Effects of therapeutic ultrasound on osteoblast gene expression

Ultrasound (US) is commonly used as a physiotherapy aid for a number of types of injury to soft connective tissues and for fracture healing. However, the precise effects of therapeutic US on tissue healing processes are not clearly understood, although they are likely to involve changes in key cellular functions. The present study has therefore examined the effects of several US intensity levels on the activity of two bone-associated proteins, alkaline phosphatase (ALP) and osteopontin (OP) in a human cell line, MG63, using RT-PCR. ALP showed progressively higher expression with increasing US intensities, whereas OP responded differently, showing down-regulation at 120 mW/cm2, the lowest US exposure. OP expression was considerably less affected overall compared with the relative response of ALP to the same US doses. The results show that there is a differential response to therapeutic levels of US, since ALP and OP clearly exhibited gene-specific response profiles. These findings suggest that modifying the parameters of US exposure could be used to improve repair and regeneration processes and enhance the clinical efficacy of implanted biomaterials for tissue engineering.     

基于拓扑优化的平面2-DOF柔顺并联机构构型设计

目的运用拓扑优化技术设计一种平面2-DOF(Degrees-of-Freedom)柔顺并联机构,使其具有微米级别的微运动特性。方法依据平面2-DOF并联原型机构的受力情况进行运动特性分析,基于同构封闭矢量映射原理构建平面2-DOF柔顺并联机构的微分运动Jacobian矩阵,表达多自由度的柔顺并联机构从输入到输出间各关节的运动关系。定义柔度为优化目标函数,微分运动Jacobian矩阵为运动条件,材料体积分数为约束条件,构建平面2-DOF柔顺并联机构材料属性的有理近似模型,并运用移动渐进线法优化求解。结果优化后的平面2-DOF柔顺并联机构构型在x方向的位移为-0.0089mm,在y方向的位移为0.0053 mm,同时,其沿x方向和y方向的微位移理论值为-0.0031 mm和0.0067 mm。结论与并联原型机构的运动特性相比,平面2-DOF柔顺并联机构优化后的微运动特性具有一致性,均为微米级。     

Detection of alkaline phosphatase using surface-enhanced Raman spectroscopy

A new approach was developed to detect the activity of alkaline phosphatase (ALP) enzyme at ultralow concentrations using a surface-enhanced Raman scattering (SERS) technique. The approach is based on the use of gold nanoparticles as a SERS material whereas 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used as a substrate of ALP. The enzymatic hydrolysis of BCIP led to the formation of indigo dye derivatives, which were found to be highly SERS active. For the first time, we were able to detect ALP at a concentration of approximately 4 x 10(-15) M or at single-molecule levels when ALP was incubated with BCIP for 1 h in the Tris-HCl buffer. The same technique also was successfully employed to detect surface-immobilized avidin, and a detection limit of 10 ng/mL was achieved. This new technique allows the detection of both free and labeled ALP as a Raman probe in enzyme immunoassays, immunoblotting, and DNA hybridization assays at ultralow concentrations.     

Monitoring of cure kinetic prepreg and cure cycle modeling

Cure kinetic model is an integral part of composite process simulation, which is used to predict the degree of curing and the amount of the generated heat. The parameters involved in kinetic models are usually determined empirically from isothermal or dynamic differential scanning calorimetry (DSC) data. In this work, DSC and rheological techniques were used to investigate some of the kinetic parameters of cure reactions of carbon/F 161 epoxy prepreg and to evaluate the cure cycle used to manufacture polymeric composites for aeronautical applications. As a result, it was observed that the F 161 prepreg presents cure kinetic with total order 1.2–1.9.     

Self-similar solutions for axisymmetric jets using two turbulence models

Similarity solutions for incompressible axisymmetric jets using a k–? and a constant eddy diffusivity turbulence models are considered. For the k–? model, the governing equations are very complex. Therefore, a transformation that simplifies these equations and makes them amenable to efficient numerical solution is used. Results for the velocity, turbulent kinetic energy and dissipation rate are obtained. Also, velocity decay rate, growth rate, entrainment and kinetic energy decay rate are determined. A comparison with experimental data and other works utilizing a parabolic marching asymptotic solution to the full partial differential equations is made. This comparison shows that similarity solutions are more accurate than solutions using numerical marching procedures.     

