中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (4): 809-817.doi: 10.12307/2025.227

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

富血小板纤维蛋白复合甲基丙烯酰化明胶水凝胶的促成骨性能

赵红霞1，孙政伟2，韩  阳3，吴学超4，韩  静5   

  1. 沈阳药科大学，1制药工程学院，3中药学院，5功能食品与葡萄酒学院，辽宁省沈阳市  110016；2山东大学制药工程，山东省烟台市  264000；4华中农业大学生命科学与技术学院，湖北省武汉市  430070
  • 收稿日期:2023-12-05 接受日期:2024-01-20 出版日期:2025-02-08 发布日期:2024-06-03
  • 通讯作者: 韩静，博士，教授，沈阳药科大学功能食品与葡萄酒学院，辽宁省沈阳市 110016
  • 作者简介:赵红霞，女，1987年生，河北省石家庄市人，汉族，硕士，主要从事生物制药工程研究。
  • 基金资助:
    辽宁省自然科学基金计划项目“博士启动基金”(2022-BS-154)，项目负责人：韩阳

Osteogenic properties of platelet-rich fibrin combined with gelatin methacryloyl hydrogel

Zhao Hongxia1, Sun Zhengwei2, Han Yang3, Wu Xuechao4 , Han Jing5   

  1. 1School of Pharmaceutical Engineering, 3School of Traditional Chinese Materia Medica, 5Faculty of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province, China; 2Pharmaceutical Engineering, Shandong University, Yantai 264000, Shandong Province, China; 4College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China
  • Received:2023-12-05 Accepted:2024-01-20 Online:2025-02-08 Published:2024-06-03
  • Contact: Zhao Hongxia, Master, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province, China
  • About author:Han Jing, PhD, Professor, Faculty of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province, China
  • Supported by:
    “Doctoral Initiation Fund” of Natural Science Foundation of Liaoning Province, No. 2022-BS-154 (to HY)

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摘要：


文题释义：

富血小板纤维蛋白：是2000年CHOUKROUN报道的第2代血小板浓缩物，含有大量的生长因子，可以刺激胶原蛋白的产生、启动血管内部生长、诱导细胞分化、控制局部炎症反应，促进组织愈合。
甲基丙烯酰化明胶：是甲基丙烯酸酐修饰明胶的氨基侧链获得的聚合物，属于一种经过改良修饰的天然细胞外基质，具有天然与人工双重材料的优势，在提升明胶稳定性的同时保留了主链上RGD序列、基质金属蛋白酶序列，具有优异的生物相容性、细胞黏附性与细胞重塑性。
背景：富血小板纤维蛋白具有制备简单、制作费用低、安全性高等诸多优势，目前已被广泛应用于口腔颌面外科骨缺损修复研究中，但存在降解速度太快、生长因子释放时间短等问题。
目的：将富血小板纤维蛋白负载于甲基丙烯酰化明胶水凝胶内，通过体内外实验分析其促成骨性能。
方法：①抽取新西兰大白兔静脉血，制备富血小板纤维蛋白。分别制备含0，0.05，0.075，0.1 g富血小板纤维蛋白的甲基丙烯酰化明胶水凝胶，分别记为GelMA、GelMA/PRF-0.05、GelMA/PRF-0.075、GelMA/PRF-0.1，表征水凝胶的微观形貌与体外缓释性能。②将4种水凝胶分别与MC3T3-E1细胞共培养，以单独培养的细胞为对照，检测细胞增殖活性；成骨诱导后，检测细胞碱性磷酸酶活性、矿化能力、成骨相关基因(骨钙素、骨桥蛋白、RUNX2)mRNA与蛋白表达，ERK1/2-p38 MAPK通路蛋白mRNA与蛋白表达。③取15只新西兰大白兔。在每只兔颅骨部分别制备4个直径8 mm的全层骨缺损，其中1处缺损不植入任何材料(空白对照组)，另3处缺损分别植入GelMA水凝胶、富血小板纤维蛋白、GelMA/PRF-0.1水凝胶。术后4，8，12周，进行骨缺损部位Micro-CT扫描与骨组织形态观察。

