Production of a Human Growth Plate Organ-Chip Model of Skeletal Development

生长板是儿童骨骼末端的一块组织，决定着骨骼的发育。它代表着骨骼和软骨之间的过渡，由血管支撑。由于生长板的重要性，人们有相当大的兴趣研究这个器官及其在发育、衰老和疾病中的作用。要做到这一点，就需要健壮的、可重现的和生理上相关的实验“模型”系统。目前，许多研究依赖于动物，通常是小鼠和大鼠。由于已知机械力会影响骨骼在体内的发育，一些动物模型包括在动物还活着的情况下控制生长板上的机械力的技术。这些广泛使用的动物模型往往重复性有限，无法代表人类的关键行为，也引发了伦理问题。因此，迫切需要用非动物的人类实验模型来取代其中的一些动物模型。这将提高科学的严谨性和与人类生理学的相关性，并减少在科学中使用动物。以前报道的生长板的非动物模型未能复制器官的关键特征，包括骨-软骨组织的梯度、机械载荷和连接到骨的血管的结合。因此，我们将利用芯片上器官技术开发和验证一种新的人体生长板模型。器官芯片是一种实验模型系统，在这种系统中，人类细胞可以在相互连接的通道中生长，通过这些通道可以泵送液体，为细胞提供必要的营养以维持它们的生命。我们将开发一种具有以下关键特征的生长板器官芯片：-从骨到软骨的复合组织-复制血管的通道-正在发育的骨-软骨生长板的机械载荷-一个可重现、可扩展的系统，可以很容易地被其他研究人员采用来实现上述目标，我们将使用从骨髓中扩增的人成年干细胞。这些干细胞将在器官芯片的其中一个通道中的3D凝胶材料中生长。然后使用自然生长因子将细胞分化为骨和软骨细胞，以产生分级的骨-软骨生长板组织。我们还将创建一个血管通道，里面排列着形成血管壁的人内皮细胞。这个血管通道将提供一条管道，将营养和激素输送到正在发育的生长板，就像在体内发生的那样。为了确保重复性和可伸缩性，器官芯片模型将使用商业上可获得的人类干细胞和内皮细胞以及由Eulate公司提供的商业上可获得的器官芯片来创建。该芯片由两个通道组成，由半透膜隔开，允许在一个通道中发育的骨-软骨生长板组织和血管通道之间的相互作用。此外，我们将利用模拟器官芯片的能力，为复制体内机械环境的通道提供受控的机械负荷。通过这种方式，我们的愿景是创建一种高度可重复性和经验证的人类血管生长板器官芯片模型，该模型可以容易地被科学界采用，减少对动物模型的依赖，并提高健康和疾病中骨骼发育研究的质量。

The growth plate is an area of tissue at the ends of bones in children which determines the development of the skeleton. It represents a transition between bone and cartilage and is supported by blood vessels. Due to the importance of the growth plate, there is considerable interest in studying this organ and its role in development, ageing, and disease. To do so necessitates robust, reproducible and physiologically relevant experimental 'model' systems. Currently many studies rely on animals, typically mice and rats. Since mechanical forces are known to influence skeletal development in the body, some of the animal models include techniques to control mechanical forces on the growth plate whilst the animal is still alive. These widely used animal models often have limited reproducibility and fail to represent key behaviour in humans as well as prompting ethical concerns. There is therefore an urgent need to replace some of these animal models with non-animal, human experimental models. This would improve scientific rigour and relevance to human physiology as well as reducing the use of animals in science. Previously reported non-animal models of the growth plate fail to replicate the key features of the organ including a gradient of bone-cartilage tissue, mechanical loading and incorporation of blood vessels linking to the bone. Therefore, we will develop and validate a new human growth plate model using organ-on-a-chip technology. An organ-chip is an experimental model system in which human cells can be grown within interconnected channels through which fluid can be pumped providing the cells with the necessary nutrients to keep them alive. We will develop a growth plate organ-chip with the following key features: - A composite tissue graded from bone to cartilage - A channel replicating the blood vessels - Mechanical loading of the developing bone-cartilage growth plate- A reproducible, scalable system that can easily be adopted by other researchers To achieve the above, we will use human adult stem cells expanded from bone marrow. These stem cells will be grown within a 3D gel material in one of the channels in an organ-chip. The cells will then be differentiated into bone and cartilage cells using natural growth factors to create the graded bone-cartilage growth plate tissue. We will also create a blood vessel channel lined with human endothelial cells that form blood vessel walls. This blood vessel channel will provide a conduit for nutrient and hormone delivery to the developing growth plate as occurs in the body. To ensure reproducibility and scalability, the organ-chip model will be created with commercially available human stem cell and endothelial cells and within the commercially available organ-chip provided by Emulate Inc. This chip consists of two channels separated by a semi-permeable membrane allowing interaction between the developing bone-cartilage growth plate tissue in one channel and the blood vessel channel. In addition, we will utilise the ability of the Emulate organ-chip to provide controlled mechanical loading to the channels replicating the mechanical environment within the body. In this way, our vision is to create a highly reproducible and validated human vascularised growth plate organ-chip model which can be readily adopted by the scientific community reducing reliance on animal models and improving the quality of research into skeletal development in health and disease.