Decreased Bone Volume and Bone Mineral Density in the Tibial Trabecular Bone Is Associated with Per2 Gene by 405 nm Laser Stimulation.

Low-level laser therapy/treatment (LLLT) using a minimally invasive laser needle system (MILNS) might enhance bone formation and suppress bone resorption. In this study, the use of 405 nm LLLT led to decreases in bone volume and bone mineral density (BMD) of tibial trabecular bone in wild-type (WT) and Per2 knockout (KO) mice. Bone volume and bone mineral density of tibial trabecular bone was decreased by 405 nm LLLT in Per2 KO compared to WT mice at two and four weeks. To determine the reduction in tibial bone, mRNA expressions of alkaline phosphatase (ALP) and Per2 were investigated at four weeks after 405 nm laser stimulation using MILNS. ALP gene expression was significantly reduced in the LLLT-stimulated right tibial bone of WT and Per2 KO mice compared to the non-irradiated left tibia (p < 0.001). Per2 mRNA expression in WT mice was significantly reduced in the LLLT-stimulated right tibial bone compared to the non-irradiated left tibia (p < 0.001). To identify the decrease in tibial bone mediated by the Per2 gene, levels of runt-related transcription factor 2 (Runx2) and ALP mRNAs were determined in non-irradiated WT and Per2 KO mice. These results demonstrated significant downregulation of Runx2 and ALP mRNA levels in Per2 KO mice (p < 0.001). Therefore, the reduction in tibial trabecular bone resulting from 405 nm LLLT using MILNS might be associated with Per2 gene expression.

Tunable hydrogel-microsphere composites that modulate local inflammation and collagen bulking.

Injectable biomaterials alone may alter local tissue responses, including inflammatory cascades and matrix production (e.g. stimulatory dermal fillers are used as volumizing agents that induce collagen production). To expand upon the available material compositions and timing of presentation, a tunable hyaluronic acid (HA) and poly(lactide-co-glycolide) (PLGA) microsphere composite system was formulated and assessed in subcutaneous and cardiac tissues. HA functionalized with hydroxyethyl methacrylate (HeMA) was used as a precursor to injectable and degradable hydrogels that carry PLGA microspheres (~50 µm diameter) to tissues, where the HA hydrogel degradation (~20 or 70 days) and quantity of PLGA microspheres (0-300 mgml(-1)) are readily varied. When implanted subcutaneously, faster hydrogel degradation and more microspheres (e.g. 75 mgml(-1)) generally induced more rapid tissue and cellular interactions and a greater macrophage response. In cardiac applications, tissue bulking may be useful to alter stress profiles and to stabilize the tissue after infarction, limiting left ventricular (LV) remodeling. When fast degrading HeMA-HA hydrogels containing 75 mgml(-1) microspheres were injected into infarcted tissue in sheep, LV dilation was limited and the thickness of the myocardial wall and the presence of vessels in the apical infarct region were increased ~35 and ~60%, respectively, compared to empty hydrogels. Both groups decreased volume changes and infarct areas at 8 weeks, compared to untreated controls. This work illustrates the importance of material design in expanding the application of tissue bulking composites to a range of biomedical applications.

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice.

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.

Enhanced release of small molecules from near-infrared light responsive polymer-nanorod composites.

Stimuli-responsive materials undergo structural changes in response to an external trigger (i.e., pH, heat, or light). This process has been previously used for a range of applications in biomedicine and microdevices and has recently gained considerable attention in controlled drug release. Here, we use a near-infrared (NIR) light responsive polymer-nanorod composite whose glass transition temperature (T(g)) is in the range of body temperature to control and enhance the release of a small-molecule drug (<800 Da). In addition to increased temperature and resulting changes in molecule diffusion, the photothermal effect (conversion of NIR light to heat) adjusts the composite above the T(g). Specifically, at normal body temperature (T < T(g)), the structure is glassy and release is limited, whereas when T > T(g), the polymer is rubbery and release is enhanced. We applied this heating system to trigger release of the chemotherapeutic drug doxorubicin from both polymer films and microspheres. Multiple cycles of NIR exposure were performed and demonstrated a triggered and stepwise release behavior. Lastly, we tested the microsphere system in vitro, reporting a ∼90% reduction in the activity of T6-17 cells when the release of doxorubicin was triggered from microspheres exposed to NIR light. This overall approach can be used with numerous polymer systems to modulate molecule release toward the development of unique and clinically applicable therapies.

Combined passive and active microrheology study of protein-layer formation at an air-water interface.

