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

生长板是儿童骨骼末端的一个组织区域，它决定了骨骼的发育。它代表骨和软骨之间的过渡，并由血管支持。由于生长板的重要性，人们对研究这个器官及其在发育、衰老和疾病中的作用非常感兴趣。要做到这一点，需要强大的，可重复的和生理相关的实验“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身体中的骨骼发育，因此一些动物模型包括在动物仍然活着时控制生长板上的机械力的技术。这些广泛使用的动物模型通常具有有限的可重复性，无法代表人类的关键行为，也无法引发伦理问题。因此，迫切需要用非动物的人类实验模型取代这些动物模型中的一些。这将提高科学的严谨性和与人类生理学的相关性，并减少动物在科学中的使用。以前报道的生长板的非动物模型不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并入。因此，我们将使用器官芯片技术开发和验证新的人类生长板模型。器官芯片是一种实验模型系统，其中人类细胞可以在相互连接的通道内生长，通过这些通道可以泵送流体，为细胞提供必要的营养以保持它们的存活。我们将开发具有以下关键特征的生长板器官芯片：-从骨到软骨分级的复合组织-复制血管的通道-发育中的骨软骨生长板的机械负载-可重复的，可扩展的系统，可以很容易地被其他研究人员采用为了实现上述目标，我们将使用从骨髓中扩增的人类成体干细胞。这些干细胞将在器官芯片的一个通道中的3D凝胶材料内生长。然后使用天然生长因子将细胞分化成骨和软骨细胞，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类内皮细胞。这种血管通道将提供一个管道，用于将营养和激素输送到体内发育的生长板。为了确保可重复性和可扩展性，器官芯片模型将使用市售的人干细胞和内皮细胞并在Emulate Inc.提供的市售器官芯片内创建。该芯片由两个通道组成，两个通道由半渗透膜隔开，允许一个通道中的发育中的骨软骨生长板组织与血管通道之间相互作用。此外，我们将利用仿真器官芯片的能力，为复制体内机械环境的通道提供受控的机械载荷。通过这种方式，我们的愿景是创建一个高度可重复和验证的人类血管化生长板器官芯片模型，该模型可以很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的研究质量。

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.