This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1 물 / 오일 / 물 에멀젼을 두 번 미소 구 생산 <ol><li> 제 2 % 및 탈 이온수에 폴리 비닐 알코올 (PVA)의 w / v 용액 0.5 %를 준비한다. 클리어 할 때까지 50 ° C에서 솔루션을 저어,이 몇 시간이 걸릴 수 있습니다. 탈 이온수에서 2 %의 v / V 이소 프로필 알코올의 용액을 준비합니다. </li><li> 바람직한 친수성 ​​단백질 수용액을 준비한다. 아래의 표는 예 제제를 제공한다. </li></ol><img alt="표 1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> 표 1. 예 단백질 해법은 다음과 같은 단백질 용액을 성공적으로 캡슐화 전기 방사는이 프로토콜을 이용하고있다. 필요에 따라 기타 친수성 ​​단백질 용액을 사용할 수있다. <ol start="3"><li> 50 ㎖의 원심 분리 관에 0.5 % PVA 수용액 40 ㎖에 넣고 옆으로 설정합니다. </li><li> 15 ㎖의 원심 분리 튜브에서 65:35의 300 mg을 용해폴리 디클로로 메탄 3 ㎖ 중의 (락트산 - 코 - 글리콜 산) (PLGA). 볼텍스 믹서는 PLGA의 분해를 촉진하는데 사용될 수있다. </li><li> 단백질 용액을 200 μL 및 2 % PVA 용액 4 μl를 겸용. PLGA 용액에 단백질 혼합물을 따르 (1.4 단계). 솔루션은 대부분 별도의 유지됩니다. </li><li> 얼음 물을 비커에 넣고, 튜브를 놓습니다. ~ 10 와트 (RMS)에 지팡이 초음파기를 사용하여, 균일 한 유백색 에멀젼이 생성 될 때까지 몇 초간 (5-10)에 대한 용액을 교반. </li><li> 0.5 % PVA (단계 1.3)를 함유하는 50 ㎖의 튜브에 에멀젼을 붓는다. ~ 20 초간 볼텍스 혼합기에서 고속으로 용액을 혼합한다. 이 솔루션은 흐린 모양을 개발할 것입니다. </li><li> 2 분 동안 350 rpm으로 저어 접시에 200 ㎖의 비이커과 장소에 에멀젼을 전송합니다. 볶음 접시에 비커에 2 % 이소 프로필 알코올 50 ㎖를 추가합니다. 혼합물을 강화하기 위해 DCM이 증발 할 수 있도록 1 시간의 최소 및 PLGA 동안 교반을 계속 할 수 있습니다. </li><li> 전송 t그는 원심 분리기 튜브에 솔루션을 마이크로 스피어. </li><li> 3 분 동안 425 XG에 원심 분리기. 미소 구체는 튜브의 바닥에 수집하고, 흰색 나타난다. 조심스럽게 미소 위에 튜브에서 뜨는을 제거하고, 500 ㎖의 병에 저장합니다. </li><li> 전체 튜브 세 분기 채우고 그것을 액체에 미소 재배포 진탕 탈 이온수로 린스 미소. </li><li> 반복 1.10 및 1.11 네 번 단계를 반복합니다. </li><li> 마지막 헹굼에 따라, 다른 샘플을 500 ㎖의 병에 다시 장소 뜨는을 제거합니다. 후 최소 24 시간 동안 동결 건조, 원심 분리 관에서 수집 한 미소를 동결. </li><li> 가벼운 현미경 또는 주사 전자 현미경으로 미세 시각화. 60 μm의보다 더 큰 마이크로 스피어는 효과적인 전기 방사를위한 것입니다. 미소 구가 너무 큰 경우, 이상 초음파 처리 또는 텍싱 시간은 단계 1.6 또는 1.7에서 요구 될 수있다. </li> &lt;리&gt; -20 ° C 냉동고에있는 말린 미소를 저장합니다. </li><li> 옵션 : 1.10 단계 30에서 500 ㎖의 병에 단백질의 양을 테스트하기 위해, 제조업체의 지침에 따라, 단백질 분석을 사용합니다. 이는 미소 구에 캡슐화 된 단백질의 제조 방법에 사용 된 것과 용액의 양을 뺌으로써 퍼센트를 계산하는데 사용된다. </li></ol> . 마이크로 스피어에서 단백질의 위치가 PLGA 용액 31에 로다 민에게 ㎖의 2 μg /를 추가하고 FITC 복합 단백질을 캡슐화 시각화하는 일이 예를 보여줍니다 :합니다. 마이크로 스피어와 2 전기 방사 <ol><li> 이전 전기 방사 용액을 제조에, 37 ° C에서 용해시켜 탈 이온수 광개시제 0.5 % / V w 솔루션을 만들 수 있습니다. 이 프로세스는 몇 시간이 걸릴 수 있습니다. </li><li> 히알루 론산 (MEHA)를 methacrylated 29 절, 3 % (버딕 외. 합성 참조) / w 2 % 만들기/ V 900 kDa의 폴리 (에틸렌 옥사이드) (PEO)와 w 0.05 % / 탈 이온수 V 사진 개시제 용액 w. <ol><li> 원하는 볼륨 MEHA와 PEO의 정확한 금액을 계산합니다. 예를 들어, 솔루션을 전기 방사의 10 ㎖를 MEHA의 200 밀리그램 및 PEO의 300 밀리그램을 필요로한다. </li><li> 원하는 최종 부피 (이 예 9 ㎖)의 90 %를 탈 이온수에 PEO 녹인다. 이것은 몇 시간이 걸릴 수 있으며, 37 ℃에서 가열 교반 플레이트 또는 수욕은 프로세스를 가속화하는데 사용될 수있다. </li><li> 다음 MEHA을 추가하고 명확 때까지 저어하는 와류 믹서를 사용합니다. 