Bye Frazer J, Wang Linge, Bullock Anthony J, Blackwood Keith A, Ryan Anthony J, MacNeil Sheila
Materials Science and Engineering, University of Sheffield.
J Vis Exp. 2012 Aug 9(66):4172. doi: 10.3791/4172.
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.
静电纺丝是一种常用且通用的方法，用于生产用于三维组织工程的支架（通常是可生物降解的）。（1,2,3）随着儿童成长，体内许多组织会经历不同程度的双轴扩张，如皮肤、膀胱、盆底，甚至硬腭。为实现这些目的而生产支架时，需要开发具有适当生物力学性能的支架（无论是否有细胞参与），并且这些支架要无菌以用于临床。本文的重点不是如何确定基本的静电纺丝参数（因为关于静电纺丝已有大量文献），而是如何在生产后对纺制的支架进行改性，使其适合组织工程目的——这里考虑了厚度、机械性能和灭菌（临床使用所需），我们还描述了如何在支架上培养细胞并使其承受双轴应变，以使其适应特定应用。静电纺丝往往会产生薄片；随着静电纺丝收集器被绝缘纤维覆盖，它会变成不良导体，导致纤维不再沉积在上面。因此，我们描述了通过加热或蒸汽退火来生产更厚结构的方法，这会增加支架的强度，但不一定增加弹性。还描述了通过顺序纺制不同聚合物的支架来获得复杂支架的方法。灭菌方法可能会对支架的强度和弹性产生不利影响。我们比较了三种方法对聚乳酸 - 乙醇酸共聚物（PLGA）静电纺丝支架生物力学性能的影响。描述了支架上细胞的成像以及支架上细胞产生细胞外基质（ECM）蛋白的评估。在体外支架上培养细胞可以提高支架的强度和弹性，但组织工程文献表明，在静态条件下培养时，细胞通常无法产生适当的ECM。市面上很少有商业系统能够让人们在动态条件下在支架上培养细胞——其中一个例子是Bose Electroforce 3100，它可用于对置于充满培养基的腔室内、由机械夹具固定的支架中的细胞施加调节程序。（4）描述了一种用于二维控制变形的经济型细胞培养生物反应器的方法。我们表明在这些条件下可以诱导细胞产生弹性蛋白。最后描述了对有或没有细胞培养的加工支架的生物力学性能评估。
