壳聚糖/海藻酸钠水凝胶顺序释放BMP/WNT信号通路激活剂促成骨细胞分化的体外实验研究

doi:10.3969/j.issn.1005-4979.2021.01.002

《口腔颌面外科杂志》 ›› 2021, Vol. 31 ›› Issue (1): 9-15. doi: 10.3969/j.issn.1005-4979.2021.01.002

壳聚糖/海藻酸钠水凝胶顺序释放BMP/WNT信号通路激活剂促成骨细胞分化的体外实验研究

王晓¹(), 郝新青², 王小萌², 闫广兴³^,⁴, 叶佳朋⁵, 齐春光², 孙宏晨⁴^,⁶, 史册³^,⁴(), 黄洋²()

¹ 青岛大学附属青岛市口腔医院儿童口腔科，山东青岛 266000
² 吉林大学口腔医院儿童口腔科
³ 吉林省牙发育及颌骨重塑与再生重点实验室
⁴ 吉林大学口腔医院病理科
⁵ 吉林大学口腔医院口腔颌面外科，吉林长春 130000
⁶ 中国医科大学口腔医院病理科，辽宁沈阳 110000

收稿日期:2020-04-06 修回日期:2020-12-03 出版日期:2021-02-28 发布日期:2021-02-25
通讯作者: 黄洋，教授. E-mail: huangyang1960@163.com；史册，副主任医师. E-mail: shice1004@gmail.com
作者简介:
王晓（1992—），女，山东潍坊人，硕士研究生. E-mail: 1357041404@qq.com
基金资助:
国家自然科学基金项目(81970903); 国家自然科学基金项目(81600843); 国家自然科学基金项目(81600823); 中国博士后科学基金项目(2018T110258); 中国博士后科学基金项目(2017M621219); 吉林省财政厅科技项目(JCSZ20193786); 青岛市医疗卫生重点学科建设项目(2020-2022)

Sequential Release of BMP/WNT Signaling Pathway Activators by Chitosan/Sodium Alginate Hydrogel to Promote Osteogenic Differentiation of Osteoblasts in Vitro

WANG Xiao¹(), HAO Xinqing², WANG Xiaomeng², YAN Guangxing³^,⁴, YE Jiapeng⁵, QI Chunguang², SUN Hongchen⁴^,⁶, SHI Ce³^,⁴(), HUANG Yang²()

¹ Department of Pediatric Dentistry, Qingdao Municipal Hospital of Stomatology, Qingdao University, Qingdao 266000, Shandong Province
² Department of Pediatric Dentistry, Hospital of Stomatology, Jilin University
³ Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling
⁴ Department of Pathology, Hospital of Stomatology, Jilin University
⁵ Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Jilin University, Changchun 130000, Jilin Province
⁶ Department of Pathology, Hospital of Stomatology, China Medical University,Shenyang 110000, Liaoning Province, China

