Osteochondral tissue engineering using novel 3D printed scaffolds and multi-layered cell sheet technology

骨软骨组织损伤或丢失是最常见的疾病之一，是由于创伤、软骨组织的自然退化、老化、关节炎或手术引起的。这些临床症状不仅包括关节软骨的严重损伤，而且还包括潜在的钙化的软骨下骨。传统的治疗方法包括自体移植、同种异体移植、骨髓刺激和清创。自体移植的库存有限，同种异体移植与免疫排斥或疾病传播的风险有关，而骨髓刺激治疗只是姑息治疗，不能完全治愈。因此，骨软骨缺损的治疗能力是临床上的一大需求。在过去的几十年里，组织工程学方法已经被用于再生关节软骨。然而，这些措施的产出仍然不能令人满意。部分原因可能是由于关节软骨独特而复杂的结构(例如，它没有血管、淋巴管和神经，但它处于生物力学环境中)。在过去的几年里，研究人员提出，一个健康的软骨下床对软骨组织再生的成功起着关键作用。在这个项目中，将利用多学科方法将材料科学、机械工程(贾晓东博士)和干细胞生物学(江博士)结合起来，用于骨软骨组织工程(杨博士)。多层细胞片(MLCS)技术与东京女子医科大学根据正式合作协议密切合作，使我们能够收获完整的细胞片，对细胞的损害最小，并最大限度地保留细胞-细胞连接、细胞外基质和基质中嵌入的生长因子。这项技术可能有助于干细胞的处理和新型胶原支架的开发，以模拟关节软骨的自然结构。此前，我们已经展示了使用表观遗传学方法在不改变基因组的情况下控制干细胞功能的潜力。新型组蛋白脱乙酰酶抑制剂(HDACi)MI192将用于人骨髓间充质干细胞的预处理，以增强其成骨分化潜能，促进新骨形成。骨相和软骨相的结合将为临床相关动物模型的骨软骨修复/修复提供新的解决方案。本项目的目的是开发一种新型的天然胶原支架，并将其与3D打印支架、表观遗传修饰干细胞以及多层细胞片技术相结合，用于体外和体内的骨软骨组织工程。该项目非常适合EPSRC的医疗保健和技术主题-开发未来疗法。利用MLCS技术开发新型胶原支架。B)利用表观遗传修饰干细胞、MLCS技术、新型胶原支架在体外构建软骨相。C)利用表观遗传修饰干细胞、MLCS技术和PepGEN P-15增强型3D打印多孔聚合物支架(与奥塔哥大学、伦敦大学和曼彻斯特大学合作)在体外构建骨相。d)利用以上形成的骨和软骨结构在体内进行骨软骨组织工程。MLCS技术是一种新的工程方法。将开发出一种新型的天然胶原支架，其结构类似于自然关节软骨，可修复。MLCS技术结合MLCS技术，将开发出一种新型的天然胶原支架新型胶原支架和3D打印聚合物支架将为复杂组织工程提供一种新的途径。

Osteochondral tissue damage or loss is one of the most common diseases due to traumatic injuries, natural degradation of cartilaginous tissue with aging, arthritis or surgery. These clinical situations encompass serious damage to not only articular cartilage but also the underlying calcified subchondral bone. The conventional therapeutic approaches include autografts, allografts, stimulation of bone marrow and debridement. Autografts have limited stock and allografts are associated with the risk of immune rejection or disease transmission, while bone marrow stimulation treatments are only palliative and not completely curative. Therefore, the ability to treat osteochondral defects is a major clinical need. Over the last decades, tissue engineering approaches have been utilised for regenerating articular cartilage. However the output of these is still not satisfactory. Part of the reason may be due to the unique and complex structure of articular cartilage (e.g. it does not have blood vessels, lymphatics and nerves, but it is under a biomechanical environment). Over the last a few years, researchers have suggested that a healthy subchondral bed plays a key role for the success of cartilage tissue regeneration. In this project, a multidisciplinary approach will be utilised to combine material science, mechanical engineering (Dr Xiaodong Jia) with stem cell biology (Dr Jiang) for osteochondral tissue engineering (Dr Yang). The multi-layered cell sheet (MLCS) technology, in close collaboration with Tokyo Women's Medical University under a formal collaborative agreement, allows us to harvest intact cell sheet with minimum damage to the cells and maximum retention of cell-cell junctions, extracellular matrix and growth factors embedded in the matrix. This technology could be useful for stem cell handling and the development of novel collagen scaffolds to mimic natural structure of articular cartilage. Previously we have showed the potential of using epigenetic approaches to control stem cell function without change the genome. A novel histone deacetylase inhibitors (HDACi), MI192 will be used for the pre-treatment of human MSCs to enhance their osteogenic differentiation potential for new bone formation. The combination of the bone phase and cartilage phase will provide a novel solution for repair/restoration of osteochondral defect in clinical relevant animal models.The aim of this project is to develop a novel natural collagen scaffold and combine these scaffolds with 3D printed scaffolds, epigenetic modified stem cells as well as multi-layered cell sheet technology for osteochondral tissue engineering in vitro and in vivo.The project fits very well within EPSRC's Healthcare and Technologies Theme - Developing Future Therapies. The objectives include:A) Development of novel collagen scaffolds using MLCS technology.B) Fabricate cartilage phase in vitro using epigenetic modified stem cell, MLCS technology, novel collagen scaffolds.C) Fabricate bone phase in vitro using epigenetic modified stem cell, MLCS technology and PepGEN P-15 enhanced 3D printed porous polymer scaffold (in collaboration Otago University, UCL and University of Manchester).D) Osteochondral tissue engineering in vivo using the bone and cartilage constructs formed above.MLCS technology is a novel engineering method.A novel natural collagen scaffolds will be developed with unique architecture similar to natural articular cartilage, which could be patentable.A combination of MLCS technology, novel collagen scaffolds and 3D printed polymer scaffolds will provide a novel approach for complex tissue engineering.