Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression
Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression
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DOI：
10.1021/acsbiomaterials.9b00975
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发表时间：
2019-10-01
影响因子：
5.8
通讯作者：
Lewis, Mark P.
中科院分区：
文献类型：
--
作者：
Rimington, Rowan P.;Capel, Andrew J.;Lewis, Mark P.
生物工程骨骼肌组织受益于动态培养环境，有利于适当提供营养和去除细胞废物。生物相容的灌注系统具有通过动态培养增强体外组织的生理仿生的潜力，此外还提供了用于分析细胞发育的分析测试和活细胞成像的技术进步。为了满足这种不同的要求，灌注系统需要并入单层和生物工程组织的多个载有细胞的构建体的能力和适应性。这项工作报告了使用增材制造技术生产的灌注系统，用于单层和生物工程组织中肌源性前体细胞的原位表型发育。使用立体光刻（SL）、激光烧结（LS）和PolyJet打印的系统的生物相容性概述了SL和LS器械内的优先形态发育。当暴露于单层中的间歇灌注时，C2 C12细胞的延迟但生理上代表性的细胞增殖、MyoD和肌细胞生成素转录是明显的。单层培养物的长期（8天）间歇灌注概述了生肌发育的活细胞成像的可行形态和遗传原位分化。连续灌注培养（13天）的生物工程骨骼肌组织概述了原位成肌分化，形成成熟的多核肌管。在此，观察到IL-1 β和TNF-α炎性细胞因子、肌肉生长抑制素和MuRF-1萎缩mRNA表达的减少。比较肌球蛋白重链（MyHC）亚型转录谱之间的条件是明显的，但是，总mRNA表达减少灌注条件。MuRF 1转录的减少和随后的MyHC蛋白泛素化的减少暗示了对MyHC同种型转录本转录的需求减少。总之，这些数据似乎表明，3D打印灌注系统引起培养环境的稳定性增强，从而降低了生物工程骨骼肌组织中MyHC基因表达的基础需求。
Bioengineered skeletal muscle tissues benefit from dynamic culture environments which facilitate the appropriate provision of nutrients and removal of cellular waste products. Biologically compatible perfusion systems hold the potential to enhance the physiological biomimicry of in vitro tissues via dynamic culture, in addition to providing technological advances in analytical testing and live cellular imaging for analysis of cellular development. To meet such diverse requirements, perfusion systems require the capacity and adaptability to incorporate multiple cell laden constructs of both monolayer and bioengineered tissues. This work reports perfusion systems produced using additive manufacturing technology for the in situ phenotypic development of myogenic precursor cells in monolayer and bioengineered tissue. Biocompatibility of systems 3D printed using stereolithography (SL), laser sintering (LS), and PolyJet outlined preferential morphological development within both SL and LS devices. When exposed to intermittent perfusion in the monolayer, delayed yet physiologically representative cellular proliferation, MyoD and myogenin transcription of C2C12 cells was evident. Long-term (8 days) intermittent perfusion of monolayer cultures outlined viable morphological and genetic in situ differentiation for the live cellular imaging of myogenic development. Continuous perfusion cultures (13 days) of bioengineered skeletal muscle tissues outlined in situ myogenic differentiation, forming mature multinucleated myotubes. Here, reductions in IL-1 beta and TNF-alpha inflammatory cytokines, myostatin, and MuRF-1 atrophic mRNA expression were observed. Comparable myosin heavy chain (MyHC) isoform transcription profiles were evident between conditions; however, total mRNA expression was reduced in perfusion conditions. Decreased transcription of MuRF1 and subsequent reduced ubiquitination of the MyHC protein allude to a decreased requirement for transcription of MyHC isoform transcripts. Together, these data appear to indicate that 3D printed perfusion systems elicit enhanced stability of the culture environment, resulting in a reduced basal requirement for MyHC gene expression within bioengineered skeletal muscle tissue.