Individual cell bioprinting to generate multi-tissue type condensations for osteochondral tissue regeneration

摘要 膝关节骨软骨缺损在世界范围内很常见，但对于患有骨软骨缺损的患者， 受损的骨软骨组织，因为目前的治疗不能一致地再生功能组织。的 骨软骨缺损修复标准护理是关节镜下微骨折手术，但该手术通常 导致形成机械性较差的纤维软骨形成。为了克服这个和其他的局限性， 外科手术，组织工程策略，如细胞负载的生物材料支架，是有前途的 替代方法来处理这些缺陷。然而，基于支架的策略面临着一些挑战，例如 干扰关键的细胞-细胞相互作用、对支架的潜在免疫和/或炎症反应 及其降解副产物，以及支架降解速率与新组织形成速率不同步。 没有支架的新的细胞浓缩策略解决了这些问题，然而，仍然难以 精确控制工程组织的结构，以模拟复杂的三维（3D） 天然骨软骨组织结构和组织及其结构衍生功能。最近，3D 生物打印已被应用于组织工程，具有创建复杂，高分辨率3D的潜力。 结构.此外，我们还设计了第一种能够3D打印纯细胞生物墨水的技术， 保持印刷的结构，这是形成细胞凝聚所必需的。这个提议的假设 精确定义的基于细胞凝聚的预血管化骨软骨组织构建物 几何形状可以通过3D生物打印直接与人类干细胞和内皮细胞组装成 可光固化的类液体固体、剪切稀化和快速自修复的微凝胶浆料， 组织特异性生长因子的表达。印刷后的微凝胶光交联将提供暂时的 在培养过程中打印的构建体的机械稳定性，以允许细胞凝聚形成。这个牢房 只有生物打印策略将被实施，以打印无缝连续的两相骨软骨组织 具有预血管化骨相和软骨相的构建体。具体而言，本建议旨在（1） 确定微凝胶特性对仅细胞3D打印构建体的分辨率和保真度的作用，（2） 用单个仅细胞生物墨水通过时空方法工程化预血管化骨软骨构建体 血管生成、成骨和软骨生成生长因子的受控递送，和（3）确定临床 通过评价新的骨软骨组织，3D打印预血管化骨软骨构建物的潜力 形成并与宿主血管网络整合，以及全层骨和软骨修复 兔骨软骨缺损模型。这一平台战略有可能极大地改善那些 患有骨软骨缺损，并可能使其他复杂的功能组织的工程化， 身体

Abstract Osteochondral defects of the knee are common worldwide, yet there are few viable options for patients with damaged osteochondral tissue as current treatments do not consistently regenerate functional tissue. The standard of care for osteochondral defect repair is arthroscopic microfracture surgery, but this procedure often results in formation of mechanically inferior fibrocartilage formation. To overcome limitations of this and other surgical procedures, tissue engineering strategies, such as cell-laden biomaterial scaffolds, are promising alternative approaches to treat these defects. However, scaffold-based strategies face several challenges, such as interference with critical cell-cell interactions, potential immune and/or inflammatory reaction to the scaffold and its degradation byproducts, and unsynchronized scaffold degradation rate with that of new tissue formation. New cellular condensation strategies without a scaffold address these issues, however, it is still difficult to precisely control the architecture of the engineered tissues to mimic the sophisticated three-dimensional (3D) structure and organization of natural osteochondral tissues and their structure-derived functions. Recently, 3D bioprinting has been applied in tissue engineering with the potential to create complicated, high-resolution 3D structures. In addition, we have engineered the first technology capable of 3D printing a cell-only bioink and maintaining the printed structure, which is necessary to form cell condensations. The hypothesis of this proposal is that cellular condensation-based prevascularized osteochondral tissue constructs of precisely defined geometries can be directly assembled with human stem cells and endothelial cells via 3D bioprinting into a photocurable liquid-like solid, shear-thinning and rapid self-healing microgel slurry with spatially controlled presentation of tissue specific growth factors. Microgel photocrosslinking after printing will provide temporary mechanical stability for the printed constructs during culture to permit cellular condensation formation. This cell- only bioprinting strategy will be implemented to print seamlessly continuous two-phase osteochondral tissue constructs with a prevascularized bone phase and a cartilage phase. Specifically, this proposal aims to (1) determine the role of microgel properties on the resolution and fidelity of the cell-only 3D printed constructs, (2) engineer prevascularized osteochondral constructs with individual cell-only bioinks by spatiotemporally controlled delivery of vasculogenic, osteogenic and chondrogenic growth factors, and (3) determine the clinical potential of the 3D printed prevascularized osteochondral constructs by evaluation of new osteochondral tissue formation and integration with the host vascular networks and bone and cartilage repair in a full-thickness osteochondral rabbit defect model. This platform strategy has the potential to greatly enhance the lives of those suffering from osteochondral defects and may enable the engineering of other complex functional tissues in the body.