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

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.

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Phase separation of both a plant virus movement protein and cellular factors support virus-host interactions

10.1101/2021.05.11.443547 ◽

2021 ◽

Author(s):

Shelby L Brown ◽

Jared P. May

Keyword(s):

Phase Separation ◽

Mosaic Virus ◽

Self Assembly ◽

Electrostatic Interactions ◽

Movement Protein ◽

Virus Movement ◽

In Planta ◽

Ribonucleoprotein Complexes

Phase separation concentrates biomolecules, which should benefit RNA viruses that must sequester viral and host factors during an infection. Here, the p26 movement protein from Pea enation mosaic virus 2 (PEMV2) was found to phase separate and partition in nucleoli and G3BP stress granules (SGs) in vivo . Electrostatic interactions drive p26 phase separation as mutation of basic (R/K-G) or acidic (D/E-G) residues either blocked or reduced phase separation, respectively. During infection, p26 must partition inside the nucleolus and interact with fibrillarin (Fib2) as a pre-requisite for systemic trafficking of viral RNAs. Partitioning of p26 in pre-formed Fib2 droplets was dependent on p26 phase separation suggesting that phase separation of viral movement proteins supports nucleolar partitioning and virus movement. Furthermore, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 RNA were formed via phase separation in vitro and could provide the basis for self-assembly in planta . Interestingly, both R/K-G and D/E-G p26 mutants failed to support systemic trafficking of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana suggesting that p26 phase separation, proper nucleolar partitioning, and systemic movement are intertwined. p26 also partitioned in SGs and G3BP over-expression restricted PEMV2 accumulation >20-fold. Expression of phase separation-deficient G3BP only restricted PEMV2 5-fold, demonstrating that G3BP phase separation is critical for maximum antiviral activity.

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Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo

Journal of Virology ◽

10.1128/jvi.76.20.10264-10269.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10264-10269 ◽

Cited By ~ 12

Author(s):

Yu-Zhang Wu ◽

Jian-Ping Zhao ◽

Ying Wan ◽

Zheng-Cai Jia ◽

Wei Zhou ◽

...

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Electrostatic Interactions ◽

Cytotoxic T Lymphocyte ◽

Interleukin 12 ◽

Antigen Delivery ◽

Intracellular Pathogens ◽

Virus Size

ABSTRACT CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine—mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg28-39, with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg28-39-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

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Spaciotemporal Association and Bone Morphogenetic Protein Regulation of Sclerostin and Osterix Expression during Embryonic Osteogenesis

Endocrinology ◽

10.1210/en.2003-1492 ◽

2004 ◽

Vol 145 (10) ◽

pp. 4685-4692 ◽

Cited By ~ 50

Author(s):

Yoshio Ohyama ◽

Akira Nifuji ◽

Yukiko Maeda ◽

Teruo Amagasa ◽

Masaki Noda

Keyword(s):

Bone Morphogenetic Protein ◽

Bone Growth ◽

Expression Patterns ◽

Cranial Bone ◽

Nodule Formation ◽

Endogenous Expression ◽

Sost Gene ◽

Morphogenetic Protein

Abstract Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.

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Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-β signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103087118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2103087118

Author(s):

Robert J. Tower ◽

Zhu Li ◽

Yu-Hao Cheng ◽

Xue-Wei Wang ◽

Labchan Rajbhandari ◽

...

Keyword(s):

Signaling Pathway ◽

Sensory Nerve ◽

Positive Association ◽

Sensory Nerves ◽

Cranial Bone ◽

Morphogenetic Protein ◽

Proliferation And Differentiation ◽

Microfluidic Chambers

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

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Gene-activated matrix harboring a miR20a-expressing plasmid promotes rat cranial bone augmentation

Regenerative Biomaterials ◽

10.1093/rb/rbaa060 ◽

2021 ◽

Vol 8 (2) ◽

Author(s):

Rena Shido ◽

Yoshinori Sumita ◽

Masahito Hara ◽

Mayumi Iwatake ◽

Shun Narahara ◽

...

Keyword(s):

Target Genes ◽

Osteoblastic Differentiation ◽

Cranial Bone ◽

Bone Marrow Mscs ◽

Bone Augmentation ◽

Bone Surface ◽

Naked Plasmid ◽

Rat Bone

Abstract Gene-activated matrix (GAM) has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established. In this study, we investigated whether an atelocollagen-based GAM containing naked-plasmid (p) DNAs encoding microRNA (miR) 20a, which may promote osteogenesis in vivo via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells (MSCs), facilitates rat cranial bone augmentation. First, we confirmed the osteoblastic differentiation functions of generated pDNA encoding miR20a (pmiR20a) in vitro, and its transfection regulated the expression of several of target genes, such as Bambi1 and PPARγ, in rat bone marrow MSCs and induced the increased expression of BMP4. Then, when GAMs fabricated by mixing 100 μl of 2% bovine atelocollagen, 20 mg β-TCP granules and 0.5 mg (3.3 μg/μl) AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface, the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation, as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation. Thus, GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.

