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Background: Anti-sclerostin antibodies are among the most efficient drugs for the treatment of osteoporosis, and have been also expected for the treatment of local bone disorders. We have previously developed porous microparticles of hydroxyapatite and chondroitin sulfate loading anti-sclerostin antibodies formulated with zinc cations. However, the biological behavior and concentration dependence of anti-sclerostin antibodies in vitro released from the microparticles remain unclear. Objective: Bolus administration and the subsequent release of anti-sclerostin antibodies from the microparticles formulated with or without zinc cations were investigated; bone-resorptive inhibitory effects on mouse MC3T3-E1 osteoblast function were revealed by cell culture using a cell culture insert plate. Methods: Differentiation induction culture of osteoblasts was performed after maintaining the concentrations of anti-sclerostin antibodies and sclerostin at previously reported concentrations of 5.0 and 1.0 µg/mL for the first 3 days. Subsequently, the medium was replaced with fresh medium that did not contain anti-sclerostin antibodies but microparticles loading anti-sclerostin antibodies (20 or 80 µg/mg) with or without zinc cations in the cell culture insert. After 11 days of incubation, the bioactivity of the osteoblasts was evaluated using the polymerase chain reaction method. Results: The formulation using zinc cations showed an increase of anti-sclerostin antibodies released from the microparticles, which increased the expression of receptor activator of the nuclear factor kappa-B ligand in the osteoblasts on day 14. This result indicates the inhibition of sclerostin-mediated bone resorption. However, the increase of loading amounts of anti-sclerostin antibodies extremely enhanced the subsequent release of anti-sclerostin antibodies, which decreased the inhibition of bone resorption contrary to expectations. Conclusion: The moderately sustained release of anti-sclerostin antibodies from the microparticles can promote the inhibition of bone resorption in osteoblasts, supporting the potential of this formulation for the treatment of localized bone disorders.

In this study, three types of porous gelatin/HAp composite-based scaffolds were prepared and their microstructure, mechanical property, and in vitro biocompatibility were evaluated. FD-gelatin/HAp scaffolds were prepared via a conventional freeze-drying (FD) method, while DCPD-bound gelatin/HAp (DCPD-gelatin/HAp) microsphere scaffolds and HAp-bound gelatin/HAp (HAp-gelatin/HAp) microsphere scaffolds were prepared via the combined method of emulsion technique and novel microsphere-packing method. It was found that the microstructure and mechanical property of porous gelatin/HAp scaffolds were highly dependent on the preparation method and type of scaffolds. FD-gelatin/HAp scaffolds had few pores with a comparatively flat surface and had a compressive strength was 24 ± 1 MPa. DCPD-gelatin/HAp microsphere scaffolds had a porous structure with the average pore size of 121 ± 12 μm and had the compressive strength of 50 ± 3 MPa. HAp-gelatin/HAp microsphere scaffolds also had a porous structure with the average pore size of 190 ± 26 μm and had the compressive strength of 17 ± 4 MPa. In vitro biocompatibility was performed by culturing the osteoblast-like MC3T3-E1 cells with all types of scaffolds. The results showed that all types of scaffolds were biocompatible to support cell attachment and proliferation. However, a significant difference in cell behavior was clearly observed. Most cells were grown on the surface of FD-gelatin/HAp scaffolds due to the lack of the porous structure, while those were grown on the surface and inside the pore of both DCPD-gelatin/HAp microsphere scaffolds and HAp-gelatin/HAp microsphere scaffolds due to the presence of the porous structure. Thus, the porous gelatin/HAp microsphere-based scaffolds have a potential as novel bone tissue regenerative materials.

