1 Department of Genetics, Cell Biology, and Anatomy, University Nebraska Medical Center , Omaha, Nebraska.
2 Department of Biological Systems Engineering, University Nebraska Lincoln , Lincoln, Nebraska.
Tissue Eng Part A. 2018 Jan;24(1-2):94-105. doi: 10.1089/ten.tea.2017.0091. Epub 2017 May 18.
Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein-Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs.
通过精确控制组织生长的标量和向量分量来定义工程组织的最终大小和形状，这是再生医学的必要基准，但事实证明，这对于组织工程师来说是一个重大挑战。促进长骨伸长的生长板软骨是研究形态发生机制的良好模型系统，因为软骨由单一细胞类型——软骨细胞组成；软骨细胞在培养中易于维持；并且生长轨迹主要在单一向量上。在这种软骨中，通过空间和时间上受到由长程和短程信号机制组成的相互连接网络调节的分化程序产生生长，这些机制共同导致功能不同的细胞区的形成。为了促进对各向异性生长机制的研究，我们使用包封在藻酸盐水凝胶珠中的新生小鼠生长板软骨细胞开发了生长板软骨的体外模型。在珠培养物中，包封的软骨细胞表现出高活力、软骨基质沉积、低水平的软骨细胞肥大以及在培养的 7 天内细胞增殖的逐渐增加。使用外源性因子测试甲状旁腺相关蛋白-印度刺猬（PTHrP-IHH）信号相互作用的功能，这是调节生长的关键反馈回路。与体内观察结果一致，外源性 PTHrP 刺激细胞增殖并抑制肥大，而 IHH 信号刺激软骨细胞肥大。重要的是，用 IHH 或甲状腺素处理藻酸盐珠培养物导致形成一个离散的肥大细胞区域，模拟天然生长板软骨的组织结构。总之，这些研究首次证明了一种可调节的体外系统，可模拟诱导生长板软骨中区域结构所需的信号网络相互作用，该系统也可用于生长特定几何形状的软骨培养物以满足个性化患者的需求。