Respirometric kinetic parameter calculations of a batch jet loop bioreactor treating leachate and oxygen uptake rate estimation by DTM

A novel circulating jet loop bioreactor adapted for organic matter oxidation has been designed and constructed. In this study, the input was leachate samples collected from Kemerburgaz Odayeri waste landfill site located on the European side of Istanbul. Controlling the jet loop bioreactor to realize high rates of purification depends on maintaining the appropriate loadings and operating conditions. This requires collecting various system data to estimate the dynamics of the system satisfactorily with the aim of keeping certain parameters within the specified range. The differential transform method (DTM) based solution of the state equations reveals the current state of the process so that any deviation in the system parameters can be immediately detected and regulated accordingly. The respirometric method for kinetic parameter calculations for biodegradation has been used for some time. In many studies, the respirometer was designed separately, usually in bench-scale. However, when a separate respirometer is used, the scale effect and parameters that affect the hydrodynamic structure of the system should be taken into consideration. In this study, therefore, the jet loop reactor itself was used as a respirometer. Thus, the kinetic parameters found reflecting the characteristics of microorganisms used for biodegradation would be more realistic. If the main reactor, here the jet loop reactor, would be used as the respirometer, the kinetic parameter changes can easily be monitored in the long run. Using the bioreactor as a respirometer, the most important kinetic parameters, Ks, kd and micromax were found to be 11,000 mg L(-1), 0.019 day(-1), and 0.21 day(-1), respectively. The stoichiometric coefficient, Y, was found to be 0.28 gr gr(-1) for the present system.     

Simultaneous activity assay of two transglutaminase isozymes, blood coagulation factor XIII and transglutaminase 2, by use of fibrinogen arrays

We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.     

Comparison between the polymerization behavior of a new bone cement and a commercial one: modeling and in vitro analysis

The polymerization behavior of a new bone cement based on poly(ethylmethacrylate), hydroxyapatite powder and n-butylmethacrylate monomer and a commercial cement have been studied. Polymerization kinetics were analyzed by means of differential scanning calorimetry (DSC). DSC data have been used to evaluate a phenomenological model describing the cure kinetics of this new bone cement. The kinetic model coupled with the energy balance was then used to obtain temperature and degree of conversion profiles in the bone–cement–prosthesis system, under non-isothermal conditions, as function of initial temperature and thickness of the cement. Material properties, boundary and initial conditions and the kinetic behavior were the input data for the numerically solved heat-transfer model. The modeling results have been compared with in vitro results. © 1998 Kluwer Academic Publishers     

General approach to high-efficiency simulation of affinity capillary electrophoresis

The differential equation describing electrophoretic migration can be evaluated with various finite difference schemes (FDSs). However, the accuracy and efficiency can be dramatically different depending on the FDS chosen and the way the algorithm is implemented in a computer simulation program. The monotonic transport scheme is used as the algorithm for the hyperbolic part of the differential equation, and the first-order fully explicit scheme is used for the parabolic part of the equation. The combination of these algorithms minimizes the errors and maintains high efficiency. A circular arrangement of the cells in the computer's memory is used in the implementation of the algorithms, and the use of concentration thresholds to enable and disable cells along the capillary makes the new algorithm highly efficient. Either thermodynamic or kinetic constants can be used in this program to simulate binding interactions between two species for equilibrium and nonequilibrium affinity CE. Simulation results with various parameters are presented. The simulated peak with proper parameters for an equilibrium affinity CE experiment has shape and position similar to that of the experimental peak. The simulated electropherograms for a nonequilibrium affinity CE experiment also show characteristics of the experimental electropherograms.     