Apatite is a biocompatible material widely used to encapsulate iron-oxide nanoparticles (IONPs) for biomedical applications, such as drug-delivery or fluorescent probe agent. Apatite-coated IONPs are commonly fabricated by initially incubating carboxylate-functionalized IONPs in calcium solution and directly adding phosphate solution to initiate apatite precipitation (direct-addition method). Apatite precipitation took place not only on IONPs surface but also in the bulk solution, resulting in apatite-IONPs mixture instead of coated structure. In this study, robust apatite-coated IONPs structure were aimed by modifying steps in direct-addition method. Initially, carboxylate-functionalized IONPs were incubated in calcium solution, physically separated from the incubating calcium solution by external magnet, and then separately reacted with phosphate solution to induce apatite deposition (alternate-addition method). Fourier-transform infrared (FTIR) analysis showed that a calcium solution at a concentration of 0.8 mol/L was required to initiate the formation of the calcium-carboxylate (Ca-COO) complex. The formation of non-stoichiometric apatite was confirmed for IONPs with Ca-COO complex, as evidenced by X-ray diffraction and FTIR analysis. The alternate-addition method produced apatite coating in the form of flake-like structures, which also exhibited strong adhesion to IONPs surface. In contrast, direct-addition method mainly produced agglomerate of apatite particles that weakly associated with IONPs. Both of apatite-coating methods did not alter the magnetic properties of IONPs. The simple modification of reaction steps in the widely used apatite-coating method was demonstrated to be beneficial in producing robust apatite-coated IONPs structure.

Multi-layered scaffolds of apatite and collagen for the healing of osteochondral defects have been investigated for optimized regenerative cues by distinct layers; however, multi-layered scaffolds exhibit delamination after implantation in the host body. In this study, as an alternative to multi-layered scaffolds, collagen sponges with an apatite content gradient were fabricated via an electrolytic method. Electrolysis at a constant current mode of i = 22 A m−2 with different electrode distances of 2, 3, or 4 cm was conducted for an acidic solution at pH 3.0, including collagen at 0.5 wt% and hydroxyapatite at 0.1 wt%. During electrolysis, a high pH pocket and collagen hydrogel were concurrently formed in the vicinity of the cathode, whereas a low pH pocket was formed near the anode due to the generation of hydroxyl and hydrogen ions, respectively. The thickness of the hydrogels gradually grew to approximately one-third of the electrode distance. Subsequently, the hydrogels were freeze-dried to form sponges. Low-crystalline apatite was precipitated, and particularly, the hydrogel region closest to the cathode was more mineralized than the region farthest from the cathode. The sponge region closest to the cathode exhibited a ratio of apatite/collagen of 0.34 ± 0.04, which gradually decreased with the distance from cathode to 0.22 ± 0.04, 0.20 ± 0.05, and 0.14 ± 0.02. The biocompatibility of collagen was unaltered by the electrolysis treatment, as evidenced by the attachment and proliferation of pre-osteoblastic cells in the electrolyzed sponges. In conclusion, the electrolysis method is a powerful tool for the fabrication of collagen sponges with a continuous graded apatite composition.

Lidocaine has been used as a local anesthetic by injection. The controlled release of lidocaine loaded into nanospheres is necessary to reduce the onset time of the anesthetic effect or increase the anesthetic analgesia duration. In this study, mesoporous silica nanoparticles (MSNs) with a large specific surface area were prepared by a sol-gel method, and the interfacial interaction between MSNs and lidocaine positively charged in aqueous solutions at different concentrations was investigated by adsorption tests, Fourier-transformed infrared spectroscopy, thermogravimetry-differential thermal analysis, and Brunauer-Emmett-Teller (BET) measurements. The electrostatic interaction between Si-OH on MSNs and lidocaine-NH+ was of importance for the adsorption phenomenon in aqueous solutions, indicating the monolayer adsorption of lidocaine. BET measurements also supported the decrease of pore volumes, and the hysteresis loop of the isotherm curve was not closed since the condensation of lidocaine in the mesopores formed micropores of less than 1.5 nm in size. The release profiles in phosphate buffered saline containing calcium and magnesium ions showed a rapid and higher release of lidocaine compared with that in phosphate buffered saline without divalent cations. The released lidocaine concentrations were sufficient for the expression of the anesthetic effect in dental anesthesia.

