Differential effects of kartogenin on chondrogenic and osteogenic differentiation of rat and rabbit bone marrow mesenchymal stem cells

doi:10.12307/2025.568

Abstract

Abstract: BACKGROUND: Bone marrow mesenchymal stem cells possess multipotent differentiation potential and are an important source of cells for cartilage and bone tissue regeneration research. Kartogenin is a small-molecule drug that has been demonstrated to promote stem cell differentiation towards chondrogenesis. However, the ability to promote osteogenic differentiation is still controversial, and the specific effects of kartogenin on the chondrogenic and osteogenic differentiation of stem cells of different species have not been fully elucidated.
OBJECTIVE: To investigate the effects of kartogenin on chondrogenic and osteogenic differentiation in rabbit-derived and rat-derived bone marrow mesenchymal stem cells.
METHODS: Rabbit-derived and rat-derived bone marrow mesenchymal stem cells were obtained through whole bone marrow separation and adherence methods, and were treated with varying concentrations of kartogenin. Cell proliferation was detected using the CCK-8 assay. Chondrogenic and osteogenic differentiation was assessed via toluidine blue and alizarin red staining, respectively. After screening for the optimal concentration of kartogenin, alkaline phosphatase staining, qRT-PCR, and western blot assay were employed to analyze the expression of osteogenic and chondrogenic-related genes and proteins.
RESULTS AND CONCLUSION: (1) Within the concentration range of 0 to 10 000 nmol/L, kartogenin did not significantly affect the proliferation of rabbit-derived or mouse-derived bone marrow mesenchymal stem cells. (2) The differentiation effect of kartogenin on rabbit-derived and rat-derived bone marrow mesenchymal stem cells showed significant differences. In rabbit-derived bone marrow mesenchymal stem cells, kartogenin predominantly promoted chondrogenic differentiation while inhibiting osteogenic differentiation. This was evident from positive toluidine blue staining, whereas alkaline phosphatase and alizarin red staining exhibited no significant changes. qRT-PCR analysis revealed upregulation of chondrogenic-related genes (Col2a1 and Sox9) and downregulation of genes associated with osteogenesis (Alpl, Col1a1, Runx2, and Bglap). Similar results were found in the western blot assay. (3) In contrast, kartogenin in mouse-derived bone marrow mesenchymal stem cells promoted both chondrogenic and osteogenic differentiation. While toluidine blue staining remained largely unchanged, alkaline phosphatase and alizarin red staining revealed increased positivity, indicative of enhanced osteogenesis. qRT-PCR analysis showed upregulation of not only chondrogenic-related genes (Col2a1 and Sox9) but also genes linked to osteogenesis (Alpl, Col1a1, Runx2, and Bglap). The western blot assay results showed similar results. These findings suggest that kartogenin exerts differential regulatory effects on the differentiation of rabbit and rat bone marrow mesenchymal stem cells, potentially stemming from variations in gene expression profiles and underlying signaling pathways.

Key words: bone marrow mesenchymal stem cell, kartogenin, chondrogenic differentiation, osteogenic differentiation, gene expression, cell proliferation, regenerative medicine, engineered stem cell

CLC Number:

Liu Chengyuan, Guo Qianping. Differential effects of kartogenin on chondrogenic and osteogenic differentiation of rat and rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(35): 7490-7498.

Figures/Tables 8

2.1 骨髓间充质干细胞的形态和流式分析结果采用全骨髓分离贴壁法分离并培养兔和大鼠源骨髓间充质干细胞，培养5 d后，可以观察到细胞形态非常相似，兔源骨髓间充质干细胞为长梭形，大鼠源骨髓间充质干细胞为梭形，两者都表现出强烈的贴壁生长特性。此外，细胞核较大，呈椭圆形或圆形，核仁清晰可见，显示出明显的干细胞特征，见图1A，B。为了验证培养的骨髓间充质干细胞的纯度，流式细胞仪检测结果显示，第2代兔源骨髓间充质干细胞高表达特异性表面抗原CD90和CD29，阳性率分别达到95.3%和92.1%；同时，这些细胞低表达或不表达骨髓成纤维细胞、造血干细胞表面抗原CD45和CD34，阳性率分别为1.92%和5.22%。同样，鼠源骨髓间充质干细胞也高表达CD90和CD29，阳性率分别为95.6%和94.9%，而CD45和CD34低表达或不表达，阳性率分别为1.18%和7.91%，见图1C，D。 2.2 KGN对细胞增殖的影响采用CCK-8法评估不同浓度KGN(0，100，1 000，10 000 nmol/L)对兔和大鼠源骨髓间充质干细胞增殖的影响。培养1，3，5，7 d后，各组之间的细胞增殖数量无显著统计学差异，这表明在0-10 000 nmol/L的浓度范围内，KGN对两种骨髓间充质干细胞的增殖均无明显影响，见图2。 2.3 不同浓度KGN对兔源骨髓间充质干细胞成骨和成软骨分化的影响通过甲苯胺蓝染色和茜素红染色来表征细胞的成软骨和成骨分化情况，见图3。在甲苯胺蓝染色图像中，细胞形态为长梭形，无明显聚集现象，KGN处理组的细胞核呈深蓝色，胞质为浅蓝色。其中，10 000 nmol/L KGN组染色极为明显，表现出最显著的促成软骨分化效果。在茜素红染色图像中，各组细胞均未着色，未观察到钙结节的沉积，表明在这些条件下未出现明显的成骨分化。 2.4 不同浓度KGN对大鼠源骨髓间充质干细胞成骨和成软骨分化的影响由图4可见，在甲苯胺蓝染色图像中，大鼠源骨髓间充质干细胞形态为梭形，具有明显的聚集现象，形成软骨小节。低浓度KGN对细胞聚集没有明显影响，但"