이것은 단지 몇 분 정도 걸립니다. </li><li> 마지막으로 나머지 10 %의 볼륨 (이 예를 들어 1 ml)에 채우기 위해 0.5 % 광개시제 솔루션을 추가합니다. </li></ol></li><li> 400 ㎎ / ㎖까지 원하는 농도에 미소를 추가합니다. 미세 균일 용액에 분산 될 때까지 소용돌이 혼합기에 용액을 혼합한다. </li><li> 주사기에 대한 해결책을 전송하고 6 인치 18 게이지 (BL)을 첨부UNT 팁 바늘입니다. </li><li> 주사기 펌프에 주사기를 놓고는 1.2 ㎖ / 시간에 분배하도록 설정합니다. </li><li> 수집 판 또는 맨드릴에 알루미늄 호일 층을 테이프입니다. 이것은 완성 된 지지체를 쉽게 정리 및 저장이 가능합니다. 회전하는 맨드릴은 정렬 된 섬유를 만드는 데 사용됩니다. 평판이나 고정 맨드릴은 무작위로 배열 된 섬유에서 발생합니다. </li><li> 수집 장치에 고전압 전원으로부터 접지 와이어를 연결한다. 바늘에 긍정적 인 리드를 연결합니다. </li><li> 니들 팁과 수집 표면 사이 15cm가되도록 주사기 펌프 및 수집 표면을 조정한다. </li><li> 이 솔루션은 주사기의 끝에서 볼 때, 고분자 펌핑을 시작 전압 소스를 켜고 24 kV의주의에 전압을 설정합니다.을 시스템의 금속 부분을 만지지 마십시오에 전압이 전원을 켜면. 요금은 피부에 전기 부분에서 짧은 거리를 이동할 수 있습니다. </li><li> D까지 솔루션을 실행esired 비계 두께는 달성된다. 전체 전압 소스와 주사기 펌프를 해제합니다. </li><li> 비계가 붙어과 호일을 제거합니다. 단백질을 함유 완료 발판은 -20 ° C 냉동고에 저장됩니다. </li></ol> 3 단백질 생물 작용 (Bioactivity) 테스트 <ol><li> 세포 배양 매체를 준비합니다. 추가 10 % v / V를 소 태아 혈청 둘 베코 변형 이글의 중간에, 1 %의 v / V의 L-글루타민, 1 % V / V 페니실린 - 스트렙토 마이신. </li><li> 잘 판에 완전히 맞게 선택 유리 커버 슬립. </li><li> 제조자에 의해 설명 된 커버 슬립을 치료 3- (트리 메 톡시 실릴) 프로필 메타 크릴 레이트를 사용한다. 메타 크릴은 커버 슬립에 비계 준수를 강화합니다. </li><li> 전기 방사하기 전에 이동식 양면 테이프로 electrospinner의 수집 지역에 methacrylated 된 커버를 부착합니다. 커버 슬립에 스피닝 처리 및 시청을 용이하게합니다. </li><li> 전술 한 바와 같이 소망의 두께로 Electrospin. </li><li>따고 조심스럽게 맨드릴에서 커버 슬립을 제거 전기 방사. 명확한 질소 챔버로 비계에게 코팅 된 커버를 놓고 모든 산소가 제거되어 있는지 확인합니다. </li><li> 15 분 동안 10 mW의 / cm 2 365 nm의 빛 아래 챔버와 비계를 놓습니다. 적절한 크기의 잘 플레이트로 장소를 가교 후. 발판면이 위를 향하도록합니다. </li><li> 30 분 동안 살균 램프 아래 장소의 발판 살균합니다. 원하는 피브로넥틴 또는 기타 단백질은 세포 부착을 향상시키기 위해 코팅으로서 사용되는 경우. 코트 발판으로 제조업체의 지침을 따르십시오. </li><li> 수확 등쪽 뿌리 신경절 (DRG)이 아니라 이전에 Hollenbeck (32)에 의해 설명했다. 한 DRG는 각 시험 비계 덮여 coverslip에 대한 필요합니다. </li><li> 잘 플레이트의 각 발판 용지의 100 ~ 200 μl를 놓습니다. 조심스럽게 미디어 방울의 각 지지체에 한 DRG를 놓습니다. 두꺼운 비계에 대한 더 많은 미디어가 필요할 수있다; DRG 완전히 잠긴 floa하지해​​야팅. </li><li> 세포가 지지체에 부착 할 수 있도록 4 시간 동안 37 ° C에서의 비계와 DRG를 품어. </li><li> 잘에 대한 적절한 수준으로 미디어를 입력하고 인큐베이터에 배치합니다. 4-6일 배양하고 계속합니다. </li><li> 잠복기 후 신중 아니라 각에서 용지를 제거하고 부드럽게 PBS로 한번 씻어. / V 파라 포름 알데히드 와트 4 %를 사용하여 30 분 동안 세포를 고정합니다. </li><li> 신경 미세 섬유에 대한 항체 염색을 사용하여 얼룩 세포. 이 정량 신경 돌기 생성의 시각화 수 있습니다. DAPI 또한 세포의 핵을 표시하는데 사용될 수있다. 예를 염색 프로토콜은 Sundararaghavan 및 동료 (14)에 의해 설명되었다. </li><li> 형광 현미경을 사용하여 세포를 시각화. <ol><li> 현미경의 무대에 웰 플레이트를 놓습니다. </li><li> DAPI에 대한 필터 및 여기 설정을 사용하여 세포 질량을 찾습니다. </li><li> 셀되면 확장 된 신경 돌기를 시각화 FITC에 필터를 전환합니다. U수집하고 전체 구조를보고 필요한만큼의 이미지를 결합 현미경 스티치 기능을 노래. DAPI, FITC 밝은 필드에 대해이 단계를 반복합니다. </li></ol></li></ol>

