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首页> 外文会议>World biomaterials congress >Effect of micro-fiber modulus on mesenchymal stem cell morphology and function in model micro-fiber/hydrogel composites
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Effect of micro-fiber modulus on mesenchymal stem cell morphology and function in model micro-fiber/hydrogel composites

机译：超细纤维模量对模型超细纤维/水凝胶复合材料中间充质干细胞形态和功能的影响

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Introduction: Ligament ruptures afflict thousands of Americans annually, often requiring surgical replacement with autologous or allogeneic grafts to restore knee joint mechanics. However, both slow healing and resulting abnormal biomechanics can accelerate joint degeneration. Tissue engineering holds promise in overcoming limitations of existing treatments through the fabrication of scaffolding materials that support cell infiltration, proliferation, and differentiation into an organized tissue. Electrospun scaffolds with different properties (e.g., topographical, mechanical, chemical, biological) can support mesenchymal stem cell (MSC) proliferation and guide deposition of an anisotropic extracellular matrix (ECM). Since the high fiber densities of electrospun meshes impede cell infiltration, we have developed composites consisting of sparse aligned micro-fibers within a collagen hydrogel. This system permits MSC migration and proliferation while providing anisotropic mechanical and topographic cues to guide MSC morphology and function. Based on a growing body of evidence that matrix stiffness affects stem cell fate, the goal of this study was to determine how the tensile modulus of electrospun micro-fibers influences MSC morphology and function. Methods: Micro-fiber meshes with varying modulus were prepared by electrospinning mixtures of polycaprolactone (PCL) and polyurethane (PUR) onto a rotating mandrel. Next, sparse micro-fiber/hydrogel composites were assembled by the encapsulation of thin (-5μm) fiber layer within a collagen gel (Figure 1). MSCs were cultured for up to 14 days in composites and the cell morphology and spatial distribution analyzed by confocal microscopy. Cell number and mRNA expression of ligaments markers were measured by Picogreen and PCR. Results and Discussion: Elastic moduli of 31, 15, and 5.6 MPa (Figure 2a) were obtained for 0.6-0.7 μm diameter fiber meshes comprised of PCL, PUR, and a 25/75 wt% PCL/PUR blend, respectively. MSCs - combined with collagen and cast atop the micro-fiber layer - oriented with the underlying fiber network (Figure 2c) but remained randomly oriented in the absence of fibers (Figure 2d). After 14 days, the 5.6 MPa composites, possessed elongated and oriented cells throughout the collagen bulk. In contrast, the 31 MPa composites, exhibited randomly oriented polygonal cells in the collagen bulk. PCR at day 14 indicated that the tendon/ligament transcription factor Scleraxis and the contractile protein α-smooth muscle actin (a marker of a regenerative phenotype) were elevated for cells in the 5.6 MPa composites compared to collagen controls, while collagen 1α1 was elevated in the 31 MPa (Figure 2b). Thus, MSCs sense the mechanical properties conferred by electrospun fibers, with the softer fibers leading more promising outcomes for ligament applications: higher levels of ligament markers and better cell alignment.
机译：简介：韧带破裂每年折磨着成千上万的美国人，常常需要用自体或同种异体移植物进行手术置换，以恢复膝关节的力学性能。但是，缓慢的愈合和由此引起的异常生物力学都会加速关节变性。组织工程技术有望通过制造支持细胞浸润，增殖和分化为有组织组织的支架材料来克服现有治疗方法的局限性。具有不同特性（例如，地形，机械，化学，生物学）的电纺支架可以支持间充质干细胞（MSC）增殖并指导各向异性细胞外基质（ECM）的沉积。由于电纺网的高纤维密度会阻碍细胞浸润，因此我们开发了由胶原蛋白水凝胶中稀疏排列的微纤维组成的复合材料。该系统允许MSC迁移和增殖，同时提供各向异性的机械和地形线索来指导MSC的形态和功能。基于越来越多的证据表明基质刚度会影响干细胞的命运，这项研究的目的是确定电纺微纤维的拉伸模量如何影响MSC的形态和功能。方法：通过将聚己内酯（PCL）和聚氨酯（PUR）的混合物静电纺丝到旋转的心轴上来制备具有不同模量的微纤维网。接下来，通过将薄的（-5μm）纤维层封装在胶原蛋白凝胶中来组装稀疏的微纤维/水凝胶复合物（图1）。将MSCs在复合物中培养长达14天，并通过共聚焦显微镜分析细胞形态和空间分布。通过Picogreen和PCR检测韧带标志物的细胞数目和mRNA表达。结果与讨论：分别由PCL，PUR和25/75 wt％PCL / PUR共混物组成的直径为0.6-0.7μm的纤维网，其弹性模量分别为31、15和5.6 MPa（图2a）。 MSC与胶原蛋白结合并浇铸在微纤维层之上，与下面的纤维网络定向（图2c），但在没有纤维的情况下仍保持随机定向（图2d）。 14天后，5.6 MPa的复合材料在整个胶原蛋白块体中均具有细长且定向的细胞。相比之下，31 MPa的复合材料在胶原蛋白主体中表现出随机取向的多边形细胞。第14天的PCR结果表明，与胶原蛋白对照组相比，5.6 MPa复合材料中的细胞的腱/韧带转录因子Scleraxis和收缩蛋白α-平滑肌肌动蛋白（可再生表型的标志物）升高，而胶原蛋白1α1在胶原蛋白中的含量则升高。 31 MPa（图2b）。因此，MSC感觉到了电纺纤维所赋予的机械性能，而较软的纤维则为韧带应用带来了更有希望的结果：更高水平的韧带标记和更好的细胞排列。

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