Growth factors regional patterned and photoimmobilized scaffold applied to bone tissue regeneration.

Bone diseases such as osteomalacia, osteoporosis, and osteomyelitis are major illnesses that threaten the health of human. This study aimed to provide an idea at the molecular level of material properties determined with UV specific surface approaches. The tert-butyl hydroperoxide (t-BHP) exposure aging model bone mesenchymal stem cells (BMSCs) were reverted by using a poly-hybrid scaffold (PS), which is a carbon nanotube (CNT) coated polycaprolactone (PCL) and polylactic acid (PLA) scaffold, combined with insulin-like growth factor-1 (IGF). Then, the region-specific PS photo-immobilized with different growth factors (GFs) was obtained by interference and diffraction of ultraviolet (UV) light. Additionally, the reverted BMSCs were regionally pattern differentiated into three kinds of cells on the GF immobilized PS (GFs/PS). In vivo, the GFs/PS accelerate bone healing in injured Sprague-Dawley (SD) rats. The data showed that GFs/PS effectively promoted the differentiation of reverted BMSCs in the designated area on 21st day. These results suggest region-specific interface immobilization of GFs concurrently differentiating reverted BMSCs into three different cells in the same scaffold. This method might be considered as a short-time, low cost, and simple operational approach to scaffold modification for tissue regeneration in the future.

Crosslinker length dictates step-growth hydrogel network formation dynamics and allows rapid on-chip photoencapsulation.

Hydrogels formed via free radical-mediated thiol-ene step-growth photopolymerization have been developed for a broad range of tissue engineering and regenerative medicine applications. While the crosslinking mechanism of thiol-ene hydrogels has been well-described, there has been only limited work exploring the physical differences among gels arising from variations in crosslinker properties. Here, we show that the character of linear polyethylene glycol (PEG) dithiols used to crosslink multi-arm polyethylene glycol norbornene (PEGNB) can be used as a facile strategy to tune hydrogel formation kinetics, and therefore the equilibrium hydrogel network architecture. Specifically, we report the dramatic effect of crosslinker length on PEGNB hydrogel formation kinetics and the formed hydrogel properties. It is shown that the hydrogel formation kinetics and formed hydrogel properties can be tuned by solely varying the crosslinker length. It was hypothesized that under identical reaction conditions, a more accessible 3.5 k PEG dithiol crosslinker would improve network ideality relative to a shorter 1.5 k crosslinker. Longer linkers consequently promote significantly more rapid macromer crosslinking and therefore gelation. Accelerated gel formation satisfies an urgent unmet need for rapid polymerization in droplet microfluidics. Using long linkers, we demonstrate the ability to photopolymerize PEGNB microgels under flow on a microfluidic chip, with reliable control over microgel size and shape in a high-throughput manner. To further validate the potential of this platform to produce novel, microstructured cell carrier vehicles, 3T3 fibroblasts were successfully encapsulated and cultured over 14 days with excellent cell viability. This study demonstrates that PEGNB hydrogel dynamics could be readily customized to fulfill a variety of needs in tissue engineering, controlled cell delivery, or drug release applications.

Photoresponsive Cucurbit[8]uril-Mediated Adhesion of Bacteria on Supported Lipid Bilayers.

In this work, the development of a photoresponsive platform for the presentation of bioactive ligands to study receptor-ligand interactions has been described. For this purpose, supramolecular host-guest chemistry and supported lipid bilayers (SLBs) have been combined in a microfluidic device. Quartz crystal microbalance with dissipation monitoring (QCM-D) studies on methyl viologen (MV)-functionalized oligo ethylene glycol-based self-assembled monolayers, gel and liquid-state SLBs have been compared for their nonfouling properties in the case of ConA and bacteria. In combination with bacterial adhesion test, negligible nonspecific bacterial adhesion is observed only in the case of methyl-viologen-modified liquid-state SLBs. Therefore, liquid-state SLBs have been identified as most suitable for studying specific cell interactions when MV is incorporated as a guest on the surface. The photoswitchable supramolecular ternary complex is formed by assembling cucurbit[8]uril (CB[8]) and an azobenzene-mannose conjugate (Azo-Man) onto MV-functionalized liquid-state SLBs and the assembly process has been characterized using QCM-D and fluorescence techniques. Mannose has been found to enable binding of E. coli via cell-surface receptors on the nonfouling supramolecular SLBs. Optical switching of the azobenzene moiety allows us to "erase" the bioactive surface after bacterial binding, providing the potential to develop reusable sensors. Localized photorelease of bacterial cells has also been shown indicating the possibility of optically guiding cellular growth, migration, and intercellular interactions.

Immobilization technique for enhanced production of the immunosuppressant mycophenolic acid by ultraviolet and gamma-irradiated Penicillium roqueforti.

AIMS: Different entrapment matrices were screened to immobilize two strains of Penicillium roqueforti (AG101 and LG109) for more effective production of mycophenolic acid (MPA). Further improvement in the MPA productivity from immobilization of spores and mycelia was adopted by UV and gamma irradiation. METHODS AND RESULTS: Penicillium roqueforti strains were immobilized in different entrapping carriers and used for MPA production in shake flask cultures. Maximum MPA production was achieved on using an alginate concentration of 3·0% (w/v) and a mycelial fresh weight of 10% (w/v). MPA produced by alginate-immobilized spores and mycelia was almost double in comparison to the free system. The MPA-producing ability of immobilized AG101 and LG109 strain was significantly enhanced by mutagenesis through irradiation by UV (254 nm) for 120 and 90 min, respectively and gamma rays at 0·75 KGy. The feasibility of MPA production in a semi-continuous form by immobilized cells as affected by irradiation was adopted. CONCLUSIONS: MPA production by immobilized spores and mycelia was more intensified by UV and gamma irradiation. Moreover, the immobilized cell culture was superior to free-cell culture. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.

