Dataset Information

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 | 2018 Feb

REPOSITORIES: biostudies-literature

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

Dou Ce C Ding Ning N Luo Fei F Hou Tianyong T Cao Zhen Z Bai Yun Y Liu Chuan C Xu Jianzhong J Dong Shiwu S

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20171204 2

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 ...[more]

PMID: 29619305

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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:Graphene oxide (GO) is a promising material for bone tissue engineering, but the validation of its molecular biological effects, especially in the context of clinically applied materials, is still limited. In this study, we compare the effects of graphene oxide framework structures (F-GO) and reduced graphene oxide-based framework structures (F-rGO) as scaffold material with a special focus on vascularization associated processes and mechanisms in the bone. Highly porous networks of zinc oxide tetrapods serving as sacrificial templates were used to create F-GO and F-rGO with porosities >99% consisting of hollow interconnected microtubes. Framework materials were seeded with human mesenchymal stem cells (MSC), and the cell response was evaluated by confocal laser scanning microscopy (CLSM), deoxyribonucleic acid (DNA) quantification, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase activity (ALP) to define their impact on cellular adhesion, osteogenic differentiation, and secretion of vascular growth factors. F-GO based scaffolds improved adhesion and growth of MSC as indicated by CLSM and DNA quantification. Further, F-GO showed a better vascular endothelial growth factor (VEGF) binding capacity and improved cell growth as well as the formation of microvascular capillary-like structures in co-cultures with outgrowth endothelial cells (OEC). These results clearly favored non-reduced graphene oxide in the form of F-GO for bone regeneration applications. To study GO in the context of a clinically used implant material, we coated a commercially available xenograft (Bio-Oss® block) with GO and compared the growth of MSC in monoculture and in coculture with OEC to the native scaffold. We observed a significantly improved growth of MSC and formation of prevascular structures on coated Bio-Oss®, again associated with a higher VEGF binding capacity. We conclude that graphene oxide coating of this clinically used, but highly debiologized bone graft improves MSC cell adhesion and vascularization.

Dataset Information

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

Publications

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

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