Bioinks development in order to explore different biomimetic strategies for cartilage regenerative medicine

我博士工作的主要目的是开发和评估可扩展的工艺，用于生产体外健康和病理模型，代表软骨组织作为测试新型药物治疗的平台。本研究的相对目标是：目的1：综述国内外关于体外健康和病理软骨模型的制备和维持方法的文献及不足；OBJ2：建立体外制造软骨组织的方法；OBJ3：开发可扩展的生物打印工艺，用于体外软骨模型的生成，并评估组织质量，并将其用于进一步的药物筛选。体外模型的使用有望增加我们对生理、生物学和疾病进展的理解，从而用于药物筛选系统。在过去的十年中，创建体外3D组织模型，发展到活结构的水平，可以在高通量平台中密切模仿天然组织环境。药物发现是一个效率低下的过程，失败率高，财政费用高。从兼容性的角度来看，动物研究的结果在人体试验中并不总是可信的，随着时间的推移，监管环境也越来越严格。从道德的角度来看，应该尝试减少根据3R原则（替代、减少和改进）进行的动物研究的数量，以更合乎道德地使用动物进行实验。虽然已经使用了几种制造技术来开发这些模型，但3D生物打印技术的优势在于其低成本和效率、高通量、出色的可重复性以及创建软骨组织等复杂几何形状的能力。目前的软骨组织工程策略还不足以复制出与健康和病理软骨相当的组织。目前，人们对研究软骨基质和细胞组成的带状差异有了更大的兴趣，生物打印为构建分层支架提供了一种有吸引力的工具，特别是在个体病变的特定尺寸和形状方面，可以控制空间分辨率、形状和机械性能。因此，复制和比较骨关节炎和健康软骨组织的3D模型，以验证体外新的替代治疗（即药物筛选），可能是一件有趣的事情。方法我将使用天然聚合物，如硫酸软骨素或壳聚糖与结冷胶或海藻酸盐混合，比较不同的生物墨水性能，以找到在可打印性、细胞活力和组织形成方面的最佳解决方案。选择的生物制造技术是一种基于挤压的3D生物打印机，ROKIT Invivo，它允许用户创建多功能的3D细胞负载结构，多层不同的材料和广泛的设计。打印系统由聚合物挤出机、温度控制在-4-80℃范围内的生物分配器和热气动分配器（高达350℃）组成。合成生物聚合物和各种水凝胶可用于支架生成，因此Rokit是许多生物医学研究的优化工具，其模块化系统和无菌环境，因为提供了H14级Hepa过滤器，用于细胞培养的外部空气过滤基础。

Aim and objectives The main aim of my PhD work is to develop and evaluate scalable process for the production of in vitro healthy and pathological models representative of cartilage tissue as platforms for testing novel pharmaceutical treatments. The relative objectives (OBJ) are:OBJ1: To review the literature on current methods for the production and maintenance of in vitro healthy and pathological cartilage models and their lack to be filled;OBJ2: To establish protocols for manufacturing in vitro cartilage tissue;OBJ3: To develop scalable bioprinted processes for the generation of cartilage models in vitro and to assess the quality of the tissues and use them for further drug screening.BackgroundThe use of in vitro models is promising in increasing our understanding mainly on physiology, biology, and progression of diseases in order to be used as drug screening systems.In the last decade creating in vitro 3D tissue models, developed to a level in which living constructs, can closely mimic the native tissue environment in a high-throughput platform. Drug discovery is an inefficient procedure with a high failure rate and an extreme financial expense. From the compatibility point of view, studies on animals do not always are trustable in terms of results in human trials, and the regulatory environment is becoming stricter as time progresses. From the moral perspective, attempts should be made to reduce the number of animal studies conducted according to the 3R's principles (Replacement, Reduction and Refinement) based on a more ethical use of animals in testing. Although several fabrication techniques have been used to develop these models, 3D bioprinting technologies are advantageous owing to their low cost and efficiency, high throughput, excellent reproducibility, and ability to create complex geometries as the cartilage tissue. Currently cartilage tissue engineering strategies are insufficient for reproducing tissue that is equivalent to healthy and pathological cartilage. At the moment there has been greater interest in studying the zonal differences found in cartilage matrix and cellular composition and bioprinting presents an appealing tool for constructing stratified scaffolds, especially in patient-specific size and shape of individual lesions with control over spatial resolution, shape, and mechanical properties. Thus, it could be interesting to reproduce and compare 3D models of osteoarthritic and healthy cartilage tissue in order to validate in vitro new alternative treatments (i.e. drug screening).MethodsI am going to use natural-based polymers, such as chondroitin sulphate or chitosan blended with gellan gum or alginate for comparing different bioinks properties with the aim of finding the best solution in terms of printability, cells viability and tissue formation. The selected biofabrication technique is an extrusion-based 3D Bioprinter, ROKIT Invivo which allows users to create versatile 3D cell-laden structures, multi-layered with different materials and extensive designs. The printing system is made of a polymer extruder, a biodispenser with controlled temperature in the range of -4-80 C and an hot pneumatic dispenser (up to 350 C). Synthetic biopolymer as well as various hydrogel for scaffold generation can be used and for this reason Rokit is an optimized tool for many biomedical research with its modular system and sterile environmental, because provided with a H14 grade Hepa filter for external air filtration fundamental for cell culture.