International audience; Major progress in the field of regenerative medicine is expected from the design of artificial scaffolds that mimic both the structural and functional properties of the ECM. The bionanocomposites approach is particularly well fitted to meet this challenge as it can combine ECM-based matrices and colloidal carriers of biological cues that regulate cell behavior. Here we have prepared bionanocomposites under high magnetic field from tilapia fish scale collagen and multifunctional silica nanoparticles (SiNPs). We show that scaffolding cues (collagen), multiple display of signaling peptides (SiNPs) and control over the global structuration (magnetic field) can be combined into a unique bionanocomposite for the engineering of biomaterials with improved cell performances. View Full-Text

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.

An artificial bone that mimics the chemical composition of biological apatite is one of the promising implants for the treatment of bone defects and is useful as a scaffold for tissue-engineered bone. There are three types of carbonate substitution in hydroxyapatite (HAp) at hydroxyl groups (A-type), phosphate groups (B-type), and both groups (AB-type). Less is known about the role of A-type carbonate apatite (CAp) in influencing the bone regeneration process and there are no comparative studies of these apatites. The present study aims to elucidate osteoconductivity and degradability of porous sintered bodies of stoichiometric HAp, A-type CAp (A-CAp), and B-type CAp (B-CAp) with a constant carbonate content at 5.0 wt%. Both CAp with 75% porosity showed similar ionic dissolution rates which were faster than HAp with 67% porosity. A-CAp had the largest affinity for albumin from time-dependent adsorption tests. The lowest attachment of MC3T3-E1 cells was observed for A-CAp; however, the proliferation rate was similar of both CAp, which was lower than HAp. Early osteoblastic marker (alkaline phosphatase activity) was significantly upregulated for CAp at day 3 relative to HAp. After two weeks of implantation, A-CAp and HAp showed scarce bone formation encapsulated by fibrous tissue. In contrast, B-CAp showed significant bone ingrowth and closely adjoined to the surrounding bone tissue. The rapid degradation of CAp was attributed to osteoclast activities, in which A-CAp showed better capacity in promoting osteoclast maturation. It is concluded that a specific carbonate substitution type significantly impacts osteoconductivity or degradability of artificial bones.

Conducting porous substrates of high hydrophilicity are advantageous in applications of electrochemical biosensors as host electrodes, offering not only fast charge transport and large sensing surface areas but also necessary wettability in aqueous analytes. In this study, inexpensive, highly hydrophilic (contact angle < 5°), conducting (sheet resistance of 17 Ω □−1) porous fluorine-doped tin oxide (FTO) glass is fabricated from commercial FTO glass with a novel, simple, one-step Sn4+-based anodic treatment process, and used as a host electrode for electrochemical biosensors. We demonstrate its superior performance with hydrogen peroxide sensing. The hydrogen peroxide sensor is fabricated by simply depositing Pt nanoparticles onto the surface of the porous FTO substrate (PFTO) with a polyol process. Pt-decorated commercial FTO (CFTO) is also investigated as a control. The sensitivity achieved with the Pt-decorated PFTO is almost one order of magnitude higher than that of the Pt-decorated CFTO (25.8 vs. 2.69 mA M−1), and the response time is shortened from 36 s for the Pt-decorated CFTO to 1 s for the Pt-decorated PFTO. The PFTO proves to be a promising electrode substrate for host functional materials in electrochemical biosensors and can be readily applied to sensing of a wide variety of biosubstances.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

BACKGROUND: Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of ‘matured’ constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. METHODS: Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. RESULTS: Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. CONCLUSION: Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.

Alpha-alumina is a ceramic with excellent chemical stability, mechanical property, high melting point, and insulating property; however, it shows poor workability due to its low fracture toughness. There are a lot of molding processes for a-alumina, such as press molding and extrusion methods. 3D printing is rapidly growing technology to make complex and precious moldings. However, there are still only a few descriptions on 3D ink-jet powder laminating printings for a-alumina due to no self-hydration hardening property. To achieve a-alumina moldings with the 3D ink-jet printers, powder fluidity of α-alumina powders and binders for bonding the powders should be investigated. The powders mixed with a-alumina at 20, 3.4 and 0.4 µm in sizes were adjusted to improve powder fluidity and packing density. Polyvinyl alcohol (PVA) or polyallylamine (PAA) including other additives was used as an ink. The addition of PVA on the adjusted powders made no chemical interaction of powders and no retention of shapes, but that of PAA formed the printed moldings. The relative packing density and compressive strength of the printed moldings were 40 % and 8.2 kPa, which was clearly depended on the printed directions due to the nozzle structure of printer head. Sintering the moldings at 1500°C caused near-net zero shrinkage and improved the maximum compressive strength at 3.6 MPa.

