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

이 연구는 국가 방위 정부 (MAB-105-070;에 의해 지원 되었다 MAB-106-077; MAB-107-032; MAB-107-065)의 과학 및 기술 (대부분 107-2320-B016-016), 세 배 서비스 종합 병원, 국가 방위 의료 센터, 대만 (TSGH-C106-046; TSGH-C106-115; TSGH-C107-041), 쳉-Hsin 종합 병원 그리고 국가 방위 의료 센터 협력 (CH-NDMC-107-8).

지방 조직으로는 제도적 검토 보드의 세 배 서비스 종합 병원에 의해 인증 정형 외과 수술에서 가져온, 타이 페이, 대만, R.O.C. 절차 동물 주제와 관련 된 국가에서 동물 관리 위원회에 의해 승인 되었습니다. 국방 의료 센터, 대만 (R.O.C)입니다.
1. 젖은 프로세스 회전
<ol><li>
솔루션 준비
	<ol>
<li>5% (w/v) 솔루션 농도를 이중 증 류 물 100 mL에 젤라틴 가루 5g을 분해.</li>...

<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>

우리는 간단한 젖은 조직 재생에 대 한 천연 고분자의 연구에 적용할 수 있는 기술 회전을 사용 하 여 젤라틴 기반 생체 재료의 개발을 제시. 이 작품 자체는 젤라틴의 속성을 최적화 하는 목적으로 다른 소스를 추가 하지 않고 훌륭한 단백질 소스로 젤라틴 제조의 가능성을 보여주었다. 젤라틴-기반 생체 재료의 개발 실 온 (22-26 ° C)에서 완전히 실행 되었다. 정확한 솔루션 농도 유지 하 고 모든 ?...

