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

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

Abstract

Abstract:

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

Shicong Zheng, Lijuan Song, Pengyu Chen, Bo Bai. Osteoinductive effect of bone morphogenetic protein 2 secreted by microencapsulated cell complexes[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2025, 19(06): 684-690.

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URL: https://zhgjwkzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-134X.2025.06.006

https://zhgjwkzz.cma-cmc.com.cn/EN/Y2025/V19/I06/684

Figures/Tables 8

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

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

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

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

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

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

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

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）

References 21

[1]	Putra NE, Xu J, Leeflang MA, et al. Additively manufactured biodegradable porous FeMn-akermanite scaffolds for critical-size bone defects: the first in vivo evaluation[J/OL]. Mater Today Bio, 2025, 34: 102123. DOI: 10.1016/j.mtbio.2025.102123.
[2]	Manawar S, Myrick E, Awad P, et al. Use of allograft bone matrix in clinical orthopedics[J]. Regen Med, 2024, 19（5）: 247-256.
[3]	Mauffrey C, Barlow BT, Smith W. Management of segmental bone defects[J]. J Am AcadOrthopSurg, 2015, 23（3）: 143-153.
[4]	March A, Castillo Aguirre SH, Eliseev R, et al. Long-term tissue engineered periosteum-mediated allograft healing is hindered due to persistent fibrosis and limited allograft remodeling[J/OL]. Bone Rep, 2025, 26: 101865. DOI: 10.1016/j.bonr.2025.101865.
[5]	Lv N, Li H, Hong L, et al. An electrospun membrane incorporating lipocalin 2-enhanced exosomes effectively facilitated the repair of critical bone defects[J/OL]. Int J BiolMacromol, 2025, 322（Pt 1）: 146674. DOI: 10.1016/j.ijbiomac.2025.146674.
[6]	Putra NE, Leeflang MA, Klimopoulou M, et al. Extrusion-based 3D printing of biodegradable, osteogenic, paramagnetic, and porous FeMn-akermanite bone substitutes[J]. Acta Biomater, 2023, 162: 182-198.
[7]	Yan J, Qi S, Zhao Y, et al. 3D-printed triply periodic minimal surface ceramic scaffold loaded with bone morphogenetic protein-2 and zoledronic for cranium defect repairment[J/OL]. J Tissue Eng Regen Med, 2025, 2025: 9964384. DOI: 10.1155/term/9964384.
[8]	Surisaeng T, Wisitrasameewong W, Champaiboon C, et al. BMP-2 mRNA-transfected BMSCs promote superior calvarial bone regeneration[J/OL]. Sci Rep, 2025, 15（1）: 15022. DOI: 10.1038/s41598-025-99979-6.
[9]	Panos JA, Coenen MJ, Nagelli CV, et al. Segmental defect healing in the presence or absence of recombinant human BMP2: Novel insights from a rat model[J]. J Orthop Res, 2023, 41（9）: 1934-1944.
[10]	Bukharova TB, Nedorubova IA, Mokrousova VO, et al. Adenovirus-based gene therapy for bone regeneration: a comparative analysis of in vivo and ex vivo BMP2 gene delivery[J/OL]. Cells, 2023, 12（13）: 1762. DOI: 10.3390/cells12131762.
[11]	Watson-Levings RS, Palmer GD, Levings PP, et al. Gene therapy in orthopaedics: progress and challenges in pre-clinical development and translation[J/OL]. Front BioengBiotechnol, 2022, 10: 901317. DOI: 10.3389/fbioe.2022.901317.
[12]	Du X, Huang F, Zhang S, et al. Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene-modified BMSCs for controlled BMP-2 release in vitro[J]. Artif Cells NanomedBiotechnol, 2017, 45（8）: 1710-1720.
[13]	Li X, Asuncion F, Ominsky M, et al. High-dose romosozumab promoted bone regeneration of critical-size ulnar defect filled with demineralized bone matrix in nonhuman Primates[J]. J OrthopTranslat, 2025, 54: 1-7.
[14]	Zhang M, Matinlinna JP, Tsoi JKH, et al. Recent developments in biomaterials for long-bone segmental defect reconstruction: a narrative overview[J]. J Orthop Translat, 2019, 22: 26-33.
[15]	Min Q, Tan R, Zhang Y, et al. Multi-crosslinked strong and elastic bioglass/chitosan-cysteine hydrogels with controlled quercetin delivery for bone tissue engineering[J/OL]. Pharmaceutics, 2022, 14（10）: 2048. DOI: 10.3390/pharmaceutics14102048.
[16]	Betz RR. Limitations of autograft and allograft: new synthetic solutions[J]. Orthopedics, 2002, 25（5 Suppl）: s561-s570.
[17]	Zhang X, Jiang W, Xie C, et al. Msx1（+） stem cells recruited by bioactive tissue engineering graft for bone regeneration[J/OL]. Nat Commun, 2022, 13（1）: 5211. DOI: 10.1038/s41467-022-32868-y.
[18]	Chen X, Li W, Ma Y, et al. Nano-assisted dynamically assembled hydrogels with strong tissue adhesion and proactive immunomodulation for bone defect repair[J]. Bioact Mater, 2025, 53: 480-494.
[19]	Sarkar A, Gallo MC, Bell JA, et al. Ex vivo regional gene therapy compared to recombinant BMP-2 for the treatment of critical-size bone defects: an in vivo single-cell RNA-sequencing study[J/OL]. Bioengineering, 2025, 12（1）: 29. DOI: 10.3390/bioengineering12010029.
[20]	MegleiAY, Nedorubova IA, Basina VP, et al. Collagen-platelet-rich plasma mixed hydrogels as a pBMP2 delivery system for bone defect regeneration[J/OL]. Biomedicines, 2024, 12（11）: 2461. DOI: 10.3390/biomedicines12112461.
[21]	Haidar ZS, Hamdy RC, Tabrizian M. Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part A: Current challenges in BMP delivery[J]. Biotechnol Lett, 2009, 31（12）: 1817-1824.

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