Mesoporous silica thin films were fabricated on gold substrates with a sol-gel method including a cationic surfactant. The interface of gold and silica thin films was fixed with a mercaptosilane. The surface roughness of the thin films and their grain sizes of silica were both increased with the elimination of the cationic surfactant and polyethylene glycol (PEG) modification on its surface. The surface reaction, adsorption of bovine serum albumin (BSA), was monitored with surface plasmon resonance (SPR). Although the adsorption amount of BSA at 1 mg/mL were 7.9 ng/cm2 on the mesoporous silica, the PEG modification can completely desorb BSA. The mesoporous silica thin films on gold substrates fabricated will be a good platform for analyzing surface reaction.

Medical products comprised of devices and drugs have been known as a combination product. The biodegradable collagen (Col) sponges impregnated with recombinant human bone morphogenetic protein can make bone formation hasten. It is expected further features by a combination of various growth factors and artificial bone materials. The binding properties of such factors to Col and hydroxyapatite (HAp) have not been elucidate to achieve a controlled release. In this study, we investigated artificial periosteum-like membranes made from tilapia fish Col and HAp composites as a drug carrying support. The Col-HAp composites with three different compositions in weight ratio of 2:8, 5:5 and 8:2 were made and crosslinked by irradiation of gamma-ray in wet condition. The tensile strengths of the membranes in wet or dry were depended on the compositions; however the strengths of the membranes in wet were apparently weaker at 1/10 or less than those in dry at a maximum 90 MPa. The adsorption ability of proteins, bovine serum albumin (BSA) or lysozyme (LSZ), on the membranes exhibited different tendency; the membranes including higher weight ratio of HAp adsorbed BSA rather than LSZ. These results indicated that the artificial Col-HAp membranes would be the suitable materials for biomedical device as combination products.

For the development of next-generation protein-based biosensor surfaces, it is important to understand how functional proteins, such as fibrinogen (FBG), interact with polar substrate surfaces in order to prepare highly sensitive points of medical care diagnostics. FBG, which is a fibrous protein with an extracellular matrix, has both positively and negatively charged regions on its 3-dimensional surface, which makes interpreting how it effectively binds to polarized surfaces challenging. In this study, single-crystal LiNbO3 (LNO) substrates that have surface charges were used to investigate the adsorption of FBG protruding polar fragments on the positively and negatively charged LNO surfaces. We performed a combination of experiments and multi-scale molecular modeling to understand the binding of FBG in vacuum and water-solvated surfaces of LNO. XPS measurements showed that the FBG adsorption on LNO increased with increment in solution concentration on surfaces independent of charges. Multi-scale molecular modeling employing Quantum Mechanics, Monte Carlo, and Molecular Mechanics addressed the phenomenon of FBG fragment bonding on LNO surfaces. The binding simulation validated the experimental observation using zeta potential measurements which showed presence of solvated medium influenced the adsorption phenomenon due to the negative surface potential.

The antibacterial properties are useful to restrain inflammatory response caused by bacterial infection after implantation. The composites of hydroxyapatite (HAp) and silver nano-dots, silver oxide or silver phosphate have been investigated; however there are still some disadvantage in sintering; 1) silver nano-dots grow large, and are not homogenously distributed, 2) silver nano-dots melt and remove, and 3) silver phosphate and silver oxide formed exhibit higher solubility than metal silver. In this study, the distribution of silver nano-dots in HAp microparticles sintered was controlled at grain boundary with a modified silver mirror reaction as a novel route. HAp microparticles adsorbed formaldehyde by a vapor deposition method were soaked in an ammoniacal silver nitrate solution and were then sintered. There was a single phase of HAp including metal silver at 6.4 wt% even after sintering. The silver nano-dots were homogeneously distributed inside the microparticles. The release profiles of silver ions in phosphate buffer saline were compared with a reference; the HAp microparticles were soaked into silver nitrate solution and were then sintered. The distribution of silver in the reference was not homogeneous and large silver microparticles were grown outside the particles at 6.3wt%. The elution amount of silver ions from the microparticles at 12 hours was one-eighteenth of that from the reference. These results suggest that the HAp microparticles including silver nano-dots at grain boundary will be suitable for a long-term antibacterial material.

