DNA hydrogels for bone regeneration.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

DDR1 associates with TRPV4 in cell-matrix adhesions to enable calcium-regulated myosin activity and collagen compaction.

Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the ß1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.

Uniaxial Hydroxyapatite Growth on a Self-Assembled Protein Scaffold.

Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection. Mineralized tissues are composed of hierarchically organized hydroxyapatite crystals, with a limited capacity to regenerate when demineralized or damaged past a critical size. Thus, the development of protein-based materials that act as artificial scaffolds to guide hydroxyapatite growth is an attractive goal both for the design of ordered nanomaterials and for tissue regeneration. In particular, amelogenin, which is the main protein that scaffolds the hierarchical organization of hydroxyapatite crystals in enamel, amelogenin recombinamers, and amelogenin-derived peptide scaffolds have all been investigated for in vitro mineral growth. Here, we describe uniaxial hydroxyapatite growth on a nanoengineered amelogenin scaffold in combination with amelotin, a mineral promoting protein present during enamel formation. This bio-inspired approach for hydroxyapatite growth may inform the molecular mechanism of hydroxyapatite formation in vitro as well as possible mechanisms at play during mineralized tissue formation.

Comparative analysis of polymerization shrinkage of different resin composites.

This study sought to compare the shrinkage of 3 resin composites after polymerization, using different curing modes and 2 methods of analysis, with 45 samples in each group. To evaluate free linear shrinkage, specimens were prepared in Teflon molds (8 mm diameter x 2 mm thick) with 1 of 2 methacrylate-based resins or a silorane-based resin. To evaluate wall-to-wall shrinkage, cavities (1.5 mm thick x 3 mm diameter) were prepared in 45 healthy bovine incisors and then restored. In both tests, the same curing lights were used: conventional quartz-tungsten-halogen, a conventional light-emitting diode (LED), and an exponential LED. Gaps were measured microscopically, and the gap percentage was calculated. The results were analyzed by 2-way ANOVA and Tukey's test (α = 0.05). Curing modes differed significantly in the free linear shrinkage test group, while resin composites did not. In the wall-to-wall shrinkage group, there were significant differences between the resin composites.

Precision polymers and 3D DNA nanostructures: emergent assemblies from new parameter space.

Polymer self-assembly and DNA nanotechnology have both proved to be powerful nanoscale techniques. To date, most attempts to merge the fields have been limited to placing linear DNA segments within a polydisperse block copolymer. Here we show that, by using hydrophobic polymers of a precisely predetermined length conjugated to DNA strands, and addressable 3D DNA prisms, we are able to effect the formation of unprecedented monodisperse quantized superstructures. The structure and properties of larger micelles-of-prisms were probed in depth, revealing their ability to participate in controlled release of their constituent nanostructures, and template light-harvesting energy transfer cascades, mediated through both the addressability of DNA and the controlled aggregation of the polymers.

An efficient and modular route to sequence-defined polymers appended to DNA.

Inspired by biological polymers, sequence-controlled synthetic polymers are highly promising materials that integrate the robustness of synthetic systems with the information-derived activity of biological counterparts. Polymer-biopolymer conjugates are often targeted to achieve this union; however, their synthesis remains challenging. We report a stepwise solid-phase approach for the generation of completely monodisperse and sequence-defined DNA-polymer conjugates using readily available reagents. These polymeric modifications to DNA display self-assembly and encapsulation behavior-as evidenced by HPLC, dynamic light scattering, and fluorescence studies-which is highly dependent on sequence order. The method is general and has the potential to make DNA-polymer conjugates and sequence-defined polymers widely available.

Chemical and Ultrastructural Characterization of Dentin Treated with Remineralizing Dentifrices.

The aim of this study is to investigate dentin chemical and ultrastructural changes upon exposure to remineralizing dentifrices. Dentin disks were obtained from permanent human molars and treated for 7 days with the dentifrices: (1) C group-control (no dentifrice); (2) S group-Sensodyne Repair & Protect; (3) D group-Dentalclean Daily Regenerating Gel; and (4) DB group-D group + Dentalclean regenerating booster. Afterwards, samples were submitted to an additional 7 days of toothbrushing associated with daily acidic challenge. Samples were imaged and analyzed (days 1, 7, and 14) for Young's modulus by atomic force microscopy (AFM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). SEM and AFM revealed precipitate deposition on dentin surfaces in groups S, D, and DB, formed as early as day 1. Surface elemental analysis showed a Si increase on all brushed surfaces. Similar surface morphology was maintained after the acidic challenge period. Bright-field TEM/SAED revealed the formation of nanocrystalline hydroxyapatite inside the dentin tubules of groups S, D, and DB after day 7. Group C presented a gradual reduction of Young's modulus from days-1-14, whereas all remaining groups had increased values. All evaluated dentifrices led to successful formation of hydroxyapatite and increased dentin stiffness.

