Biomimetic Hydroxyapatite Crystallization in Gelatin Nanoparticles Synthesized Using a Miniemulsion Process

Here, we report a novel biomimetic strategy to synthesize hydroxyapatite (HAP) inside of crosslinked gelatin nanoparticles, which serve as a nanoenvironment for crystal growth in the aqueous phase. The synthesis of gelatin nanoparticles with the inverse miniemulsion technique is very intriguing because of the flexibility offered by the technique in tailoring the properties of the gelatin nanoparticles. It can be shown that the nanoenvironment promotes a different growth environment for the crystal because of the confinement inside the particle. The formation of HAP inside the particles follows Ostwald's rule of stages. At first an amorphous phase is formed, which itself has a great potential to be used as a resorbable bone substitute. This further transforms into single crystalline HAP via an octacalcium phosphate intermediate. The solution‐mediated transformation into the HAP phase without any calcination step is studied in detail using transmission electron microscopy (TEM) and X‐ray diffraction (XRD) measurements.     

Encapsulation of Nanoparticles Using Nitrilotriacetic Acid End‐Functionalized Polystyrenes and Their Application for the Separation of Proteins

The use of nitrilotriacetic acid end‐functionalized polystyrenes (NTA‐PS) as a multifunctional nanocarrier for the aqueous dispersion of CdSe, γ‐Fe₂O₃ and gold nanoparticles (NPs) is described. When the amphiphilic end‐ functionalized polystyrenes and NPs are dissolved together in tetrahydrofuran, the addition of water causes the spontaneous formation of micellar aggregates, resulting in the successful encapsulation and aqueous dispersion of NPs. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), photoluminescence (PL) spectroscopy, and vibrating sample magnetometer (VSM) are used to characterize the structure and properties of the NPs‐containing micellar aggregates (nanocarrier). After complexation of Ni²⁺ with NTA on the surface of the nanocarrier containing γ‐Fe₂O₃, specific binding between Ni‐NTA complex and histidine‐tagged (His‐tagged) proteins enables selective separation of His‐tagged proteins using a magnet.     

Structural Evolution of Self‐Assembling Nanohybrid Thin Films from Functionalized Urea Precursors

Hybrid organic‐inorganic thin films exhibiting patterned structuring on the nanometer scale have been prepared through the controlled hydrolysis‐condensation of enantiomerically pure chiral urea‐based silyl compounds. The thin films are obtained by spin‐coating of sols obtained via acid‐ or base‐catalyzed hydrolytic condensation of these molecular precursors. A self‐templating process is demonstrated via atomic force and transmission electron microscopy, showing the formation of nanometer size aggregates consisting of interconnected spherulates under acidic condition and of assembled fibers under basic conditions.     

Nanofiber Generation of Gelatin–Hydroxyapatite Biomimetics for Guided Tissue Regeneration

The development of biomimetic bone matrices is one of the major goals in the bone‐regeneration and tissue‐engineering fields. Nanocomposites consisting of a natural polymer and hydroxyapatite (HA) nanocrystals, which mimic the human bone matrix, are thus regarded as promising bone regenerative materials. Herein, we developed a biomimetic nanocomposite with a novel nanofibrous structure by employing an electrospinning (ES) method. The HA precipitate/gelatin matrix nanocomposites are lyophilized and dissolved in an organic solvent, and then electrospun under controlled conditions. With this process, we can successfully generate a continuous fiber with a diameter of the order of hundreds of nanometers. The internal structure of the nanofiber features a typical nanocomposite, i.e., HA nanocrystals well distributed within a gelatin matrix. These nanocomposite fibers improve the bone‐derived cellular activity significantly when compared to the pure gelatin equivalent. This method of generating a nanofiber of the biomimetic nanocomposite was effective in producing a biomedical membrane with a composition gradient, which is potentially applicable in the field of guided tissue regeneration (GTR).     