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The authors have nothing to disclose.

많은 연구는 신경 세포가 지형적 큐들 (섬유 배향) 및 화학 신호 (성장 인자) 1,2,10,11,35로 지향 될 수 있음을 보여 주었다. 전기 방사는 정렬 된 섬유를 만들 수있는 손쉬운 방법이다. 성장 인자는 신경 성장하지만 신경 도관 (NC)로 포함하기 위해, 지속 방출을위한 방법이 요구된다 바랍니다. 두 신호와 함께보다 강력한 시스템을 만들려면 다음이 신호가 결합되어야한다. 여러 가지 방법으로...

신경 조직 공학의 지속적인 과제 중 하나는 신경이 자연적으로 성장 세포 외 매트릭스를 모방 한 신경 도관 (NC)를 만드는 것입니다. 연구는 세포가 기계, 지형, 접착제 및 화학 신호 1-3 포함한 환경에서 여러 가지 요인에 반응 것으로 나타났습니다. 이 분야의 주된 문제점 중 하나는 신호들의 적절한 조합을 결정하고 세포의 성장을 지원하기 위해 4 장기간 큐들을 유지할 수있는 시스템을 제조한다. 말초 신경은 연질 기판을 선호하는 것으로 알려져있다, 섬유 배향에 의해 지시되는, 신경 성장 인자 (NGF)에 대응 5-7. 주 화학적 신호를 제공 할 수있는 나노 결정은 가까운 이식, 신경 수리 8,9 현재 금 표준의 기능 회복에 대한 개선을 제공하는 것으로 나타났다. 재료 및 제조 방법은 각종 기계적 및 지형 생성하는데 사용될 수있다알은 10-13 큐들. 기계 단서 1,13 중요한 애플리케이션에 대한 적절한 재료의 선택을, 선택된 물질에 고유하다. 생산 방법은 지형적 단서가 상분리, 자기 조립 및 1,13 전기 방사를 포함 제어한다. 마이크로 애플리케이션, 마이크로 유체, 포토 패터닝, 에칭, 염 침출 또는 가스 발포체를 들어도 14-17을 사용할 수있다. 전기 방사로 인해 유연성 및 생산 13,18-23의 용이성에 조직 배양을위한 섬유 기판을 설계하는 가장 인기있는 방법으로 떠오르고있다. 전기 방사 나노 섬유는 그 자체 격퇴 24를 방전 짧은 갭을 가로 질러 스트레칭 일으키는 고분자 용액에 고전압을인가함으로써 제조된다. 정렬 비계 접지 회전 맨드릴에 섬유를 수집하여 생성 할 수 있습니다 및 비동맹 비계는 고정 플레이트 (25)에 수집됩니다. 접착 시그널링은 섬유질 골격 재치 코팅함으로써 달성 될 수있다H의 피브로넥틴 또는 26을 전기 방사 전에 HA 그러한 RGD 같은 접착 펩타이드, 컨쥬 게이트. 그들이 제어 방출을위한 소스가 필요하기 때문에 이러한 성장 인자와 같은 화학 신호는, 장기간에 걸쳐 유지하는 것이 가장 어렵다. 대부분의 시스템은 성공의 다양한 수준으로 전기 방사 섬유 네트워크 제어 방출을 추가하려고 시도하고있다. 이러한 방법은 혼합 전기 방사, 에멀젼 전기 방사, 코어 쉘 전기 방사 및 단백질 접합 (27)을 포함한다. 또한, 전기 방사는 전통적 따라서 단백질의 생활 성이 고려되어야 유지, 단백질 (28)의 생존에 영향을 미칠 수 휘발성 용매에서 수행된다. 이 접근법은 특히 말초 신경 성장 동조 지지체를 작성하기 위해 기계적, 지형, 화학적 및 접착 신호를 결합 다룬다. 비계 역학 정밀 합성에 의해 제어된다methacrylated 히알루 론산 (HA). 