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.

Combination of silica component with other materials is one of the current strategies to design bone regenerative materials. In this study, novel reduced graphene oxide (RGO)-aminosilica hybrid nanosheets with enhanced osteo-compatibility were synthesized from a mixture of 3-aminopropyltriethoxysilane (APTES), graphene oxides (GO) and water. The presence of APTES in the mixture not only caused the conversion of GO to RGO, but also led to the hydrolysis and condensation of itself. It was for the first time reported the reducing role of APTES in the conversion of GO to RGO. It was found that the silicon (IV) ions were released from the hybrid nanosheets in a sustained way. The in vitro osteo-compatibility was evaluated by incubating the hybrid nanosheets with osteoblast MC3T3-E1 cells. A water soluble tetrazolium salt assay quantitatively indicated that the hybrid nanosheets had no significant toxicity and exhibited good biocompatibility. An alkaline phosphatase assay quantitatively indicated that the hybrid nanosheets enhanced the osteoblast differentiation compared to the GO nanosheets. An immunochemical assay further qualitatively indicated that the hybrid nanosheets stimulated the production of osteopontin as typical marker for osteoblast differentiation. Thus, the resultant hybrids nanosheets had a potential application in the bone regeneration.

Calcium phosphate cements show self-hardening reaction upon mixing with liquids to form calcium-deficient hydroxyapatite (CDHA) or dicalcium phosphate dihydrate. The effects of particle sizes, crystallinities, and natural polymers such as tilapia scale collagen (Col) and hyaluronic acid as a dispersant on the mechanical properties of alpha tricalcium phosphate (TCP) cements mixed with citric acid (CA) as an additive were investigated. Three types of alpha TCP particles were fabricated with spray-dry (SD; 14 μm), freeze-dry (FD; 45 μm), and cold isostatic press (CIP; 134 μm) methods, followed by sintering at 1300°C and ground/crushed. The amounts of Ca dissolution from these particles were in the order of SD > FD > CIP. The CA liquid was added to the powders of SD-FD or SD-CIP, and kneaded under different liquid/powder ratios. The cements containing CIP particles showed lower compressive strength at 22.9 ± 1.5 MPa than those containing FD particles at 28.3 ± 2.5 MPa, even though the apparent densities of the cements containing CIP material was higher. Although the packing density of powders is an important factor on the mechanical properties of cements, the dissolution of Ca ion has a greater impact on the mechanical properties. The addition of Col into the cements increased the mechanical properties at 33.6 ± 2.5 MPa at 1 day to enhance the re-precipitation of CDHA.

Detection of volatile organic compounds (VOCs) emitted from cancerous tumor cells in exhaled human breath allows for early diagnosis of various types of cancers. 3D graphene with a large surface area is considered a suitable material for creating novel sensitive VOCs sensors. In this study, 3D graphene and 3D graphene oxide were synthesized from graphene oxide suspension, hydroquinone and formaldehyde by employing polymerization and reduction. The capability of VOC gas sensing was evaluated by measuring the electrical current response in flowing N2 gas over a range of concentrations of acetone or 1-butanol at room temperature. It was observed that the device current correlated well with the VOC concentration. The adsorption of acetone decreased the current, but the adsorption of 1-butanol increased the current during sensing. 3D graphene oxide device was more sensitive than 3D graphene device because of the high concentration of oxygen-containing functional groups on the surface. These results indicated that 3D graphene and 3D graphene oxide may be the suitable materials for VOCs sensing devices.