Magnetic Nanoparticle Chains in Gelatin Ferrogels: Bioinspiration from Magnetotactic Bacteria

Inspired by chains of ferrimagnetic nanocrystals (NCs) in magnetotactic bacteria (MTB), the synthesis and detailed characterization of ferrimagnetic magnetite NC chain‐like assemblies is reported. An easy green synthesis route in a thermoreversible gelatin hydrogel matrix is used. The structure of these magnetite chains prepared with and without gelatin is characterized by means of transmission electron microscopy, including electron tomography (ET). These structures indeed bear resemblance to the magnetite assemblies found in MTB, known for their mechanical flexibility and outstanding magnetic properties and known to crystallographically align their magnetite NCs along the strongest <111> magnetization easy axis. Using electron holography (EH) and angular dependent magnetic measurements, the magnetic interaction between the NCs and the generation of a magnetically anisotropic material can be shown. The electro‐ and magnetostatic modeling demonstrates that in order to precisely determine the magnetization (by means of EH) inside chain‐like NCs assemblies, their exact shape, arrangement and stray‐fields have to be considered (ideally obtained using ET).     

Biomimetic Formation of Hydroxyapatite Nanorods by a Single‐Crystal‐to‐Single‐Crystal Transformation

Uniform nanorods of hydroxyapatite (HAP) with an unusual orthorhombic shape have been synthesized from homogeneous solutions of Ca²⁺ and HPO₄^2– in the presence of gelatin and urea. The lengths of the nanorods are in the range of hundreds of micrometers, and the widths are about 100 nm. The HAP phase is generated by the transformation from its precursor phase of octacalcium phosphate (OCP), which has been monitored by X‐ray diffraction, NMR spectroscopy, scanning electron microscopy, and transmission electron microscopy. The rise in pH due to the decomposition of urea drives the OCP transformation to HAP. In the presence of gelatin, nanorods of OCP phase formed first and then transformed into the HAP phase, preserving the single‐crystal morphology. On the other hand, blade‐like OCP crystals form from the solution in the absence of gelatin. On increasing the pH of the solution, the large, blade‐like OCP crystals tend to crash into irregular, hexagonal HAP crystallites. A single‐crystal‐to‐single‐crystal topochemical transformation may be attributed to the evolution of HAP nanorods from the precursor OCP phase. This gives a strong indication as to the OCP to HAP transformation mechanism in the mineralization of biological apatite in tooth enamel and bone.     

Facilitation of Gene Transfection and Cell Adhesion by Gelatin‐Functionalized PCL Film Surfaces

Efficient local gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration. In this work, the gelatin‐functionalized polycaprolactone (PCL) film surfaces are prepared via surface‐initiated atom transfer radical polymerization of glycidyl methacrylate. The resultant covalent attachment of gelatin could enhance the cell‐adhesion and local gene transfection properties. The gelatin‐functionalized PCL film surfaces exhibit excellent cell‐adhesion ability to both adherent and suspension cells. The attached adherent cells demonstrate the characteristic elongated morphologies with good spreading capability, while the attached suspension cells can maintain the original status of the round morphologies without spreading. More importantly, the gelatin coupled on the PCL surface could be used to absorb the cationic vector/plasmid deoxyribonucleic acid (pDNA) complexes via electrostatic interaction. The local gene transfection property on the immobilized cells is dependent on both the density of the immobilized cells and the loading types of pDNA complexes. The transfection efficiency of different assemble methods of pDNA complex was compared. With the pre‐ and post‐loading sandwich‐like gene transfection, the gelatin‐functionalized PCL film surface can substantially enhance the transfection properties to different cell lines. The present study is very useful to spatially control local gene delivery within PCL‐based tissue scaffolds.     

A new model for PCBM phase segregation in P3HT:PCBM blends

The phase segregation in P3HT:PCBM blend films has been investigated from an experimental and theoretical viewpoint. Optical microscopy, atomic force microscopy, scanning electron microscopy and X-ray diffraction show that thermal annealing of P3HT:PCBM blend films leads to the formation of PCBM aggregates. These aggregates are composed of dense randomly packed ∼50 nm PCBM crystallites with an overall aggregate density of ∼0.85 g cm⁻³. By applying the critical radius of nucleation for PCBM and the Stokes-Einstein equation for mobility of PCBM in a P3HT matrix, a model is developed which explains the formation of both crystallites and aggregates.     

