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Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation.

ABSTRACT: We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

SUBMITTER: Hara M

PROVIDER: S-EPMC8621468 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1?mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and ?-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1?mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1?mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1?mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1?mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.

Project description:The contraceptive effectiveness of intrauterine devices has been attributed in part to effects of a foreign body reaction on the endometrium. We performed this study to identify compare the effects on the endometrial transcriptome of intrauterine devices and combined oral contraceptives, to better understand their mechanisms of action. We collected endometrial and cervical biopsies from women using the levonorgestrel-intrauterine device, copper intrauterine device or levonorgestrel-containing combined oral contraceptives, and from women not using contraceptives (control group). Transcriptional profiling was performed with Affymetrix arrays, Principal Component Analysis and the bioconductor package limma. Pathway analysis was performed using EnrichR and Reactome 2016. In endometrial samples from copper intrauterine device users (n=13), there were no genes with statistically significant differential expression compared to controls (n=11), whereas in levonorgestrel-intrauterine device users (n=11), 2509 genes were significantly dysregulated and mapped onto several immune and inflammatory pathways. In combined oral contraceptive users (n=12), 133 genes were significantly dysregulated and mapped predominantly onto pathways involving regulation of metal ions. Both levonorgestrel-intrauterine device and combined oral contraceptive use were associated with significant downregulation of members of the metallothionein gene family. In cervical samples, none of the groups showed statistically significant differential gene expression compared to controls. In conclusion, hormonal and copper intrauterine devices differ significantly in their effects on the endometrial transcriptome, with endometrium from copper intrauterine device users being indistinguishable from luteal phase endometrium. These results suggest that the contraceptive mechanisms of intrauterine devices are unlikely to rely on a common pathway involving a foreign body reaction in the endometrium.

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Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation.

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