Name: electrospun nanofiber scaffolds with gradations in fiber organization
Uploaded: 2015-04-19T15:00:00.000+00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

이 작품은 네브라스카 의료 센터와 국립 보건 연구소 (허가 번호 1R15 AR063901-01)의 대학에서 시작 펀드에서 부분적으로 지원되었다.

해결 방법 1. 준비 <ol><li> 100 ㎎ / ㎖의 대략적인 농도 폴리 (ε 카프로 락톤) (PCL) (w M = 80,000g / 몰)의 용액을 준비합니다. (4)의 비율로 디클로로 메탄 (DCM)과 N, N-dimethlyformamide (DMF)의 혼합물에 녹이고 PCL : 1 (v / v)로 10 %의 농도를 갖는 (W / V). </li><li> 혼합하는 20 ㎖ 유리 튜브에서 용액을 놓는다. 30 분 동안 초음파 세척기에 유리 튜브를 배치, 또는 솔루션은 반투명 때까지. </li></ol> 2. 장치 준비 <ol><li> 부착 된 21 게이지 무딘 바늘로 5 ML의 주사기에 준비 PCL 솔루션을 추가합니다. </li><li> 그림 1 항에있어서, 수직 전기 방사 위치에 주사기 펌프를 놓습니다. </li><li> 2cm x를 정렬 섬유 기판 5cm의 열린 공간을 스테인리스 갭 수집기를 사용합니다. 컬렉터에게 바늘 끝에서 12cm를 놓습니다. </li><li> 에 직류 (DC) 고전압 전원을 접속바늘과 콜렉터 접지. 집이 개별적으로 다른 실험실 장비없이 연락처와 접지되어 있는지 확인합니다. </li></ol> 3. 전기 방사 <ol><li> 제거 할 때 바늘 끝에서 형성 방울까지 1.50 ㎖ / 시간으로 설정 주사기 펌프는 즉시 대체됩니다. 그런 다음, 0.50 ㎖ / 시간에 유량을 설정합니다. </li><li> 12 kV의에 전압 연산자를 돌립니다. </li><li> Electrospin는 일축 정렬 섬유는 완전히 집에 격차를 커버 할 때까지. </li><li> 작은 유리판의 가장자리에 접착제를 적용하고, 유리판에 갭 수집기로부터 섬유를 옮긴다. 유리판과 같은 크기를 가지며 접지 된 알루미늄 호일 조각 위에 유리판을 놓고. </li><li> 그림 3에 따른 제 2 주사기 수집기를 배치합니다. </li><li> 집 위의 두 번째 주사기 펌프 및 위치는 2 mm의 플라스틱 마스크를 연결합니다. </li><li> (W / W) PCL 솔루션의 경우 할머니 1 %에서 쿠마린 추가 6oencapsulation는 desired-하고, 용액이 투명해질 때까지 혼합한다. </li><li> 무딘 21 게이지 바늘로 5 ml의 바늘에 PCL 또는 PCL / 쿠마린 (6) 솔루션을로드합니다. 0.50 ㎖ / 시간으로 수직 펌프를 설정하고 9 ㎖ / 시간 또는 1mm / 분의 대략적인 속도로 당길 수평. </li><li> 마스크까지 Electrospin 콜렉터 거의 완전히 떨어져 이동하지만, 아직 마스크 엣지 영역으로하고있다. </li></ol> 4. 섬유 특성 <ol><li> 40mA에서 스퍼터 코터를 이용하여 40 초 동안 백금과 금속 스터드와 코트에 양면 전도성 테이프와 장소 샘플. <ol><li> 우리의 이전 연구 17, 18에 따른 주 사형 전자 현미경 (SEM)을 통해 섬유를 조사한다. </li><li> 15 kV로의 가속 전압에서 이미지를 수집합니다. </li></ol></li><li> 섬유 배향을 측정하는 별도의 섬유 샘플을 고속 푸리에 변환 (FFT) 분석을 수행한다. FFT에 의한 측정 섬유 정렬에 대한 자세한 내용은 t 참조 할 수 있습니다이전의 연구 (19, 20) 오. </li></ol> 5. 시드 줄기 세포. <ol><li> 2 시간 동안 70 % 에탄올 용액에 담가 섬유 샘플을 소독. 그런 다음, 오염물을 제거하고 증류수로 세척 섬유. </li><li> 95 % 공기 / 5 % CO 2의 분위기에 37 ° C에서 25cm 2 플라스크에 인간 ADSCs과 문화 세포를 얻습니다. 세포 배양 배지를 매일 10을 변경합니다. </li><li> 세포를 Trypsinize 세포의 개수를 카운트. 구체적으로, 세포 배양 플라스크에서 모든 배지를 제거하고 인산 완충 식염수 회 (PBS)으로 세포를 세척 하였다. 그런 다음 세포 단층을 포함 2 ~ 3 분 동안 세포 배양 인큐베이터에서 플라스크를 부화 (37 ° C에 물을 욕조에 미리 따뜻한) 0.25 % 트립신 - EDTA 용액의 1m 리터를 추가합니다. 4 ml의 배지를 추가하고 모든 표면에 매체를 피펫 팅에 의해 표면에서 모든 세포를 씻어. 세포 현탁액을 원심 분리 재 분산 번째배양액 E 세포 펠릿. hemacytometer를 통해 세포를 계산합니다. 와 35mm의 - 문화 접시에 배치 된 나노 섬유 지지체에 1 × 104 세포 주위에 씨는 3, 7 일 동안 배양한다. </li><li> 형광 다음 디 아세테이트 (아세톤 5 ㎎ / ㎖) (FDA) 이미지 형광 현미경을 이용하여 샘​​플을 사용하여 얼룩 세포. 구체적으로, 배양 접시에 FDA 용액 100 ㎕를 추가하고 30 분 동안 배양한다. 세 번 형광 이미징 전에 PBS로 세포를 씻으십시오. 우리의 이전 연구 (10)에 따라 사용자 정의 매트 실험실 프로그램에 의해 세포의 방향을 분석합니다. </li></ol>

