Garvin Joanne, Qi Jie, Maloney Melissa, Banes Albert J
Curriculum of Applied and Materials Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Tissue Eng. 2003 Oct;9(5):967-79. doi: 10.1089/107632703322495619.
Cells cultured in three-dimensional collagen gels express a more native state phenotype because they form a syncytial network that can be mechanically loaded. Moreover, cells remodel their matrix by eliminating water, and by reorganizing and aligning the collagen fibrils. Last, the ability to subject cells to mechanical loading in a native matrix is desirable because cells, in tissues as well as the matrix, bear strains and alter their expression profile consistent with either immobilization, moderate activity, or repetitive loading. This is the first report of a model bioreactor system to fabricate and culture tendon cell-populated, linear, tethered matrix constructs that can be mechanically loaded by a computer-driven, pressure-controlled system. Bioartificial tissues (BATs) as tendon constructs were molded in a novel, rubber bottom Tissue Train culture plate bearing nonwoven nylon mesh anchors at the east and west poles of each culture well. Mechanical loading was achieved by placing an Arctangle loading post (an Arctangle is a rectangle with curved short ends) beneath each well of the six-well culture plate and using vacuum to displace the flexible membrane downward, resulting in uniaxial strain on the BAT. BATs populated with avian flexor tendon cells expressed collagen genes I, III, and XII as well as aggrecan, fibronectin, prolyl hydroxylase, and tenascin, consistent with expression levels of cells grown on collagen-bonded two-dimensional surfaces or in native, whole, avian flexor tendon. Likewise, cells in BATs established a morphology of linearly arranged cells aligned with the principal strain direction as in fasicles of whole tendons. Last, BATs that were mechanically loaded had an ultimate tensile strength that was nearly 3-fold greater than that of nonloaded BATs in the first week of culture. Taken together, these results indicate that tendon cells fabricated in a mechanically loaded, linear collagen gel construct assume a phenotype that is similar to that of a native tendon in terms of appearance and expression and are stronger than nonexercised counterparts yet far weaker than native adult tendons. This technique represents a novel approach to culturing cells in a mechanically active, three-dimensional culture environment that can be readily used for the fabrication of tissue simulates for drug testing or tissue engineering.
在三维胶原蛋白凝胶中培养的细胞表现出更接近天然状态的表型，因为它们形成了一个可承受机械负荷的多核网络。此外，细胞通过排出水分、重新组织和排列胶原纤维来重塑其基质。最后，在天然基质中对细胞施加机械负荷的能力是可取的，因为组织中的细胞以及基质会承受应变，并根据固定、适度活动或重复负荷改变其表达谱。这是关于一种模型生物反应器系统的首次报道，该系统用于制造和培养可由计算机驱动的压力控制系统进行机械加载的、含有肌腱细胞的线性、系留基质构建体。作为肌腱构建体的生物人工组织（BATs）在一种新型的、底部为橡胶的组织培养板中成型，该培养板在每个培养孔的东、西两极带有非织造尼龙网锚。通过将一个弧形加载柱（弧形是指短边为弧形的矩形）放置在六孔培养板每个孔的下方，并利用真空使柔性膜向下移动，从而在BAT上产生单轴应变，实现机械加载。接种了禽屈肌腱细胞的BATs表达了I、III和XII型胶原蛋白基因以及聚集蛋白聚糖、纤连蛋白、脯氨酰羟化酶和腱生蛋白，这与在胶原结合的二维表面或天然的完整禽屈肌腱中生长的细胞的表达水平一致。同样，BATs中的细胞形成了与整个肌腱束中一样的、沿主应变方向线性排列的细胞形态。最后，在培养的第一周，经过机械加载的BATs的极限抗拉强度比未加载的BATs高出近3倍。综上所述，这些结果表明，在机械加载的线性胶原凝胶构建体中培养的肌腱细胞在外观和表达方面呈现出与天然肌腱相似的表型，并且比未进行锻炼的对应物更强，但远弱于天然成年肌腱。这项技术代表了一种在机械活性三维培养环境中培养细胞的新方法，可轻易用于制造用于药物测试或组织工程的组织模拟物。
