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. 
Fu...

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 border="0" cellpadding="0" cellspacing="0">
	<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>&#945;-Modified Eagle&#39;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 여기...

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

Research

JoVE Journal

Bioengineering

9.7K 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

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 ...

연구

생체공학

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 ...

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 ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

상피 세포 다시 채우기 및 신장 조직 개발을위한 설치류 세포 외 기질 비계의 준비

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

의 생명 공학에 대한 각질 기반의 나노 섬유의 합성

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

손쉬운 및 환경 친화적 인 경로는 그레이드 기공 크기와 폴리 (젖산) 공사장 공중 발판을 제조

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

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 and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

감염 성병 제조 및 방지를 위한 Griffithsin 수정 섬유 장비의 특성

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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 ...