Embryonic States of Fluorapatite–Gelatine Nanocomposites and Their Intrinsic Electric‐Field‐Driven Morphogenesis: The Missing Link on the Way from Atomistic Simulations to Pattern Formation on the Mesoscale

The shape development of fluorapatite (FAP)–gelatine nanocomposites is revealed by means of HRTEM investigations starting from molecular dimensions up to the formation of mesoscaled (elongated) hexagonal prisms. The composite nature of the aggregates is proved by IR spectroscopy and by chemical analyses on all states of shape development. The initial states are characterized by triple‐helical fiber protein bundles, which are mineralized step‐by‐step forming and fixing nanoplatelets of FAP in a mosaic arrangement. After being fully mineralized the bundles form elongated composite nanoboards. In the next step of the growth process the boards aggregate to bundles of boards which are in a more or less parallel alignment with respect to each other. By adding up more and more composite nanoboards a critical size is reached and an electric field is developed, which takes over control and directs the further development of the aggregates. This kind of electric‐field‐directed growth of the elongated polar nanoboards additionally leads to the formation and inclusion of protein nanofibrils into the growing composite aggregate. By this method, cone‐like nanofibril structures develop along the long axis of the aggregates accompanied by more perfect parallel alignment of the composite boards within the aggregates. Further shape development is characterized by adding up composite nanoboards, in particular to increase the third dimension in volume. This thickening process preferably takes place in the middle part of the elongated aggregates and finally proceeds to their basal ends until a perfect hexagonal prismatic seed is formed, which then is ready for further shape development on the micrometer scale.     

Tissue Engineering: Scaffold‐Mediated 2D Cellular Orientations for Construction of Three Dimensionally Engineered Tissues Composed of Oriented Cells and Extracellular Matrices (Adv. Funct. Mater. 7/2009)

Various hydrogels, such as poly(γ‐glutamic acid) (γ‐PGA), gelatin (GT), alginic acid (Alg), and agarose (Aga), with 3D interconnected and oriented fibrous pores (OP gels) are prepared for 3D polymeric cellular scaffolds by using silica fiber cloth (SC) as template. After the preparation of these hydrogels with the SC templates, the latter are subsequently removed by washing with hydrofluoric acid solution. Scanning electron microscopy (SEM) clearly shows OP structures in the hydrogels. These various types of OP gels are successfully prepared in this way, independently of the crosslinking mechanism, such as chemical (γ‐PGA or GT), coordinate‐bonded (Alg), or hydrogen‐bonded (Aga) crosslinks. SEM, confocal laser scanning microscopy, and histological evaluations clearly demonstrate that mouse L929 fibroblast cells adhere to and extend along these OP structures on/in γ‐PGA hydrogels during 3D cell culture. The L929 cells that adhere on/in the oriented hydrogel are viable and proliferative. Furthermore, 3D engineered tissues, composed of the oriented cells and extracellular matrices (ECM) produced by the cells, are constructed in vitro by subsequent decomposition of the hydrogel with cysteine after 14 days of cell culture. This novel technology to fabricate 3D‐engineered tissues, consisting of oriented cells and ECM, will be useful for tissue engineering.     

