Adipose tissue-derived mesenchymal stem cell conditioned medium combined with platelet-rich fibrin repairs skin injury in mice

doi:10.3969/j.issn.2095-4344.1520

Abstract

Abstract:

BACKGROUND: Platelet-rich fibrin can promote soft tissue healing, and soluble factors secreted by mesenchymal stem cells can promote skin regeneration, but their combination effects on skin repair are little reported.
OBJECTIVE: To investigate the effect of conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin on the healing of skin wound in mice.
METHODS: Full-thickness skin injury models were established in C57/BL6 mice and randomly divided into four groups, 10 mice in each group. After the modeling, each group was smeared with the specific medium of adipose tissue-derived mesenchymal stem cells, the conditioned medium of adipose tissue-derived mesenchymal stem cells, the platelet-rich fibrin, the conditioned medium of adipose tissue-derived mesenchymal stem cells combined with platelet-rich fibrin, thrice daily for 7 consecutive days. The wound size was measured at 1, 3, 7 and 10 days after treatment and the wound healing rate was calculated at 3 and 7 days after treatment. Histological analysis of wound healing was performed at 14 days after treatment.
RESULTS AND CONCLUSION: Compared with the specific culture medium of adipose tissue-derived mesenchymal stem cells, the wound healing time in the other three groups was shorter. The wound healing rate of adipose-derived mesenchymal stem cell conditioned medium combined with platelet-rich fibrin group was significantly higher than that in the other three groups at 3 and 7 days after treatment (P < 0.05). The adipose-derived mesenchymal stem cell conditioned medium group and platelet-rich fibrin group showed higher healing rate than the adipose-derived mesenchymal stem cell specific medium group (P < 0.05). Histological findings showed that the aggregation of white blood cells and fibroblasts, angiogenesis, granulation tissue remodeling and epithelization in the conditioned medium of adipose mesenchymal stem cells combined with the platelet-rich fibrin group were more obvious than those in the other three groups, in which the best wound healing effect was found. These results suggest that the combination of adipose tissue-derived mesenchymal stem cell conditioned medium and platelet-rich fibrin can effectively promote the repair of skin injury in mice, and the effect is better than that of conditioned medium or platelet-rich fibrin alone.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Skin, Wounds and Injuries, Adipose Tissue, Mesenchymal Stem Cells, Culture Media, Conditioned, Platelet-Rich Plasma, Tissue Engineering

CLC Number:

Huang Min, Yan Hong. Adipose tissue-derived mesenchymal stem cell conditioned medium combined with platelet-rich fibrin repairs skin injury in mice[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 18-23.

Figures/Tables 1

2.1 小鼠ADSCs的原代培养及鉴定结果将由小鼠脂肪组织分离获得的ADSCs接种到培养皿中4 h后，倒置相差显微镜下观察发现贴壁细胞呈梭形，培养5 d后呈漩涡状排列的细胞形成较大集落，铺满培养皿底90%，见图1A。约5 d传1代，取第3代ADSCs成脂诱导14 d，油红O染色显示细胞内布满染成红色的脂滴，大小不一，见图1B；成骨诱导21 d，茜素红染色显示深红色结节散在分布于细胞集落中，见图1C，提示分离培养的ADSCs具有多向分化能力。流式细胞术检测结果显示，分离培养的小鼠ADSCs表达CD90和CD44，不表达CD31和CD34，见图1D，提示分离培养的细胞具有间充质干细胞的特性。 2.2 实验小鼠数量分析 40只小鼠在造模和治疗过程中均无死亡等异常发生，所有实验数据均计入结果分析。 2.3 创面愈合过程中的组织学分析 ADSCs条件培养液+ PRF组小鼠创面的愈合效果优于其他3组，治疗14 d后，肉芽组织表面的上皮增厚并分层，结构与周围正常皮肤相似，真皮层细胞聚集明显，血管增多，说明创面发生肉芽组织重塑和上皮化；ADSCs专用培养基组只有成纤维细胞和胶原纤维组成的细密结缔组织，表层上皮未明显分层；ADSCs条件培养液组和PRF组局部增厚，肉芽组织较少，见图2。 2.4 小鼠创面愈合情况及创面愈合率各组小鼠的创面在7 d内形成痂皮，14 d内创面均愈合，在治疗过程中均未发生感染，ADSCs条件培养液+PRF组在治疗后10 d可见完整的组织修复，而ADSCs专用培养基组约在治疗后14 d完成修复，ADSCs条件培养液组与PRF组均在治疗后12 d左右完成修复；治疗7 d后，与ADSCs专用培养基组相比，其他3组小鼠创面面积均有缩小，ADSCs条件培养液+PRF组创面面积缩小最明显，见图3A；为了进一步比较对创面愈合的影响，计算各组治疗3 d和7 d的创面愈合率，ADSCs条件培养液+PRF组创面愈合率高于其他3组，差异有显著性意义(P < 0.05)，见图3B。"

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