Reduced Graphene Oxide Nanohybrid-Assembled Microneedles as Mini-Invasive Electrodes for Real-Time Transdermal Biosensing.

A variety of nanomaterial-based biosensors have been developed to sensitively detect biomolecules in vitro, yet limited success has been achieved in real-time sensing in vivo. The application of microneedles (MN) may offer a solution for painless and minimally-invasive transdermal biosensing. However, integration of nanostructural materials on microneedle surface as transdermal electrodes remains challenging in applications. Here, a transdermal H2 O2 electrochemical biosensor based on MNs integrated with nanohybrid consisting of reduced graphene oxide and Pt nanoparticles (Pt/rGO) is developed. The Pt/rGO significantly improves the detection sensitivity of the MN electrode, while the MNs are utilized as a painless transdermal tool to access the in vivo environment. The Pt/rGO nanostructures are protected by a water-soluble polymer layer to avoid mechanical destruction during the MN skin insertion process. The polymer layer can readily be dissolved by the interstitial fluid and exposes the Pt/rGO on MNs for biosensing in vivo. The applications of the Pt/rGO-integrated MNs for in situ and real-time sensing of H2 O2 in vivo are demonstrated both on pigskin and living mice. This work offers a unique real-time transdermal biosensing system, which is a promising tool for sensing in vivo with high sensitivity but in a minimally-invasive manner.

Liquid-like polymer-based self-cleaning coating for effective prevention of liquid foods contaminations.

Liquid food containers commonly suffer from inevitable contamination and even biofilm formation due to the adhesion of food residuals or saliva, which requires detergents to clean. Although previously reported superhydrophobic and omniphobic coatings can resist the adhesion of liquids, the requirements of specific nanostructures or infused lubricants limit their applications in food containers. Here, by grafting smooth glass containers with "liquid like" polydimethylsiloxane brushes, we developed a unique approach for preparing a slippery coating that could exhibit highly robust repellency to various liquid foods. The coating was highly transparent and did not induce a significant alteration of the smooth surface. The "liquid like" coating could effectively prevent the adhesion of various liquid foods and inhibit the formation of bacterial biofilms, without the use of detergents for cleaning. Moreover, this coating could resist mechanical damage from friction, and displayed high biocompatibility with biological cells. The slipperiness, smoothness, robustness and biocompatibility of the "liquid like" coating was highly beneficial to practical applications as self-cleaning glass container, which has been challenging to achieve by conventional superhydrophobic or omniphobic coatings. Our study introduced a versatile strategy to functionalize biocompatible surfaces for food containers which reduced the contamination of residues and the use of detergents, and may be beneficial to human and environmental health.

Liquid-like Polymer Coating as a Promising Candidate for Reducing Electrode Contamination and Noise in Complex Biofluids.

Biosensors that can automatically and continuously track fluctuations in biomarker levels over time are essential for real-time sensing in biomedical and environmental applications. Although many electrochemical sensors have been developed to quickly and sensitively monitor biomarkers, their sensing stability in complex biofluids is disturbed by unavoidable nonspecific adhesion of proteins or bacteria. Recently, various substrate surface modification techniques have been developed to resist biofouling, yet functionalization of electrodes in sensors to be anti-biofouling is rarely achieved. Here, we report an integrated three-electrode system (ITES) modified with a "liquid-like" polydimethylsiloxane (PDMS) brush that can continuously and stably monitor reactive oxygen species (ROS) in complex fluids. Based on the slippery "liquid-like" coating, the modified ITES surface could prevent the adhesion of various liquids as well as the adhesion of proteins and bacteria. The "liquid-like" coating does not significantly affect the sensitivity of the electrode in detecting ROS, while the sensing performance could remain stable and free of bacterial attack even after 3 days of incubation with bacteria. In addition, the PDMS brush-modified ITES (PMITES) could continuously record ROS levels in bacterial-rich fluids with excellent stability over 24 h due to the reduced bacterial contamination on the electrode surface. This technique offers new opportunities for continuous and real-time monitoring of biomarkers that will facilitate the development of advanced sensors for biomedical and environmental applications.

Protection of Nanostructures-Integrated Microneedle Biosensor Using Dissolvable Polymer Coating.

Real-time transdermal biosensing provides a direct route to quantify biomarkers or physiological signals of local tissues. Although microneedles (MNs) present a mini-invasive transdermal technique, integration of MNs with advanced nanostructures to enhance sensing functionalities has rarely been achieved. This is largely due to the fact that nanostructures present on MNs surface could be easily destructed due to friction during skin insertion. In this work, we reported a dissolvable polymer-coating technique to protect nanostructures-integrated MNs from mechanical destruction during MNs insertion. After penetration into the skin, the polymer could readily dissolve by interstitial fluids so that the superficial nanostructures on MNs could be re-exposed for sensing purpose. To demonstrate this technique, metallic and resin MNs decorated with vertical ZnO nanowires (vNWs) were employed as an example. Dissolvable poly(vinyl pyrrolidone) was spray-coated on the vNW-MNs surface as a protective layer, which effectively protected the superficial ZnO NWs when MNs penetrated the skin. Transdermal biosensing of H2O2 biomarker in skin tissue using the polymer-protecting MNs sensor was demonstrated both ex vivo and in vivo. The results indicated that polymer coating successfully preserved the sensing functionalities of the MNs sensor after inserting into the skin, whereas the sensitivity of the MN sensor without a coating protection was significantly compromised by 3-folds. This work provided unique opportunities of protecting functional nanomodulus on MNs surface for minimally invasive transdermal biosensing.