Usefulness of ultrasonic strain measurement-based shear modulus reconstruction for diagnosis and thermal treatment

We previously reported an ultrasonic strain measurement-based one-dimensional (1-D) shear modulus reconstruction technique using a regularization method for differential diagnosis of malignancies on human superficial tissues (e.g., breast tissues). Here, ultrasonic strain measurement-based 2-D and 3-D shear modulus reconstruction techniques are described, and the 1-D technique is reviewed and subsequently applied to various human in vivo tissues, including deeply situated tissues (e.g., liver). Because soft tissues are deformed in 3-D space by externally situated arbitrary mechanical sources, the accuracy of the low-dimensional (i.e., 1-D or 2-D) reconstructions is lower to that of 3-D reconstruction due to occurrence of erroneous reconstruction artifacts (i.e., the reconstructed modulus is different than reality). These artifacts are confirmed on simulated inhomogeneous cubic phantoms containing a spherical homogenous inclusion using numerically calculated deformation data. The superiority of quasi-real-time imaging of the shear modulus is then demonstrated by comparing it with conventional B-mode imaging and strain imaging from the standpoints of monitoring the effectiveness of minimally invasive thermal therapy as well as differential diagnosis. Because the 2-D and 3-D techniques require special ultrasonic (US) equipment, the 1-D technique using conventional US imaging equipment is used, even though erroneous artifacts will occur. Specifically, the 1-D technique is applied as a diagnostic tool for differentiating malignancies in human in vivo liver and breast tissue, and a monitoring technique for determining the effectiveness of interstitial electromagnetic wave (micro and rf) thermal therapy on human in vivo liver and calf in vitro liver. Even when using the 1-D technique, reconstructed shear moduli were confirmed to be a suitable measure for monitoring thermal treatment as well as differential diagnosis. These results are encouraging in that they will promote use of 2-D and 3-D reconstruction techniques.     

Testing the hypothesis that amphiphilic antineoplastic lipid analogues act through reduction of membrane curvature elastic stress

The alkyllysophospholipid (ALP) analogues Mitelfosine and Edelfosine are anticancer drugs whose mode of action is still the subject of debate. It is agreed that the primary interaction of these compounds is with cellular membranes. Furthermore, the membrane-associated protein CTP: phosphocholine cytidylyltransferase (CCT) has been proposed as the critical target. We present the evaluation of our hypothesis that ALP analogues disrupt membrane curvature elastic stress and inhibit membrane-associated protein activity (e.g. CCT), ultimately resulting in apoptosis. This hypothesis was tested by evaluating structure–activity relationships of ALPs from the literature. In addition we characterized the lipid typology, cytotoxicity and critical micelle concentration of novel ALP analogues that we synthesized. Overall we find the literature data and our experimental data provide excellent support for the hypothesis, which predicts that the most potent ALP analogues will be type I lipids.     

Boltzmann schemes for continuum gas dynamics

Many problems arising in the aerodynamic design of aerospace vehicles require the numerical solution of the Euler equations of gas dynamics. These are nonlinear partial differential equations admitting weak solutions such as shock waves and constructing robust numerical schemes for these equations is a challenging task. A new line of research called Boltzmann or kinetic schemes discussed in the present paper exploits the connection between the Boltzmann equation of the kinetic theory of gases and the Euler equations for inviscid compressible flows. Because of this connection, a suitable moment of a numerical scheme for the Boltzmann equation yields a numerical scheme for the Euler equations. This idea called the “moment method strategy” turns out to be an extremely rich methodology for developing robust numerical schemes for the Euler equations. The richness is demonstrated by developing a variety of kinetic schemes such as kinetic numerical method, kinetic flux vector splitting method, thermal velocity based splitting, multidirectional upwind method and least squares weak upwind scheme. A 3-D time-marching Euler code calledbheema based on the kinetic flux vector splitting method and its variants involving equilibrium chemistry have been developed for computing hypersonic reentry flows. The results obtained from the codebheema demonstrate the robustness and the utility of the kinetic flux vector splitting method as a design tool in aerodynamics. The work presented in this paper is based on the research work done by several graduate students at our laboratory and collaborators from research and development organizations within the country.     