Focusing on the Native Matrix Proteins in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is a widespread valvular heart disease affecting people in aging societies, primarily characterized by fibrosis, inflammation, and progressive calcification, leading to valve orifice stenosis. Understanding the factors associated with CAVS onset and progression is crucial to develop effective future pharmaceutical therapies. In CAVS, native extracellular matrix proteins modifications, play a significant role in calcification in vitro and in vivo. This work aimed to review the evidence on the alterations of structural native extracellular matrix proteins involved in calcification development during CAVS and highlight its link to deregulated biomechanical function.

Effect of Odanacatib on the release of NTX (Amino Terminal Telopeptide) from LPS contaminated type I dentin collagen.

OBJECTIVE: To evaluated the Odanacatib inhibitor treatment on lipopolysaccharide (LPS) contamination effect on cathepsin-K mediated dentin degradation by analysis of type I collagen C- and N-termini telopeptides. METHODS: Pulverized and disks of human dentin were demineralized and LPS contaminated, or stored in deionized water (DW) for 12 h. Samples were challenged with lactic acid (LA). Aliquots of dentin powder were treated with 1 mL Odanacatib or stored in DW for 30 min. Dentin collagen degradation was determined by sub-product release of C-terminal (ICTP and CTX) and N-terminal (NTX) telopeptides, normalized to total protein (tp) concentration (n = 3). Dentin matrix was evaluated for gravimetric (n = 8) and ultrastructural changes. Data were analyzed by Student t-test, one-way ANOVA and Tukey's test (α = 5 %). RESULTS: LA incubation significantly increased telopeptide release compared with DW (p < 0.05). In untreated groups, significantly higher CTXtp, NTXtp telopeptide rates were observed for LA+LPS samples compared with DW (p < 0.01). Odanacatib significantly reduced ICTPtp, CTXtp, and NTXtp telopeptide release for LPS, LA, and LA+LPS conditions. In untreated groups, LPS and LA+LPS challenge significantly increased dentin weight loss (p = 0.02). Within each storage condition, Odanacatib treatment did not affect weight change (p > 0.05) of dentin disks. SIGNIFICANCE: This study showed that LPS contamination resulted in significantly higher rates of NTX than CTX from dentin matrix. Odanacatib significantly reduced telopeptide release rates of LPS contaminated dentin matrix.

Rolling circle amplification-templated DNA nanotubes show increased stability and cell penetration ability.

DNA nanotubes hold promise as scaffolds for protein organization, as templates of nanowires and photonic systems, and as drug delivery vehicles. We present a new DNA-economic strategy for the construction of DNA nanotubes with a backbone produced by rolling circle amplification (RCA), which results in increased stability and templated length. These nanotubes are more resistant to nuclease degradation, capable of entering human cervical cancer (HeLa) cells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation and release behavior. As such, they represent a potentially unique platform for the development of cell probes, drug delivery, and imaging tools.

Three-dimensional organization of block copolymers on "DNA-minimal" scaffolds.

Here, we introduce a 3D-DNA construction method that assembles a minimum number of DNA strands in quantitative yield, to give a scaffold with a large number of single-stranded arms. This DNA frame is used as a core structure to organize other functional materials in 3D as the shell. We use the ring-opening metathesis polymerization (ROMP) to generate block copolymers that are covalently attached to DNA strands. Site-specific hybridization of these DNA-polymer chains on the single-stranded arms of the 3D-DNA scaffold gives efficient access to DNA-block copolymer cages. These biohybrid cages possess polymer chains that are programmably positioned in three dimensions on a DNA core and display increased nuclease resistance as compared to unfunctionalized DNA cages.

Peptide-Decorated DNA Nanostructures Promote Site-Specific Hydroxyapatite Growth.

