Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (30): 4812-4818.doi: 10.12307/2022.760
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Huang Gao, Xu Jun, Chen Wenge
Received:
2021-07-06
Accepted:
2021-09-04
Online:
2022-10-28
Published:
2022-03-29
Contact:
Chen Wenge, Chief physician, Minda Hospital Affiliated to Hubei University for Nationalities, Enshi 445000, Hubei Province, China
About author:
Huang Gao, Attending physician, Master, Minda Hospital Affiliated to Hubei University for Nationalities, Enshi 445000, Hubei Province, China
Supported by:
CLC Number:
Huang Gao, Xu Jun, Chen Wenge. Implantation of bone marrow mesenchymal stem cells-loaded platelet-rich plasma combined with extracorporeal shock wave in the repair of bone defects[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4812-4818.
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2.1 各组骨髓间充质干细胞的增殖、活性和迁移能力分析 将第4代骨髓间充质干细胞以2.0×104/孔种植在24孔培养板中,分别培养1,4,7 d后使用CCK-8法测定骨髓间充质干细胞的增殖情况,见图1A。结果表明:随着培养时间的延长,各组细胞增殖均发生明显增加。在种植当天(即第1天)时,各组间骨髓间充质干细胞的吸光度值差异无显著性意义(P > 0.05)。但是,在第4,7天,体外冲击波+富血小板血浆组与其他组比较差异均有显著性意义;富血小板血浆组细胞增殖也均优于对照组和体外冲击波组(P < 0.05)。单纯体外冲击波处理组与未处理对照组相比,在第4天时未见明显变化,但这种差异在第7天时变得显著(P < 0.05)。以上结果提示体外冲击波和富血小板血浆处理均有利于细胞增殖,尤其是联合两种处理方式时可显著促进骨髓间充质干细胞在体外增殖。 在培养3 d后进行钙黄绿素/碘化丙啶活-死细胞染色,见图1B,荧光图片清楚地显示,各组细胞均多为绿染的活细胞,红染的死细胞数量极少。结果显示不管是体外冲击波处理还是与富血小板血浆共培养,均不会降低细胞活性。"
2.2 各组骨髓间充质干细胞的成骨能力 除了影响骨髓间充质干细胞的数量和活性外,成骨分化是骨再生的关键指标。经体外成骨诱导培养14 d后,体外冲击波+富血小板血浆组的碱性磷酸酶活性显著高于对照组(P < 0.001)、体外冲击波组(P < 0.01)和富血小板血浆组(P < 0.05)。同时,体外冲击波组和富血小板血浆组的碱性磷酸酶活性也显著高于对照组,说明不管是体外冲击波还是富血小板血浆处理都可以显著上调骨髓间充质干细胞的碱性磷酸酶活性,见图3A,B。茜素红染色是一种用于矿化的组织学染色,它可以识别沉积的钙结节。大体图像显示体外冲击波和/或富血小板血浆处理组的钙沉积斑点较对照组更明显。对茜素红染色结果进行半定量分析进一步证实了这一变化:即在富血小板血浆组和体外冲击波+富血小板血浆组检测到明显的钙沉积量,见图3C,D。 成骨诱导培养7,14 d后,体外冲击波 +富血小板血浆组的Runx2和OCN基因表达量均较其他组明显上调,差异有显著性意义;而体外冲击波组和富血小板血浆组Runx2和OCN的基因表达量也显著高于对照组;成骨相关基因表达的差异仅在第14天时富血小板血浆组高于体外冲击波组 (P < 0.05),以上研究结果说明体外冲击波和/或富血小板血浆处理可以促进骨髓间充质干细胞成骨相关基因的表达,见图3E,F。"
2.4 影像学和组织学评估节段性骨缺损的再生情况 术后12周采用Micro-CT进行影像学检查,未处理组骨再生情况最差,出现明显节段性骨不连;单纯骨髓间充质干细胞组骨再生也相对受限,出现部分再生后两断端髓腔封闭,也出现骨不连情况;骨髓间充质干细胞/体外冲击波组一侧骨皮质连续再生,另一侧皮质再生不完全;骨髓间充质干细胞/富血小板血浆组的节段性骨缺损修复较好,仅残留少量缺损;而骨髓间充质干细胞/体外冲击波+富血小板血浆组几乎完全修复,并实现髓腔再通,见图6A。各组的骨体积/总体积比值分别为(5.93±1.67)%,(10.72±2.24) %,(16.52±2.16)%,(18.26±2.27)%和(23.53±2.15)%,见图6B。 苏木精-伊红染色组织学评价显示,未处理组和单纯骨髓间充质干细胞组的骨缺损断端间隙被纤维组织填充,术后12周原始骨与纤维组织的界面清晰可见;相比之下,骨髓间充质干细胞/体外冲击波组和骨髓间充质干细胞/富血小板血浆组缺损处再生的皮质骨部分连续,可见缺损部位少量成熟骨痂;而骨髓间充质干细胞/体外冲击波+富血小板血浆组双侧皮质骨再生明显,形成连续结构,缺损区见完全骨性愈合,骨组织成熟,与周围原始骨组织结构相似,实现较好的再生,见图6C。这些结果表明,骨髓间充质干细胞、体外冲击波和富血小板血浆均可部分促进骨质疏松节段性骨缺损的再生,3者的联合使用能获得一个较为满意的骨愈合效果。"
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