中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (34): 5469-5476.doi: 10.12307/2023.557

• 组织工程骨材料 tissue-engineered bone • 上一篇    下一篇

构建载免疫调节肽/miR-26a复合物微球缓释体系的体内成骨

李欣伦1，朱昱树1，杨乙苓2，何思齐3，文  楠3，牟雁东1，3   

  1. 1西南医科大学口腔医学院，四川省泸州市  646000；2川北医学院，四川省南充市  637000；3四川省医学科学院·四川省人民医院，四川省成都市  610072
  • 收稿日期:2022-09-13 接受日期:2022-10-28 出版日期:2023-12-08 发布日期:2023-04-20
  • 通讯作者: 牟雁东，主任医师，西南医科大学口腔医学院，四川省泸州市 646000；四川省医学科学院·四川省人民医院， 四川省成都市 610072
  • 作者简介:李欣伦，男，1997年生，四川省攀枝花市人，汉族，西南医科大学在读硕士，医师，主要从事口腔种植研究。
  • 基金资助:
    国家自然科学基金面上项目(82071168)，项目负责人：牟雁东；四川省科技厅重点研发项目(2021YFS0009)，项目负责人：牟雁东

Construction and in vivo osteogenesis of microspheres loaded with immunomodulatory peptide/miR-26a complexes for slow release

Li Xinlun1, Zhu Yushu1, Yang Yiling2, He Siqi3, Wen Nan3, Mu Yandong1, 3   

  1. 1School of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2North Sichuan Medical College, Nanchong 637000, Sichuan Province, China; 3Sichuan Academy of Medical Sciences · Sichuan Provincial People’s Hospital, Chengdu 610072, Sichuan Province, China
  • Received:2022-09-13 Accepted:2022-10-28 Online:2023-12-08 Published:2023-04-20
  • Contact: Mu Yandong, Chief physician, School of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Sichuan Academy of Medical Sciences · Sichuan Provincial People’s Hospital, Chengdu 610072, Sichuan Province, China
  • About author:Li Xinlun, Master candidate, Physician, School of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    General Program of National Natural Science Foundation of China, No. 82071168 (to MYD); Key Research & Development Project of Sichuan Provincial Department of Science and Technology, No. 2021YFS0009 (to MYD)

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摘要：


文题释义：

免疫调节肽：是由生物体内产生的多肽，具有分子质量小、结构稳定性高、对特定细胞器或细胞选择性小、毒性小、成本低等优点。
临界骨缺损：骨缺损大小达到临界值后，在自然状态下缺损区域无法形成完全的骨性愈合。

背景：课题组前期研究证明，免疫调节肽DP7-C具有良好的免疫调节功能，并可高效转染递送miRNA进入细胞发挥调控功能。
目的：制备并筛选不同尺寸的多孔聚乳酸-羟基乙酸共聚物微球，装载免疫调节肽DP7-C/miR-26a复合物，构建并优化缓释体系，验证其生物相容性及体内成骨能力。
方法：①微球制备：以碳酸氢铵作为造孔剂，通过双乳化法制备多孔聚乳酸-羟基乙酸共聚物微球，通过调整转速制备3种不同尺寸的多孔微球，将微球分散于免疫调节肽DP7-C/miR-26a复合物溶液中，制备载药微球，通过微球物理性能表征、投入产出比、包封率及缓释性能，筛选出最佳载药多孔聚乳酸-羟基乙酸共聚物微球，用于后续实验。②体外细胞实验：将不同质量浓度的载药与未载药微球溶液分别与大鼠骨髓间充质干细胞共培养，采用CCK-8法检测微球的生物相容性。③动物体内实验：取9只成年SD大鼠，按照随机数字表法分为空白组、对照组、实验组，每组3只。3组均建立直径5 mm颅骨缺损模型，空白组不进行干预，对照组植入未载药微球，实验组植入载药微球，术后8周，分别进行主要脏器及颅骨缺损区病理形态、碱性磷酸酶免疫组化染色检测。

