Institute of Reconstructive Plastic Surgery, New York University School of Medicine, 560 First Avenue, New York, NY, 10016, USA.
J Tissue Eng Regen Med. 2012 Nov;6(10):769-76. doi: 10.1002/term.478. Epub 2011 Nov 3.
Encased in lacunae, osteocytes receive nutrition and biomechanical signals through the lacunocanalicular system. We have developed a novel flow-perfusion bioreactor designed to support lacunocanalicular fluid flow. We hypothesize that ex vivo fluid flow can maintain endochondral bone viability and, ultimately, serve as a novel model to study bone biology in vitro. Sprague-Dawley rat femurs were harvested, stripped of soft tissue, loaded into a custom-designed bioreactor and perfused with osteogenic culture medium. After 14 days of flow-perfusion or static culture, the bones were harvested, fixed, decalcified, embedded, sectioned and stained with haematoxylin and eosin. Fresh long bone samples were similarly processed for comparison. Osteocyte viability and function were also evaluated, using thiazolyl blue tetrazolium bromide (MTT), fluorospectrophotometric DNA quantification, alkaline phosphatase (ALP) colorimetric assay and fluorochrome labelling of mineralizing surfaces. All samples remained free of infection throughout the study period. After 14 days of flow perfusion, histological analysis showed normal-appearing bony architecture, with 72% of lacunae being osteocyte-filled compared with 93% in freshly harvested samples and only 36% in static samples. MTT staining and assay confirmed osteocyte viability in the flow-perfusion samples as well as in fresh samples. DNA quantification demonstrated DNA to be preserved in flow-perfused samples when compared with freshly harvested samples. ALP activity in flow-perfusion explants was upregulated compared with fresh and static samples. Fluorochrome-labelled mineralizing surfaces were seen throughout the explanted flow-perfused samples. This is the first demonstration that flow perfusion provides adequate chemotransportation to explanted murine endochondal bones.
被包裹在腔隙内的骨细胞通过骨陷窝-骨小管系统接收营养和生物力学信号。我们开发了一种新型的灌注式生物反应器，旨在支持骨陷窝-骨小管内的液体流动。我们假设，体外液流可以维持骺软骨内骨的活力，并最终成为研究骨生物学的体外新型模型。从 Sprague-Dawley 大鼠股骨中取出，去除软组织，装入定制的生物反应器中，并用成骨培养基进行灌注。经过 14 天的灌注或静态培养后，取出骨骼，固定、脱钙、包埋、切片并进行苏木精和伊红染色。新鲜的长骨样本也进行了类似的处理以作比较。使用噻唑蓝溴化四氮唑（MTT）、荧光分光光度法 DNA 定量、碱性磷酸酶（ALP）比色测定和矿化表面的荧光标记来评估骨细胞的活力和功能。在整个研究过程中，所有样本均未发生感染。经过 14 天的灌注后，组织学分析显示出正常的骨结构，与新鲜样本相比，有 72%的骨陷窝内充满了骨细胞，而静态样本中只有 36%。MTT 染色和测定也证实了灌注样本中的骨细胞活力，以及新鲜样本中的活力。与新鲜样本相比，DNA 定量显示在灌注样本中 DNA 得到了保存。与新鲜和静态样本相比，灌注样本中的 ALP 活性升高。在整个灌注样本中都能看到荧光标记的矿化表面。这是首次证明灌注可以为体外骺软骨内骨提供足够的化学转运。
