Toward tissue engineering of the knee meniscus

描述(申请人提供)：该更新方案旨在组织工程一种各向异性半月板结构，该结构还能捕捉天然组织中存在的区域变化。随后，该构建物将被植入Leporine模型，使用同种和异种细胞来源，以实现半月板修复和替换。假设：1)通过优化细胞培养和无支架培养条件，可以设计出区域变异的、各向异性的半月板形状的结构；2)某些合成代谢和分解代谢刺激的协同和战略性的时间应用将增强成熟的半月板结构的功能特性；3)异体和异种结构都可以成功地植入Leporine模型。这些假说将通过以下三个具体目标得到验证：1)创建模仿天然组织的各向异性半月板结构，2)通过协同的、时间上协调的外源刺激来增强结构的功能和组织性能，3)开发组织结构外科固定技术，并在兔体内植入同种和异种结构。在之前的资助中，发现自然半月板在形态和生物力学上都是高度各向异性和区域变异的，这促使我们目前的组织工程学方法来模拟这些特征。因此，目标1将使用共培养、半月板特定的霉菌和新颖的播种技术来实现这一目标。在上一笔赠款中也发现了对支架方法有积极影响的刺激，但在平行研究中，清楚地证明了由差异黏附假说驱动的无支架方法更优越。该提案的目标2将使用这种无支架方法，结合静水压力、拉伸-压缩、良好限制时间、转化生长因子-1、低氧和软骨素酶-ABC来创建组织工程化构建物。此外，之前的拨款强调了可用于活体研究的半月板细胞的稀缺性。因此，在这项提案中，为了避免使用原代细胞，我们将研究传代的同种异体细胞的使用，并首次研究异种细胞来源在半月板体内修复和替换中的应用(目标3)。通过对同种和异种细胞来源的研究，这项建议试图避免组织稀缺的问题(无论是自体或同种异体半月板和软骨)，并旨在为半月板再生这一复杂的问题提供解决方案。与公共卫生相关：建立组织工程和植入半月板组织的方法对每年接受半月板手术的100多万美国人来说是个好兆头。然而，在天然组织中观察到的高度各向异性的力学特性和形态区域差异使得概括这些结构/功能关系成为一个复杂的问题。目前的组织工程学方法试图模仿这些特征来产生各向异性、不均匀的同种和/或异种结构，以恢复膝关节半月板的功能特性。这项提议的成功完成将为未来的无支架半月板再生尝试建立一个框架，使用替代细胞来源修复和替换半月板组织。

DESCRIPTION (provided by applicant): This renewal proposal aims to tissue engineer an anisotropic meniscus construct that also captures the regional variations present in the native tissue. Subsequently, the construct will be implanted in a leporine model, using both allogenic and xenogenic cell sources, to achieve both meniscus repair and replacement. It is hypothesized that: 1) regionally variant, anisotropic, meniscus-shaped constructs can be engineered by optimizing cell culture and scaffoldless culture conditions; 2) the synergistic and strategic temporal application of certain anabolic and catabolic stimuli will enhance the functional properties of the maturing meniscus construct; and 3) both allogenic and xenogenic constructs can be successfully implanted in a leporine model. These hypotheses will be tested via the following three specific aims: 1) to create an anisotropic meniscus construct with regional variations mimicking native tissue, 2) to enhance functional and organizational properties of constructs via synergistic, temporally coordinated exogenous stimulation, and 3) to develop tissue-construct surgical fixation techniques and implant allogenic and xenogenic constructs in the rabbit. In the previous grant, the native meniscus was found to be highly anisotropic and regionally variant both morphologically and biomechanically, motivating our current tissue engineering approach to mimic these characteristics. Aim 1 will thus use co-cultures, meniscus-specific molds, and novel seeding techniques to accomplish this goal. Also identified in the previous grant were stimuli that had positive effects on a scaffold- based approach, but in parallel studies, it was clearly demonstrated that a scaffoldless approach, driven by the differential adhesion hypothesis, was superior. Aim 2 of this proposal will use such a scaffoldless approach, in conjunction with hydrostatic pressure, tension-compression, well confinement time, TGF-1, hypoxia, and chondroitinase-ABC to create tissue engineered constructs. Furthermore, the previous grant underscored the scarcity of meniscus cells that could be used in an in vivo study. Thus, in this proposal, to avoid the use of primary cells, we will investigate the use of passaged allogenic cells and, for the first time, a xenogenic cell source in the in vivo repair and replacement of the meniscus (Aim 3). By examining both allogenic and xenogenic cell sources, this proposal seeks to obviate the issue of tissue scarcity (either autologous or allogenic meniscus and cartilage) and aims to provide a solution to the complex problem of meniscus regeneration. PUBLIC HEALTH RELEVANCE: Establishing the means to tissue engineer and implant meniscus tissue would bode well for over one million Americans that undergo meniscal procedures annually. However, the highly anisotropic mechanical properties and morphological regional variance observed in native tissue render recapitulating these structure/function relationship a complex problem. The current tissue engineering approach seeks to mimic these characteristics to produce anisotropic, inhomogeneous allogenic and/or xenogenic constructs that can restore the functional properties of the knee meniscus. Successful completion of this proposal will establish a framework for future scaffoldless meniscus regeneration attempts using alternate cell sources for the repair and replacement of meniscus tissue.