Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30 mW/cm² is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30 mW/cm² are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30 mW/cm² and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30 mW/cm² but also 2 and 15 mW/cm², modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30 mW/cm², alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2 mW/cm². As the intensity increased to 15 and 30 mW/cm², the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30 mW/cm² continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.     

Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors

Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte–osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5 MHz sine wave, I_SATA = 30 mW/cm²) was applied to osteocytes (mechanosensor) at three axial distances: 0 mm (near field), 60 mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more β-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the acoustic differences of LIPUS at various axial distances. Furthermore, we found that the soluble factors secreted by far field LIPUS exposed osteocytes could further promote pre-osteoblasts cell migration, maturation (transition of cell proliferation into osteogenic differentiation), and matrix calcification. In summary, our results of this present study indicated that axial distance beyond near field could transmit ultrasound energy to osteocyte more efficiently. The LIPUS exposed osteocytes conveyed mechanical signals to pre-osteoblasts and regulated their osteogenic cellular activities via paracrine factors secretion. The soluble factors secreted by far field exposed osteocytes led to promotion in migration and maturation in pre-osteoblasts. This finding demonstrated the positive effects of far field LIPUS on stimulating osteocytes and promoting mechanotransduction between osteocytes and osteoblasts.     

Applications of low-intensity pulsed ultrasound to increase monoclonal antibody production in CHO cells using shake flasks or wavebags

Many technologies, such as cell line screening and host cell engineering, culture media optimization and bioprocess optimization, have been proposed to increase monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. Unlike the existing biochemical approaches, we investigated stimulation using low-intensity pulsed ultrasound (LIPUS) as a purely physical approach, offering enhanced scalability, contamination control and cost-efficiency, while demonstrating significantly increased cell growth and antibody production. It was found that daily ultrasound treatments at 40 mW/cm² for 5 min during cell culture increased the production of human anti-IL-8 antibody by more than 30% using 10 or 30 mL shake flasks. Further increasing the ultrasound dosage (either intensities or the treatment duration) did not appreciably increase cell growth or antibody production, however feeding the culture with additional highly-concentrated nutrients, glucose and amino acids (glutamine in this case), did further increase cell growth and antibody titer to 35%. Similar ultrasound treatments (40 mW/cm², 5 min per day) when scaled up to larger volume wavebags, resulted in a 25% increase in antibody production. Increased antibody production can be attributed to both elevated cell count and the ultrasound stimulation. Theoretical study of underlying mechanisms was performed through the simulations of molecular dynamics using the AMBER software package, with results showing that LIPUS increases cell permeability. The significance of this study is that LIPUS, as a physical-based stimulation approach, can be externally applied to the cell culture without worrying about contamination. By combining with the existing technologies in antibody production, LIPUS can achieve additional mAb yields. Because it can be easily integrated with existing cell culture apparatuses, the technology is expected to be more acceptable by the end users.     

Investigation of rat bone fracture healing using pulsed 1.5 MHz, 30 mW/cm burst ultrasound – Axial distance dependency

This study investigated the effect of LIPUS on fracture healing when fractures were exposed to ultrasound at three axial distances: z = 0 mm, 60 mm, and 130 mm. We applied LIPUS to rat fracture at these three axial distances mimicking the exposure condition of human fractures at different depths under the soft tissue. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). We asked whether different positions of the fracture within the ultrasound field cause inconsistent biological effect during the healing process. Closed femoral fractured Sprague–Dawley rats were randomized into control, near-field (0 mm), mid-near field (60 mm) or far-field (130 mm) groups. Daily LIPUS treatment (plane, but apodized source, see details in the text; 2.2 cm in diameter; 1.5 MHz sine waves repeating at 1 kHz PRF; spatial average temporal average intensity, I_SATA = 30 mW/cm²) was given to fracture site at the three axial distances. Weekly radiographs and endpoint microCT, histomorphometry, and mechanical tests were performed. The results showed that the 130 mm group had the highest tissue mineral density; and significantly higher mechanical properties than control at week 4. The 60 mm and 0 mm groups had significantly higher (i.e. p < 0.05) woven bone percentage than control group in radiological, microCT and histomorphometry measurements. In general, LIPUS at far field augmented callus mineralization and mechanical properties; while near field and mid-near field enhanced woven bone formation. Our results indicated the therapeutic effect of LIPUS is dependent on the axial distance of the ultrasound beam. Therefore, the depth of fracture under the soft tissue affects the biological effect of LIPUS. Clinicians have to be aware of the fracture depth when LIPUS is applied transcutaneously.     