The guiding principle for mineralized tissue formation is that mineral growth occurs through the interaction of Ca2+ and phosphate ions with extracellular matrix (ECM) proteins. Recently, nanoengineered DNA structures have been proposed as mimics to ECM scaffolds. However, these principles have not been applied to mineralized tissues. Here, we describe DNA nanostructures, namely, a DNA nanotube and a DNA origami rectangle that are site specifically functionalized with a mineral-promoting "SSEE" peptide derived from ECM proteins present in mineralized tissues. In the presence of Ca2+ and phosphate ions (mineralizing conditions), site-specific calcium phosphate formation occurred on the DNA nanostructures. Amorphous calcium phosphate or hydroxyapatite was formed depending on the incubation time, shape of the DNA nanostructure, and amount of Ca2+ and phosphate ions present. The ability to design and control the growth of hydroxyapatite through nanoengineered scaffolds provides insights into the mechanisms that may occur during crystal nucleation and growth of mineralized tissues and can inspire mineralized tissue regeneration strategies.

DNA nanostructures as templates for biomineralization.

Nature uses extracellular matrix scaffolds to organize biominerals into hierarchical structures over various length scales. This has inspired the design of biomimetic mineralization scaffolds, with DNA nanostructures being among the most promising. DNA nanotechnology makes use of molecular recognition to controllably give 1D, 2D and 3D nanostructures. The control we have over these structures makes them attractive templates for the synthesis of mineralized tissues, such as bones and teeth. In this Review, we first summarize recent work on the crystallization processes and structural features of biominerals on the nanoscale. We then describe self-assembled DNA nanostructures and come to the intersection of these two themes: recent applications of DNA templates in nanoscale biomineralization, a crucial process to regenerate mineralized tissues.

Long-range assembly of DNA into nanofibers and highly ordered networks using a block copolymer approach.

A simple method to introduce the long-range order achieved by block copolymers into DNA structures is described. This results in the hierarchical assembly of short DNA strands into a new one-dimensional material, with high aspect ratio and the ability to further align into highly ordered surfaces over tens of micrometers. Fibers derived from biological materials have a wide range of potential applications, such as scaffolds for nanowires and one-dimensional (1D) materials, templates for tissue growth, and ligand display tools for multivalent biological interactions. Fibers derived from short DNA strands are an attractive class of materials, as they combine long-range 1D ordering with the programmability of DNA, and its ability to undergo structure switching with specifically added DNA strands. Here, we present the first examples of long fibers self-assembled from short (10-20 base-pairs), blunt-ended DNA strands. This was accomplished by covalently attaching a dendritic oligoethylene glycol (OEG) unit to a DNA strand to form a dendritic DNA molecule (D-DNA). Hybridization of this unit with complementary DNA creates a block copolymer/double-stranded DNA architecture, which readily undergoes self-assembly into long fibers upon the addition of a selective solvent. These fibers can further align into parallel rows, to yield highly ordered micrometer-sized surfaces. We demonstrate that a DNA nanotechnology motif, a three-helix DNA bundle, can also be readily induced to form long fibers upon incorporation of D-DNA. Thus, this provides a straightforward method to introduce hierarchical long-range ordering into DNA motifs, simply through hybridization with short D-DNA strands.

Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation.

The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.

Functionalized DNA nanostructures as scaffolds for guided mineralization.

The field of DNA nanotechnology uses synthetic DNA strands as building blocks for designing complex shapes in one-, two- and three-dimensions. Here, we investigate whether DNA nanostructures are feasible platforms for the precise organization of polyaspartic acid (pAsp), a known mineral carrier, with a goal towards biomimetic mineralization for enamel regeneration. We describe the preparation of DNA-pAsp conjugates and their subsequent assembly into ordered nanostructures. Covalent attachment of pAsp to DNA was noted to hinder DNA nanostructure formation past a certain threshold (50% pAsp) when tested on a previously published DNA system. However, a simplified double stranded DNA system (3sDH system) was more robust and efficient in its pAsp incorporation. In addition, the 3sDH system was successful in organizing mineral inducing groups in one dimension at repeating intervals of 28.7 ± 4.0 nm, as determined by atomic force microscopy. Our results demonstrate that DNA nanostructures can be functionalized with pAsp and act as a platform to investigate guided mineralization.

Bioactivity and properties of an adhesive system functionalized with an experimental niobium-based glass.

