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

目的和目的我博士工作的主要目的是开发和评估可扩展的过程，以生产代表软骨组织的体外健康和病理模型，作为测试新药物治疗的平台。相关目标(OBJ)是：OBJ1：综述目前健康和病理软骨模型的制作和维护方法及其不足的文献：OBJ2：建立体外软骨组织的制作方案；目的：开发可扩展的生物打印方法，用于体外软骨模型的建立，评估组织的质量，并将其用于进一步的药物筛选。背景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了解，以便用作药物筛选系统。在过去的十年中，体外三维组织模型的建立已经发展到可以在高通量平台上接近模拟自然组织环境的水平。药物发现是一种效率低下的过程，失败率高，经济成本高。从兼容性的角度来看，动物研究在人体试验结果方面并不总是可信的，而且监管环境随着时间的推移而变得更加严格。从道德的角度来看，应该努力减少根据3R原则(替换、减少和改进)进行的动物研究的数量，这是基于在测试中更合乎道德地使用动物。虽然已经使用了多种制造技术来开发这些模型，但3D生物打印技术具有成本低、效率高、产量高、重复性好以及能够像软骨组织一样创造复杂几何形状的优势。目前，软骨组织工程策略不足以复制出相当于健康和病态软骨的组织。目前，人们对研究软骨基质和细胞组成的地带性差异更感兴趣，生物打印是构建分层支架的一种有吸引力的工具，特别是在控制空间分辨率、形状和机械性能的情况下，针对患者特定的单个病变的大小和形状。因此，复制和比较骨关节炎和健康软骨组织的3D模型可能是有趣的，以便在体外验证新的替代治疗方法(即药物筛选)。方法我将使用基于天然的聚合物，如硫酸软骨素或壳聚糖与结冷胶或海藻酸盐混合，比较不同生物墨水的性能，目的是从印刷性、细胞活力和组织形成方面找到最佳解决方案。所选的生物制造技术是一种基于挤压的3D生物打印机ROKIT Invivo，它允许用户创建多种3D细胞负载结构，使用不同的材料和广泛的设计来创建多层结构。打印系统由聚合物挤出机、温度控制在-4-80摄氏度范围的生物分配器和热气动分配器(高达350摄氏度)组成。可以使用合成生物聚合物以及用于支架生成的各种水凝胶，因此，Rokit凭借其模块化系统和无菌环境，成为许多生物医学研究的优化工具，因为它配备了H14级高效空气过滤器，用于细胞培养的外部空气过滤。

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.