We investigate the mechanical properties of layers of the protein beta-lactoglobulin during their formation at the air-water interface using a combination of passive and active microrheological techniques. The passive microrheology, which employs multiple particle tracking measurements using spherical colloids, indicates that the interfacial rheology evolves over time through three stages as protein adsorbs at the interface: (i) an increase in viscosity, (ii) a period of spatial heterogeneity in which the interface contains elastic and viscous regions, and (iii) the development of a uniformly rigid elastic film. Varying solution pH between pH = 5.2, the isoelectric point of beta-lactoglobulin, and pH = 7.0 has no qualitative effect on this mechanical evolution. The active microrheology, which employs ferromagnetic nanowires rotating in response to magnetic torques, similarly shows an increasing interfacial viscosity at early times and evidence of mechanical heterogeneity at intermediate times. However, at late times, the nanowire mobility becomes strongly pH dependent. For pH = 5.2, the layer responds as a rigid elastic film to the stress imposed by the wire. For pH = 7.0, it displays a viscous response that contrasts with the passive measurements. We associate this contrast with a nonlinear response to the wire at late times that reflects a low yield stress of the film at higher pH. This ability to compare passive and active measurements demonstrates the advantage of applying multiple microrheological methods to resolve ambiguity in any single approach.

Microfluidic fabrication of stable nanoparticle-shelled bubbles.

We introduce a microfluidic approach to generating monodisperse, stable nanoparticle-shelled bubbles using air-in-oil-in-water (A/O/W) compound bubbles as templates. The oil phase of the A/O/W compound bubbles comprises a volatile organic solvent and a hydrophobic silica nanoparticle. Upon evaporation of the organic solvent, the nanoparticles in the oil layer form a stiff shell at the air-water interface, which drastically enhances the stability of the bubbles against dissolution and coarsening. On the basis of this approach, we demonstrate that it is also possible to generate functional bubbles stabilized by composite shells that are composed of mixtures of hydrophobic materials and nanoparticles with unique properties.

Interfacial hydrodynamic drag on nanowires embedded in thin oil films and protein layers.

We investigate the motion of ferromagnetic nanowires confined to nanometer-scale oil films at an air/aqueous interface in response to the application of external magnetic fields and field gradients. By varying the oil viscosity, film thickness, and wire length, we cover two regimes of response suggested by theory: one where the surface viscosity is expected to dominate the wire's motion and one where the subphase viscosity is expected to dominate [Levine, A. J.; Liverpool, T. B.; MacKintosh, F. C. Phys. Rev. E 2004, 69, 021503]. For wire motion parallel to the long axis of the wire, the observed drag agrees reasonably with theoretical predictions. However, the drag on wires moving perpendicular to their long axis or rotating about a short axis is unexpectedly insensitive to the film properties over the full range of measurements. This behavior is in contrast to the rotational and translational drag on nanowires in molecularly thin protein layers, which follow theoretical expectations. The observations in the oil films, which are explained in terms of the manner in which the wire immerses dynamically in the film and subphase, demonstrate how the effective drag viscosity of an aspherical particle confined to a fluid interface can depend on its direction of motion.

Response of a colloidal gel to a microscopic oscillatory strain.

We study the microscopic mechanical response of colloidal gels by manipulating single probe particles within the network. For this work, we use a refractive index and density-matched suspension of polymethylmethacrylate (PMMA) particles with nonadsorbing polymer: polystyrene. As the polymer concentration increases, a dynamically arrested, space-filling network is formed, exhibiting structural transitions from a clusterlike to a more homogeneous stringlike gel phase, consistent with observations by Dibble and co-workers [C. J. Dibble, M. Kogan, and M. J. Solomon, Phys. Rev. E 74, 041403 (2006)]. In a gel, probe particles are oscillated with an optical trap, creating the local strain field in the network. We find that the micromechanics correlate strongly with the gel structure. At high polymer concentration, the average deformation field decays as 1/r to a distance quite close to the probe particle, as expected for a purely elastic material. In contrast, at lower polymer concentrations, gels exhibit anomalous strain fields in the near field; the strain plateaus, indicating that many particles move together with the probe. By rescaling the probe size in the theoretical model, we obtain a micromechanical gel correlation length, which is consistent with the structural difference in terms of "clusterlike" and "stringlike."

Formation and evolution of sediment layers in an aggregating colloidal suspension.

