Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea.
ACS Appl Mater Interfaces. 2022 Jan 26;14(3):3773-3783. doi: 10.1021/acsami.1c20585. Epub 2022 Jan 11.
Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor β subunit (CBFβ) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFβ and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.
叶酸被报道能显著促进间充质干细胞的软骨分化。基于类似的作用机制，我们研究了美国食品和药物管理局批准的临床用抗叶酸类药物，将其作为促进扁桃体来源间充质干细胞软骨形成的化合物。聚乙二醇-聚丙氨酸温敏水凝胶被用作三维细胞培养基质，干细胞和抗叶酸类药物可在热诱导的原位溶胶-凝胶转变过程中同时掺入。通过温敏水凝胶的药物持续释放，可以在几天内提供抗叶酸类药物。最初，根据细胞活力和 II 型胶原（COL II）的典型软骨形成生物标志物的 mRNA 水平，对七种抗叶酸类药物进行了预筛选。然后，在第二轮筛选中选择了氨苯砜、培美曲塞和甲氧苄啶作为候选化合物，并对 COL II、性别决定区 Y 框转录因子 9 和聚集蛋白聚糖等各种软骨形成生物标志物的 mRNA 和蛋白表达进行了详细研究。在不存在软骨形成促进剂化合物的情况下，以及在卡托醌（KGN）存在的情况下，在温敏水凝胶中进行了干细胞的三维培养，分别作为阴性对照和阳性对照。与阴性对照组相比，在含有选定的抗叶酸类药物的系统中，软骨形成生物标志物的表达显著增加，而 I 型胶原（成骨生物标志物）的表达没有增加。与阳性对照（KGN）相比，培美曲塞是诱导干细胞软骨分化的最佳化合物。抗叶酸类药物处理后核心结合因子 β 亚基（CBFβ）的核易位和核 runt 相关转录因子 1（RUNX1）的增强提示，软骨形成增强的机制是由 CBFβ 和 RUNX1 介导的。计算机模拟研究证实了这一机制，证明培美曲塞与细丝蛋白 A 的靶蛋白的结合亲和力高于其他抗叶酸类药物候选物。总之，培美曲塞被重新发现为一种先导化合物，并且包含培美曲塞和间充质干细胞的多肽温敏水凝胶可以成为促进干细胞软骨分化的非常有效的系统，并且可能用于软骨修复的可注射组织工程。
