21EBTA Driving Pluripotent Stem Cell Osteogenesis with Light for Tissue Engineering

影响骨骼和关节的骨骼问题，如无法愈合的骨折或骨关节炎，是45岁以上人群功能失调、疼痛和残疾的主要原因。如果在适当的时间将合适的试剂添加到培养皿中的细胞中，人类多能干细胞可以形成不同的组织，包括骨骼。然而，产生这样的组织是昂贵的，依赖于一种叫做生长因子的蛋白质的添加，尤其是一种叫做骨形态发生蛋白（BMP）的蛋白质。bmp在骨的制造过程中很重要，但这种蛋白质价格昂贵，而且不同批次的蛋白质活性不同，这降低了实验方案的可重复性。在我们的研究方案中，我们将通过改造干细胞，使其包含对特定波长的可见光作出反应的受体，而不是BMP蛋白，以触发BMP对细胞的作用，从而取代BMP的使用。我们将根据光脉冲的强度和时间来评估细胞对光的反应，看看它是否复制了BMP蛋白对细胞的作用。除了节省成本，这将使我们更准确地控制促骨信号，而不是通过每天添加由细胞分解的蛋白质来获得。因此，我们将开发一种新方法，利用光驱动干细胞形成骨骼。首先，这将在一个盘子里完成，然后我们将把它转移到三维格式；我们将使用3D打印机打印光反应细胞，通过将它们结合到含有胶原蛋白的凝胶中来制作3D结构，胶原蛋白存在于骨骼中，羟基磷灰石会促进骨骼矿化。我们将把它和一个更硬的支架结合起来促进骨的形成。我们将监控打印和培养参数，以获得最真实的骨组织并对其进行表征。在建立了这个系统之后，未来我们将在3个方面向前推进：1)制造适合愈合骨损伤的类骨结构；2)结合光驱动、多能干细胞生成的软骨细胞，构建可用于研究关节形成和关节疾病因素的结构体；3)用作发现促进骨愈合或纠正骨或关节疾病异常的新药的模型系统。这些光驱动工程细胞也可以应用于其他依赖bmp的人类干细胞生成的组织，以了解发育和疾病，并且该管道可用于设计适用于人类健康和制造过程的其他生长因子反应。因此，这标志着合成生物学这一充满希望的领域迈出了第一步，开启了光与工程人类多能干细胞和生物打印的结合，以推动不同的应用。

Skeletal problems affecting bones and joints, such as fractures that don't heal or osteoarthritis are a major cause of disfunction, pain and disability in the over 45s. Human pluripotent stem cell can make different tissues, including bone, if the right reagents are added to the cells in a dish at appropriate times. However, generating such tissues is costly, relying on addition of proteins called growth factors- in particular one called bone morphogenetic protein (BMP). BMPs are important in development for making bone, but the protein is expensive and different batches have different activity, reducing the reproducibility of protocols. We will replace the use of BMP in our protocol by engineering stem cells to contain receptors which respond to a particular wavelength of visible light, instead of the BMP protein, to trigger the effect of BMP on the cells. We will evaluate the cell-response to light, in terms of intensity and timing of light pulses, to see if it replicates what the BMP protein does to the cells. As well as saving costs, this will give us much more accurate control of the bone-encouraging signals, compared to that obtainable by daily addition of the protein which is broken down by the cells. We will thus develop a novel method, driving the stem cells to form bone using light. First this will be done in a dish but then we will transfer this to a 3 dimensional format; we will use a 3D printer to print the light responsive cells to make a 3D construct by incorporating them into a gel containing collagen, found in bone, and hydroxyapatite which will encourage mineralisation of the bone. We will combine this with a stiffer scaffold to encourage bone formation. We will monitor the printing and culture parameters to give the most authentic bone tissue and characterise this. Having established this system, in the future we will take this forward in 3 ways 1) to make bone-like constructs suitable for healing bone lesions; 2) to combine with light driven, pluripotent stem cell-generated, cartilage cells and make a construct which can be used to investigate the formation of the joint and factors causing joint disease; 3) to use as a model system for discovering new drugs which enhance bone healing or correct bone or joint disease abnormalities. These light-driven engineered cells can also be applied to other BMP-dependent human stem cell generated tissues, for understanding development and disease, and the pipeline can be used to engineer other growth factor responses applicable to human health and manufacturing processes. Thus this marks some of the first step in this promising area of synthetic biology, opening up the use of light with engineered human pluripotent stem cells and bioprinting to drive different applications.