Effects of platelet-derived growth factor-DD on proliferation and multilineage differentiation of human tendon-derived stem cells

doi:10.12307/2025.537

Abstract

Abstract: BACKGROUND: Chronic rotator cuff injury is often companied by tendon degeneration and impaired function of tendon-derived stem cells. As am important cytokine, platelet-derived growth factor-DD has a regulatory effect on the proliferation and differentiation of tendon-derived stem cells.
OBJECTIVE: To investigate the effect of platelet-derived growth factor-DD on the proliferation and multilineage differentiation of tendon-derived stem cells in human chronic rotator cuff injury.
METHODS: Tendon-derived stem cells were isolated from human chronic rotator cuff injury tissue and cultured in vitro. Immunofluorescence staining was used to observe the cytoskeletal morphology of tendon-derived stem cells. Flow cytometry was used to identify the phenotype of tendon-derived stem cells. Tendon-derived stem cells were divided into two groups. The control group did not receive any intervention. The platelet-derived growth factor-DD group was treated with 5 μg/mL platelet-derived growth factor-DD. The effect of platelet-derived growth factor-DD on the proliferation and multilineage differentiation of tendon-derived stem cells was evaluated by cell proliferation assay and three-lineage differentiation assay.
RESULTS AND CONCLUSION: (1) The number of EdU-positive cells in the platelet-derived growth factor-DD group was significantly increased compared with the control group (P < 0.05). Tendon-derived stem cells entered the rapid proliferation phase earlier, and the cell growth was logarithmic. (2) The positive areas of Oil Red O staining, Alcian Blue staining, and Alizarin Red staining in the platelet-derived growth factor-DD group were significantly larger than those in the control group (P < 0.05). (3) The above results show that platelet-derived growth factor-DD significantly promotes the proliferation and adipogenic, osteogenic, and chondrogenic differentiation of tendon-derived stem cells.

Key words: tendon stem cell, tendon-derived stem cell, platelet-derived growth factor-DD, rotator cuff injury, rotator cuff repair, engineered stem cell

CLC Number:

Wen Huawei, Zhang Qingsong, Tang Ming, Li Yanan, Tan Hongfei, Fang Yushun. Effects of platelet-derived growth factor-DD on proliferation and multilineage differentiation of human tendon-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(31): 6649-6655.

Figures/Tables 1

2.1 肌腱干细胞骨架形态和流式鉴定结果免疫荧光结果显示，肌腱干细胞轮廓清晰可见，细胞形态整齐，类似于纤维状或梭形。肌腱干细胞胞质内部呈现出强烈的荧光信号且分布均匀，显示出明显的细胞骨架，微丝蛋白等标记物沿着细胞骨架形成纤维状结构，见图1A。流式检测结果显示，绝大多数细胞表达CD44和CD90，阳性率超过98%，而CD34阳性细胞仅占有总细胞数的极少部分，阳性率为0.5%，CD106表达则未检测到，见图1B。 2.2 血小板衍生生长因子DD对肌腱干细胞增殖的影响 EdU结果显示，培养48 h后，两组均可以观察到高荧光强度的细胞，表明有少量细胞正在进行DNA复制，处于增殖状态，但两组正在进行DNA复制的细胞分布不均。此外，与对照组相比，血小板衍生生长因子DD组肌腱干细胞显示出更高强度的荧光，具有更高的增殖活性，见图2A。免疫荧光定量分析结果显示，血小板衍生生长因子DD组的EdU细胞阳性率明显高于对照组(P < 0.01)，见图2B。 CCK-8结果显示，正常条件下生长的肌腱干细胞在接种后第1天便缓慢进入了增殖期，从第3天开始进入快速增殖期，细胞呈对数增殖，直至第6天增殖速度减慢，进入平台期。而血小板衍生生长因子DD干预后的肌腱干细胞在接种后第1天处于潜伏期，从第2天开始进入快速增殖期，细胞呈对数增殖，直至第6天增殖变得缓慢。从第3天开始直至第8天，与对照组相比，血小板衍生生长因子DD组吸光度值显著升高，差异有显著性意义 (P < 0.05)，见图2C。 2.3 血小板衍生生长因子DD对肌腱干细胞三系分化的影响见图3。空白对照组肌腱干细胞在高糖DMEM/F12完全培养基中培养7 d后，油红O染色未见脂滴形成；对照组肌腱干细胞在成脂诱导分化完全培养基中培养7 d后，油红O染色可见少量脂滴形成，呈橘红色，分布不均；血小板衍生生长因子DD组肌腱干细胞在含有血小板衍生生长因子DD的成脂诱导分化完全培养基中培养7 d后，油红O染色可见中等量脂滴形成，呈橘红色，分布较为均匀，部分脂肪细胞聚集成团。空白对照组肌腱干细胞在高糖DMEM/F12完全培养基中培养7 d后，阿利新蓝染色未见软骨形成，可见大量酸性黏液物质，但未见完整胞质蓝染；对照组肌腱干细胞在成软骨诱导分化完全培养基中培养7 d后，阿利新蓝染色可见中等量软骨形成，呈蓝色，分布均匀；血小板衍生生长因子DD组肌腱干细胞在含有血小板衍生生长因子DD的成软骨诱导分化完全培养基中培养7 d后，阿利新蓝染色可见大量软骨形成，蓝色软骨细胞分布均匀。空白对照组肌腱干细胞在高糖DMEM/F12完全培养基中培养7 d后，茜素红染色未见钙盐沉积；对照组肌腱干细胞在成骨诱导分化完全培养基中培养7 d后，茜素红染色可见少量钙盐沉积，呈红色或橘红色，部分钙盐聚集呈结节状；血小板衍生生长因子DD组肌腱干细胞在含有血小板衍生生长因子DD的成骨诱导分化完全培养基中培养7 d后，茜素红染色可见中等量钙盐沉积，整体呈红色，结节状钙盐沉积明显增多，部分骨细胞聚集成块。染色区域定量分析结果显示，血小板衍生生长因子DD组的油红O染色、阿利新蓝染色、茜素红染色阳性面积较对照组明显增加，差异有显著性意义(P < 0.001)。"

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