Evaluation and modeling of thermal kinetic degradation for PVA doped PbS quantum dot

The kinetic analysis of the thermogravimetric curves for the thermal decomposition processes of PVA/PbS was performed. The samples were heated in nitrogen, with three different heating rates: 10, 20 and 30 °C min⁻¹. Various forms of non-isothermal methods of analysis for determining the kinetic parameters were used. The differential and integral models were used to calculate the activation energies. Comparing with pure PVA, the results showed that the maximum activation energy of thermal degradation is achieved for PVA/PbS nanocomposite. Isoconversion model is used for predicting the thermal degradation acceleration. The results showed that the acceleration of thermal degradation for pure PVA was faster than PVA/PbS nanocomposite.     

Pharmacological evaluation of mangiferin herbosomes for antioxidant and hepatoprotection potential against ethanol induced hepatic damage

Context: Fatty liver is the first stage of alcoholic damage which is reversible with abstinence from alcohol. Mangiferin (MF) showed potent scavenging activity on diphenyl-1-picrylhydrazyl radicals which stimulate liver regeneration in various liver injuries.

Objective: Although, MF shows hepatoprotection against various liver disorders but due to rapid clearance and limited solubility in lipoid environment, there is problem of its poor absorption from intestine hence poor bioavailability. Owing to which there is a need to develop MF herbosomes to resolve the problem of poor bioavailability to enhance the therapeutic potential.

Methods: Successfully prepared MF herbosomes through complexation with phospholipids were characterized by physicochemical, chromatography, spectroscopy (differential scanning calorimetry (DSC), infrared (IR), and nuclear magnetic resonance (NMR)), ex vivo absorption using everted small intestine sac technique and in vivo studies using ethanol inducing hepatotoxicity in albino rats and comparing the results against plain MF.

Results: Ex vivo study showed significant increased absorption of MF from prepared MF herbosomes as compared to plain MF. The hepatoprotective potential of MF herbosomes evaluated by in vivo study revealed significantly decreased levels of serum glutamate oxaloacetate transminase (SGOT), serum glutamate pyruvate transminase (SGPT), total bilirubin, and alkaline phosphatase (ALP) in MF herbosomes as compared to plain MF. MF herbosomes also showed significantly decreased level of malonyl dehydrogenase along with increased levels of reduced glutathione, superoxide dismutase (SOD) and catalase as compared to plain MF which was also comparable to the standard drug, silymarin (SL).

Conclusion: The above mentioned results showed that hepatoprotective and antioxidant potency of MF enhanced due to the preparation of its herbosomes.     

Spatially addressed deposition and imaging of biochemically active bead microstructures by scanning electrochemical microscopy

A new procedure is described to deposit paramagnetic beads on surfaces to form microscopic agglomerates. By using surface-modified beads, microscopic structures with defined biochemical activity are formed. The shape and size of agglomerates were characterized by scanning electron microscopy (SEM), and the biochemical activity was mapped with scanning electrochemical microscopy (SECM). This approach is demonstrated using beads modified with anti-mouse antibodies (Ab). After allowing them to react with a conjugate of mouse IgG and alkaline phosphatase (ALP), the beads were deposited as agglomerates of well-defined size and shape. The biochemical activity was recorded in the generation-collection SECM mode by oxidizing 4-aminophenol formed in the ALP-catalyzed hydrolysis of 4-aminophenyl phosphate at the surface of the beads. The signal height correlated with both the amount of beads present in one agglomerate and the proportion of Ab binding sites saturated with the ALP mouse IgG conjugate. The feedback mode of the SECM was used to image streptavidin-coated beads after reaction with biotinylated glucose oxidase.     

A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer.

A simple and rapid means of enzyme kinetic analysis was achieved using electrospray ionization mass spectrometry and a one-point normalization factor. The model system used, glutathione S-transferase from porcine liver, is a two-substrate enzyme catalyzing the conjugation of glutathione with a variety of compounds containing an electrophilic center. An internal standard that is structurally similar to the product was added to the reaction quench solution, and a single-point normalization factor was used to determine the product concentration without the need of a calibration curve. Kinetic parameters, such as Km, Vmax and Ki (for thyroxine), obtained by electrospray mass spectrometry agreed with those obtained from traditional UV-vis spectroscopy, and competitive vs noncompetitive inhibition reactions could be delineated via mass spectrometry. These results suggest that our method can be applied to enzymatic processes in which spectrophotometric or spectrofluorometric assays are not feasible or when the relevant substrates do not incorporate chromophores or fluorophores. This new method is competitive with traditional UV assays in that it is facile and it involves very little analysis time.     

