微囊细胞复合体分泌骨形态蛋白2的成骨诱导作用

doi:10.3877/cma.j.issn.1674-134X.2025.06.006

目的

验证前期构建的微囊细胞复合体分泌骨形态发生蛋白2（BMP2）对周围骨髓间充质干细胞（BMSCs）的成骨影响。

方法

通过酶联免疫吸附试验检测诱导组（应用含强力霉素诱导剂的培养基）及对照组（应用不含强力霉素诱导剂的培养基）的微囊细胞复合体的BMP2释放；通过实时定量聚合酶链反应（qRT-qPCR）、蛋白质印迹（western blot）实验验证BMSC（-）、BMSC7 d、BMSC14 d各组的微囊细胞复合体对周围rBMSCs成骨基因、蛋白表达的影响；通过免疫组化验证对照组、未转染组、未诱导组、诱导组各组的微囊细胞复合体对周围rBMSCs的成骨诱导能力；茜红素染色、von Kossa染色验证空白组、对照组、诱导组各组微囊细胞复合体对周围rBMSCs的成骨分化影响。本研究所有实验重复3次；定量数据以±s描述，应用重复方差分析、单因素方差分析统计各组间差异。

结果

经强力霉素（DOX）诱导后诱导组的hBMP2蛋白分泌逐渐升高（F=234.6，R²= 0.9902，P<0.05，各组间Tukey多重检验均为P<0.05），第21天停止DOX诱导后hBMP2蛋白分泌较前下降（各组间Tukey多重检验：P<0.05）；qRT-PCR实验证实经DOX诱导后微囊细胞复合体周围的rBMSCs细胞BMP2（F=26.7，P<0.05）、runt相关转录因子2‌（RUNX2）（F=115.9，P<0.05）、骨钙蛋白（OCN）（F=1652，P<0.05）表达均随时间延长而上调；western blot结果提示蛋白层面上经DOX诱导后微囊细胞复合体周围的rBMSCs细胞分泌BMP2、RUNX2蛋白；免疫组化证实该复合体能增强周围rBMSCs的RUNX2（F=110.3，P<0.05）、OCN（F=125.6，P<0.05）、Ⅰ型胶原蛋白‌（COL1）表达（F=157.2，P<0.05）；染色实验进一步证实该复合体能增强周围rBMSCs的钙结节和钙盐沉积。

结论

该微囊细胞复合体可实现BMP2的按需释放，有效诱导周围BMSCs成骨分化，为基因增强型骨组织工程提供了新策略，具有潜在的临床应用价值。

关键词: 骨形态发生蛋白质2, 细胞封装, 骨生成

Objective

To verify the effect of bone morphogenetic protein 2 (BMP2) secreted by the previously constructed microcapsule cell complex on osteogenic differentiation of surrounding bone mesenchymal stem cells (rBMSCs).

Methods

Enzyme-linked immunosorbent assay (ELISA) was used to detect BMP2 release from the microcapsule cell complex in the induction group (using medium containing doxycycline inducer) and the control group (using medium without doxycycline inducer). Real-time quantitative polymerase chain reaction (qRT-qPCR) and western blot experiments were used to verify the effect of the microcapsule cell complex in the BMSC(-), BMSC7 d, and BMSC14 d groups on the osteogenic gene and protein expression of surrounding rBMSCs. Immunohistochemistry was used to verify the osteogenic induction ability of the microcapsule cell complex in the control group, untransfected group, uninduced group, and induced group on surrounding rBMSCs. Alizarin staining and von Kossa staining were used to verify the effect of the microcapsule cell complex in the blank group, control group, and induced group on the osteogenic differentiation of surrounding rBMSCs. All experiments in this study were repeated three times; quantitative data were discribed as ±s. Repeated measurement analysis of variance (ANOVA) and one-way ANOVA were used to statistically analyze differences between groups.

Results

After doxycycline (DOX) induction, hBMP2 protein secretion gradually increased in the induced group (F=234.6, R²=0.9902, P<0.05, Tukey multiple test among groups: all P<0.05). After DOX induction stopped on day 21, hBMP2 protein secretion decreased compared with the previous data (Tukey multiple test among groups: all P<0.05). qRT-PCR experiments confirmed that after DOX induction, the expression of BMP2 (F=26.7, P<0.05), runt-related transcription factor 2 (RUNX2) (F=115.9, P<0.05), and osteocalcin (OCN) (F=1652, P<0.05) in rBMSCs surrounding the microcapsule cell complex was upregulated over time. Western blot results indicated that at the protein level, hBMP2 secretion in rBMSCs surrounding the microcapsule cell complex after DOX induction.BMP2 and RUNX2 proteins were detected. Immunohistochemistry confirmed that the complex enhanced the expression of RUNX2 (F=110.3, P<0.05), OCN (F=125.6, P<0.05), and type I collagen (COL1) in surrounding rBMSCs (F=157.2, P<0.05). Staining experiments further confirmed that the complex enhanced calcium nodules and calcium salt deposition in peripheral rBMSCs.

Conclusion

This microcapsule cell complex can achieve on-demand release of BMP2, effectively inducing osteogenic differentiation of peripheral BMSCs, providing a new strategy for gene-enhanced bone tissue engineering, and has potential clinical application value.