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The authors have nothing to disclose.

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Furthermore, the protocol allows for fabrication of discrete gradations in fiber organization. However, this protocol yields variable thicknesses across the scaffold, which could potentially limit its mechanical applications. The area with increased random fiber deposition will be thicker than the aligned region. This protocol also does not extend to the recreation of the chemical composition at tendon-to-bone insertion site. Construction of mineral gradients is needed to successfully recreate the native mineral contents at the insertion sites. Fabrication of fiber scaffolds with gradients in mineral composition and organizational structure will be further explored to better replicate the complete native environment in a tendon-to-bone interfacial tissue. 
As of now, this is the first protocol for fabrication of an electrospun nanofiber scaffold with an organizational gradient. Previous studies have fabricated scaffolds with both random and aligned fibers, but only as two separate sections, with an immediate change between the two organizations9. Gradient structures should allow for tailoring cell orientation and their extracellular matrix deposition and thus contribute to graded mechanical properties throughout the tissue constructs. Native tendon-to-bone insertion sites have reduced stiffness by decreasing the fiber alignment on one side2, which can be replicated with our scaffold. Additionally the organizational gradient offers increased biomimicry of the extracellular matrix with the new ability to replicate the native fiber orientations. This structure offers more biomedical applications by the formation of chemical gradients through nanoencapsulation. New chemical gradients facilitate areas of study in cellular behavior, high throughput screening, and implant or implant coating design10. Additionally bone morphogenetic protein2 (BMP2), which is resembled by a dye molecule Coumarin-6 because of its ease of detection using fluorescence microscopy, could be encapsulated to further promote cellular differentiation at the end made of random nanofibers10. Encapsulation within the fiber gradient provides a method for control of its chemical concentration, as BMPs are only needed at the site of bone tissues.

