Toward tissue engineering of the knee meniscus

描述（由申请人提供）：本更新提案旨在组织工程一个各向异性半月板结构，也捕获了本地组织中存在的区域差异。随后，使用同种异体和异种细胞源将构建物植入麻素模型，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，可以构建区域性、各向异性、半月板形状的构建体；2)某些合成代谢和分解代谢刺激的协同和战略性时间应用将增强成熟半月板结构的功能特性；3)异体和异种构建体均可成功植入麻素模型。这些假设将通过以下三个具体目标进行验证：1)创建具有区域差异的模拟天然组织的各向异性半月板构建体；2)通过协同、时间协调的外源刺激增强构建体的功能和组织特性；3)开发组织构建体手术固定技术并在兔体内植入同种异体和异种构建体。在之前的研究中，我们发现天然半月板在形态和生物力学上都具有高度的各向异性和区域差异，这促使我们目前的组织工程方法来模拟这些特征。因此，Aim 1将使用共培养、半月板特异性霉菌和新颖的播种技术来实现这一目标。在之前的研究中也发现了对基于支架的入路有积极影响的刺激，但在平行研究中，明确证明了由差异粘附假设驱动的无支架入路更优越。该提案的目标2将使用这种无支架的方法，结合静水压力、张力压缩、井约束时间、TGF-1、缺氧和软骨素酶- abc来创建组织工程构建体。此外，先前的拨款强调了可用于体内研究的半月板细胞的稀缺性。因此，在本提案中，为了避免使用原代细胞，我们将研究传代同种异体细胞的使用，并首次将异种细胞来源用于半月板的体内修复和替换（目的3）。通过研究同种异体和异种细胞来源，本研究旨在解决组织缺乏（自体或同种异体半月板和软骨）的问题，并旨在为半月板再生的复杂问题提供解决方案。公共卫生相关性：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，在原生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再现这些结构/功能关系成为一个复杂的问题。目前的组织工程方法试图模仿这些特征来产生各向异性、非均质异体和/或异种结构，以恢复膝关节半月板的功能特性。该建议的成功完成将为未来使用替代细胞来源修复和替换半月板组织的无支架半月板再生尝试建立一个框架。

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.