Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes and gradients of composition and biological cues. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the micron and the molecular level. Herein, we introduce a bioink containing the tyramine derivative of hyaluronan (HA; henceforth known as THA) and collagen (Col) type 1. In this bioink, similar to connective tissues, Col is present in the fibrillar form, and HA functions as a viscoelastic space filler. THA was enzymatically cross-linked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. The THA-Col composite displayed synergistic properties in terms of storage modulus and shear thinning, translating into good printability. Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second-harmonic generation. Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on human bone marrow–derived mesenchymal stromal cell (hMSC) migration. Anisotropic HA-Col showed cell-instructive properties modulating hMSC adhesion, morphology, and migration from micropellets stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded in aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and ECM production, hMSCs embedded in the isotropic bioink displayed chondrogenic differentiation comparable with standard pellet culture by means of proteoglycan production (safranin O staining and proteoglycan quantification). The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.

生物制造为科学家和临床医生提供了生产具有所需形状、成分梯度和生物线索的工程组织的能力。基于挤出的生物打印实现的典型分辨率是在宏观水平上。然而，为了捕获细胞外基质 (ECM) 的纤维性质，有必要在更小的尺度上排列 ECM 成分，直至微米和分子水平。在此，我们介绍了一种含有透明质酸酪胺衍生物 (HA；以下称为 THA) 和 1 型胶原蛋白 (Col) 的生物墨水。在这种生物墨水中，与结缔组织类似，Col 以纤维状形式存在，HA 充当粘弹性空间填充物。 THA 在温和条件下进行酶促交联，允许同时发生 Col 原纤维，从而在基于粘弹性 HA 的基质内实现 Col 原纤维的均匀分布。 THA-Col 复合材料在储能模量和剪切稀化方面表现出协同特性，转化为良好的印刷适性。通过免疫荧光和二次谐波产生，实现了剪切诱导的 Col 原纤维沿打印方向的排列并进行量化。打印并表征了无细胞和充满细胞的构建体，分析了受控的微观各向异性对人骨髓源性间充质基质细胞（hMSC）迁移的影响。各向异性 HA-Col 显示出细胞指导特性，可调节 hMSC 粘附、形态以及由 Col 纤维的存在和方向刺激的微球的迁移。肌动蛋白丝染色显示，嵌入对齐构建体中的 hMSC 沿原纤维方向显示出增加的细胞骨架对齐。 基于软骨/骨标记物的基因表达和 ECM 产生，嵌入各向同性生物墨水中的 hMSC 通过蛋白多糖产生（番红 O 染色和蛋白多糖定量）显示出与标准颗粒培养物相当的软骨分化。通过控制微观排列来打印矩阵组件的可能性使生物制造更接近捕获天然组织的复杂性。