Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington, USA.
Department of Chemistry, Morehouse College, Atlanta, Georgia, USA.
Biotechnol Prog. 2024 Jan-Feb;40(1):e3404. doi: 10.1002/btpr.3404. Epub 2023 Nov 20.
This work is focused on designing an easy-to-use novel perfusion system for articular cartilage (AC) tissue engineering and using it to elucidate the mechanism by which interstitial shear upregulates matrix synthesis by articular chondrocytes (AChs). Porous chitosan-agarose (CHAG) scaffolds were synthesized and compared to bulk agarose (AG) scaffolds. Both scaffolds were seeded with osteoarthritic human AChs and cultured in a novel perfusion system with a medium flow velocity of 0.33 mm/s corresponding to 0.4 mPa surfice shear and 40 mPa CHAG interstitial shear. While there were no statistical differences in cell viability for perfusion versus static cultures for either scaffold type, CHAG scaffolds exhibited a 3.3-fold higher (p < 0.005) cell viability compared to AG scaffold cultures. Effects of combined superficial and interstitial perfusion for CHAG showed 150- and 45-fold (p < 0.0001) increases in total collagen (COL) and 13- and 2.2-fold (p < 0.001) increases in glycosaminoglycans (GAGs) over AG non-perfusion and perfusion cultures, respectively, and a 1.5-fold and 3.6-fold (p < 0.005) increase over non-perfusion CHAG cultures. Contrasting CHAG perfusion and static cultures, chondrogenic gene comparisons showed a 3.5-fold increase in collagen type II/type I (COL2A1/COL1A1) mRNA ratio (p < 0.05), and a 1.3-fold increase in aggrecan mRNA. Observed effects are linked to NF-κB signal transduction pathway inhibition as confirmed by a 3.2-fold (p < 0.05) reduction of NF-κB mRNA expression upon exposure to perfusion. Our results demonstrate that pores play a critical role in improving cell viability and that interstitial flow caused by medium perfusion through the porous scaffolds enhances the expression of chondrogenic genes and extracellular matrix through downregulating NF-κB1.
这项工作专注于设计一种易于使用的新型灌注系统，用于关节软骨（AC）组织工程，并利用该系统阐明间质切应变通过关节软骨细胞（AChs）上调基质合成的机制。合成了多孔壳聚糖-琼脂糖（CHAG）支架，并与块状琼脂糖（AG）支架进行了比较。两种支架均接种了骨关节炎人类 AChs，并在新型灌注系统中培养，该系统的培养基流速为 0.33mm/s，对应于 0.4mPa 表面切应变和 40mPa CHAG 间质切应变。虽然对于两种支架类型，灌注培养与静态培养相比，细胞活力均无统计学差异，但 CHAG 支架的细胞活力比 AG 支架培养物高 3.3 倍（p<0.005）。对于 CHAG 的表面和间质联合灌注的影响表明，与 AG 非灌注和灌注培养物相比，总胶原（COL）分别增加了 150 倍和 45 倍（p<0.0001），糖胺聚糖（GAGs）分别增加了 13 倍和 2.2 倍（p<0.001），与非灌注 CHAG 培养物相比，分别增加了 1.5 倍和 3.6 倍（p<0.005）。与 CHAG 灌注和静态培养相比，软骨形成基因比较显示，COL2A1/COL1A1 信使 RNA 比值增加了 3.5 倍（p<0.05），聚集蛋白信使 RNA 增加了 1.3 倍。观察到的效果与 NF-κB 信号转导通路的抑制有关，因为在灌注暴露时 NF-κB mRNA 表达降低了 3.2 倍（p<0.05）。我们的结果表明，孔在提高细胞活力方面起着关键作用，并且通过多孔支架中的培养基灌注产生的间质流通过下调 NF-κB1 增强了软骨形成基因和细胞外基质的表达。