조직 재생의 최신 개발 조직 공학, 의료 치료에 새로운 치료 전략의 개선에 대 한 도전을 나타내는 응용 프로그램을 포함 한다. 예를 들어 신 경계 재생, 다음 부상 또는 질병의 제한 가능성 전세계 중요 한 건강 문제가 포즈지 않습니다. 신 경계와 관련 pathophysiological 프로세스의 복잡성 때문에 안정화 수술의 구현 또는 전통적인 autograft 사용 표시 되었습니다 기능 결과에 혜택을 제공 하지만 대 한 강력한 증거가 없다 척추 고정 수술1,2의 효과. 손상된 된 영역에서 조직 손실 이며 hypertrophically 유도 이다3, 결국 밀도 glial 흉터4,5형성으로 바뀝니다. 이 매트릭스는 블록 신경의 복구6,7 을 작동 하 고, 따라서, 크게 방해 재생 방 벽으로 작동 합니다. 따라서, 적당 한 충전 간격 재 조직의 손실을 방지 하 고 신경 세포의 직접 교체를 제공 하 여 뿐만 아니라 손상된 된 영역 무결성을 유지 관리 하 여 흉터에 관련 된 결합 조직 형성을 줄일 것으로 예상 되 고 축 삭 재생을 촉진 하는 회로가
고분자 생체 조직 재생 치료, 자연 세포 외 기질 (ECM) 지원을 통해 셀 또는 축 삭 행동과 조직 진행의 규정에 따라 건설 기계로 선호 되었습니다. 섬유 포맷은 다양 한 재료 때문에 그것의 1 차원 구조8대 한 빌딩 블록으로 일반적으로 간주 됩니다. 섬유는 용융 압출 또는 습식 방법; 회전에 의해 일반적으로 얻을 수 있습니다. 그러나, 큰 크기와 비용의 장비와 이러한 방법을 수행 하는 데 어려움은 도전. 또한, 고분자 섬유에 관련 된 일의 대부분은 합성 또는 복합 재료에 집중 되었습니다. 천연 고분자 소재의 원천으로 인간의 신체에 대 한 더 나은 생체 적합성 속성을 제공합니다. 그럼에도 불구 하 고, 자연 고분자 섬유의 맞춤을 상대적으로 더 어렵습니다 보다 합성 폴리머 소스9. 따라서, 소재 섬유에 단백질의 풍부한 원천으로 자연적인 중합체의 변환은 중요 한 전략은-뿐만 아니라 소재 섬유 수 직접 따라서, 단위체에는 불필요 한 변환을 피하 원료에서 분리 하지만 단백질 섬유는 또한 좋은 모양 및 유리한 특성10있다.
이와 관련는 조직 공학에 대 한 실험실 규모에 구현할 수 있는 젖은 회전의 기본 개념을 통해 천연 고분자 섬유의 제조에 대 한 저렴 한 처리 방법에 설명 합니다. 젖은 회전 압출 및 응고 폴리머 솔루션의 적당 한 폴리머 nonsolvent에 의해 수행 됩니다. 응고 중에 첨가 하는 적절 한, 점성 솔루션 폴리머 분자 분해를 하면 됩니다. 위상 전환을 통해는 필 라 멘 트 다음 그들의 용 해도 잃게 되 고 고체 폴리머 단계11의 형태로 침전 된다. 이 개념을 언급 하는, 우리가 다음 확장 튜브 형태로 젤라틴의 개발 성형 프로세스, 조직 재생 응용 프로그램에 대 한 적절 한 간주 됩니다. 또한, 본질적으로, 우리는 또한 개발할 수 있습니다 젤라틴 섬유에서 자료의 어떤 모양 든 지 (예를 들어, 젤라틴 도관 올리고 여러 젤라틴 섬유에서), 다른 응용 프로그램을 원하는.
젤라틴, 생 분해성 천연 폴리머, 변성 및 분해 된 콜라겐, 콜라겐12의 모든 semicrystalline, 비정 질, 또는 3 배 나선형 상태를 포함 하 여에서 형성 된다. 그것은 잘 알려져 그 콜라겐은 척추 동물 및 무척 추 동물13,14, 신경 성장 유도 주요 ECM의 단백질 구조와 비슷한의 모든 결합 조직에 필수적인 구조 단백질, 동시에, glycosaminoglycan 척수 부상 중 분 비의 다량을 대체 합니다. 따라서, 소스로 젤라틴을 사용 하 여 모든 의료 차량에 대 한 좋은 선택 것입니다. 되 고 게다가 저렴 한 소스, 젤라틴은 또한 생 분해성 및 cytocompatible 및 임시 제거 필러15수를 임상적으로 입증 된. , 튜브 형태로 개발 여기 설명 하는 생체 외에서 그리고 vivo에서 테스트 젤라틴은 우수한 생체 적합성 고 미래의 조직 엔지니어링 응용 프로그램에 대 한 적합성을 보여줍니다. 인간 지방 줄기 세포 배양, 젤라틴 튜브 신경 셀 표식으로 긍정적인 nestin 얼룩을 사용 하 여 신경 조상 세포로 세포 분화를 향상. 또한,이 연구에서 설립 하는 방법에 의해 생산 격차 자료 작성으로 젤라틴 관리 하 고 안전 하 고 크게 혜택을 현재 조직의 향상을 위한 상호 천연 고분자를 개발 하는 조직 엔지니어 예정입니다. 재생 전략입니다.

<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

이 문서는 monofilament 섬유 또는 다른 적절 한 형태의 젤라틴, 천연 폴리머로 조작 하는 저렴 한 메서드를 제공 합니다. 회전 방법 젖은 통해 젤라틴 섬유는 적당 한 응고 매체에 부드럽게 밀어 남에 의해 생산 됩니다. 이 젤라틴 섬유 및 직물의 기능을 모방 하는 능력의 기능 표면 증가, 젤라틴이이 개념을 참조 하 여 튜브 형태로 성형 수 있습니다. 생체 외에서 그리고 vivo에서 테스트 검사, 젤라틴 튜브 조직 공학에서 응용 프로그램에 대 한 좋은 잠재력을 보여 줍니다. 행동으로 적당 한 충전 간격 재, 젤라틴 (예를 들어, 긴장 또는 심장 혈관 시스템에), 손상된 된 영역에 조직을 대체할 뿐 아니라 줄기 세포와 신경 회로의 직접 교체를 제공 하 여 재생을 촉진 하 튜브를 사용할 수 있습니다. 이 프로토콜은 천연 폴리머에 따라 소재를 만들기 위한 자세한 절차를 제공 합니다 그리고 구현 크게 조직 재생 전략을 실현 하는 데 도움이 상호 천연 고분자의 개발 혜택을 받을 것으로 예상 된다.