Scanning electron microscopy subsequent to a combined treatment of NaOCl and EDTA in some non-collagenous calcified matrixes

Using backscattered electron (BSE) imaging and scanning electron microscopy, subsequent to a combined treatment of sodium hypochlorite (NaOCl) and ethylenediamine tetra-acetic acid (EDTA) or only with EDTA etching, we observed some structures of non-collagenous calcified matrixes with the aim of revealing the correlation of deposition between calcification degree and organic amount. In human tooth enamel, the NaOCl-EDTA method eroded more intensively the hypocalcified prisms of enamel tufts containing a relatively large amount of EDTA-insoluble organic matter than the hypercalcified normal prismatic enamel containing a small amount of the organic matter. Afibrillar cementum, one of the non-collagenous calcified tissues similar to the enamel, has been reported to consist of organic-rich and poor incremental lamellae. The BSE imaging showed an alternation pattern of hypocalcification and hypercalcification. The hypocalcified lamellae were retained by EDTA etching, while the hypercalcified lamellae showed a resistance against the NaOCl-EDTA method. In the non-collagenous calcareous concretions of human pineal body, organic-rich and poor, and hyper- and hypocalcified incremental lamellae have been reported. The deposition pattern of calcification degree and organic amount was similar to that in afibrillar cementum, and the hypercalcified lamellae showed a resistance against the NaOCl-EDTA method. In conclusion, the high and the lower calcified regions of non-collagenous calcified matrixes contained smaller and larger amounts of EDTA-insoluble organic matter respectively. Moreover, scanning electron microscopy subsequent to the NaOCl-EDTA method corresponding to the BSE imaging clearly showed fine calcified structures compared with the BSE imaging.     

Gold Nanoparticles Stabilized by Acetylene‐Functionalized Multidentate Thioether Ligands: Building Blocks for Nanoparticle Superstructures

Aiming at the formation of inorganic/organic hybrid gold nanoparticle superstructures, the design and synthesis of acetylene‐monofunctionalized multidentate thioether ligands and their ability to stabilize gold nanoparticles are presented. Rather monodisperse gold particles with diameters of about 1 nm are obtained, which are coated by a small number of ligands, each comprising a silyl‐protected acetylene. The acetylene is attached at the end of a rigid ethynylene‐phenylene unit of variable length, leading to functionalized gold nanoparticles carrying acetylenes at different distances from the nanoparticle surface. These particles are interlinked by diacetylene formation and are investigated by transmission electron microscopy and UV/vis spectroscopy, revealing the formation of nanoparticle aggregates and small superstructures such as dimers or trimers while the nanoparticles themselves retain their integrity. The interparticle distance in small nanoparticle superstructures reflects the ethynylene‐phenylene spacer length corroborating the wet chemical interlinking as the origin of these organic/inorganic hybrid structures.     

Bioinspired Hydrogel Electrospun Fibers for Spinal Cord Regeneration

Fully simulating the components and microstructures of soft tissue is a challenge for its functional regeneration. A new aligned hydrogel microfiber scaffold for spinal cord regeneration is constructed with photocrosslinked gelatin methacryloyl (GelMA) and electrospinning technology. The directional porous hydrogel fibrous scaffold consistent with nerve axons is vital to guide cell migration and axon extension. The GelMA hydrogel electrospun fibers soak up water more than six times their weight, with a lower Young's modulus, providing a favorable survival and metabolic environment for neuronal cells. GelMA fibers further demonstrate higher antinestin, anti‐Tuj‐1, antisynaptophysin, and anti‐CD31 gene expression in neural stem cells, neuronal cells, synapses, and vascular endothelial cells, respectively. In contrast, anti‐GFAP and anti‐CS56 labeled astrocytes and glial scars of GelMA fibers are shown to be present in a lesser extent compared with gelatin fibers. The soft bionic scaffold constructed with electrospun GelMA hydrogel fibers not only facilitates the migration of neural stem cells and induces their differentiation into neuronal cells, but also inhibits the glial scar formation and promotes angiogenesis. Moreover, the scaffold with a high degree of elasticity can resist deformation without the protection of a bony spinal canal. The bioinspired aligned hydrogel microfiber proves to be efficient and versatile in triggering functional regeneration of the spinal cord.     