OBJECTIVE: This study evaluated the incorporation of niobophosphate bioactive glass (NbG) fillers into a commercial adhesive resin. MATERIALS AND METHODS: The silanized (NbGs) or non-silanized (NbG) NbG was added to the commercial adhesive system One Step (OS) at 30% by weight; unfilled adhesive served as control. The bioactivity of adhesives was analyzed by SEM and FTIR/ATR after 28 days in PBS. The adhesives were evaluated as regards microtensile bond strength immediately and after six months (n = 6); degree of conversion (n = 3), microhardness (n = 5); and radiopacity (n = 3). Data from each test were submitted to ANOVA and Tukey tests (P <0.05). RESULTS: FTIR/ATR analysis showed phosphate and carbonate precipitates on the NbG adhesive specimen surface. Statistical analysis of microtensile bond strength values showed that material x time interaction was not significant, but NbG group values were similar to those of unfilled adhesive (p <0.05). Addition of NbG did not alter the degree of conversion, but did increase microhardness and radiopacity values of the adhesive systems compared with those of the control group (OS). Incorporation of NbG into the adhesive system did not compromise the properties of the adhesive. CONCLUSION: A smart adhesive system with bioactive properties, high radiopacity, microhardness, and similar bond strength and degree of conversion was obtained by incorporating 30% by weight of NbG.

The role of protease inhibitors on the remineralization of demineralized dentin using the PILP method.

Mineralized and sound dentin matrices contain inactive preforms of proteolytic enzymes that may be activated during the demineralization cycle. In this study, we tested the hypothesis that protease inhibitors (PI) preserve demineralized collagen fibrils and other constituents of the dentin matrix and thereby affect the potential for remineralization. Artificial carious lesions with lesion depths of 140 µm were created with acetate buffer (pH = 5.0, 66 hours), and remineralized using a polymer-induced-liquid-precursor (PILP) process (pH = 7.4, 14 days) containing poly(aspartic acid) (pAsp) as the process-directing agent. De- and remineralizing procedures were performed in the presence or absence of PI. Ultrastructure and mechanical recovery of demineralized dentin following PILP remineralization were examined and measured in water with atomic force microscopy (AFM) and nanoindentation. Nanomechanical properties of hydrated artificial lesions had a low elastic modulus (ER <0.4 GPa) extending about 100 µm into the lesion, followed by a sloped region of about 140 µm depth where values reached those of normal dentin (18.0-20.0 GPa). Mapping of mineral content by both micro-FTIR and micro x-ray computed tomography correlated well with modulus profiles obtained by nanoindentation. Tissue demineralized in the presence of PI exhibited higher elastic moduli (average 2.8 GPa) across the lesion and comprised a narrow zone in the outer lesion with strongly increased modulus (up to 8 GPa; p < 0.05), which might be related to the preservation of non-collagenous proteins that appear to induce calcium phosphate mineral formation even under demineralizing physical-chemical conditions. However, mechanical aspects of remineralization through the elastic modulus change, and the micromorphological aspects with SEM and TEM observation were almost identical with PILP treatments being conducted in the presence or absence of PI. Thus, the application of the protease inhibitors (PI) seemed to be less effective in promoting the remineralization of demineralized dentin.

Adhesives Doped with Bioactive Niobophosphate Micro-Filler: Degree of Conversion and Microtensile Bond Strength.

To evaluate the effect of incorporating niobium phosphate bioactive glass (NbG) into commercial etch-and-rinse adhesive systems, with and without silane, on their degree of conversion (DC) (%) and microtensile bond strength (µTBS). The NbG micro-filler was added to two etch-and-rinse adhesive systems: One Step (OS) and Prime & Bond (PB) at 40% concentration. The following groups were formed: control without glass addition OS; addition of unsilanized NbG (OSNbG); addition of silanized NbG (OSNbGS); control without glass PB; addition of unsilanized NbG (PBNbG); addition of silanized NbG (PBNbGS). The DC was determined using total Fourier spectroscopy reflection (FTIR/ATR). For µTBS testing, 48 human third molars (n=8) were restored and sliced to obtain specimens (0.8 mm2) and they were tested at two different time intervals: immediately and after 6 months. The fracture mode was evaluated with a stereoscopic loupe (40×) and by scanning electron microscopy (SEM). The data were subjected to ANOVA and Tukey tests (a=0.05). NbG addition did not compromise the adhesive system DC values (p>0.05). Furthermore, the NbG added to the adhesive systems did not affect µTBS values (p>0.05). Fracture occurred predominantly at the dentin-adhesive interface. NbG bioactive glass did not affect the DC or microtensile bond strength results.

Amyloid-like ribbons of amelogenins in enamel mineralization.

Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into ß-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development.