2.5 KGN在早期对兔和大鼠源骨髓间充质干细胞成骨的影响因10 000 nmol/L KGN对兔源和大鼠源骨髓间充质干细胞的成骨分化具有不同的调控效果，进一步使用含10 000 nmol/L KGN的培养基对兔源和大鼠源骨髓间充质干细胞培养1周，通过碱性磷酸酶染色和定量分析KGN对不同种属骨髓间充质干细胞成骨早期的影响。兔源骨髓间充质干细胞对照组中碱性磷酸酶染色较弱，经KGN处理后碱性磷酸酶阳性染色进一步减弱，定量分析结果也显示出相同的趋势，见图5A，B。然而，在大鼠源骨髓间充质干细胞中，对照组表现出较强的碱性磷酸酶阳性染色，而经KGN处理后碱性磷酸酶阳性染色进一步增强，定量分析结果同样支持这一现象，见图5A，C。 2.6 KGN在晚期对兔和大鼠源骨髓间充质干细胞成骨的影响使用含10 000 nmol/L KGN的培养基对兔源和大鼠源骨髓间充质干细胞培养3周，通过碱性磷酸酶染色和定量分析KGN对不同种属骨髓间充质干细胞成骨晚期的影响。兔源骨髓间充质干细胞的对照组和KGN组均表达碱性磷酸酶，但阳性率较低，且定量结果显示两组之间无显著性差异，见图6A，B。相反，在大鼠源骨髓间充质干细胞中，无论是对照组还是KGN组，碱性磷酸酶表达均较高，其中KGN组的阳性染色面积更大，定量结果显示碱性磷酸酶含量更高，见图6A，C。这些结果表明，与兔源骨髓间充质干细胞相比，大鼠源骨髓间充质干细胞本身表达更高的碱性磷酸酶，而在KGN作用下大鼠源骨髓间充质干细胞表现出更强的成骨潜能。 2.7 KGN对兔和大鼠源骨髓间充质干细胞的软骨诱导作用高密度微团(Micromass)法是一种高密度细胞培养系统，能够模拟软骨组织的细胞环境，促进细胞的自然聚集和分化，从而展示软骨细胞的特征。采用Micromass培养法进一步证实KGN的软骨诱导作用，将兔源和大鼠源骨髓间充质干细胞置于含KGN的软骨诱导培养基中进行培养。经过1周的诱导处理后，番红染色结果显示，细胞中出现大量软骨细胞外基质的沉积，并伴随明显的细胞团聚现象，见图7。这些结果表明，KGN能够有效诱导兔源和大鼠源骨髓间充质干细胞分化为软骨细胞。 2.8 KGN对兔和大鼠源骨髓间充质干细胞成软骨/成骨分化基因和蛋白表达的影响由图8可见，qRT-PCR结果显示，在KGN处理的兔源骨髓间充质干细胞中，成软骨相关基因Col2a1和Sox9 的表达显著上调；同时成骨相关基因Alpl、Col1a1、Runx2和Bglap的表达水平降低或无显著变化。Western blot结果显示，KGN可以促进兔源骨髓间充质干细胞成软骨蛋白Ⅱ型胶原的表达上调，同时成骨相关蛋白Ⅰ型胶原的表达无明显变化。这些结果表明KGN在兔源骨髓间充质干细胞中主要促进软骨分化并抑制成骨分化。相反，在大鼠源骨髓间充质干细胞中，KGN上调了成软骨相关基因 Col2a1 和成软骨相关蛋白Ⅱ型胶原的表达，同时还显著上调了成骨相关基因 Alpl、Col1a1、Bglap和成骨相关蛋白Ⅰ型胶原的表达。这些结果表明，KGN在鼠源骨髓间充质干细胞中能同时促进成软骨和成骨分化。"

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