Design Principle of Conjugated Polyelectrolytes to Make Them Water‐Soluble and Highly Emissive

The correlation between the molecular design of a conjugated polyelectrolyte (CPE) and its aggregated structure and the emissive properties in water is systematically investigated by means of UV–vis spectrometry, fluorescence spectroscopy, and scanning/transmission electron microscopy. Five different and rationally designed CPEs having carboxylic acid side chains are synthesized. All five conjugated polyelectrolytes are seemingly completely soluble in water in visual observation. However, their quantum yields are dramatically different, changing from 0.45 to 51.4%. Morphological analysis by electron microscopy combined with fluorescence spectrophotometry reveals that the CPEs form self‐assembled aggregates at the nanoscale depending on the nature of their side chains. The feature of the self‐assembled aggregates directly determines the emissive property of the CPEs. The nature and the length of the spacer between the carboxylic acid group and the CPE backbone have a strong influence on the quantum yield of the CPEs. Our study demonstrates that bulky and hydrophilic side chains and spacers are required to achieve complete water‐solubility and high quantum yield of CPEs in water, providing an important molecular design principle to develop functional CPEs.     

Cover Picture: Preparation of Rectangular WO3·H2O Nanotubes Under Mild Conditions (Adv. Funct. Mater. 11/2007)

The synthesis of WO₃ · H₂O nanotubes under mild conditions is reported by Limin Wu and co‐workers, of Fudan University, P.R. China on p. 1790. The synthesis is carried out with the aid of polyaniline (PANI). The PANI molecules are intercalated into tungsten oxide layers to provide a driving force for the formation of nanotubes from nanosheets. By this approach, the nanosheets can be directly rolled into nanotubes. This method could be applied to many other materials that possess layered/lamellar structures for forming nanotubes. WO₃·H₂O nanotubes are successfully synthesized with the aid of intercalated polyaniline (PANI) under relatively mild conditions. More interestingly, the WO₃·H₂O nanotubes have a rectangular cross‐section structure formed through a rolling mechanism. Scanning electron microscopy, transmission electron microscopy, X‐ray diffraction, thermogravimetric analysis, Fourier‐transform infrared analysis, UV‐vis‐near‐IR spectroscopy, selected‐area electron diffraction, and vibrating‐sample magnetometry analysis are employed to characterize the morphology, structure, and properties of the nanotubes.     

Electron microscopical evaluation of connective tissue healing to periodontally involved teeth.

The present study was undertaken to examine if our novel root treatment for human periodontally involved teeth can facilitate a new connective tissue attachment. The novel treatment was in two parts: 1) planning and surface decalcification with citric acid on the coronal quarter of the exposed root corresponding to pocket, and 2) curettage of superficial cementum of the apical three quarters of the exposed root. The interface of the root surface and regenerating connective tissue was observed 2 and 3 weeks after surgery by light and electron microscopy. The apical migration of junctional epithelium was effectively prevented. The fibroblasts synthesized new fibrillar materials and collagen fibrils towards the curetted cementum. The results suggested the possibility that this treatment for periodontally involved teeth would provide ideal root surface conditions for the formation of new fibrous attachment.     

Protein‐Mediated Synthesis of Uniform Superparamagnetic Magnetite Nanocrystals

Magnetite nanocrystals are synthesized in the presence of a recombinant Mms6 protein thought to be involved in the biomineralization of bacterial magnetite magnetosomes, the mammalian iron‐storage protein, ferritin, and two proteins not known to bind iron, lipocalin (Lcn2) and bovine serum albumin (BSA). To mimic the conditions at which magnetite nanocrystals are formed in magnetotactic bacteria, magnetite synthesis is performed in a polymeric gel to slow down the diffusion rates of the reagents. Recombinant Mms6 facilitates formation of ca. 30 nm single‐domain, uniform magnetite nanocrystals in solution, as verified by using transmission electron microscopy analysis and magnetization measurements. The nanocrystals formed in the presence of ferritin, Lcn2, and BSA, do not exhibit the uniform sizes and shapes observed for those produced in the presence of Mms6. Mms6‐derived magnetite nanoparticles show the largest magnetization values above the blocking temperature, as well as the largest magnetic susceptibility compared to those of the nanomaterials synthesized with other proteins. The latter is indicative of a substantial effective magnetic moment per particle, which is consistent with the presence of magnetite with a well‐defined crystalline structure. The combination of electron microscopy analysis and magnetic measurements confirms our hypothesis that Mms6 promotes the shape‐selective formation of uniform superparamagnetic nanocrystals. This provides a unique bioinspired route for synthesis of uniform magnetite nanocrystals.     