结果与结论：①扫描电镜下可见4组水凝胶都具有蜂窝状的孔隙结构，并且随着水凝胶中富血小板纤维蛋白含量的增加，水凝胶的孔径轻微减小，但组间比较无明显差异；3组GelMA/PRF水凝胶均能以一定的速率释放转化生长因子β1与胰岛素样生长因子1，随着时间的延长，转化生长因子β1与胰岛素样生长因子1累计释放量显著增加。②CCK-8检测与活/死染色显示，3组GelMA/PRF水凝胶可促进MC3T3-E1细胞的增殖；碱性磷酸酶染色、茜素红染色及成骨基因检测结果显示，GelMA/PRF水凝胶可促进MC3T3-E1细胞的成骨分化，同时可抑制ERK1/2-p38 MAPK通路蛋白的表达，并且呈现富血小板纤维蛋白含量依赖性。③Micro-CT扫描显示，GelMA/PRF-0.1水凝胶组兔骨缺损处骨矿物质密度与骨体积分数均高于其他3组(P < 0.05)；苏木精-伊红染色显示，相较于其他3组，GelMA/PRF-0.1水凝胶组兔骨缺损处新骨形成更快、更成熟。④结果表明，GelMA/PRF水凝胶在体内外均具有良好的促成骨性能，该作用可能与抑制ERK1/2-p38 MAPK通路蛋白表达有关。

https://orcid.org/0009-0003-9658-7554(赵红霞)


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: 骨损伤, 富血小板纤维蛋白, 甲基丙烯酰化明胶, 水凝胶, 颅骨缺损

Abstract: BACKGROUND: Platelet-rich fibrin (PRF) has many advantages, such as simple preparation, low production cost, and high safety, and has been widely used in the study of bone defect repair in oral and maxillofacial surgery, but there are problems such as too fast degradation rate and short release time of growth factors.
OBJECTIVE: PRF was loaded into gelatin methacryloyl (GelMA) hydrogel and its osteogenic properties were analyzed by in vivo and in vitro experiments. 
METHODS：(1) New Zealand white rabbit venous blood was extracted to prepare PRF. GelMA hydrogels containing 0, 0.05, 0.075, : and 0.1 g PRF were prepared, respectively, and were recorded as GelMA, GelMA/PRF-0.05, GelMA/PRF-0.075, and GelMA/PRF-0.1, respectively, to characterize the micromorphology and in vitro slow-release properties of the hydrogels. (2) Four kinds of hydrogels were co-cultured with MC3T3-E1 cells, respectively, and the cell proliferation activity was detected with the single cultured cells as the control. After osteogenic induction, alkaline phosphatase activity, mineralization ability, mRNA and protein expression levels of osteogenic genes (osteocalcin, osteopontin, RUNX2), ERK1/2-p38 MAPK pathway protein mRNA and protein expression levels were detected. (3) Fifteen New Zealand white rabbits were taken. Four full-layer bone defects of 8 mm diameter were prepared in the skull of each rabbit, one of which was implanted without any material (blank control group), and the other three were implanted with GelMA hydrogel, PRF, and GelMA/PRF-0.1 hydrogel, respectively. The bone defect was detected by Micro-CT and bone morphology was observed at 4, 8, and 12 weeks after operation.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy observed that all the hydrogels of the four groups had honeycomb pore structure, and the pore size of the hydrogels decreased slightly with the increase of PRF content, but there was no significant difference between the groups. The three groups of GelMA/PRF hydrogel could release transforming growth factor β1 and insulin-like growth factor 1 at a certain rate, and the cumulative release of transforming growth factor β1 and insulin-like growth factor 1 increased significantly with the extension of time. (2) CCK-8 assay and live/dead staining showed that GelMA/PRF hydrogel could promote the proliferation of MC3T3-E1 cells. The results of alkaline phosphatase staining, alizarin red staining, and osteogenic gene detection showed that GelMA/PRF hydrogel could promote the osteogenic differentiation of MC3T3-E1 cells, and inhibit the expression of ERK1/2-p38 MAPK pathway protein, and showed a PRF content dependence. (3) Micro-CT scan showed that the bone mineral density and bone volume fraction in the bone defect of GelMA/PRF-0.1 hydrogel group were higher than those in the other three groups (P < 0.05). Hematoxylin-eosin staining showed that compared with the other three groups, GelMA/PRF-0.1 hydrogel group had faster and more mature new bone formation at the bone defect. (4) These findings indicate that GelMA/PRF hydrogel has good osteogenic activity both in vivo and in vitro, which may be related to inhibiting the expression of ERK1/2-p38 MAPK pathway protein.

Key words: bone injury, platelet-rich fibrin, gelatin methacryloyl, hydrogel, skull defect

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