이 연구에서 우리는 성공적으로 섬유 (그림 2A)에 젤라틴과 사용자 친화적인 젖은 회전 개념을 통해 튜브 (그림 2B, C)을 개발. 이 젤라틴 기반 물자는 그들의 모양에 따라 어떤 의료 도구로 활용할 수 있습니다. 우리 생체 외에서 그리고 vivo에서 테스트를 수행 하 여 젤라틴 튜브의 생체 적합성 시험 기능 ...

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

우리가 개발 하 고 젤라틴 기반 생체 조직 공학의 응용 프로그램에 대 한 사용의 건설에 대 한 젖은 회전 개념에 따라 프로토콜을 설명.

조직 재생에 대 한 젤라틴의 젖은 회전-기반 성형 과정

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

이 프로토콜은 단일 섬유 스트립에서 원하는 의료 응용 프로그램에 사용할 수있는 다양한 스트립으로 재료를 개발하기 위해 습식 방적의 기본 개념을 향상시킵니다. 이 기술은 간단하고 저렴하며, 관심있는 물질을 위한 단백질 자원에 천연 폴리머의 사용자 정의를 위한 합성 폴리머를 첨가할 필요가 없습니다. 이 기술은 중추 또는 말초 신경계 치료, 혈관 교체 또는 직접 요로 재건과 같은 감기 치료를 죽이는 생체 물질 적 개발로 확장 될 수 있습니다.이 방법은 조직 신경화 또는 재생 의학을 위한 전략으로 포함하여 의학 응용을 위한 것입니다. 이 기술은 초보자가 쉽게 구현 할 수 있습니다. 여러 성형 반복 후 최적의 형성을 유지하기 위해 솔루션의 품질을 항상 확인해야 합니다.젖은 회전 튜브를 형성하기 위해 먼저 24 게이지를 3 분기 인치 주변 정맥 카테터를 갓 준비 된 젤라틴 용액에 3 초 동안 담근 다음 갓 준비 된 아세톤 솔루션에 1 분 동안 침지합니다. 이 단계를 20번 반복합니다. 마지막 아세톤 침지 후, 성형 튜브를 실온에서 5분 동안 건조하게 하고, 하층 온도에서 2.5%의 폴리카폴락톤 디클로로메탄 용액을 사용하여 카테터에서 젤라틴 튜브를 유리 파스퇴르 파이펫으로 조심스럽게 옮기도록 한다.다음 아침, 75%에탄올에 튜브를 잠깐 잠정적으로 잠정적으로 잠정하기 전에 자외선 아래에서 2 시간 동안 젤라틴을 살균하십시오. 그런 다음 줄기 세포 배지로 튜브를 두 번 세척하여 잔류 에탄올을 제거합니다. 세포 배양으로 젤라틴 튜브를 육성하기 위해 배양 플라스크에서 인간 지방 줄기 세포의 수피네이트 끝을 0.25%의 1밀리리터로 대체하여 섭씨 37도및 이산화탄소 5%에서 3.5분 배양에 대한 트립신-EDTA를 1밀리리터로 대체한다.세포가 분리되면 태아 소 혈청의 1 밀리리터로 반응을 체포하고 세포 현탁액을 미세 원심 분리 튜브로 옮기십시오. 원심분리에 의해 세포를 수집하고 줄기 세포 매체의 1 밀리리터에서 펠릿을 재보힙. 그런 다음 젤라틴 튜브를 줄기 세포 배지 3 밀리리터를 포함하는 6웰 플레이트의 1웰에 놓습니다.세포를 세은 후, 세포 배양 배양기에서 2주 동안 튜브내로 줄기세포를 4번 10번 시드한다. 8주된 스프라그 Dawley 쥐의 발가락 핀치에 대한 반응 부족으로 적절한 수준의 침착을 확인한 후, 트라페지오 부위에서 모발을 면도하고 포비도네 요오드 용액으로 노출된 피부를 살균한다. 수술 부위의 세균 오염을 줄이기 위해 멸균 수술 커튼으로 쥐를 덮고 수술 가위를 사용하여 얕고 2센티미터 긴 피부 절개를 합니다.젤라틴 튜브를 근막과 근육 사이의 층에 직접 넣고 나일론 봉합사를 사용하여 상처를 닫습니다. 그런 다음 포비도오드 요오드 용액으로 절개를 살균합니다. 케토프로펜과 세파졸린의 근육 내 주입을 통해 쥐를 유도하고 7 일 동안 무균 조건하에서 동물을 유지합니다.이 사용자 친화적 인 습식 방사 개념은 섬유와 튜브로 젤라틴을 개발하는 데 사용할 수 있습니다. 