A reverse micro-emulsion method has been investigated to control crystal morphology in a nanometer region and to increase specific surface area for calcium phosphate. The nanocrystals with the control of its morphology is a candidate of drug delivery carriers. This study investigated the effects of mixing volume ratios of two surfactants, tween80 (T) and aliquate 336 (A) in kerosene as an oil phase, and pH values in the nano-region on crystalline phases and specific surface area of calcium phosphate synthesized by the reverse micro-emulsion method. A di-ammonium hydrogen phosphate solution including phosphoric acid at pH of 6.3 and a calcium nitrate solution at pH of 5.7 were adjusted, and both the solutions were separately added into the kerosene with the surfactants. Both the emulsions were then mixed at the same volume and the Ca/P ratio of 1.0, and stirred at room temperature for 24 hours. The crystalline phases were dependent on the T amounts; pure DCPD with the specific surface area of 6.7 to 12 m2/g was obtained at the T/A ratio of 4, the mixture ofDCPD and DCPA with that of 48 to 162 m2/g was at the ratios of 5 to 8, and a low crystalline HAp with 163 m2/g was at the ratio of 9. These specific surface areas of DCPD (T/A=4) and HAp (T/A=9) were apparently higher than those prepared with a wet method, 7.8 times and 1.8 times respectively. DCPA with 43 m2/g was successfully produced to decrease the pH of phosphate solution at T/A of 9. The change of crystalline phases would be explained as follows; the increase of T amount decreased the micro-emulsion sizes to reduce bulk water to be DCPA, and increased the pH to precipitate HAp nanocrystals.

Pore alignment is a vital step to obtain bone-mimicking microstructure in collagen-based scaffold. The porous structure can be controlled by altering the configuration of ice crystals formed in collagen solution. In this study, movements of magnetite/hydroxyapatite composite particles were used to alter the configuration of pore alignment formed in 1.0 and 1.5wt% collagen solutions (HCol 1.0 and HCol 1.5 respectively) by applying moving external magnetic field. In case of HCol 1.0 pores were aligned parallel to the magnetic field, while for HCol 1.5 pores were aligned at curve shape. Different results of pore alignment were thought to originate from differences in collagen fiber alignment caused by magnetic particles movement paths. This result thus opens up the possibility of controlling pore alignment by utilizing magnetic particle movement.

The present study investigates how effectively lidocaine ions are transported across a cellophane membrane through the application of either a direct current (DC) or alternating current (AC). The cellophane membrane was set on a parallelplate- type acrylic cell with platinum electrodes at both ends, filled with a donor cell of a 1 % aqueous solution of lidocaine and a receptor cell with distilled water. Lidocaine concentrations were measured for 60 min while the following voltages were applied, with changes every 10 min: 3 V DC and 7.5 V sine wave AC; frequency at 1 kHz. As a result, lidocaine concentrations in the receptor cell increased in a time-dependent manner. Significant increases in lidocaine concentrations were observed in groups where the voltage combination consisted of DC 30 min/AC 30 min, DC 50 min/AC 10 min, DC 60 min and AC 10 min/DC 50 min, compared with the passive diffusion group or in groups where voltage application was performed for 20, 30 , 40, 50 and 60 min. Significant increases were also observed in groups where the voltage combination consisted of A C 6 0 min, D C 10 min/AC 5 0 min, AC 3 0 min/ DC 30 min and AC 50 min/DC 10 min, compared with the passive diffusion group or in groups where voltage application was performed for 40, 50 and 60 min. These results suggest that lidocaine was delivered more rapidly with DC than with AC, and that its ions are transported faster when voltage is switched from DC to AC than from AC to DC, which is presumably due to the contribution of electrorepulsion by DC voltage application and the vibration energy infiltration mechanism owning to AC. Iontophoresis in combination with DC and AC was found to enable highly efficient drug delivery that shares the benefits of both forms of current application.

We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs.