메타 크릴 사이트는 사진 반응 가교제를 연결하는 데 사용됩니다. 가교 물질은 더 이상 수용성없는 독점적 효소 (29)에 의해 분류됩니다. 가교제의 양이 열화 속도, 기계 및 기타 물질의 물성을 변화시킨다. ~ 500 Pa의 인장 탄성률이 30 % 메타 크릴로 HA를 사용하여 신경 조직의 기본 역학에 가까운 일반적으로 신경 세포 26,29 선호하는 부드러운 기판을 만듭니다. 회전하는 맨드릴에 전기 방사하는 지형 큐에 대한 정렬 된 섬유를 만드는 데 사용됩니다. 미소와 함께 전기 방사를 사용하면 확장 된 기간 동안 비계 내에서 화학적 신호를 제공한다. 화학적 신호를 생성하는 데 사용되는 NGF를 함유하는 미세 신경 돌기 성장을 지원하기 위해. NGF 생산시 솔벤트가 발생하지 않도록 대부분의 전기 방사 물질과는 달리 HA는 물에 용해합니다. , SCA를 접착제 신호를 추가하려면ffold는 피브로넥틴으로 코팅된다. NGF는 피브로넥틴 (접착제)로 코팅 된 마이크로 스피어 (화학)를 해제 소프트 (기계) 정렬 (지형) 섬유 : 완성 된 시스템은 전술 한 신호의 네 종류가 포함되어 있습니다. 생산 및이 시스템의 테스트는이 프로토콜에 설명되어있다. 프로세스는 유중 수중 이중 에멀젼 미립자의 제조로 시작된다. 에멀젼은 계면 활성제, 폴리 비닐 알코올 (PVA)로 안정화된다. 내부 수상은 단백질이 포함되어 있습니다. 이를 디클로로 메탄 (DCM)에 용해 PLGA 쉘 물질을 함유하는 유상에 첨가 될 때, 계면 활성제는 DCM로부터 단백질을 보호 상 사이의 장벽을 생성한다. 이 미소 에멀젼의 외부 표면을 생성하기 위해 PVA를 함유하는 다른 수상에 분산보다. 안정한 에멀젼을 DCM가 증발 할 수 있도록 교반 하였다. 세정 및 동결 건조 후에는 건조 미소의 계속 남아 있습니다단백질 aining. 미소가 완료되면 그들은 발판으로 전기 방사 할 준비가 된 것입니다. 먼저 전기 방사 용액을 제조. 용액의 점도는 적절한 섬유 형성에 중요하다. 순수 HA의 솔루션은 이러한 요구 사항을 충족하지 않는; PEO는 전기 방사를 허용하는 담체 중합체로 추가된다. 마이크로 전반에 걸쳐 분산 미소와 섬유 지지체의 결과로 솔루션 및 전기 방사에 추가됩니다. 제작이 완료되면, 단백질은 그 가능성을 검증하기 위해 테스트되어야한다. 이를 위해, NGF에 응답 일차 전지가 사용될 수있다. 이 프로토콜은 8 ~ 10 일 이전 닭 배아에서 등쪽 뿌리 신경절 (DRG)를 사용합니다. 셀 번들은 NGF 또는 비어있는 것들로 가득 미소를 포함하는 발판에 씨앗을 품고있다. NGF는 여전히 실행 가능한 경우에는 NGF 포함 비계에 강화 된 신경 돌기의 성장을 볼 수 있습니다. NGF가 더 이상 실행 가능한 없으면 그것 것하지 확장하는 신경 돌기를 촉진하고 컨트롤과 유사하게 나타납니다. 본원에 설명 된 정확한 순서는 단순한 변형 재료, 전기 방사 방법 및 시스템은 다양한 세포 및 조직 종류에 최적화 될 수있는 단백질로, 그러나, 신경 지지체 상에 집중된다.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospinning growth factor releasing microspheres into fibrous scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

