Henshaw D Ross, Attia Erik, Bhargava Madhu, Hannafin Jo A
Laboratory for Soft Tissue Research, Sports Medicine and Shoulder Service, The Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021, USA.
J Orthop Res. 2006 Mar;24(3):481-90. doi: 10.1002/jor.20050.
Tissue-engineered ligament substitutes have the potential to become an alternative graft source for ligament reconstruction. If this approach is to become viable, one must first understand and define the mechanisms responsible for creation, maintenance, and remodeling of the native anterior cruciate ligament. It is well accepted that mechanical load alters fibroblast phenotypic expression in a variety of cell sources; however, the mechanosensitive pathways responsible for alteration in matrix production, remodeling, and alignment are unknown. We hypothesize that cell surface integrins play a role in this mechanotransduction process, and as such respond to application of cyclic tensile load. Linear 3D collagen gels containing canine ACL fibroblasts were created in Flexercell Tissue-Train Culture Plates. Gels were untethered (control), tethered without external strain (tethered), or tethered and exposed to 2.5% cyclic strain for 2 h per day for 4 days (strain). Quantitation of alpha1, alpha5, and beta1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of alpha5 and beta1 integrin subunits was increased significantly in fibroblasts in tethered and strained 3D collagen gels compared with the control, unloaded constructs (p < 0.05). These integrins are known to function as mechanotransducers in other tissues, implicating a similar role in mechanotransduction in ACL fibroblasts. Histologic analysis of the tethered and strained gels demonstrated a linear arrangement of cells and parallel collagen fibril architecture. In contrast, cell distribution and collagen alignment were disorganized in the control, unloaded gels. The alignment of cells and collagen in the 3D gels parallel to applied strain is similar to the in vivo state. These data add to our understanding of the behavior of ACL fibroblasts in vitro. The ability to manipulate signal transduction pathways may enhance our ability to engineer implantable ACL grafts or to modify ACL healing response.
组织工程韧带替代物有可能成为韧带重建的一种替代移植物来源。如果这种方法要可行，首先必须了解并确定负责天然前交叉韧带的形成、维持和重塑的机制。机械负荷会改变多种细胞来源中的成纤维细胞表型表达，这一点已得到广泛认可；然而，负责基质产生、重塑和排列改变的机械敏感途径尚不清楚。我们假设细胞表面整合素在这个机械转导过程中起作用，因此会对周期性拉伸负荷的施加做出反应。在Flexercell组织培养板中制作了含有犬前交叉韧带成纤维细胞的线性三维胶原凝胶。凝胶未系留（对照）、系留但无外部应变（系留）或系留并每天暴露于2.5%的周期性应变下2小时，持续4天（应变）。使用流式细胞术对α1、α5和β1整合素亚基进行定量。使用光学显微镜、偏振光显微镜和荧光显微镜研究细胞和基质的排列。与对照的未加载构建体相比，系留和应变的三维胶原凝胶中的成纤维细胞中α5和β1整合素亚基的表达显著增加（p < 0.05）。已知这些整合素在其他组织中作为机械转导器发挥作用，这意味着在 ACL 成纤维细胞的机械转导中也有类似作用。对系留和应变凝胶的组织学分析显示细胞呈线性排列且胶原纤维结构平行。相比之下，对照的未加载凝胶中的细胞分布和胶原排列是紊乱的。三维凝胶中细胞和胶原与施加应变平行的排列类似于体内状态。这些数据增进了我们对 ACL 成纤维细胞体外行为的理解。操纵信号转导途径的能力可能会增强我们构建可植入 ACL 移植物或改变 ACL 愈合反应的能力。
