Engineering biomimetic knee menisci with zonal and anisotropic variations

项目摘要 该建议旨在组织工程化各向异性的新半月板，也捕获区域变化 存在于天然组织中。随后，将同种异体新骨结构植入兔体内， 模型，以实现半月板修复和置换。假设：1）区域差异， 通过优化细胞培养和无支架培养， 条件; 2）多水平刺激的策略性时间应用（在细胞、分子和结构上）; 水平）将允许在不同水平的作用之间产生协同作用，以增强药物的功能特性。 成熟的新血管;和3）同种异体构建体可以成功植入兔模型中。这些 假设将通过以下三个具体目标进行测试：1）为了创建具有以下特征的各向异性新半月板， 模仿天然组织的区域变化; 2）增强新组织的功能和组织特性， 半月板通过多层次的外源性刺激协同时间协调;和3）发展 手术固定技术，并将新骨瓣植入家兔体内。以前，原生半月板是 发现在形态和生物力学上都是高度各向异性和区域变化的，这促使我们 目前的组织工程方法来模仿这些特征。同种异体兔细胞将用于 在目标1中形成组织上和区域上模仿的新概念结构;这一目标将得以实现 通过使用新颖的空间和时间变化的播种技术。各向异性和组织 然后，通过操纵分子、细胞和分子水平来增强工程化新半月板的性质。 Aim 2中的构建级目标。具体而言，TGF-β1和静水压力将作用于细胞水平， 增加基质产生;溶血磷脂酸和软骨素酶-ABC将用于排列和压实 在分子水平上的矩阵;和特定的机械刺激将直接在各向异性 结构水平。在这个建议中，为了避免使用原代细胞，我们将研究使用传代细胞。 同种异体细胞用于半月板的体内修复和置换（目标3）。成功演示后 在兔模型中的修复/替换，我们将确定方法，同样扩大绵羊和人类 细胞为未来的研究。这种方法试图解决组织稀缺的问题，并旨在提供一种 解决半月板修复和置换的复杂问题。

PROJECT SUMMARY This proposal aims to tissue engineer an anisotropic neo-meniscus that also captures the regional variations present in the native tissue. Subsequently, allogeneic neo-meniscal constructs will be implanted in a leporine model 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 scaffold-free culture conditions; 2) the strategic temporal application of multi-level stimuli (at cellular-, molecular-, and construct- levels) will allow for synergisms across the different levels of action to enhance the functional properties of the maturing neo-menisci; and 3) allogeneic 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 neo-meniscus with regional variations mimicking native tissue; 2) to enhance functional and organizational properties of the neo- meniscus via multi-level exogenous stimulation synergized by temporal coordination; and 3) to develop surgical fixation techniques and implant the neo-menisci in the rabbit. Previously, 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. Allogeneic leporine cells will be used to form organizationally and regionally mimetic neo-meniscal constructs in Aim 1; this goal will be accomplished via the use of novel spatial and temporally variant seeding techniques. The anisotropic and organizational properties of the engineered neo-meniscus will then be enhanced by manipulating molecular-, cellular-, and construct-level targets in Aim 2. Specifically, TGF-β1 and hydrostatic pressure will act on the cellular level to increase matrix production; lysophosphatidic acid and chondroitinase-ABC will be used to align and compact the matrix at the molecular level; and meniscus-specific mechanical stimulation will direct anisotropy at the construct level. In this proposal, to avoid the use of primary cells, we will investigate the use of passaged allogeneic cells toward in vivo repair and replacement of the meniscus (Aim 3). Upon successful demonstration of repair/replacement in the leporine model, we will determine methods to likewise expand sheep and human cells for future studies. This approach seeks to address the issue of tissue scarcity and aims to provide a solution to the complex problem of meniscus repair and replacement.