The coupled aggregation and sedimentation of colloidal particles with short-range attractions are investigated. Nonadsorbing polymer is used to induce depletion interactions between the hard-sphere particles. Gravitational forces, caused by a density mismatch between the particles and the suspending fluid, result in the sedimentation of particles and aggregates, as well as the compaction and rearrangement of the final sediment layer. At low polymer concentrations CP, or low initial volume fractions phio, clusters formed during the period of fast sedimentation are small, and the structure of the final sediment is dense. Conversely, at high CP, or high phio, large clusters form during sedimentation, and the resulting sediment structure is significantly less dense, with large void volumes. The size of the presediment aggregates depends on CP and phio with a functional form that resembles other thermally activated barrier hopping processes in colloidal systems, such as the delayed sedimentation of colloidal gels. Finally, when the particles are weakly attractive, gravitational stresses are found to induce compaction of the sediment over long periods of time. However, sediments composed of particles that are strongly attractive resist rearrangements and compaction, even when the sediment layers have a relatively large amount of free volume.

Synthesis of monodisperse fluorescent core-shell silica particles using a modified Stober method for imaging individual particles in dense colloidal suspensions.

Core-shell silica particles, with a diameter of 1.5 mum, containing a dye fluorescein isothiocyanate (FITC), are synthesized by the hydrolysis and condensation of tetraethylorthosilicate (TEOS). Sodium dodecyl sulfate (SDS) is added to synthesize fluorescent core particles with the diameter of approximately 1 mum. In the addition of SDS, the surface charge reduced by counterions (Na+) of the surfactant leads to a higher degree of aggregation of the primary particles and the formation of larger secondary particles. The particle growth kinetics confirms the aggregation growth model for the synthesis of monodisperse silica particles, and also shows the dependence of final particle size on colloidal stability resulting from the addition of SDS. Light and X-ray scattering data reveal that the final particles have compactly packed structures with smooth surfaces. The seeded growth technique is then used to form a silica shell layer on the fluorescent core. The added amount of water and NH4OH has significant effects on shell formation. Finally, the final core-shell silica particles are modified by chemisorption of octadecanol at the surface to be dispersed in organic solvents. Octadecyl-coated silica particles are sterically stabilized in silica index-matching solvents such as chloroform and hexadecane to directly image separate particles using confocal microscopy. In chloroform, the organophilic silica particles disperse well, whereas in hexadecane they form a volume-filling gel structure at room temperature.

Preparation of Silica Particles Encapsulating Retinol Using O/W/O Multiple Emulsions.

Retinol, a cosmetic ingredient, was entrapped within inorganic microspheres obtained from sol-gel reaction of TEOS in o/w/o multiple emulsions as microreactors. In o/w/o multiple emulsions, the retinol was emulsified as an internal oil phase in a aqueous solution of 3.0 wt% Tween 20 prior to emulsification into an external oil phase. The multiple emulsions appeared to be stable enough in the presence of HPC polymer in the external oil phase. In sol-gel reaction, the hydrolysis and condensation rate of TEOS were greatly dependent upon the catalyst and the molar ratio of H(2)O to TEOS (R(W)). In this study, sphere-like microspheres entrapping retinol were best formed with the addition of NH(4)OH as a catalyst when the concentration of TEOS was at the R(W) value of 4. Microspheres obtained under these conditions were 15-40 &mgr;m with very dense surfaces containing a few globules 1.17-2.35 &mgr;m. Also, they showed the slower release of retinol into the external ethanol phase and higher loading and encapsulation efficiency. Copyright 2001 Academic Press.

Preparation of Eu-Doped Y(2)O(3) Luminescent Nanoparticles in Nonionic Reverse Microemulsions.

In this study, Y(2)O(3):Eu luminescent nanoparticles were prepared by precipitation of aqueous yttrium nitrate/europium nitrate solution using ammonium hydroxide in the reverse microemulsions based on polyoxyethylene (5) nonylphenyl ether/polyoxyethylene (9) nonylphenyl ether, cyclohexane, and water. With Eu-doped Y(2)O(3) nanoparticles obtained, particle size, shape, chemical composition, crystalline formation rate, crystallinity, and photoluminescence were measured and compared with those of particles formed by a bulk precipitation method. The nanoparticles synthesized in microemulsion showed a narrow size distribution, spherical shape, fast crystalline formation rate, high crystallinity, and strong photoluminescence. This stronger photoluminescence of particles formed in a microemulsion might be attributed to more densely packed particles with very few voids and higher crystallinity at a relatively low temperature than those synthesized through a bulk precipitation method. Copyright 2000 Academic Press.