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Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly

eLife ◽

10.7554/elife.46207 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 14

Author(s):

Sudeep Banjade ◽

Shaogeng Tang ◽

Yousuf H Shah ◽

Scott D Emr

Keyword(s):

Saccharomyces Cerevisiae ◽

Self Assembly ◽

Electrostatic Interactions ◽

Membrane Remodeling ◽

Lateral Interactions ◽

Vesicle Biogenesis ◽

Flexible Architectures ◽

Functional Defects

Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.

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Amphiphilic QP(DMAEMA-co-LMA)-b-POEGMA Random-Block Terpolymers as Nanocarriers for Insulin

Biomedicines ◽

10.3390/biomedicines8100392 ◽

2020 ◽

Vol 8 (10) ◽

pp. 392

Author(s):

Martha Kafetzi ◽

Stergios Pispas ◽

Xiaoyan Bao ◽

Ping Yao

Keyword(s):

Surface Charge ◽

Conformational Changes ◽

Self Assembly ◽

Electrostatic Interactions ◽

Aqueous Media ◽

Physicochemical Characteristics ◽

In Vitro Cytotoxicity ◽

Assembly Features

We report on the utilization of the amphiphilic poly[quaternized (2-(N,N-dimethylamino) ethyl methacrylate)]-co-(lauryl methacrylate))-b-poly[(oligo ethylene glycol) methyl ether methacrylate] QP(DMAEMA-co-LMA)-b-POEGMA cationic diblock terpolymer aggregates as nanocarriers for insulin delivery applications. QP(DMAEMA-co-LMA)-b-POEGMA random diblock terpolymer is derived from the chemical modification of the precursor amino diblock copolymer via quaternization, producing permanent positive charges on the macromolecular chain. The QP(DMAEMA-co-LMA)-b-POEGMA diblock terpolymer as well as its amino precursor investigated self-assemble in aqueous media, forming aggregates. In vitro cytotoxicity and in vivo biocompatibility studies on QP(DMAEMA-co-LMA)-b-POEGMA and its amino precursor aggregates, showed good cytocompatibility and biocompatibility. QP(DMAEMA-co-LMA)-b-POEGMA aggregates were chosen to be complexed with insulin due to their self-assembly features and the permanent positive charge in each amino group. QP(DMAEMA-co-LMA)-b-POEGMA aggregates were complexed with insulin through electrostatic interactions. Light scattering techniques were used in order to study the ability of the polymer aggregates to complex with insulin, to determine critical physicochemical parameters such as size, mass, and surface charge of the stable complexes and study the effect of salt addition on their properties. The results showed that in both cases, the complexation process was successful and as the insulin concentration increases, nanosized complexes of different physicochemical characteristics (mass, size, surface charge) and spherical morphology are formed. UV-Vis and fluorescence spectroscopy studies showed that no conformational changes of insulin occurred after the complexation.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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Biomimetic reduced graphene oxide coated collagen scaffold for in situ bone regeneration

Scientific Reports ◽

10.1038/s41598-021-96271-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sajad Bahrami ◽

Nafiseh Baheiraei ◽

Mostafa Shahrezaee

Keyword(s):

Graphene Oxide ◽

Reduced Graphene Oxide ◽

Cranial Bone ◽

Drying Methods ◽

Porous Scaffolds ◽

Alizarin Red ◽

Reduced Graphene ◽

Coated Collagen

AbstractA variety of bone-related diseases and injures and limitations of traditional regeneration methods require new tissue substitutes. Tissue engineering and regeneration combined with nanomedicine can provide different natural or synthetic and combined scaffolds with bone mimicking properties for implantation in the injured area. In this study, we synthesized collagen (Col) and reduced graphene oxide coated collagen (Col-rGO) scaffolds, and we evaluated their in vitro and in vivo effects on bone tissue repair. Col and Col-rGO scaffolds were synthesized by chemical crosslinking and freeze-drying methods. The surface topography, and the mechanical and chemical properties of scaffolds were characterized, showing three-dimensional (3D) porous scaffolds and successful coating of rGO on Col. The rGO coating enhanced the mechanical strength of Col-rGO scaffolds to a greater extent than Col scaffolds by 2.8 times. Furthermore, Col-rGO scaffolds confirmed that graphene addition induced no cytotoxic effects and enhanced the viability and proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) with 3D adherence and expansion. Finally, scaffold implantation into rabbit cranial bone defects for 12 weeks showed increased bone formation, confirmed by Hematoxylin–Eosin (H&E) and alizarin red staining. Overall, the study showed that rGO coating improves Col scaffold properties and could be a promising implant for bone injuries.

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