나노 섬유는 때문에 구조와 상대적 크기 1의 세포 외 기질을 모방 할 수있는 능력의 조직 공학에 대한 인기 유틸리티입니다. 그러나, 이러한 힘줄 투 뼈 삽입 부위의 조직 일부 원시 인터페이스, 힘줄을 향해 정렬 증가 뼈 사이트 2-5로 감소 변수 조직 구조를 나타낸다 콜라겐 섬유를 함유한다. 따라서, 효과적인 조직 재생이 효과적으로 구조 구배를 모방 할 수 지지체를 제조 할 필요가있다. 섬유 조성물의 점진적인 변화를 실시 이전 연구되고있다, 즉, 미네랄 함량 6. 그러나, 결합 조직의 구조 부품을 다시하면 크게 비경 남아있다. 이전 연구는 쥐의 두 개관 골 모세포의 증식 표면 실리카 입자 밀도의 효과를 연구함으로써 형태학 그라디언트 검사하고 인버 발견실리카 입자 밀도, 세포 증식 (7) 사이 SE 관계. 그러나 이전의 연구에서 세포 증식을 매개 형태 학적 변화는 섬유 조직 변경 7, 8을 흉내 낸의 능력을 결여 표면 거칠기에 주로 관련이 있었다. 최근의 한 연구는 9 전기 방사에 대한 새로운 컬렉터를 사용하여 고유의 콜라겐 섬유 방향을 모방 발판을 제작하려고했습니다. 이 연구는 모두 정렬 랜덤 섬유와 발판을 생산하는 데 성공하지만, 네이티브 조직에 전시 점진적인 변화를 모방하는 데 실패했습니다. 또한, 임의의 방향에 정렬에서 즉각적인 변화, 별도의 부품을 생산,이 지지체의 생체 역학적 특성은 유의하게 감소 하였다. 아니 이전의 작품은 정렬 된 임의의 섬유 방향에 연속 계조로 적용 나노 섬유 지지체를 생산할 수 없었다. 우리의 최근의 연구는 나노 섬유 지지체의 성공적인 휴양을 보여 주었다잠재적 건 - 투 - 뼈 삽입 (10)에서 네이티브 콜라겐 조직을 모방 할 수 섬유 조직의 계조와. 이 작업은 밀접 네이티브 힘줄 투 뼈 조직 인터페이스에서 섬유 조직이 유사한 구조를 갖는 나노 섬유 지지체의 제조에 사용되는 프로토콜을 제공하는 것을 목적으로한다. 그라데이션 나노 섬유 구조는 잠재적으로 다양한 분야에서 응용 프로그램을 광범위한했다. 우리는 이미 다양한 기판 11-14 조직 재생을 위해 사용된다 지방 유래 줄기 세포 (ADSCs) 우리의 발판을 결합하여 건 - 투 - 뼈 삽입 부위의 조직 공학에 응용 프로그램에 초점을 맞추었다. 또한, 다 능성 ADSCs는 측면에서 골수 줄기 세포를 본질적으로 매우 유사하며, 이들 자원은 지방 흡입 간단한 절차 (15, 16)를 사용하여 수거 할 수있는 풍부하다. 더 계조 나노 섬유 지지체에 이들 세포를 심는 것은 자신의 TIS을 향상잠재적으로 다양한 조직으로 분화 할 수있는 세포의 제어 배포 허용함으로써 엔지니어링 응용 프로그램을 고소. 줄기 세포를 시딩 외에 나노 파이버는 세포 반응의 조절에 신호 분자로 캡슐화 될 수있다. 이러한 비계의 조직 그라데이션 nanoencapsulation 커플 링은 세포 행동이나 가능한 임플란트 디자인과 코팅의 연구 수 있습니다. 조골 세포 분화를 유도 15,16 도시 된 뼈 형태 형성 단백질 2 (BMP2), 작용 성 분자의 캡슐화는 또한 이들 지지체 (10)의 조직 공학 응용 프로그램을 향상 할 수있다.