전자 현미경 을 스캔하여 젤라틴 튜브의 형태학적 관측은 200 마이크로미터 의 내부 직경과 약 20 마이크로미터의 두께로 매우 매끄러운 표면을 나타낸다. 신경세포 마커 발현을 위한 인간 지방 줄기 세포 배양 젤라틴 튜브의 염색은 세포가 젤라틴 튜브에 침투및 부착하고 신경 전구 세포로 분화할 수 있음을 시사하는 다중 검출된 핵과 함께 마커에 대한 긍정적인 염색을 보여준다.젤라틴 튜브를 근막층으로 이식하면 적색, 붓기 또는 기타 염증 증상이 국소 조직 내에서 관찰되지 않고 잘 발달된 결합 조직으로 둘러싸여 있기 때문에 그 안전성 및 생체 적합성을 확인합니다. 이 절차를 2점 1로 시도하는 동안 각 솔루션의 특정 코팅 시간에 금형의 침지를 보장하는 것은 정확한 튜브 형태를 구축하는 데 매우 중요합니다. 이 절차에 따라, 우리의 실험은 조직 재생 전략에 대한 생체 재료의 결과를 정확하게 조사하기 위해 수행 될 수 있습니다 인간 질환의 동물 모델을 사용합니다.개발 후, 이 기술은 결정된 의료 응용을 위한 물질의 어떤 모형으로 간단한 상관 관계 특성 폴리머를 개발하는 연구원을 위한 도로를 포장했습니다. 정상적인 사용 조건하에서 폴리카폴톤 디클로로메탄 용액은 최소한의 환경 적 위험을 제시하는 것으로 간주되지만 적절한 생물 안전 캐비닛에서이 화학 물질로 어떤 단계를 수행하는 것을 잊지 마십시오.

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.

신경 조직의 재생을위한 가용성 및 불용성 바이오틴 단백질의 발현, 분리 및 정제

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

연구

생체공학

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.

액상 사출 성형을위한 3D 인쇄 금형을 사용하여 의료 기기의 신속하고 저렴한 프로토 타이핑

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.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

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.

전기 방사를 적응하는 것은 트라이 계층에 대한 세 가지 독특한 환경을 제공하기 위해<em&gt; 체외</em&gt;기도 벽의 모델

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.

비행 시간 형 질량 분석과 2D 가스 크로마토 그래피를 사용하여 조류의 열수 액화에서 수성 분수의 질적 특성

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.

제어 약물 릴리스에 대한 자기장 응답 하이브리드 젤라틴 마이크로 겔을 교류

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.

쌍된 구슬 및 Microwells 가변 오목한 형상의 성형에 대 한 자석 배열

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.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

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.

조직 수준 역학을 제어하기 위해 준수 기판에 인간 배아 줄기 세포 식민지의 기하학적 패터닝

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.

젤라틴 메타크릴 하이드로겔 기반 바이오잉크의 3D 바이오프린팅 프로토콜

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