Measurement of nonlinear absorption coefficients in GaAs, InP and Si by an optical pump THz probe technique

An optical pump terahertz (THz) probe method for measuring carrier mobility and multiphoton absorption coefficients in semiconductors is demonstrated. A THz probe pulse is used to detect the transient photoconductivity generated by an optical pump pulse. The change in transmission coefficient at THz frequencies due to a pump pulse with photon energy greater than the band gap energy is used to determine the sum of electron and hole mobilities. The weak nonlinear absorption of a pump pulse with photon energy less than the band gap energy produces an approximately uniform free carrier distribution. The THz transmission coefficient vs. pump fluence, and the mobility, are used in a bulk photoconductivity model to determine the multiphoton absorption coefficients. For GaAs, InP and Si we find two photon absorption coefficients at 1305 nm of 42.5 ± 11, 70 ± 18 and 3.3 ± 0.9 cm/GW, respectively. For GaAs and InP we determine three photon absorption coefficients at 2144 nm of 0.19 ± 0.07 and 0.22 ± 0.08 cm³/GW².     

Influence of low-intensity pulsed ultrasound on osteogenic tissue regeneration in a periodontal injury model: X-ray image alterations assessed by micro-computed tomography

Objective

This study was conducted to evaluate, with micro-computed tomography, the influence of low-intensity pulsed ultrasound on wound-healing in periodontal tissues.

Methods

Periodontal disease with Class II furcation involvement was surgically produced at the bilateral mandibular premolars in 8 adult male beagle dogs. Twenty-four teeth were randomly assigned among 4 groups (G): G1, periodontal flap surgery; G2, periodontal flap surgery + low-intensity pulsed ultrasound (LIPUS); G3, guided tissue regeneration (GTR) surgery; G4, GTR surgery plus LIPUS. The affected area in the experimental group was exposed to LIPUS. At 6 and 8 weeks, the X-ray images of regenerated teeth were referred to micro-CT scanning for 3-D measurement.

Results

Bone volume (BV), bone surface (BS), and number of trabeculae (Tb) in G2 and G4 were higher than in G1 and G3 (p < 0.05). BV, BS, and Tb.N of the GTR + LIPUS group were higher than in the GTR group. BV, BS, and Tb.N of the LIPUS group were higher than in the periodontal flap surgery group.

Conclusion

LIPUS irradiation increased the number, volume, and area of new alveolar bone trabeculae. LIPUS has the potential to promote the repair of periodontal tissue, and may work effectively if combined with GTR.     

Role of network connectivity in intercellular calcium signaling

It is important to understand the coordinated performance of cells in tissue. One possible mechanism in this coordination involves intracellular Ca²⁺ signaling. The topology of intercellular connections in tissue should also play an important role in this process. It is most relevant for plane tissues, in which the interaction between cells is due to gap junctions (epithelium, blood vessels). We demonstrate the importance of the topology of intercellular connectivity by investigating the properties of a model of Ca²⁺ signaling for a small number of connected cells.     

Gap junctions in olfactory neurons modulate olfactory sensitivity

Background  

One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis.     

The far-infrared spectrum of acrolein, CH2CHCHO: The ν18 fundamental and (ν17 + ν18) − ν18 hot bands

The ν₁₈ fundamental band (∼158 cm⁻¹) of acrolein is studied at high resolution (0.0015 cm⁻¹) using synchrotron radiation from the Canadian Light Source facility and a Bruker IFS 125HR Fourier transform spectrometer. By fitting this band, together with some pure rotational transitions, molecular parameters are obtained to accurately determine the energies of the ν₁₈ = 1 state levels for values of (J, K_a) up to at least (45, 24). These parameters should be useful for future high resolution studies of acrolein hot bands. This is demonstrated here by a detailed analysis of the (ν₁₇ + ν₁₈) − ν₁₈ hot band at ∼589 cm⁻¹. The upper state (ν₁₇ + ν₁₈) of this band is found to be perturbed by Coriolis interactions analogous to those affecting the ν₁₇ state.     