<table><tbody><tr><td>Polycaprolactone</td><td>Sigma-Aldrich</td><td>440744</td><td></td></tr><tr><td>N,N-Dimethlyformamide</td><td>Fisher Chemical</td><td>D-119-1</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>AC61093-1000</td><td></td></tr><tr><td>Coumarin 6</td><td>Sigma-Aldrich</td><td>546283</td><td></td></tr><tr><td>Adipose Derived Stem Cells</td><td>Cellular engineering Technologies</td><td>HMSC.AD-100</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>&amp;alpha;-Modified Eagle&#039;s Medium</td><td>Invitrogen</td><td>a10490-01</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>s25120a</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>Glass Slides</td><td>VWR international, LLC</td><td>101412-842</td><td></td></tr><tr><td>Syringe Pump</td><td>Fisher Scientific</td><td>14-831-200</td><td>Single syringe</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>High Voltage DC Power Supply</td><td>Gamma High Voltage Research</td><td>ES30</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Nova 2300</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr></tbody></table>

electrospun nanofiber scaffolds with gradations in fiber organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

이 프로토콜을 이용하여, 조직 그라데이션 섬유 매트를 형성 하였다.도 3은 나노 섬유 지지체상의 다양한 위치에서 찍은 SEM 이미지를 도시한다. 질적으로, 그것은 6mm (도 3d)에서 랜덤 섬유 구색 0mm (도 3A)에서 일축 정렬 된 섬유에서 진행이 있는지 판단 할 수있다. FFT는 섬유 정렬을 정량적 가치를 제공, 정량적 프로세스에 대한 세부 사항은 (19)는 여기에 자세히 설명되어 있습니다. 0mm 전시 섬유 배향을 나타내고, 6mm에서 FFT 패턴은 무작위 배향을 의미 FFT에서 섬유. 점점 임의 섬유 증착 (- C 그림 3B)에 정렬 된 섬유 조직의 SEM 이미지에서 명확한 진행 (그림 3)이 있습니다. ADSCs는 나노 섬유 지지체에서의 위치에 따라 형태 변경을 시행 하였다. 그림 4 </stron7 일 (그림 4E - H) - g&gt; 3 일 (D 그림 4A)에서 형광 현미경 (자이스)로 촬영 한 이미지를 보여줍니다. 줄기 세포의 각도 분포를 정량적으로 정의 MATLAB 프로그램에 의해 평가하고, 다양한 거리에서의 콜 모고 로프 - 스 미르 노프 테스트를 사용하여 분석 하였다. 4I는 상이한 위치에서 각 셀의 분포를 도시한다. 0mm, 또는 정렬 된 섬유의 영역에서 세포의 70 %가 나노 섬유의 제조 축의 20 ° 내에 나타났다. 대조적으로, 20 ° ADSCs 내에 나타나는 세포의 20 %가이 조직 구조 결여 섬유 지지체의 임의의 부분에 시드. 마지막으로, 쿠마린 6 이용한 화학적 구배의 형성 -로드 PCL 섬유는 형광 현미경을 이용하여 연구 하였다. 화학적 구배 질적 현미경 영상 (도 5a)를 사용하여 확인 하였다. 화상을 확인 증가 CHEMI형광 이미지의 꾸준히 증가 강도에 의해 전시 된 발판에서 칼 농도. 형광 강도 (이미지 J) (도 5b)의 그래프는 발판에 걸쳐 선형 증가를 나타내는 화학적으로 농도 구배를 확인한다. <img alt="그림 1" src="/files/ftp_upload/52626/52626fig1.jpg" /> 그림 1 : 일축 정렬 섬유 기판의 제조를위한 실험 장치의 개략도를 보여줍니다. <img alt="그림 2" src="/files/ftp_upload/52626/52626fig2.jpg" /> 도 2 (A)는 경사 지지체의 제조를위한 제 주사기 펌프의 배치가도. 콜렉터 위의 마스크 (B)를 요구함. 이 수치는 [10] 문헌 [Macromol에서 재 인쇄되었습니다. Biosci., 12시에, J., 엄마, B., 마이클, PL 및 슐러, FD 파브리섬유 조직 및 그들의 잠재적 인 응용 프로그램에서의 그라데이션으로 나노 비계의 양이온. 1336년부터 1341년까지, 저작권 2012, 와일리 - VCH의 허가. <img alt="그림 3" src="/files/ftp_upload/52626/52626fig3.jpg" /> 도 3 : 0mm (A), 2mm (B), 4mm (C), 및 6mm (D)에서의 계조 PCL 나노 섬유 지지체의 SEM 이미지. 보조 이미지는 푸리에 빠른 전송 패턴 (FFT)입니다. (A)에서 패턴 정렬 섬유, (D)의 임의의 섬유 증착을 제시한다는 것이다. 이 수치는 [10] 문헌 [Macromol에서 재 인쇄되었습니다. Biosci., 12시에, J., 엄마, B., 마이클, PL 및 슐러, 섬유 조직의 변화와 그들의 잠재적 인 응용 프로그램이있는 나노 비계의 FD 제작. 1336년부터 1341년까지, 저작권 2012, 와일리 - VCH의 허가. <imgALT = &quot;그림 4&quot;SRC = &quot;/ 파일 / ftp_upload / 52626 / 52626fig4.jpg&quot;/&gt; 그림 4 : 형광 현미경 이미지를 3 일 (A - D) 배양 후 ADSCs을 보여주는 7 일 (E - H). 이미지의 계조 지지체 다른 위치에 ADSCs의 다양한 모폴로지를 나타낸다. (I) : 지지체의 다른 위치에서의 셀의 각도 분포. 세포는 훨씬 더 많은 섬유 배향 (0mm)에 나노 섬유 배향 축을 20 °까지 농축 하였다. 이 수치는 [10] 문헌 [Macromol에서 재 인쇄되었습니다. Biosci., 12시에, J., 엄마, B., 마이클, PL 및 슐러, 섬유 조직의 변화와 그들의 잠재적 인 응용 프로그램이있는 나노 비계의 FD 제작. 1336년부터 1341년까지, 저작권 2012, 와일리 - VCH의 허가. <img alt="그림 5" src="/files/ftp_upload/52626/52626fig5.jpg" /> Figu5 재 : 쿠마린 6 캡슐화 된 섬유 (A) 형광 현미경 이미지. (B)의 그래프는 발판에 걸쳐 형광 강도를 나타낸다. 선형 증가는 지지체를 통해 화학적 농도의 점진적 변화를 의미한다. 이 수치는 [10] 문헌 [Macromol에서 재 인쇄되었습니다. Biosci., 12시에, J., 엄마, B., 마이클, PL 및 슐러, 섬유 조직의 변화와 그들의 잠재적 인 응용 프로그램이있는 나노 비계의 FD 제작. 1336년부터 1341년까지, 저작권 2012, 와일리 - VCH의 허가.