The release property of osteoprotegerin (OPG) from hydroxyapatite / chondroitin sulphate (HAp/ChS) microparticles was described in this paper. The formulation was conducted by subsequent addition of zinc chloride solution into the mixture of HAp/ChS microparticles as a ceramic carrier and OPG solution. The release of OPG from the microparticles in PBS was positively correlated with soaking time without initial burst. The release rate of OPG was decreased with the increase of the amounts of zinc cation, which was not dependent of ChS wt% in the microparticles. The controlled release of OPG would provide the improvement of drug effect and the reduction of adverse effects. These findings reveal further investigation of the other antiresortive agents as a potential safer medication for osteoporosis.

Mechanochemically activated graphite (Gp) and iron particle surfaces were prepared, and the Gp/iron composite interfaces were elucidated to utilize new functions. Activated iron particles with the oxygen atoms prepared by a milling under oxygen atmosphere were shown. Alternately, the Gp particles activated by the milling under argon atmosphere easily agglomerated because of the dangling bonds on the edge surfaces. In the composites, the interfacial bonds (e.g., Fe–O–C) between the Gp and iron oxides on the iron were clearly induced while preserving the carbon atom six-ring structures. Furthermore, the molecular orbital calculations suggested that the activated oxygen atoms in the iron oxides effectively abstract hydrogen atoms from the C–H groups of the Gp edge, resulting in the active bare carbon atoms which can form the interfacial bonds; therefore, the interfacial Fe–O–C bonds for a higher adhesive strength were successfully prepared.

Luminescent europium(III)-doped nanoporous silica nanospheres (Eu:NPS) were successfully synthesized, and a folate N-hydroxysuccinimidyl ester (FA-NHS) molecule as a targeting ligand for cancer cells was immobilized on the nanosphere surfaces through mediation of the 3-aminopropyltriethoxysilane (APTES) adlayer. The ordered nanopores were preserved by the immobilization, and the specific surface area decreased only with the APTES immobilization, suggesting that the FA-NHS was predominantly immobilized on the outer surface of the nanopores. The photoluminescence of the nanospheres functionalized with folic acid (FA) exhibited a characteristic peak due to the interactions (e.g., energy transfer) between FA and Eu(3+), and further the orange luminescence could be clearly detected by fluorescence microscopy in air and water. Furthermore, the nanospheres highly dispersed in cell culture medium exhibited nontoxicity in the cellular proliferation stages of the Hela cancer cells and NIH3T3 fibroblasts and specifically bind to the Hela cells. The nanospheres after the binding and uptake also showed intense luminescence from the outer/inner cell surfaces for the culture time of 4 days. Therefore, the luminescent FA-functionalized Eu:NPS nanospheres could be used for specific targeting and imaging abilities for cancer cells.

B-type carbonate apatite (B-CAp) powders were prepared by a wet method using Ca(OH)2 suspension and H3PO4 solution including NaHCO3 as a carbonate source, and porous B-CAp ceramics with two different amounts of carbonate contents were fabricated by sintering freeze-dried mixtures of the powders and gelatin composite. The porous B-CAp ceramics prepared had three-dimensionally interconnected pores. The sinterability of B-CAp ceramics was dependent on the chemical composition, especially sodium content and vacancy of OH site, and the carbonate contents did not directly influence the dissolution rate of porous B-CAp ceramics.

Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16INK4a in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27Kip1, a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. Implications: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells. Mol Cancer Res; 11(11); 1437–47. ©2013 AACR.

BACKGROUND A number of inorganic fertilizers have been used as a slow-release fertilizer for plants but control of release rates of nutrient ions can be difficult. We have prepared a novel slow-release inorganic fertilizer consisting of zeolite NaP1/hydroxyapatite nanocomposite containing the following nutrient ions: 17.4 mg g-1 of ammonium ion, 54.6 mg g-1 of potassium ion and 24.1 mg g-1 of phosphate ion. The release behavior of nutrient ions were evaluated in distilled water. RESULTS Zeolite NaP1 was synthesized from coal fly ash by alkaline hydrothermal treatment. The zeolite surfaces were then covered with hydroxyapatite needle-like crystals of about 200 nm diameter by a hydrothermal treatment of the calcium-exchanged zeolite NaP1 in a mixed solution of potassium and ammonium phosphates at 40°C for 8 h. The release behavior of the nanocomposite was evaluated by measuring the release into distilled water of nutrient ions over the long term. Phosphate ions were slowly released by the dissolution of HA in water, ammonium and potassium ions by cation exchange with calcium co-dissolved with phosphate. CONCLUSION The zeolite NaP1/hydroxyapatite nanocomposite obtained is very useful as an inorganic fertilizer which can release nutrient ions over a long time. © 2013 Society of Chemical Industry