Non-equilibrium crystallization in freezing porous media: Numerical solution

The boundary value problem which arises during heat and moisture transfer in freezing fine-grained porous media under phase transition conditions is solved. It is assumed that the phase transition process occurs with finite rate of the water crystallization. So, the non-instantaneous kinetics is considered. Since the problem is significantly nonlinear the numerical method for the solution is applied. For the approximation of the system of differential equations the implicit two-level finite-difference scheme with central differences for space coordinate and one-side differences for time is used. The finite-difference system of equations is solved by “double sweep method.” It was shown the stability of “double sweep method” and solvability of the problem. Based on the correlation analysis, the dimensionless form for the diffusion coefficient as a function of moisture is obtained and used for the modeling. It is shown that the results for the characteristic distributions — temperature and total moisture, obtained in numerical solution, are in a good agreement with experimental investigations. The effect of the main criteria for the considered process — Lewis and Stefan numbers on the temperature, moisture, ice content and total moisture distributions is discussed. Especial attention was paid on the formation of the kinetic zone and its transformation in the course of non-equilibrium freezing. It was shown that the kinetic zone has a width of about 20–40% of the overall dimension of the system. Therefore the simulation of the phase transition zone as an infinitely thin front in freezing process, which is an approach incorporated in most theoretical models, is not suitable for the non-equilibrium water crystallization processes in fine-grained soils, and thereby conforms the validity of the kinetic approach.     

Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation

Assessing mesenchymal stem cell (MSC) differentiation status is crucial to verify therapeutic efficacy and optimize treatment procedures. Currently, this involves destructive methods including antibody‐based protein detection and polymerase chain reaction gene analysis, or laborious and technically challenging genetic reporters. Development of noninvasive methods for real‐time differentiation status assessment can greatly benefit MSC‐based therapies. This report introduces a nanoparticle‐based sensing platform that encapsulates two molecular beacon (MB) probes within the same biodegradable polymeric nanoparticles. One MB targets housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal reference, while another detects alkaline phosphatase (ALP), a functional biomarker. Following internalization, MBs are gradually released as the nanoparticle degrades. GAPDH MBs provide a stable reference signal throughout the monitoring period (18 days) regardless of differentiation induction. Meanwhile, ALP mRNA undergoes well‐defined dynamics with peak expression observed during early stages of osteogenic differentiation. By normalizing ALP‐MB signal with GAPDH‐MB, changes in ALP expression can be monitored, to noninvasively validate osteogenic differentiation. As proof‐of‐concept, a dual‐colored nanosensor is applied to validate MSC osteogenesis on 2D culture and polycaprolactone films containing osteo‐inductive tricalcium phospate.     

Synthesis and cellular biocompatibility of two nanophase hydroxyapatite with different Ca/P ratio

The cellular biocompatibility of two types of nanophase hydroxyapatites including nanophase standard hydroxyapatite (n-HA) and nanophase calcium deficient hydroxyapatite (n-CDHA) synthesized by a wet chemical method were assessed using primary cultured osteoblasts. Cytotoxicity of both materials was investigated with L929 cell line. The MTT method was used to evaluate the proliferation of osteoblasts on the third day and ALP activity assay was carried out on the fifth day. SEM was used to observe the morphology of the osteoblasts on the third day. Two types of nanophase hydroxyapatite both showed no cytotoxicity. Higher cell proliferation was observed on n-CDHA than n-HA. At the same time, cells spread more actively on the n-CDHA group. The ALP level of n-CDHA was also significantly higher on the former. Our results show that the n-CDHA is more suitable for osteoblasts growth and is also helpful for ALP synthesis.