Received:2020-04-06 Revised:2020-12-03 Online:2021-02-28 Published:2021-02-25

摘要/Abstract

摘要：

目的: 通过组织工程策略构建顺序释放骨形态发生蛋白（bone morphogenetic protein, BMP）和WNT信号通路激活剂的双层复合材料，并研究其在体外对成骨细胞分化的作用。方法: 用CCK-8法、碱性磷酸酶（alkaline phosphatase, ALP）染色法和茜素红染色法分别检测BMP信号通路激活剂他克莫司（tacrolimus, FK506）和WNT信号通路激活剂6-溴靛玉红-3-肟（6-bromoindirubin-3-oxim, BIO）对前成骨细胞系MC3T3-E1增殖活性和成骨向分化的影响，筛选出最合适的药物浓度；制备壳聚糖/海藻酸钠支架材料，采用称量法测定材料的降解曲线，制备内层壳聚糖载BIO、外层海藻酸钠载FK506的支架材料，采用紫外分光光度计测定药物的释放；制备内外单载不同药物的支架材料，用ALP染色法和茜素红染色法检测载药复合材料对前成骨细胞系MC3T3-E1成骨向分化的作用，应用实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）法检测载药支架材料对前成骨细胞系MC3T3-E1成骨相关基因表达的影响。结果: 在适宜范围内，FK506处理的细胞早期ALP活性较BIO处理的细胞更强，且FK506促进ALP活性最适合的质量浓度为2.0 μg/mL；茜素红染色结果表明，BIO在0.1 μmol/L时，细胞表现出更强的钙结节形成能力。支架材料缓慢稳定降解至第35天，期间FK506和BIO顺序释放，药物在14 d内基本释放完毕。应用载药材料浸提液ALP染色的支架材料和茜素红染色结果显示，内载BIO外载FK506的实验组相比较于其他组表现出更强的成骨向分化能力； RT-qPCR结果显示，内载BIO外载FK506的支架材料能够显著促进成骨相关基因Runx2和OCN的表达。结论: 以壳聚糖/海藻酸钠为支架材料，内层壳聚糖载WNT信号通路激活剂BIO，外层海藻酸钠载BMP信号通路激活剂FK506，两者顺序释放，即先激活BMP信号通路，后激活WNT信号通路，能够在体外促进MC3T3-E1细胞的成骨向分化，为临床治疗骨缺损，促进骨再生提供新思路。

关键词: 壳聚糖, 海藻酸钠, 水凝胶, 他克莫司, 6-溴靛玉红-3-肟, BMP/WNT信号通路, 成骨向分化

Abstract:

Objectives: To construct a double-layer composite by tissue engineering strategy, which could sequentially release the activators of bone morphogenetic protein (BMP) and WNT signaling pathway and investigate the influence on the differentiation of osteoblasts in vitro. Methods: CCK-8 assay, alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the effects of BMP signaling activator tacrolimus (FK506) and WNT signaling activator 6-bromoindirubin-3-oxim(BIO) on the cell viability and osteogenic differentiation of pre-osteoblast cell line MC3T3-E1. Then the optimal drug concentrations were screened. Chitosan/sodium alginate scaffold was prepared and the degradation curve of the material was measured by weighing method. The scaffold with inner chitosan layer loaded with BIO and outer sodium alginate layer loaded with FK506 was prepared and the drug release was determined by ultraviolet spectrophotometry. ALP staining and alizarin red staining were used to detect the ability of the drug-loading composites to promote the osteogenic differentiation of the pre-osteoblast cell line MC3T3-E1. Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the effects of the drug-loaded scaffolds on the expressions of osteogenic related genes in the pre-osteoblast cell line MC3T3-E1. Results: FK506 could promote ALP activity at an earlier stage during osteogenic differentiation of pre-osteoblast cell line MC3T3-E1, compared with BIO. The optimal concentration of FK506 to promote ALP activity was 2.0 μg/mL; the results of alizarin red staining showed that when the concentration of BIO was 0.1 μmol/L, the cells showed stronger ability of calcium nodule formation. The scaffolds slowly and stably degraded until the 35th day. FK506 and BIO were released sequentially, and the molecules were basically released within 14 days. ALP staining and alizarin red staining results showed that the experimental group with internal BIO and external FK506 showed stronger osteogenic differentiation ability compared with other groups. RT-qPCR results showed that scaffold with internal BIO and external FK506 significantly promoted the expressions of Runx2 and OCN in MC3T3-E1 cell line. Conclusion: Chitosan/sodium alginate was used as the scaffold to carry WNT signaling activator BIO inside and BMP signaling activator FK506 outside, and the sequential release of the drugs could promote the osteogenic differentiation of MC3T3-E1 cells in vitro, which opened a new approach for the treatment of bone defects and the promotion of bone regeneration clinically.

