Zhang Qiwen, Liang Ning, He Bin, Wu Siyou, Wen Depeng, Tang Xiaoyong, Shen Xiongcheng
People's Hospital of Honghuagang District, Guizhou, China.
The Third Affiliated Hospital of Zunyi Medical University (Zunyi first people's Hospital), Guizhou, China.
Turk J Biol. 2022 Dec 21;47(1):14-28. doi: 10.55730/1300-0152.2638. eCollection 2023.
Clinical observations show that the current spinal fusion with internal fixation has a nonfusion rate of 5%-35%; however, methods to promote spinal fusion are limited. This study aimed to investigate the role of SDF-1-induced directional chemotaxis of BMSCs in bone marrow chimera spinal intervertebral fusion mouse model. BMSCs were isolated from bone marrow and identified by detecting CD44/CD34 positive cells. BMSCs (GFP-BMSCs) were labeled with GFP for tracking in vivo. Mice were inoculated with GFP-BMSCs to construct bone marrow chimera spinal intervertebral fusion model, which were divided into BM-SIF model, BM-SIF+SDF-1, BM-SIF+SDF-1-Anta group. The callus area of intervertebral fusion site was detected by radiology. HE staining was used to detect trabeculae formation. Expressions of osteogenic molecules and fibroblast markers were detected by RT-PCR and Western blotting. GFP-BMSCs showed obvious osteogenic and adipogenic differentiation ability, according to oil-red O and alizarin-red staining. Bone marrow chimera spinal intervertebral fusion mouse model was successfully established, with efficient localization of GFP-BMSCs at intervertebral fusion site. SDF-1 significantly promoted bone trabeculae formation in callus at intervertebral fusion site. SDF-1 significantly increased osteogenic molecules transcription/expression in callus at intervertebral bone graft fusion site of mice; however, SDF-1-Anta (AMD3100) significantly decreased osteogenic molecules transcrition/expression, compared to those of mice from the BM-SIF model group (p < 0.05). SDF-1 markedly induced and SDF-1-Anta significantly decreased fibroblast proliferations in the callus at the intervertebral fusion site of mice, compared to those of mice from the BM-SIF model group (p < 0.05). SDF-1 enhanced expression of Wnt10b and β-catenin in callus at intervertebral fusion site of mice compared to mice of the BM-SIF model group (p < 0.05). In conclusion, SDF-1 induced directional chemotaxis of BMSCs to the intervertebral fusion site and promoted osteogenic differentiation in bone marrow chimera spinal intervertebral fusion mice by regulating Wnt/β-catenin pathway and modulating the proliferation of BMSCs.
临床观察表明，目前的脊柱内固定融合术的不融合率为5%-35%；然而，促进脊柱融合的方法有限。本研究旨在探讨基质细胞衍生因子-1（SDF-1）诱导骨髓间充质干细胞（BMSCs）定向趋化在骨髓嵌合体脊柱椎间融合小鼠模型中的作用。从骨髓中分离出BMSCs，并通过检测CD44/CD34阳性细胞进行鉴定。用绿色荧光蛋白（GFP）标记BMSCs（GFP-BMSCs）以便在体内追踪。给小鼠接种GFP-BMSCs以构建骨髓嵌合体脊柱椎间融合模型，将其分为骨髓-脊柱椎间融合（BM-SIF）模型组、BM-SIF+SDF-1组、BM-SIF+SDF-1拮抗剂组。通过放射学检测椎间融合部位的骨痂面积。采用苏木精-伊红（HE）染色检测小梁形成情况。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测成骨分子和成纤维细胞标志物的表达。根据油红O和茜素红染色结果，GFP-BMSCs表现出明显的成骨和成脂分化能力。成功建立了骨髓嵌合体脊柱椎间融合小鼠模型，GFP-BMSCs在椎间融合部位有效定位。SDF-1显著促进椎间融合部位骨痂中小梁的形成。SDF-1显著增加小鼠椎间植骨融合部位骨痂中成骨分子的转录/表达；然而，与BM-SIF模型组小鼠相比，SDF-1拮抗剂（AMD3100）显著降低成骨分子的转录/表达（p<0.05）。与BM-SIF模型组小鼠相比，SDF-1显著诱导而SDF-1拮抗剂显著降低小鼠椎间融合部位骨痂中成纤维细胞的增殖（p<0.05）。与BM-SIF模型组小鼠相比，SDF-1增强了小鼠椎间融合部位骨痂中Wnt10b和β-连环蛋白的表达（p<0.05）。总之，SDF-1诱导BMSCs向椎间融合部位定向趋化，并通过调节Wnt/β-连环蛋白信号通路和调控BMSCs的增殖，促进骨髓嵌合体脊柱椎间融合小鼠的成骨分化。
