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Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization.

ABSTRACT: The objective of this study is to design a graphene-based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR-7b overexpression plasmid. GO-PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR-7b after GO-PEI-miR-7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell-specific transmembrane protein. However, osteoclastogenesis occurs without cell-cell fusion and preosteoclast (POC) is preserved. Through preservation of POC, GO-PEI-miR-7b transfection promotes mesenchymal stem cell osteogenesis and endothelial progenitor cells angiogenesis in the coculture system. Platelet-derived growth factor-BB secreted by POC is increased by GO-PEI-miR-7b both in vitro and in vivo. In treating osteoporotic ovariectomized mice, GO-PEI-miR-7b significantly enhances bone mineral density, bone volume as well as bone vascularization through increasing CD31^hiEmcn^hi cell number. This study provides a cell-cell fusion targeted miRNA transfection drug delivery strategy in treating bone disorders with excessive osteoclastic bone resorption.

SUBMITTER: Dou C

PROVIDER: S-EPMC5826985 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hedgehog (Hh) signaling is critical in developmental osteogenesis, and recent studies suggest it may also play a role in regulating osteogenic gene expression in the post-natal setting. However, there is a void of studies directly assessing the effect of Hh inhibition on post-natal osteogenesis. This study utilized a cyclic loading-induced ulnar stress fracture model to evaluate the hypothesis that Hh signaling contributes to osteogenesis and angiogenesis during stress fracture healing. Immediately prior to loading, adult rats were given GDC-0449 (Vismodegib - a selective Hh pathway inhibitor; 50mg/kg orally twice daily), or vehicle. Hh signaling was upregulated in response to stress fracture at 3 days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1 day and 3 days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1 day, but decreased Shh expression by 37% at 3 days. GDC-0449 decreased woven bone volume (-37%) and mineral density (-17%) at 7 days. Dynamic histomorphometry revealed that the 7 day callus was composed predominantly of woven bone in both groups. The observed reduction in woven bone occurred concomitantly with decreased expression of Alpl and Ibsp, but was not associated with differences in early cellular proliferation (as determined by callus PCNA staining at 3 days), osteoblastic differentiation (Osx expression at 1 day and 3 days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1 day and 3 days), or bone resorption metrics (callus TRAP staining at 3 days, Rankl and Opg expression at 1 day and 3 days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3 days, which was associated with increased Hif1α gene expression (+30%). Dynamic histomorphometric analysis demonstrated that GDC-0449 also inhibited lamellar bone formation. Lamellar bone analysis of the loaded limb (directly adjacent to the woven bone callus) showed that GDC-0449 significantly decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) (-17% and -20%, respectively). Lamellar BFR/BS in the non-loaded ulna was also significantly decreased (-37%), indicating that Hh signaling was required for normal bone modeling. In conclusion, Hh signaling plays an important role in post-natal osteogenesis in the setting of stress fracture healing, mediating its effects directly through regulation of bone formation and angiogenesis.

Project description:Objective: The objective of this study is to compare the bone induction of five kinds of calcium phosphate (Ca-P) biomaterials implanted in mice and explore the vascularization and particle-size-related osteoinductive mechanism. Methods: The following five kinds of Ca-P biomaterials including hydroxyapatite (HA) and/or tricalcium phosphate (TCP) were implanted in the muscle of 30 BALB/c mice (n = 6): 20 nm HA (20HA), 60 nm HA (60HA), 12 µm HA (12HA), 100 nm TCP (100TCP) and 12 µm HA + 100 nm TCP (HATCP). Then, all animals were put on a treadmill to run 30 min at a 6 m/h speed each day. Five and ten weeks later, three mice of each group were killed, and the samples were harvested to assess the osteoinductive effects by hematoxylin eosin (HE), Masson's trichrome and safranine-fast green stainings, and the immunohistochemistry of the angiogenesis and osteogenesis markers CD31 and type I collagen (ColI). Results: The numbers of blood vessels were 139 ±&nbsp;29,&nbsp;118&nbsp;±&nbsp;25,&nbsp;78&nbsp;±&nbsp;15,&nbsp;65&nbsp;±&nbsp;14 in groups HATCP, 100TCP, 60HA and 20HA, respectively, which were significantly higher than that of group 12HA (12 ± 5) in week 5 (p < 0.05). The area percentages of new bone tissue were (7.33 ± 1.26)% and (8.49 ±&nbsp;1.38)% in groups 100TCP and HATCP, respectively, which were significantly higher than those in groups 20HA (3.27 ±&nbsp;0.38)% and 60HA (3.43 ±&nbsp;0.27)% (p < 0.05); however, no bone tissue was found in group 12HA 10 weeks after transplantation. The expression of CD31 was positive in new blood vessels, and the expression of ColI was positive in new bone tissue. Conclusions: Nanoscale Ca-P biomaterials could induce osteogenesis in mice muscle, and the osteoinductive effects of TCP were about 124% higher than those of 20HA and 114% higher than those of 60HA. The particle size of the biomaterials affected angiogenesis and osteogenesis. There was a positive correlation between the number of blood vessels and the area percentage of new bone tissue; therefore, osteoinduction is closely related to vascularization. Our results provide an experimental basis for the synthesis of calcium-phosphorus matrix composites and for further exploration of the osteoinductive mechanism.

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Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization.

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