Formation of two ripple modes on Si by ion erosion with simultaneous Fe incorporation

This report focuses on the self organized nanostructure formation on Si (0 0 1) by erosion with low energy Kr⁺ ions with simultaneous incorporation of metallic atoms, in particular Fe. The incorporation of Fe is thought to play an important role in the formation of some features. In the experimental set-up used here the Fe atoms come from the sputtering of a cylindrical stainless steel target situated between the source and the sample holder. It is demonstrated how the Fe flux can be regulated by operational parameters of the ion source. It is shown that two different ripple modes, one perpendicular to the ion beam projection on the surface and the other parallel, were formed at near normal incidence (α = 20°) with ion energy between 300 eV and 2000 eV and a fluence of 6.7 × 10¹⁸ cm⁻². The perpendicular mode ripples dominated the topography when E_ion = 2000 eV, while the parallel mode ripples were the main features observed when E_ion = 300 eV. The correlation of Fe concentration with ion sources parameters and resulting topography is analyzed. It is demonstrated that a certain Fe concentration is necessary for the formation of ripples that are oriented perpendicular to the ion beam and that the Fe concentration alone does not determine the evolving topography.     

30-ps impulse response monolithically waveguide coupled photo-detector for high-speed performance monitoring applications

Optical power performance functions monitoring can be demonstrated using a waveguide coupled photo-detector using a reflector and a metal coplanar waveguide. A full-width half-maximum temporal response of 30 ps is illustrated with minimal ringing from a monolithic integrated photo-detector, suitable for 10-Gb/s digital communication applications. The high-speed response of the pin photo-detector is limited by an average saturation drift velocity of 3.5 × 10⁶ cm/s and the impurity concentration of the absorbing In_0.53Ga_0.47As layer of 1.9 × 10¹⁷ cm^− 3 will be demonstrated.     

Laser synthesis of nanostructures based on transition metal oxides

Nanostructures based on iron oxides in the form of thin films were synthesized while laser chemical vapor deposition (LCVD) of elements from iron carbonyl vapors (Fe(CO)₅) under the action of Ar⁺ laser radiation (λ_L = 488 nm) on the Si substrate surface with power density about 10² W/cm² and vapor pressure 666 Pa. Analysis of surface morphology and relief of the deposited films was carried out with scanning electron microscopy (SEM) and atomic force microscopy (AFM). This analysis demonstrated their cluster structure with average size no more than 100 nm. It was found out that the thicker the deposited film, the larger sizes of clusters with more oxides of higher oxidized phases were formed. The film thickness (d) was 10 and 28 nm. The deposited films exhibited semiconductor properties in the range 170-340 K which were stipulated by oxide content with different oxidized phases. The width of the band gap E_g depends on oxide content in the deposited film and was varied in the range 0.30-0.64 eV at an electrical field of 1.6 × 10³ V/m. The band gap E_g was varied in the range 0.46-0.58 eV at an electrical field of 45 V/m. The band gap which is stipulated by impurities in iron oxides E_i was varied in the range 0.009-0.026 eV at an electrical field of 1.6 × 10³ V/m and was varied in the range 0-0.16 eV at an electrical field 45 V/m. These narrow band gap semiconductor thin films displayed of the quantum dimensional effect.     