Tuning the Dimensions of C60‐Based Needlelike Crystals in Blended Thin Films

A new ordered structure of the C₆₀ derivative PCBM ([6‐6]‐phenyl C₆₁‐butyric acid methyl ester) is obtained in thin films based on the blend PCBM:regioregular P3HT (poly(3‐hexylthiophene)). Rapid formation of needlelike crystalline PCBM structures of a few micrometers up to 100 μm in size is demonstrated by submitting the blended thin films to an appropriate thermal treatment. These structures can grow out to a 2D network of PCBM needles and, in specific cases, to spectacular PCBM fans. Key parameters to tune the dimensions and spatial distribution of the PCBM needles are blend ratio and annealing conditions. The as‐obtained blended films and crystals are probed using atomic force microscopy, transmission electron microscopy, selected area electron diffraction, optical microscopy, and confocal fluorescence microscopy. Based on the analytical results, the growth mechanism of the PCBM structures within the film is described in terms of diffusion of PCBM towards the PCBM crystals, leaving highly crystalline P3HT behind in the surrounding matrix.     

Exfoliation of Threading Dislocation‐Free,Single‐Crystalline,Ultrathin Gallium Nitride Nanomembranes

Despite the recent progress in gallium nitride (GaN) growth technology, the excessively high threading dislocation (TD) density within the GaN crystal, caused by the reliance on heterogeneous substrates, impedes the development of high‐efficiency, low‐cost, GaN‐based heterostructure devices. For the first time, the chemical exfoliation of completely TD‐free, single‐crystalline, ultrathin (tens of nanometers) GaN nanomembranes is demonstrated using UV‐assisted electroless chemical etching. These nanomembranes can act as seeding layers for subsequent overgrowth of high‐quality GaN. A model is proposed, based on scanning and transmission electron microscopy as well as optical measurements to explain the physical processes behind the formation of the GaN nanomembranes. These novel nanomembranes, once transferred to other substrates, present a unique and technologically attractive path towards integrating high‐efficiency GaN optical components along with silicon electronics. Interestingly, due to their nanoscale thickness and macroscopic sizes, these nanomembranes may enable the production of flexible GaN‐based optoelectronics.     

Novel Europium‐Complex/Nitrile‐ Butadiene Rubber Composites

The novel europium complex acrylato(1,10‐phenanthroline)bis(2‐thenoyltrifluoroacetonato)europium(III ) [Eu(tta)₂(aa)(phen)] [Eu‐AAPhen; Htta = 4,4,4‐trifluoro‐1‐(2‐thienyl)‐1,3‐butanedione, Haa = acrylic acid, phen = 1,10‐phenanthroline], which combines the excellent fluorescence properties of [Eu(tta)₃(phen)] and the reactivity of acrylic acid with radicals, has been synthesized. Various amounts of this complex (powder) are mixed with nitrile‐butadiene rubber (NBR) and peroxide to form uncured composites. These composites are vulcanized to obtain cured Eu‐AAPhen/NBR composites. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations show that the dispersion dimension for the cured composites is far finer than that for the uncured composites. Wide‐angle X‐ray diffraction (WAXD) experiments show that the crystallinity of Eu‐AAPhen in the composite decreases dramatically after the crosslinking process, implying that in‐situ reactions (including polymerization and grafting) of Eu‐AAPhen initiated by peroxide radicals should take place during the crosslinking of the NBR matrix with peroxide. The dispersion phase of the cured Eu‐AAPhen/NBR composite should be composed of nearly nanometer‐sized aggregates of poly(Eu‐AAPhen) and residual Eu‐AAPhen particles with reduced dimensions. The fluorescence properties of the cured composite are much better than those of the uncured one.