Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway

Abstract

Background: Recently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, can exert therapeutic effects in various disease models in the absence of parent cells. However, application of exosomes in bone defect repair and regeneration has been rarely reported, and little is known regarding their underlying mechanisms.

Methods: Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects, and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting, scratch assays, and qRT-PCR, respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair.

Results: We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition, the internalization of exosomes into hBMSCs could profoundly enhance the proliferation, migration, and osteogenic differentiation of hBMSCs. Furthermore, gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs.

Conclusions: We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs, which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials, and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects.

Keywords: Bone regeneration; Exosomes; Microarray; PI3K/Akt; Tricalcium phosphate; iPS-MSCs.

Fig. 1

Characterization of exosomes derived from human induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSCs). a Morphology observed by TEM. b Particle size distribution and concentration measured by TRPS. c Western blot analysis of the exosome surface markers

Fig. 2

Micro-CT and fluorochrome-labeling histomorphometrical analysis of the repaired craniums at 8 weeks post-implantation. a Three-dimensional reconstruction and sagittal images show the different reparation effects of exosomes or tricalcium phosphate (β-TCP) only. b The bone volume/tissue volume (BV/TV) and c bone mineral density (BMD) varied between the different groups. d Fluorochrome-labeling histomorphometrical analysis of new bone formation and mineralization at 8 weeks post-operation (yellow, between 2 and 4 weeks; red, between 4 and 6 weeks; green, between 6 and 8 weeks). e Percentage of each fluorochrome area for different groups. Dunnett t test; *p < 0.05 compared with the β-TCP group, ^# p < 0.05 compared with the exosome (Exos) 5 × 10¹¹ particles/mL group

Fig. 3

Histological and immunohistochemical analysis of the newly formed bone after 8 weeks of transplantation. a The un-decalcified craniums were stained with van Gieson’s picrofuchsin. The new bone area and tricalcium phosphate (β-TCP) residue are shown in red and black, respectively. b Quantitative analysis of (a). Dunnett t test; *p < 0.05 compared with the β-TCP group, ^# p < 0.05 compared with the exosome (Exos) 5 × 10¹¹ particles/mL group. c The sections were subjected to immunohistochemical analysis to inspect the distribution of OCN. OCN-positive immunostaining illustrated the bone tissues. Scale bars = 80 μm

Fig. 4

Internalization of hiPS-MSC-Exos in human BMSCs, exosome release from β-TCP, and their pro-osteogenesis effects on the recipient cells. A (a) DiO-labeled exosome release from β-TCP over 48 h. (b) Accumulated release of exosomes from β-TCP over 5 days. B Exosomes (Exos) enhanced the proliferation of hBMSCs as analyzed by Cell Counting Kit-8 assay. C Exosomes stimulated the migration of BMSCs as analyzed by migration assay. Scale bars = 250 μm. D Quantitative analysis of (C). E, F Incubation of hBMSCs with exosomes resulted in a dose-dependent increase in the alkaline phosphatase (ALP) staining and activity. Dunnett t test; *p < 0.05 compared with the control group, ^# p < 0.05 compared with the Exos 5 × 10¹¹ particles/mL group

Fig. 5

Differential expression of mRNAs between exosome-treated and control groups. a The differentially expressed (DE) genes in hBMSCs in response to exosome (Exos) stimulation are illustrated as a heat map. A p value cut-off of 0.05 and a fold-value change of ≥2 were used as a filter to identify the DE genes. b Enrichment analysis of all DE genes was performed. The top ten enriched pathways associated with exosome stimulation are shown. c A heat map of PI3K/Akt signaling pathway-related DE genes was generated. d The altered expression of PI3K/Akt signaling-related genes was confirmed by qRT-PCR analysis. ANOVA; *p < 0.05 compared with the control group

Fig. 6

Involvement of PI3K/Akt signaling in the exosome-induced osteogenic responses from BMSCs. a Exosomes (Exos) induced the activation of the PI3K/Akt signaling pathway and increased the protein levels of osteogenesis-related molecules; these effects by exosomes were abolished by the PI3K inhibitor (LY294002; 10 μM). b The exosome-treated hBMSCs showed much higher levels of alkaline phosphatase (ALP) and Alizarin red S (ARS) staining compared with the control groups on days 10 and 14, respectively; these effects were inhibited by LY294002