Biological assemblies provide novel templates for the synthesis of hierarchical structures and facilitate cell adhesion.

Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.

Identification of differentially expressed cDNA transcripts from a rat odontoblast cell line.

Odontoblasts and osteoblasts are two among the myriads of cell types present in the craniofacial complex. Both have a common ectomesenchymal origin and secrete macromolecules that are necessary for the formation of dentin and alveolar bone via matrix-mediated mechanisms. The mineralized matrices of bone and dentin differ in morphology and function but several mineral associated proteins, formerly thought to be tissue specific, have been found to be common in both tissues. To decipher the complex molecular mechanisms involved in mineralized dentin formation, the suppressive subtraction hybridization (SSH) approach has been used to identify the genes expressed by polarized odontoblasts. Employing SSH, 187 cDNA clones were identified from the subtracted cDNA library. Many of these genes have not been previously reported to be expressed by terminally differentiated odontoblasts. Genes were classified into seven groups based on the predicted function of the encoded proteins: extracellular matrix; cytoskeletal components, molecules involved in adhesion and cell-cell interaction; metabolic enzymes, transporters, ion channels; protein processing, protein transport and protein folding molecules; nuclear proteins (transcription factors, DNA processing enzymes); signaling molecules and genes of yet unknown function. Northern blot and in situ hybridization analysis performed for five putative novel genes and one new isoform of amelogenin revealed differential expression levels in the osteoblasts, ameloblasts and the odontoblasts of the developing rat molars. Some of the known genes isolated from this enriched pool were the cleavage products of dentin sialophosphoprotein (DSPP) namely, phosphophoryn (PP) and dentin sialoprotein (DSP). Interestingly amelogenin, ameloblastin and enamelin were also expressed in the odontoblasts during dentin formation.

Coligand-directed synthesis of five Co(II)/Ni(II) coordination polymers with a neutral tetradentate ligand: syntheses, crystal structures, and properties.

The solvothermal reactions of 1,1'-oxybis[3,5-di-4-pyridine]-benzene (L) and transition metal cations (Co and Ni) afford five novel coordination polymers in the presence of flexible bridging ligands (4,4'-H2nba = 4,4'-dicarboxydiphenylamine, H2cam = d-camphoric acid, 4,4'-H2sdb = 4,4'-sulfonyldibenzoic acid, H2chdc = 1,4-trans-cyclohexanedicarboxylic acid), namely {[Co2L2(OH)2(nba)]·2DMF}n (), {[CoL(cam)(H2O)]}n (), {[Co3(L)(4,4'-sdb)3(H2O)]·1.5CH3CN·4H2O}n (), {[Ni3(L)(4,4'-sdb)3(H2O)]·1.5CH3CN·4H2O}n (), and {[Ni2L2(chdc)2(H2O)2]·(H2O)3}n () (DMF = N,N-dimethylformamide). Their structures have been determined by single-crystal X-ray diffraction analyses and further characterized by elemental analyses, IR spectroscopy, and powder X-ray diffraction. Complex reveals a 2-fold interpenetrating three-dimensional (3D) framework with the Schläfli symbol {4·8·10(4)}{4·8·10} topology. Compound crystallizes in the achiral space group with the d-camphorate ligand racemized. Compounds and reveal similar structure with the {3·4(4)·6}{3(2)·4(8)·5(9)·6(9)} topology based on a linear trinuclear building block M3(OOCR)6 (M = Co(ii) or Ni(ii)). Compound is a wavy sheet, where both carboxylate and L ligands act as bidentate ligands. Moreover, UV-Visible absorption spectra of complexes , and the magnetic properties of have been investigated.

Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization.

Phosphoproteins of the organic matrix of bone and dentin have been implicated as regulators of the nucleation and growth of the inorganic Ca-P crystals of vertebrate bones and teeth. One such protein identified in the dentin matrix is phosphophoryn (PP). It is highly acidic in nature because of a high content of aspartic acid and phosphate groups on serines. The 244-residue carboxyl-terminal domain of rat PP, predominantly containing the aspartic acid-serine repeats, has been cloned, and the corresponding protein has been expressed recombinantly in Escherichia coli. This portion of PP, named DMP2 (dentin matrix protein 2), is not phosphorylated by the bacteria and thus provided a means to study the function of the phosphate groups, the major post-translational modification of native PP. The recombinant DMP2 (rDMP2) possessed much lower calcium binding capacity than native PP. Small angle x-ray scattering experiments demonstrated that PP folds to a compact globular structure upon calcium binding, whereas rDMP2 maintained an unfolded structure. In vitro nucleation experiments showed that PP could nucleate plate-like apatite crystals in pseudophysiological buffer, whereas rDMP2 failed to mediate the transformation of amorphous calcium phosphate to apatite crystals under the same experimental conditions. Collagen binding experiments demonstrated that PP favors the formation of collagen aggregates, whereas in the presence of rDMP2 thin fibrils are formed. Overall these results suggested that the phosphate moieties in phosphophoryn are important for its function as a mediator of dentin biomineralization.

