td>
			<td>VENT type</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>1209938</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Immunocytochemistry:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate-buffered saline</td>
			<td>Gibco</td>
			<td>654471</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00697</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>NP-40 surfactant (Tergitol solution)</td>
			<td>Sigma</td>
			<td>056K0151</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>20 mL vial, concentrate</td>
		</tr>
		<tr>
			<td>Nestin (primary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23927</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-fluorescein isothiocyanate (secondary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2099</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Anaspec</td>
			<td>AS-83218</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>In vivo biocompatibility test:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine+zolazepam&#160;</td>
			<td>Virbac</td>
			<td>BC91</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer korea</td>
			<td>KR03227</td>
			<td>10 mL vial</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Astar</td>
			<td>1406232</td>
			<td>2 mL vial</td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Everstar</td>
			<td>HA161202</td>
			<td>4 L barrel</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>China Chemical &#38; Pharmaceutical</td>
			<td>18P909</td>
			<td>1 g vial</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

wet-spinning-based molding process of gelatin for tissue regeneration

Bu makalede monofilament iplik veya uygun diğer biçimleri jelatin, doğal bir polimer imal etmek ucuz bir yöntem sunuyor. Islak yöntemi iplik, jelatin lifler uygun koagülasyon ortamda düzgün ekstrüzyon tarafından üretilmektedir. İşlevsel yüzey bu jelatin lifleri ve doku özelliklerini taklit yeteneğini artırmak için jelatin tüp forma bu kavramı bakarak kalıplanmış. Tüp Bebek ve içinde vivo testleri tarafından muayene, jelatin tüpler doku Mühendisliği uygulama için büyük bir potansiyel göstermek. Görevi uygun dolgu boşluğu malzemesi, jelatin Borular hasarlı bölgeyi dokusunda (örneğin, sinirli ya da Kardiyovasküler sistemde) yerine gibi doğrudan yerine kök hücreler ve nöro çevrim sağlayarak yeniden oluşturma işlemi tanıtmak için kullanılabilir. Bu protokol üzerinde doğal bir polimer esaslı bir biomaterial oluşturmak için ayrıntılı bir yönerge sağlar ve uygulanması büyük yarar doku rejenerasyonu stratejileri gerçekleştirmek için yardım Bağdaşık doğal polimerler geliştirilmesi bekleniyor.

Bu çalışmada, biz lifleri (Şekil 2A) içine jelatin ve tüpler (Şekil 2B, C) kullanıcı dostu ıslak eğirme kavramı aracılığıyla geliştirdi. Bu jelatin-esaslı malzemeler şekillerini bağlı olarak tıbbi herhangi bir aracı olarak yararlı olabilir. İşlevsel yüzey ve çerçeve gibi malzemelerin doku rejenerasyonu için daha uygun olduğunu göz önünde bulundurursak, tüp bebek...

Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

Geliştirilen ve jelatin tabanlı Biyomalzeme doku Mühendisliği uygulama için kullanılacak inşası için ıslak eğirme kavramı dayalı bir iletişim kuralı tanımlayın.