The mechanochemical surface functionalization of iron oxides with disordered lattices on bare iron (Fe) particles was investigated using simple milling processes to clarify the formation mechanism of the oxide layer and investigate the near-surface models with different states. The homogeneous α-Fe particles at the milling equilibrium were first prepared under an argon atmosphere. After the subsequent milling reaction of the particles with oxygen molecules, the surface analyses by X-ray diffraction and Raman and X-ray photoelectron spectroscopies revealed that the near-surface layers consisted of two iron oxide phases (α-Fe2O3 and Fe3O4) through oxygen atom diffusion, and the α-Fe2O3 was dominantly grown on the near surface. During the initial reaction, the signals from an electron spin resonance suggested the dangling bond formation on α-Fe2O3. The oxygen atoms effectively induce disordered lattices in the local area to form oxidized Fe3+ clusters, and the geometric distortion formed the dangling bonds, ...

The mechanochemical fabrication of the composites between carbon fibers (CFs) and nylon-6 (N6) was investigated using a milling process. By the milling, defected surfaces were generated on CFs to obtain the homogeneous length with preserving the cylindrical shape of CF. Fourier transform infrared spectra revealed that the band intensities due to C═O, C–N and O–H bondings increased with the milling. The band position of C–N was shifted to a higher wavenumber, while that of C═O did not change, implying that the main chain of N6 was dissociated at the C–N neighboring the C═O bonds by the force, and subsequently, new bonds between the N atoms of the activated N6 and the C atoms on CFs would be formed. Therefore, the efficient fabrication of the CF/N6 composite fibers with the interfacial bondings was achieved using the mechanochemical reaction.

The purposes of this study were to fabricate porous carbonate-substituted apatite (CAp) ceramics and characterize important properties such as compressive strengths and dissolution rates for an artificial bone. Porous A-type CAp (A-CAp) with uniaxial pores was fabricated by sintering porous hydroxyapatite green bodies at 1000°C in carbon dioxide gas atmosphere. B-type CAp (B-CAp) nanocrystals prepared by a wet method were mixed with an organic binder, freeze-dried and heated at 800°C in air, and AB-type CAp (AB-CAp) was fabricated by heating B-CAp porous bodies including the organic binder at 1100°C in carbon dioxide gas atmosphere. From the morphological observations with a scanning electron microscope, the porous A-CAp had uniaxially oriented pores of 104±33 μm in diameter, while the porous B-CAp and AB-CAp had large pores of 143±48 μm and 181±46 μm in diameter, of which pores were interconnected with small pores of 31±11 μm and 45±17 μm in diameter. The dissolution rates of CAps were apparently larger than that of HAp; the calcium concentrations increased in the order of AB-CAp > B-CAp > A-CAp > HAp. This is mainly attributed to carbonate content although it could be partialy depended on the different porous structures and diameters.

Calcium phosphates films were deposited onto pipes and stents of nitinol alloys by an electrolytic deposition (ELD) method. Monocalcium phosphate (Ca (H2PO4)2·H2O) solutions were used as the electrolyte, and electric depositions were carried out at the constant cathode current of 1.59 mA/cm2 at 65°C for 60 min. From the deposition on nitinol pipes, deposition rates were changed in 15 minutes and the precipitates were identified to be octacalcium phosphate (Ca8H2(PO4)6·5H2O) and dicalcium phosphate anhydrous (CaHPO4). The electrolytic depositions on the nitinol alloys were useful for the formation of calcium phosphates films on the complex shape of stents.