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

여기서 우리는 섬유의 계조 조직과 전기 방사 나노 섬유 지지체를 제작하고 세포 형태 / 방향을 조절하는 응용 프로그램을 탐구하는 프로토콜을 제시한다. 나노 섬유 지지체의 물리 화학적 특성에 관한 그라디언트는 생물 의학 분야에서 다양한 응용 프로그램을 제공합니다.

섬유 조직의 그라데이션으로 전기 방사 나노 섬유 공사장 공중 발판

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.8K 조회수.  University of Nebraska Medical Center.  여기서 우리는 섬유의 계조 조직과 전기 방사 나노 섬유 지지체를 제작하고 세포 형태 / 방향을 조절하는 응용 프로그램을 탐구하는 프로토콜을 제시한다. 나노 섬유 지지체의 물리 화학적 특성에 관한 그라디언트는 생물 의학 분야에서 다양한 응용 프로그램을 제공합니다. 

비디오: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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. ...

연구

생체공학

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

차 모터 및 Electrospun 폴리 - L - lactide Nanofiber 공사장 공중 발판을에 정의된 미디어 감각 뉴런의 문화

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

신경과학

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

건설 기계 3-차원으로 2 차원 Electrospun Nanofiber 매트의 확장

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

유전학

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

ECM 단백질 나노 섬유 및 나노 구조 엔지니어링 사용하여 표면 시작 회의

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

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.

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, ...

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 ...

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 ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

조직 공학 및 세포 배양을위한 섬유 고분자의 공사장 공중 발판을 Electrospinning

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...