Key words: chitosan, sodium alginate, hydrogel, FK506, 6-bromoindirubin-3-oxim, BMP/WNT signaling pathway, osteogenic differentiation

中图分类号:

R782

王晓, 郝新青, 王小萌, 闫广兴, 叶佳朋, 齐春光, 孙宏晨, 史册, 黄洋. 壳聚糖/海藻酸钠水凝胶顺序释放BMP/WNT信号通路激活剂促成骨细胞分化的体外实验研究[J]. 《口腔颌面外科杂志》, 2021, 31(1): 9-15.

WANG Xiao, HAO Xinqing, WANG Xiaomeng, YAN Guangxing, YE Jiapeng, QI Chunguang, SUN Hongchen, SHI Ce, HUANG Yang. Sequential Release of BMP/WNT Signaling Pathway Activators by Chitosan/Sodium Alginate Hydrogel to Promote Osteogenic Differentiation of Osteoblasts in Vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2021, 31(1): 9-15.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2021/V31/I1/9

图/表 7

表1 引物序列

Table 1 Sequences of primers

基因名称	引物序列（5′-3′）
β-actin	F: GGAGATTACTGCCCTGGCTCCTA R: GACTCATCGTACTCCTGCTTGCTC
Runx2	F: GCACAAACATGGCCAGATTCA R: AAGCCATGGTGCCCGTTAG
Sp7	F: AAGTTATGATGACGGGTCAGGTACA R: AGAAATCTACGAGCAAGGTCTCCAC
OCN	F: ATGAGAGCCCTCACACTCCTCG R: GTCAGCCAACTCGTCACAGTC

图1 不同浓度药物对细胞存活的影响 A. 不同质量浓度的FK506在1、3、5、7 d时对细胞存活的影响；B. 不同浓度的BIO在1 、3 、5 、7 d时对细胞存活的影响。#表示细胞毒性，#为P<0.05，##为P<0.01，####为P<0.000 1，与对照组比较；*表示细胞存活能力，*为P<0.05，**为P<0.01，***为P<0.001，****为P<0.000 1，与对照组比较。

Figure 1 Effects of different concentrations of drugs on cell viability

图2 不同浓度药物对MC3T3-E1细胞成骨向分化的影响 A—B. 不同浓度的BIO和FK506处理7 d和14 d后，ALP染色结果（×40）；C. 不同浓度的BIO和FK506处理21 d后，茜素红染色结果（×40）；D. 21 d时茜素红染色定量分析。*为P<0.05，**为P<0.01，***为P<0.001，****为P<0.000 1，与对照组比较。

Figure 2 Effects of different concentrations of drugs on osteogenic differentiation of MC3T3-E1 cells

图3 壳聚糖/海藻酸钠扫描电镜图红色箭头为海藻酸钠，黄色箭头为壳聚糖。

Figure 3 Scanning electron microscope image of chitosan/sodium alginate composite

图4 壳聚糖/海藻酸钠复合材料降解曲线

Figure 4 Degradation curve of chitosan/sodium alginate composite

图5 载药壳聚糖/海藻酸钠复合材料药物释放规律 A. 外层海藻酸钠单载FK506的释放曲线；B. 内层壳聚糖单载BIO的释放曲线。

Figure 5 Drug release of chitosan/sodium alginate composite loaded with drugs

图6 载药壳聚糖/海藻酸钠复合材料对MC3T3-E1细胞成骨向分化的影响 A—B. 7 d和14 d 时的ALP染色结果（×40）；C. 21 d时的茜素红染色结果（×40）；D. 21 d时的茜素红染色定量分析；E. 14 d时成骨相关基因的表达。*表示各组与对照相比较，目的基因表达量的变化，*为P<0.05，**为P<0.01，***为P<0.001，****为P<0.000 1。

Figure 6 Effects of drug-loaded chitosan/sodium alginate composites on osteogenic differentiation of MC3T3-E1 cells

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壳聚糖/海藻酸钠水凝胶顺序释放BMP/WNT信号通路激活剂促成骨细胞分化的体外实验研究

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