85 %의 단백질 캡슐화 직경에, 미소 ± 14 μm의 (50)는 지속적으로 생산 발판으로 전기 방사되고있다. 크기는 세 개의 분리 된 생산 배치에서 미소 구체의 샘플을 이미징에 의해 결정 하였다. 상업용 랩 소프트웨어를 사용하여 측정 한 광학 현미경과 길이에 캡처 이미지. 도표 1은 크기 분포의 히스토그램을 도시한다. 캡슐화 율은 생산 공정 중에 탈출 단백질을 정량화하여, 세 개의 분리 ?...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

섬유 공사장 공중 발판으로 마이크로 스피어를 해제 전기 방사의 성장 인자

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K 조회수.  Wayne State University. This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

비디오: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospinning 기본 : 최적화 솔루션 및 장치 매개 변수

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

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생체공학

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

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

탄성 PGS는 아티 조직 공학 공사장 공중 발판

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

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

액슨 스트레치 성장의 연결을 성장의 Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

표피 성장 인자를 사용하여 인간의 췌장 암 세포의 유전자 전달 및 Transfection은 다행 기반 엔지니어링된 Nanovectors를 수용체 타겟

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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

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

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

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

마우스 배아 줄기 세포에서 신경 조직 공학을위한 폴리의 전기 방사 섬유 발판 (글리세롤 dodecanedioate)

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

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

상호 현미경을 사용하여 본래 세포의 표피 성장 인자 수용체의 화학 양론을 공부

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

헤파린 결합 도메인 골 형성 성장 인자 전달을위한 고분자 전해질 복합

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

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.

신경 성장 인자 인 캡슐 레이션을위한 새로운 HDL 모방 나노 입자의 제조 및 특성 규명

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

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

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

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

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

조직 공학 응용 프로그램에 대 한 제어 형태학과 3 차원 Poly(ε-caprolactone) 장비의 전기 글 녹아

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

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

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

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

Decellularized 연골 파생 매트릭스 건설 기계 제조

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