Ultrafast Laser Micromachining to Form Capillary-Sized Networks within Bioprinted Constructs

生物打印已经成为一种很有前途的方法，用于生成功能性组织和器官，以及用于再生医学、药理学、毒理学和疾病建模的组织类似物。生物打印技术通常涉及将活细胞封装在生物相容性生物墨水（如水凝胶）中。然而，以目前的技术，毛细管大小的网络不能在生物打印结构中打印。这些毛细血管大小的网络对于维持组织结构的活力至关重要，因此是建立当前技术广泛应用的核心。超快脉冲激光器在生物打印结构中具有独特的加工毛细血管大小网络的能力，然而，超快激光加工对生物结构的影响尚不清楚。该奖项支持超快激光微加工对细胞损伤影响的基础研究。研究结果将有助于开发一种结合激光微加工和立体光刻生物打印的新型混合工艺，以生产包含毛细血管大小网络的生物打印结构。研究目的是在细胞负载明胶基水凝胶生物材料中建立超快激光变量与细胞损伤之间的关系。为了实现这一研究目标，将通过紫外线交联将间充质祖细胞包裹在甲基丙烯酸明胶水凝胶中制备水凝胶结构。飞秒激光多光子吸收工艺将用于微机械通道（直径约10微米）在细胞负载的水凝胶结构。这些结构将具有不同的细胞密度（10,000 - 10,000,000个细胞/mL），水凝胶浓度（7-18% w/v）和光引发剂浓度（0.05-0.5% w/v）。实验将在不同的超快激光参数下进行：激光流畅度为0 ~ 25 J/cm^2，脉冲能量为0 ~ 100 nJ，扫描速度为0.1 ~ 10 mm/s。水凝胶结构中的细胞损伤将通过以下参数进行评估：通过钙黄蛋白am -乙二聚体生物标志物共聚焦成像测量细胞活力的径向区域，使用Comet测定DNA损伤，以及使用EdU和Movat荧光生物标志物共聚焦成像测量功能（增殖和产生细胞外基质的能力）。

Bioprinting has emerged as a promising approach to generate functional tissues and organs, as well as tissue analogs for regenerative medicine, pharmacology, toxicology, and disease modeling. Bioprinting technologies generally involve the encapsulation of living cells within biocompatible bioinks (such as hydrogels). However, with current technologies, capillary-sized networks cannot be printed within bioprinted constructs. These capillary-sized networks are critical for maintaining viability of tissue constructs and, therefore, are at the center of establishing the widespread application of current technologies. Ultrafast pulsed lasers are uniquely capable of machining capillary-sized networks within bioprinted constructs, however the impact of ultrafast laser machining on biological constructs is not known. This award supports fundamental research on effects of ultrafast laser micromachining on cellular damage. Research results will be useful for the development of a novel hybrid process combining laser micromachining and stereolithography bioprinting to produce bioprinted constructs that contains capillary-sized networks.The research objective is to establish the relationship between ultrafast laser variables and cellular damage within cell-laden gelatin-based hydrogel biomaterials. To achieve this research objective, hydrogel constructs will be prepared by encapsulating mesenchymal progenitor cells within gelatin methacrylate hydrogel via ultraviolet crosslinking. Femtosecond laser multiphoton absorption process will be used to micromachine channels (about 10 microns in diameter) within the cell-laden hydrogel constructs. These constructs will have different cell densities (10,000 - 10,000,000 cells/mL), hydrogel concentration (7-18% w/v), and photoinitiator concentration (0.05-0.5% w/v). Experiments will be conducted under different ultrafast laser parameters: laser fluency from 0 to 25 J/cm^2, pulse energy from 0 to 100 nJ, and scanning speed from 0.1 to 10 mm/s. Cellular damage within hydrogel constructs will be evaluated by the following parameters: radial zones of cellular viability measured by confocal imaging of calcein AM-ethidium homodimer biomarkers, DNA damage measured using Comet assay, and function (ability to proliferate and produce extracellular matrix) measured using confocal imaging of EdU and Movat's fluorescent biomarkers.