结果与结论：①微球制备：通过相关检测结果得出，相较于转速500，1 200 r/min，转速1 000 r/min制备的载药微球具有均匀的尺寸、更高的产率及良好的缓释性能，可用于后续实验。②体外细胞实验：CCK-8检测结果显示，载药与未载药微球溶液对骨髓间充质干细胞的增殖无影响，无明显的细胞毒性。③动物体内实验：苏木精-伊红染色显示，各组大鼠内脏未发生与实验干预相关的病理学改变。苏木精-伊红及Masson染色显示，空白组缺损部位主要被纤维结缔组织增生占据，可见少量血管生成，未见明显新骨生成；对照组缺损部位可见少量新骨生成及不同程度纤维组织增生，可见新生毛细血管；实验组缺损区域有明显新骨生成及不同程度纤维组织增生，可见新生毛细血管。免疫组化染色显示，实验组缺损部位碱性磷酸酶表达高于对照组、空白组(P < 0.05)。④结果显示，多孔聚乳酸-羟基乙酸共聚物微球/免疫调节肽miR-26a复合物缓释体系具有良好的生物相容性及体内成骨性能，可促进大鼠颅骨临界骨缺损的再生修复。

https://orcid.org/0000-0001-6092-6073(李欣伦)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 聚乳酸-羟基乙酸共聚物, 缓释微球, 免疫调节肽, miR-26, 颅骨缺损, 骨再生修复

Abstract: BACKGROUND: Our group has demonstrated that the immunomodulatory peptide DP7-C has good immunomodulatory functions and can efficiently transfect and deliver miRNAs into cells to exert regulatory functions. 
OBJECTIVE: To prepare and screen porous poly(lactic-co-glycolic acid) microspheres of different sizes loaded with immunomodulatory peptide miR-26a complexes, construct and optimize the sustained release system, and verify its biocompatibility and osteogenic ability in vivo.
METHODS: (1) Microsphere preparation: Porous poly(lactic-co-glycolic acid) microspheres were prepared by double emulsification with NH4HCO3 as the pore-forming agent. Three different sizes of porous poly(lactic-co-glycolic acid) microspheres were prepared by adjusting the rotational speed. The microspheres were dispersed in the solution of immunomodulatory peptide DP7-C/miR-26a complex to prepare drug-loaded microspheres. The physical properties of the microspheres were characterized. The best drug-loaded porous poly(lactic-co-glycolic acid) microspheres were selected by comparing productivity, encapsulation rate and release performance for further use. (2) In vitro cell experiment: Drug-loaded and drug-free microsphere solutions with different mass concentrations were co-cultured with rat bone marrow mesenchymal stem cells. CCK-8 assay was performed to verify biocompatibility. (3) Animal in vivo experiment: Nine adult Sprague-Dawley rats were randomly divided into blank group, control group and experimental group, with three rats in each group. Cranial defect models with a diameter of 5 mm were established in all three groups. The rats of the blank group were not implanted with the material. The rats of the control group were implanted with blank microspheres. The rats of the experimental group were implanted with microspheres loaded with DP7-C/miR-26a complex. At 8 weeks after operation, the pathological morphology and immunohistochemical staining for alkaline phosphatase of the main organs and skull defects were performed. 
RESULTS AND CONCLUSION: (1) Microsphere preparation: According to the relevant detection results, the drug-loaded microspheres prepared at 1 000 r/min compared with 500 and 1 200 r/min had uniform size, higher yield and better sustained release performance, which could be used in subsequent experiments. (2) In vitro cell experiment: CCK-8 assay results exhibited that drug-loaded and drug-free microsphere solutions had no effect on the proliferation of bone marrow mesenchymal stem cells and had no obvious cytotoxicity. (3) In vivo experiments: There were no pathological changes in the viscera on hematoxylin-eosin staining related to the treatment with the intervention. Hematoxylin-eosin staining and Masson staining demonstrated that in the blank group, the defect site was mainly filled by fibrous connective tissue, with a small amount of angiogenesis, but without obvious new bone formation. In the control group, a small amount of new bone formation, fibrous tissue hyperplasia and new capillaries could be visible at the defect site. In the experimental group, there was obvious new bone formation, different degrees of fibrous tissue hyperplasia and new capillaries in the defect area. Immunohistochemical results displayed that alkaline phosphatase was highly expressed in the experimental group compared to the blank group and the control group (P < 0.05). (4) These findings have concluded that the porous poly(lactic-co-glycolic acid)/DP7-C/miR-26a composite system has good biocompatibility and in vivo osteogenic properties and can promote bone regeneration and repair of critical bone defects in the rat skull. 

Key words: poly(lactic-co-glycolic acid), slow-release microsphere, immunomodulatory peptide, miR-26a, skull defect, bone regeneration repair

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