Key words: Bone morphogenetic protein 2, Cell encapsulation, Osteogenesis

图1 BMP2、RUNX2、OCN、GADPH引物注：hBMP2-人骨形态发生蛋白2；RUNX2-runt相关转录因子2；OCN-骨钙蛋白；GAPDH-甘油醛-3-磷酸脱氢酶

Figure 1 BMP2, RUNX2, OCN, GADPH primersNote: hBMP2-human bone morphogenetic protein 2; RUNX2-runt-related transcription factor; OCN-osteocalcin; GAPDH-glyceraldehvde-3-phosphate dehydrogenase

图2 微囊细胞复合体释放hBMP2（人骨形态发生蛋白2）

Figure 2 The microcapsule cell complex releaseshBMP2(human bone morphogenetic protein 2)

图3 不同时间DOX（多西环素）（+）组微囊细胞复合体释放hBMP2（人骨形态发生蛋白2）浓度注：^*-P<0.05

Figure 3 Concentration of hBMP2 (human bone morphogenetic protein 2) released by microcapsule cell complexes in the DOX（doxycycline） (+) group at different timesNote: ^*-P< 0.05

图4 微囊细胞复合体诱导周围rBMSCs（鼠骨髓间充质干细胞）表达成骨基因。图A为周围BMSCs中hBMP2基因在不同时间点的表达；图B为周围BMSCs中RUNX2基因在不同时间点的表达；图C为周围BMSCs中OCN基因在不同时间点的表达注：hBMP2-骨形态发生蛋白2；RUNX2-runt相关转录因子；OCN-骨钙蛋白；DOX-多西环素；BMSC（-）组-普通培养基；BMSC7 d组-含10 mg/L DOX的完全培养基培养7 d；BMSC14 d组-含10 mg/L DOX的完全培养基培养14 d；^*-P<0.05；^**-P<0.01；^***-P<0.001

Figure 4 Microcapsule cell complexes induce osteogenic gene expression in peripheral rBMSCs (rat bone marrow mesenchymal stem cells). A is hBMP2 gene expression in surrounding BMSCs at different time points; B is RUNX2 gene expression in surrounding BMSCs at different time points; C is OCN gene expression in surrounding BMSCs at different time pointsNote: hBMP2-human bone mophologenetic protein 2；RUNX2-runt related transcription；OCN-osteocalcin；DOX-doxycyclin；BMSC(-) group- cultured in normal medium; BMSC7 d group- cultured in complete medium containing 10 mg/L DOX for seven days; BMSC14 d group- cultured in complete medium containing 10 mg/L DOX for 14 days；^*-P<0.05；^**-P<0.01；^***-P<0.001

图5 微囊细胞复合体诱导周围rBMSCs（鼠骨髓间充质干细胞）表达成骨蛋白

Figure 5 Microcapsule cell complexes induce osteoblast expression in surounding rBMSCs (rat bone marrow mesenchymal stem cells)

图6 微囊细胞复合体周围rBMSCs（鼠骨髓间充质干细胞）成骨相关蛋白免疫组化染色（×20）

Figure 6 Effects of microcapsule cell complex on osteogenic protein expression in surrounding rBMSCs (rat bone marrow mesenchymal stem cells) （×20）

表2 微囊细胞复合体光密度值（

±s）

Table 2 Optical density of microcapsule cell complex

组别Groups	例数Number of cases	RUNX2	OCN	COL1
对照组Control group	3	0.042±0.021	0.032±0.014	0.027±0.017
未转染组Non-transfected group	3	0.056±0.035	0.064±0.024	0.042±0.019
未诱导组Non-induced group	3	0.089±0.032	0.078±0.038	0.069±0.042
诱导组Induction group	3	0.465±0.128^abc	0.624±0.274^abc	0.389±0.187^abc
F值		110.3	125.6	157.2
P值		<0.05	<0.05	<0.05

表2 微囊细胞复合体光密度值（

±s）

Table 2 Optical density of microcapsule cell complex

组别Groups	例数Number of cases	RUNX2	OCN	COL1
对照组Control group	3	0.042±0.021	0.032±0.014	0.027±0.017
未转染组Non-transfected group	3	0.056±0.035	0.064±0.024	0.042±0.019
未诱导组Non-induced group	3	0.089±0.032	0.078±0.038	0.069±0.042
诱导组Induction group	3	0.465±0.128^abc	0.624±0.274^abc	0.389±0.187^abc
F值		110.3	125.6	157.2
P值		<0.05	<0.05	<0.05

图7 微囊细胞复合体影响周围rBMSCs（鼠骨髓间充质干细胞）细胞钙结节（茜红素染色）和钙盐沉积（von Kossa染色）（×4）

Figure 7 Microcyst cell complexes affect calcium nodules（alizarin red staining） and calcium salt deposition （von Kossa staining）in surrounding rBMSCs (rat bone marrow mesenchymal stem cells) （×4）

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Osteoinductive effect of bone morphogenetic protein 2 secreted by microencapsulated cell complexes

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Osteoinductive effect of bone morphogenetic protein 2 secreted by microencapsulated cell complexes