Gong Weimin, Li Molin, Zhao Lizhou, Wang Pengtao, Wang Xiaofang, Wang Bo, Liu Xing, Tu Xiaolin
Laboratory of Skeletal Development and Regeneration, Institute of Life Sciences, Chongqing Medical University, Chongqing, China.
Department of Orthopedics, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Chongqing, China.
Front Bioeng Biotechnol. 2023 Jul 28;11:1215233. doi: 10.3389/fbioe.2023.1215233. eCollection 2023.
The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3β inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3β activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including , , and , promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors and in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines and but decreased the ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7 days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7 days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21 days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of , a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.
Wnt信号通路的安全有效应用是开发成骨药物的一个热门话题。SB216763（S33）是一种广泛使用的高特异性GSK3β抑制剂。在此，我们表明S33通过抑制骨髓基质细胞系ST2中的GSK3β活性来启动经典Wnt信号通路，并增加成骨细胞标志物碱性磷酸酶活性、包括 、 和 在内的成骨细胞标志物基因表达，促进ST2细胞的成骨分化和矿化。此外，S33抑制ST2细胞中脂肪生成转录因子 和 的表达以抑制脂肪生成。ICRT - 14，一种Wnt信号通路的特异性转录抑制剂，逆转了S33对ST2细胞分化的影响。S33还增加了破骨细胞细胞因子 和 的表达，但降低了 比值，并具有抑制破骨细胞分化的潜力。此外，我们使用新建立的硬质材料和细胞一体化3D打印系统打印了PSCI3D（聚己内酯、S33、细胞一体化3D）支架。S33在支架水凝胶中持续释放，第1天释放25.4%，7天内释放81.7%。支架中的细胞具有良好的细胞活力。活/死细胞比例在7天内保持在94%以上，而支架中的细胞呈线性增殖，PSCI3D支架组在第4天和第7天的增殖活性分别增加了1.4倍和1.7倍。同样，在PSCI3D支架中，st2细胞的成骨分化增加。碱性磷酸酶活性在第7天和第14天分别增加了1.4倍和4.0倍，矿化在第21天增加了1.7倍。此外，PSCI3D条件培养基促进了人脐静脉内皮细胞（HUVECs）的迁移和管腔形成，并且S33上调了血管生成关键因子 的表达。总之，我们的研究表明S33在成骨、抗脂肪生成和潜在抑制破骨细胞分化中发挥作用。并且S33在PSCI3D支架中的持续释放创造了一个安全的成骨微环境，促进细胞增殖、成骨和血管生成，具有应用前景。