Spatially and temporally controlled biomineralization is facilitated by interaction between self-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution.

Bone and dentin biomineralization are well-regulated processes mediated by extracellular matrix proteins. It is widely believed that specific matrix proteins in these tissues modulate nucleation of apatite nanoparticles and their growth into micrometer-sized crystals via molecular recognition at the protein-mineral interface. However, this assumption has been supported only circumstantially, and the exact mechanism remains unknown. Dentin matrix protein 1 (DMP1) is an acidic matrix protein, present in the mineralized matrix of bone and dentin. In this study, we have demonstrated using synchrotron small-angle X-ray scattering that DMP1 in solution can undergo oligomerization and temporarily stabilize the newly formed calcium phosphate nanoparticle precursors by sequestering them and preventing their further aggregation and precipitation. The solution structure represents the first low-resolution structural information for DMP1. Atomic force microscopy and transmission electron microscopy studies further confirmed that the nascent calcium phosphate nuclei formed in solution were assembled into ordered protein-mineral complexes with the aid of oligomerized DMP1, recombinant and native. This study reveals a novel mechanism by which DMP1 might facilitate initiation of mineral nucleation at specific sites during bone and dentin mineralization and prevent spontaneous calcium phosphate precipitation in areas in which mineralization is not desirable.

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro.

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.

[Observation of the clinical effects of two kinds of resin materials for restoring the defects of maxillary incisor].

To analyse and compare the effects of the resin materials of etched and nonetched groups in restoring the defect of maxillary incisor. 120 teeth were divided into two groups and filled by two kinds of materials. One year follow up results was studied. The effects of retention had no significant difference between the two groups, but the nonetched group had a lower stimulation to the pulp than the other one in restoring middle to deep caries. Compared to the etched group, the nonetched materials of resin has lower stimulation to pulp, more adhesion and higher success rate.

Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1.

Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Acidic extracellular matrix proteins play a pivotal role during biomineral formation. However, the mechanisms of protein-mediated mineral initiation are far from understood. Here we report that dentin matrix protein 1 (DMP1), an acidic protein, can nucleate the formation of hydroxyapatite in vitro in a multistep process that begins by DMP1 binding calcium ions and initiating mineral deposition. The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Subsequently, these expand and coalesce into microscale crystals elongated in the c-axis direction. Characterization of the functional domains in DMP1 demonstrated that intermolecular assembly of acidic clusters into a beta-sheet template was essential for the observed mineral nucleation. Protein-mediated initiation of nanocrystals, as discussed here, might provide a new methodology for constructing nanoscale composites by self-assembly of polypeptides with tailor-made peptide sequences.

Dentin matrix protein 1 initiates hydroxyapatite formation in vitro.

Bone and dentin formation are interesting examples of matrix-mediated mineralization. However, factors and mechanisms regulating this process are poorly understood. Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein found in dentin and bone, and based on its amino acid composition it could be postulated to play an important role in mineralization. Our present study examines the ability of recombinant DMP1 to initiate apatite formation in vitro. A 45Ca-binding assay demonstrated that recombinant DMP1 (rDMP1) possesses calcium-binding ability under physiological conditions. The in vitro nucleation experiments when conducted with rDMP1-coated glass plates demonstrated hydroxyapatite nucleation, while amorphous mineral was deposited on blank or BSA-coated surface. This mineral deposition was found to be 10-fold higher on rDMP1-coated glass surface when compared with the control glass plates. These findings suggest that DMP1 could be considered as a nucleator for apatite deposition in vitro.

Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo.

The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, "cross-talk" between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissue in vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.

Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca2+ store.

Dentin matrix protein 1 (DMP1) is a bone- and teeth-specific protein initially identified from mineralized dentin. Here we report that DMP1 is primarily localized in the nuclear compartment of undifferentiated osteoblasts. In the nucleus, DMP1 acts as a transcriptional component for activation of osteoblast-specific genes like osteocalcin. During the early phase of osteoblast maturation, Ca(2+) surges into the nucleus from the cytoplasm, triggering the phosphorylation of DMP1 by a nuclear isoform of casein kinase II. This phosphorylated DMP1 is then exported out into the extracellular matrix, where it regulates nucleation of hydroxyapatite. Thus, DMP1 is a unique molecule that initiates osteoblast differentiation by transcription in the nucleus and orchestrates mineralized matrix formation extracellularly, at later stages of osteoblast maturation. The data presented here represent a paradigm shift in the understanding of DMP1 function. This information is crucial in understanding normal bone formation, remodeling, fracture healing, and skeletal tissue repair.