Islak eğirme esaslı kalıplama işlemi doku rejenerasyonu için jelatin

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Bu protokol, tek bir lif şeridinden herhangi bir tercih edilen tıbbi uygulama için kullanılabilecek çeşitli şeritler e bir malzeme geliştirmek için ıslak iplik temel bir kavram geliştirir. Bu teknik basit, ucuz ve ilgi malzemeleri için protein kaynağı içine doğal polimerlerin özelleştirme için sentetik polimerler eklenmesi gerektirmez. Bu teknik, merkezi veya periferik sinir sistemi tedavisi, kan damarı replasmanı veya doğrudan idrar yolu rekonstrüksiyonu gibi soğuk tedavi öldürmek için biyomalzeme gelişimine genişletilebilir.Bu yöntem doku nöralizasyon veya rejeneratif tıp için bir strateji olarak da dahil olmak üzere tıbbi uygulama için tasarlanmıştır. Bu teknik yeni başlayanlar tarafından kolayca uygulanabilir. Birkaç kalıp tekrarı sonra, çözümün kalitesi her zaman en iyi oluşumu korumak için kontrol edilmelidir unutmayın.Islak dönen tüpü oluşturmak için, ilk üç saniye için taze hazırlanmış jelatin çözeltisi içine üç saniye için üç çeyrek inç periferik venöz kateter tarafından 24 göstergesi batırın, bir dakika için taze hazırlanmış aseton çözeltisi daldırma izledi. Bu adımları 20 kez tekrarlayın. Son aseton daldırma sonra, oda sıcaklığında beş dakika boyunca kalıplı tüp kuru izin, ve dikkatle oda sıcaklığında bir gecede başlık altı kuluçka için% 2.5 polikaprolakton diklorometan çözeltisi ile cam pastörette içine kateter jelatin tüp aktarmak için bir hemostat kullanın.Ertesi sabah, jelatini ultraviyole ışık altında iki saat sterilize edin ve tüpü %75 etanol ile daldırın. Sonra artık etanol kaldırmak için kök hücre orta ile tüp iki kez yıkayın. Hücre kültürü ile jelatin tüp yetiştirmek için, 37 santigrat derece ve% 5 karbondioksit 3,5 dakika kuluçka için% 0.25 Trypsin-EDTA bir mililitre ile bir kültür şişesi insan yağ kök hücrelerinin supinate sonu değiştirin.Hücreler ayrıldığında, bir mililitre fetal sığır serumu ile reaksiyonu durdurun ve hücre süspansiyonuna mikrosantrifüj tüpüne aktarın. Santrifüj ile hücreleri toplamak ve kök hücre orta bir mililitre pelet resuspend. Daha sonra jelatin tüpünü, üç mililitre kök hücre lilitre içeren altı kuyulukbir plakanın bir kuyuya yerleştirin.Hücreleri sayarak sonra, tohum dört kez 10 dördüncü insan yağ kök hücreleri hücre kültürü kuluçka iki hafta kuluçka için tüp içine. Bir kadın sekiz haftalık Sprague Dawley sıçanda parmak kıskacına yanıt eksikliği ile sedasyon uygun düzeyde doğruladıktan sonra, trapezius bölgeden saç tıraş ve povison-iyot çözeltisi ile maruz kalan cilt sterilize. Cerrahi sitenin bakteriyel kontaminasyonunu azaltmak için steril cerrahi perdeler ile sıçan kapağı ve sığ yapmak için cerrahi makas kullanın, iki santimetre uzunluğunda deri kesi.Fasya ve kas arasındaki tabaka içine doğrudan jelatin tüp yerleştirin ve yara kapatmak için bir naylon dikiş kullanın. Sonra povison-iyot çözeltisi ile kesi sterilize. Ketoprofen ve sefazolin bir intramüsküler enjeksiyon yoluyla sıçan neden ve yedi gün boyunca aseptik koşullar altında hayvan korumak.Bu kullanıcı dostu ıslak iplik kavramı lifler ve tüpler içine jelatin geliştirmek için kullanılabilir. Elektron mikroskobu tarayarak jelatin tüpünün morfolojik gözlemleri, 200 mikrometreden daha büyük iç çapı ve yaklaşık 20 mikrometre kalınlığında çok düzgün olmayan bir yüzey ortaya koymaktadır. Nöral hücre belirteç ekspresyonu için bir insan yağ kök hücre kültürü jelatin tüp boyama hücrelerin nüfuz ve jelatin tüp etek ve yapışmak ve nöral progenitor hücreleri içine ayırt düşündüren birden fazla tespit çekirdekleri ile birlikte marker için olumlu bir boyama göstermektedir.Jelatin tüpün fasya tabakasına yerleştirilmesi, yerel dokuda kızarıklık, şişlik veya diğer iltihap belirtileri nin görülmemeve tüpün iyi gelişmiş bağ dokusu ile çevrili olması nedeniyle güvenlik ve biyouyumluluk olduğunu doğrular. Bu prosedürü iki nokta bir çalışırken, her çözeltide belirli kaplama süresi içinde kalıbın daldırma sağlanması çok hassas bir tüp formu oluşturmak için önemlidir. Bu prosedürütaki deneylerimiz, doku rejenerasyon stratejisi için biyomalzemelerin sonucunu doğru bir şekilde araştırmak için insan hastalığının hayvan modelini kullanmaktadır.Bu teknik, geliştirildikten sonra, araştırmacıların basit korelasyonlu doğa polimerleri geliştirmelerinin önünü, karar verilen bir tıbbi uygulama için her türlü malzemeye dönüştürmektedir. Normal kullanım koşulu altında, polikaprolakson diklorotan çözeltisi minimal çevresel tehlike sunmak için kabul edilir, ancak uygun bir biyogüvenlik kabine bu kimyasal ile herhangi bir adım gerçekleştirmek için unutmayın.

wet-spinning-based-molding-process-of-gelatin-for-tissue-regeneration

Research

JoVE Journal

Bioengineering

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Sinir doku rejenerasyonu için Çözünür ve Çözünmeyen Biyotinile Proteinlerin ifade, izolasyon ve saflaştırılması

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Biyomühendislik

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Sıvı Enjeksiyon için 3D Baskılı Kalıpları Kullanma Tıbbi Cihazlar hızlı ve düşük maliyetli Prototip

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Vasküler Doku Rejenerasyon için Mühendislik 3D cellularized Kollajen jeller

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Elektrospinning Süreci Uyarlama bir Tri-katmanlı için üç Benzersiz ortamlar sağlamak için<em&gt; In Vitro</em&gt; Havayolu Wall Modeli

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Zaman-of-flight Kütle Spektrometre ile 2D Gaz Kromatografisi Kullanarak Alg Hidrotermal Sıvılaşma gelen Sulu Kesir Nitel Karakterizasyonu

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

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

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

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

Kontrollü İlaç Release için Manyetik Alan-Duyarlı Hibrid Jelatin mikroj Alternatif

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

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

Eşleştirilmiş boncuk ve mıknatıs dizi Microwells değişken içbükey geometrileri ile kalıplama için

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Astrocyte ağlar gelişimsel mekanizmaları özetlemek ve sinir sistemi yeniden oluşturma işlemi kolaylaştırmak için tasarlanmış üç boyutlu doku uyumlu

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Doku Seviyesi Mekaniğinin Kontrol Altına Almak İçin Uyumlu Yüzeylerde İnsan Embriyonik Kök Hücre Kolonilerinin Geometrisinin Desenlemi

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Jelatin Metacryloyl Hidrojel Bazlı Bioinks 3D Biyobaskı Protokolleri

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...