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. 2024 Aug 23;27(9):110801.
doi: 10.1016/j.isci.2024.110801. eCollection 2024 Sep 20.

Modifying MSCs-derived EVs with esterase-responsive and charge-reversal cationic polymers enhances bone regeneration

Yihan Chen  1   2 , Bang Li  1 , Mukeshimana Christelle  1 , Nshimiyimana Eugene  1 , Wenjia Han  1 , Hong Zhou  3 , Nasha Qiu  4 , Hengguo Zhang  1 , Jianguang Xu  1
Affiliations

Modifying MSCs-derived EVs with esterase-responsive and charge-reversal cationic polymers enhances bone regeneration

Yihan Chen et al. iScience. .

Abstract

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) for the treatment of bone defects have been widely reported as a cell-free therapy because of their appropriate stability and biocompatibility. However, EV isolation is expensive and time-consuming. We developed a method of modifying EVs derived from bone marrow MSCs (BMSCs) via the cationic polymer (ERP) with characteristics of charge reversal and esterase response (ERP-EVs). When simply mixing BMSCs-EVs with ERP at a 1:1 ratio, ERP-EVs significantly enhanced the osteogenesis of BMSCs. More EVs were released by ERP in BMSCs than in fibroblasts, realizing the selective release. Last, ERP-EVs were loaded on an nHA/CS-MS scaffold and showed enhanced bone regeneration on rat calvarial bone defects in vivo. In general, this study provided an effective strategy to improve cellular uptake and selective release of BMSCs-EVs in bone-related cells, which had great potential to accelerate the clinical practice of BMSCs-EVs-based bone defect repair.

Keywords: Bioengineering; Biological sciences; Biomaterials; Drug delivery system.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Identification of rBMSCs-EVs (A) Illustration of the extraction protocol. (B) Morphology of rBMSC-EVs under a TEM. The scale bar in the left figure represents 200 nm. The scale bar in the right figure represents 50 nm. (C and D) Image and particle size of rBMSCs-EV particles detected by NTA. (E) Confocal images of cellular uptake of PKH67-labeled rBMSCs-EVs by rBMSCs, scale bar, 20 μm. (F) Western blot analysis of CD9 expression in rBMSC and EV.
Figure 2
Figure 2
EVs modified with ERP (A and B) Particle size and Zeta potential of ERP measured by DLS. (C) Cytotoxicity of ERP on rBMSCs as evaluated by CCK8 assay. (D) Cell viability of rBMSCs when co-cultured with EVs and a 1:1 mixture of ERP-EVs using CCK-8 assay. (E) The uptake efficiency of rBMSCs to ERP-rBMSCs-EVs at different microparticle ratios observed by CLSM. Scale bar, 50 μm. (F) The quantity measurement of mean fluorescence strength by ImageJ. (G) Zeta potential of rBMSCs-EVs and ERP-rBMSCs-EVs incubated with different microparticle ratios. Data are presented as mean ± SEM. Differences in expression between groups were calculated by one-way ANOVA. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
Figure 3
Figure 3
EVs were efficiently released from ERP via esterase-hydrolyzation (A) rBMSCs and L929 were treated with FDA to measure the content of cellular esterase levels. (B and C) Cellular uptake efficiency at different time points observed by inverted fluorescent microscope and their median fluorescence intensity. Data are presented as mean ± SEM. Differences in expression between groups were calculated by t test. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
Figure 4
Figure 4
ERP-EVs promoted osteoblastic differentiation of rBMSCs (A) Quantitation of ALP activity of rBMSCs co-cultured with EVs and ERP-EVs for 7 days. (B) Real-time qPCR was used to analyze the relative mRNA expression level of bone-specific markers Col-1, Sp7, and OPN. Data are presented as mean ± SEM. Differences in expression between groups were calculated by one-way ANOVA. (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (C) The effect of EVs and ERP-EVs on calcium deposition of rBMSCs after co-culture for 21 days. (D) Western blot analysis of bone-specific markers COL-1, Sp7, and OPN expressions.
Figure 5
Figure 5
Biological characterization and osteogenic effect of ERP-EVs-nHA/CS-MS (A) The adhesion of rBMSCs to nHA/CS-MS and ERP-EVs-nHA/CS-MS scaffolds via live/dead staining after co-culture with rBMSCs for 24 h. Scale bar, 100 μm. (B) The cumulative release amount of ERP-EVs by nHA/CS-MS at different time points detected by NTA. (C) CCK-8 was used to detect the effect of blank and loading scaffold on cell proliferation at 24 h. (D) The ALP activity of rBMSCs co-cultured blank and loading scaffold for 7 days was quantitatively tested. Data are presented as mean ± SEM. Differences in expression between groups were calculated by one-way ANOVA. (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
Figure 6
Figure 6
New bone regeneration in vivo after 8 weeks of implantation with ERP-EVs-nHA/CS-MS scaffold (A) Two sides of the bone defect area and sagittal planes were captured. Original bone defect margins were marked with red dotted lines. (B) Masson staining showed new bone regeneration and collagen formation respectively from the calvarial bone defect margins, new bone regenerated around the ERP-EVs-MS scaffold (yellow arrows). 20×, scale bar represents 1000 μm; 100×, scale bar represents 250 μm. (C) Quantitative analysis of BS/TV, Tb. Sp, BS/BV, BMD, Tb.N and Tb.Th values of new bone formation at 8 weeks post-implantation. Data are presented as mean ± SEM. Differences in expression between groups were calculated by one-way ANOVA. (n = 4, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
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