Laser synthesis of semiconductor nanostructures with narrow band gap

Semiconductor nanostructures with narrow band gap were synthesized by means of laser chemical vapor deposition (LCVD) of elements from iron carbonyl vapors [Fe(CO)₅] under the action of Ar⁺ laser radiation (λ_L = 488 nm) on the Si substrate surface. The temperature dependence of the specific conductivity of these nanostructures in the form of thin films demonstrated typical semiconductor tendency and gave the possibility to calculate the band gap for intrinsic conductivity (E_g) and the band gap assigned for impurities (E_i), which were depended upon film thickness and applied electrical field. Analysis of deposited films with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated their cluster structure with average size not more than 100 nm. Semiconductor properties of deposited nanostructures were stipulated with iron oxides in different oxidized phases according to X-ray photoelectron spectroscopy (XPS) analysis.These deposited nanostructures were irradiated with Q-switched YAG laser (λ_L = 1064 nm) at power density about 6 × 10⁷ W/cm². This irradiation resulted in the crystallization process of deposited films on the Si substrate surface. The crystallization process resulted in the synthesis of iron carbide-silicide (FeSi_2−xC_x) layer with semiconductor properties too. The width of the band gap E_g of the synthesized layer of iron carbide-silicide was less than for deposited films based on iron oxides Fe₂O_3−x (0 ≤ x ≤ 1).     

Growth of Cu2S/CdS nano-layered photovoltaic junctions for solar cell applications

Layered Cu₂S/CdS photovoltaic p-n junctions were fabricated via a simple and reproducible route. CdS inner layer was grown on ITO substrate using chemical bath deposition process for different times. The utilized bath consisted of cadmium sulfate and thiourea with concentrations of 0.05 M and 0.07 M, respectively. CdS layer grown for 600 min was uniform with a thickness of about 500 nm. Moreover, band gap energy of the CdS inner layers was measured as 2.40-2.44 eV depending on the thickness of the layer. Cu₂S outer layer was formed over the CdS via ion exchange chemical route, in a bath consisting of copper chloride aqueous solution. EDS, XRD, and XPS were utilized to characterize the formation of cadmium sulfide, and copper sulfide phases during the fabrication steps of the p-n junctions. Nano-layered cell, each layer 200-250 nm in thickness was fabricated with an apparent band gap of 2.22 eV. SEM imaging of both inner and the outer layers confirmed the uniformity and homogeneity of the CdS and the Cu₂S layers.     

Anisotropy of two-photon absorption in gallium selenide at 1064 nm

The Bridgman method is used to grow single crystals of ε-GaSe. The two-photon absorption (TPA) coefficient β was measured for especially un-doped crystals at room temperature for the ordinary o-ray (β_⊥) and extraordinary e-ray (β_∥) using single-wavelength excitation by a Nd:YVO₄ laser at 1.064 μm with a pulse duration of 10 ps and a repetition rate of 81 MHz. No large anisotropy was observed for the TPA coefficients between the two geometries. The TPA coefficients were found to be β_⊥ = 1.07 × 10⁻⁹ cm/W and β_∥ = 1.88 × 10⁻⁹ cm/W. It was found that doping slightly increases the TPA coefficient. The value of the TPA for crystals doped with 0.5 at.% of Tl is β_⊥ = 7.56 × 10⁻⁹ cm/W.     

Optical and electrical properties of SnS semiconductor crystals grown by physical vapor deposition technique

Tin sulfide (SnS) is a material of interest for use as an absorber in low cost solar cells. Single crystals of SnS were grown by the physical vapor deposition technique. The grown crystals were characterized to evaluate the composition, structure, morphology, electrical and optical properties using appropriate techniques. The composition analysis indicated that the crystals were nearly stoichiometric with Sn-to-S atomic percent ratio of 1.02. Study of their morphology revealed the layered type growth mechanism with low surface roughness. The grown crystals had orthorhombic structure with (0 4 0) orientation. They exhibited an indirect optical band gap of 1.06 eV and direct band gap of 1.21 eV with high absorption coefficient (up to 10³ cm⁻¹) above the fundamental absorption edge. The grown crystals were of p-type with an electrical resistivity of 120 Ω cm and carrier concentration 1.52×10¹⁵ cm⁻³. Analysis of optical absorption and diffuse reflectance spectra showed the presence of a wide absorption band in the wavelength range 300-1200 nm, which closely matches with a significant part of solar radiation spectrum. The obtained results were discussed to assess the suitability of the SnS crystal for the fabrication of optoelectronic devices.     

