Exosomes derived from human umbilical cord mesenchymal stem cells in treatment of osteoporotic femoral fractures in SD rats

doi:10.12307/2025.538

Abstract

Abstract: BACKGROUND: Exosomes derived from human umbilical cord mesenchymal stem cells are widely used for bone repair and reconstruction, significantly enhancing osteogenesis and promoting angiogenesis.
OBJECTIVE: To explore the mechanisms of exosomes derived from human umbilical cord mesenchymal stem cells in the treatment of osteoporotic fractures.
METHODS: Human umbilical cord mesenchymal stem cells were extracted using tissue block culture method. Exosomes derived from human umbilical cord mesenchymal stem cells were extracted using ultracentrifugation method for identification. Thirty 12-week-old female SD rats were randomly divided into sham-operated group (n=6) and ovariectomized group (n=24). Osteoporosis model was established by castration in the ovariectomized group. 12 weeks after modeling, 6 rats in the ovariectomized group and 6 rats in the sham-operated group were randomly selected for Micro-CT and hematoxylin-eosin staining to verify the models. After verification, the remaining 18 rats in the ovariectomized group were randomly assigned to three groups to establish osteoporotic fracture models. The fracture end was separately injected with PBS (PBS group), exosomes at 1.5×1011 particles/mL (low-concentration exosome group), and 3×1011 particles/mL (high-concentration exosome group). Four weeks after operation, fracture healing and bone angiogenesis were evaluated by imaging and histological observations.
RESULTS AND CONCLUSION: (1) In the gross specimens, compared with the PBS group, the exosome group had faster fracture healing and more callus formation. (2) The X-ray score of fracture healing in the exosome group was significantly higher than that in the PBS group, and the difference was significant (P < 0.05). (3) Micro-CT three-dimensional imaging: Compared with the PBS group, the fracture healing in the exosome group was accelerated and the callus formation was significantly increased; the bone volume fraction in the exosome group was significantly higher than that in the PBS group, and the difference was significant (P < 0.01). (4) Hematoxylin-eosin staining and Masson’s trichrome staining showed that bone trabeculae and the new bone tissue in the exosome group were more than those in the PBS group. (5) Immunohistochemical staining showed that the expression of CD31 and osteocalcin in the exosome group was significantly higher than that in the PBS group. The high-concentration exosome group had a higher density of new blood vessels, more callus formation, and faster fracture healing than the low-concentration exosome group, showing a concentration-dependent manner. The results show that exosomes derived from human umbilical cord mesenchymal stem cells can promote the repair of osteoporotic fracture by enhancing the angiogenesis and osteogenesis. The mechanism may be related to the increased expression of CD31 and osteocalcin.

Key words: osteoporotic fracture, human umbilical cord mesenchymal stem cell, exosome, fracture healing, osteocalcin, CD31

CLC Number:

Bu Xianmin, Liang Di, Zhang Bin, Xu Yingjie, Ding Hao, Wu Bin, Tian Ronghua. Exosomes derived from human umbilical cord mesenchymal stem cells in treatment of osteoporotic femoral fractures in SD rats[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(31): 6634-6641.

Figures/Tables 2

2.1 人脐带间充质干细胞传代培养及hucMSCs-exo鉴定结果经传代培养，可见人脐带间充质干细胞贴壁生长，状态良好，形态呈梭形或长梭形。纳米粒度分析仪检测外泌体粒径为40-100 nm，透射电子显微镜观察到hucMSCs-exo呈现典型的椭圆形形态，细胞膜结构完整，见图1。以上结果表明，已成功提取外泌体。 2.2 实验动物数量分析实验选用 SD大鼠30只，造模后无死亡，全部进入结果分析。 2.3 骨质疏松模型鉴定结果 Micro-CT扫描及骨组织定量分析显示，与假手术组相比，去势组大鼠骨小梁数量明显减少，结构排列紊乱，间隙增宽。与假手术组相比，骨密度、骨小梁厚度、骨小梁数量均显著减少，差异有显著性意义(P < 0.01)；骨小梁分离度增加，差异有显著性意义(P < 0.01)，见图2。苏木精-伊红染色显示，与假手术组相比，去势组大鼠骨小梁厚度降低，数量减少，骨小梁稀疏，结构紊乱，小梁间充斥大量脂滴，见图3。 2.4 各组大鼠股骨骨折端骨痂生成情况在骨折术后4周，大体观察到骨折端周围的组织愈合良好。PBS组与外泌体组之间未发现明显的愈合差异。此外，在外泌体组中未观察到免疫排斥反应现象。PBS组骨折端纤维组织连接，少量骨痂形成，骨折端无明显愈合迹象；低浓度外泌体组可见骨折端部分骨痂形成；高浓度外泌体组骨折端大量骨痂形成，骨折基本愈合，见图4。 2.5 各组大鼠骨折愈合X射线片评分 PBS组骨折线清晰可见，少量骨痂形成，骨折端无明显愈合迹象；低浓度外泌体组骨折线模糊，骨折端周围可见较多骨痂形成；高浓度外泌体组骨折线基本消失，骨折端大量骨痂形成，骨折基本愈合。PBS组、低浓度外泌体组、高浓度外泌体组骨折愈合X射线片评分分别为0.83±0.40，1.67±0.51，3.17±0.40，差异有显著性意义(P < 0.05)，见图5。"

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