Analysis and minimization of cross phase modulation in semiconductor optical amplifiers for multichannel WDM optical communication systems

A theoretical model for crosstalk in multichannel wavelength division multiplexing communication systems due to cross phase saturation in semiconductor optical amplifier structure is developed. This theoretical model is used to analyze the impact of the cross phase noise on the performance of semiconductor optical amplifiers in saturation region for WDM communication system by using differential phase shift modulation format. It is shown that by increasing the carrier life time, width and thickness while reducing the confinement factor, differential gain and bias current in the SOA structure mitigates the cross talk due to cross phase saturation. The impact of penalty and cross phase noise imposed on multichannel WDM links have been investigated for different parameters of the SOA with the variation in transmission distance. With the slight increase in differential gain of 200.2 × 10⁻¹⁸ cm² and confinement factor 0.41, the maximum transmission distance observed is 5220 km with good quality and nil power penalty for 10 × 40 Gb/s soliton RZ-DPSK WDM signals for the first time.     

Remodelling of cellular excitation (reaction) and intercellular coupling (diffusion) by chronic atrial fibrillation represented by a reaction-diffusion system

Human atrial tissue is an excitable system, in which myocytes are excitable elements, and cell-to-cell electrotonic interactions are via diffusive interactions of cell membrane potentials. We developed a family of excitable system models for human atrium at cellular, tissue and anatomical levels for both normal and chronic atrial fibrillation (AF) conditions. The effects of AF-induced remodelling of cell membrane ionic channels (reaction kinetics) and intercellular gap junctional coupling (diffusion) on atrial excitability, conduction of excitation waves and dynamics of re-entrant excitation waves are quantified. Both ionic channel and gap junctional coupling remodelling have rate dependent effects on atrial propagation. Membrane channel conductance remodelling allows the propagation of activity at higher rates than those sustained in normal tissue or in tissue with gap junctional remodelling alone. Membrane channel conductance remodelling is essential for the propagation of activity at rates higher than 300/min as seen in AF. Spatially heterogeneous gap junction coupling remodelling increased the risk of conduction block, an essential factor for the genesis of re-entry. In 2D and 3D anatomical models, the dynamical behaviours of re-entrant excitation waves are also altered by membrane channel modelling. This study provides insights to understand the pro-arrhythmic effects of AF-induced reaction and diffusion remodelling in atrial tissue.     

Synthesis and investigation of nonlinear optical properties of semiconductor ZnS nanoparticles

ZnS nanoparticles were prepared by a simple chemical method and using PVP (poly vinylpyrrolidone) as capping agent. The sample was characterized by UV-vis spectrophotometer, X-ray diffraction (XRD) and Z-scan technique. XRD pattern showed that the ZnS nanoparticles had zinc blende structure with an average size of about 2.18 nm. The value of band gap of these nanoparticles was measured to be 4.20 eV. The nonlinear optical properties of ZnS nanoparticles in aqueous solution were studied by Z-scan technique using CW He-Ne laser at 632.8 nm. The nonlinear absorption coefficient (β) was estimated to be as high as 3.2×10⁻³ cm/W and the nonlinear refractive index (n₂) was in order of 10⁻⁸ cm²/W. The sign of the nonlinear refractive index obtained negative that indicated this material exhibits self-defocusing optical nonlinearity.     

Resistive hysteresis and capacitance effect in NiFe2O4/SrTiO3: Nb(1 wt%) junctions

Epitaxial ultrathin NiFe₂O₄ films were deposited on 1 wt% Nb-doped SrTiO₃ (0 0 1) substrates by reactive cosputtering to form junctions with an area of ∼2 mm², and current-voltage curves show rectifying and asymmetrical hysteresis characteristics. The resistance calculated from the current-voltage curves is strongly voltage dependent, and the hysteretic loops with high and low resistive states were observed. The hysteretic loops are considered to stem from the capacitance effect of the highly resistive NiFe₂O₄ layer, which leads to charge accumulation at the interfaces. The results show that the interfaces of the junctions have a large areal capacitance of ∼100 nF/mm² from 300 to 120 K.