Bioconversion characteristics of Rhodopseudomonas palustris CQK 01 entrapped in a photobioreactor for hydrogen production.

The performance of the entrapped-cell photobioreactor during H2 production was assessed by using glucose as substrate in a continuous operation mode. The maximal hydrogen production rate and light conversion efficiency, 2.61 mmol/L/h and 82.3%, were obtained at a HRT of 11.4 h, an substrate loading rate of 4.2 mmol/h and an illumination of 590 nm and 6000 lux, the corresponding hydrogen yield and total energy efficiency were 0.62 mmol H2/(mmol glucose) and 4.8%, respectively. The results indicate the H2 production system illuminated at 590 nm wavelength engaged in energy storage for H2 production due to more ATP synthesized in primary reaction center, and was of higher energy recovery capacity. Furthermore, the total energy efficiency was far lower than the corresponding light conversion efficiency due to intermediates production.

Influence of blue light on Streptococcus mutans re-organization in biofilm.

Our aim was to examine the viability and structure of new biofilm formed by Streptococcus mutans that was previously exposed to blue light. S. mutans bacteria were grown to form a mature biofilm, that was exposed to blue light (wavelengths, 400-500 nm) for 1-10 min (equivalent to 68-680 J/cm(2)). Biofilm was dispersed by sonication, and then the suspended bacteria were grown to re-organize as a new biofilm. Biofilm formation after 2, 4, and 6 h, was examined by viable counts and by confocal laser scanning microscopy using live/dead bacterial staining. A significant decrease in bacterial viability was found in the 6h biofilms formed by bacteria that had been previously exposed to blue light for 7 or 10 min. Confocal microscopy images showed a decrease in the live/dead bacterial ratio after 3-10 min of light exposures. Dead bacteria were mainly at the outer layers of the biofilm. Exposure of S. mutans in biofilm to blue light affected the re-formation of a new biofilm, showing an increase in the amount of dead bacteria. This phenomenon suggests that blue light has a delayed antibacterial effect, although it does not interfere with bacterial capability to reform an initial biofilm.

Photochemical immobilization of cells onto a glass substrate for in situ DNA analysis.

A simple and robust method to immobilize cells onto a glass substrate is presented. The method employs a photochemical reaction of benzophenone, which is modified on the substrate using a standard silane coupling agent, with cells. Cells were immobilized to an area irradiated with UV light from a standard light source under an inverted microscope. The dependence of immobilization on the light power intensity and irradiation time was investigated. In situ DNA analysis within the immobilized cells was demonstrated using target-primed rolling circle amplification and fluorescent detection.

Immobilization of magnetic modified Flavobacterium ATCC 27551 using magnetic field and evaluation of the enzyme stability of immobilized bacteria.

The magnetic modified Flavobacterium sp. was prepared by covalently binding carboxylate-modified magnetic nanoparticles, and also, ionic adsorption of magnetic Fe(3)O(4) nanoparticles on the cell surface. The magnetic modified bacteria were immobilized by both internal and external magnetic fields. The pH stability and inherent resistance of the enzyme activity of the immobilized bacteria under acidic and alkaline conditions were increased. Immobilization of the magnetic modified bacteria using an external magnetic field improved the enzyme thermal stability. The results revealed that immobilization of the magnetic modified bacteria by an external magnetic field keeps 50% of the enzyme activity after 23.4, 16.6 and 6 h of incubation at 55 °C for the covalently binding of magnetic nanoparticles, the ionic adsorption of magnetic nanoparticles and the free cells, respectively. The results demonstrated the negative effect of various magnetic beads on the enzyme thermal stability of immobilized magnetic modified bacteria using an internal magnetic field.

PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids.

Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. While recent research efforts have created diverse macromer chemistry to form hydrogels, the mechanisms of hydrogel polymerization for in situ cell encapsulation remain limited. Hydrogels prepared from chain-growth photopolymerization of poly(ethylene glycol) diacrylate (PEGDA) are commonly used to encapsulate cells. However, free radical associated cell damage poses significant limitation for this gel platform. More recently, PEG hydrogels formed by thiol-ene photo-click chemistry have been developed for cell encapsulation. While both chain-growth and step-growth photopolymerizations offer spatial-temporal control over polymerization kinetics, step-growth thiol-ene hydrogels offer more diverse and preferential properties. Here, we report the superior properties of step-growth thiol-ene click hydrogels, including cytocompatibility of the reactions, improved hydrogel physical properties, and the ability for 3D culture of pancreatic ß-cells. Cells encapsulated in thiol-ene hydrogels formed spherical clusters naturally and were retrieved via rapid chymotrypsin-mediated gel erosion. The recovered cell spheroids released insulin in response to glucose treatment, demonstrating the cytocompatibility of thiol-ene hydrogels and the enzymatic mechanism of cell spheroids recovery. Thiol-ene click reactions provide an attractive means to fabricate PEG hydrogels with superior gel properties for in situ cell encapsulation, as well as to generate and recover 3D cellular structures for regenerative medicine applications.

A novel bioreactor for immobilized phototrophs.

A novel configuration of photobioreactor is described in which filaments of alginate containing immobilized cells of a leaky mutant of Dunaliella parva are wound round a central light well which is located within a glass outer chamber so that a liquid medium is caused to flow in the annular space between the outside chamber and the alginate filaments. Glycerol production by D. parva was maintained for 700 h and the highest concentration of glycerol attained was approx. 12 mg l(-1).

[Telomerase expression is not involved in aging process of human keratinocytes induced by UVB irradiation].

OBJECTIVE: To investigate the mechanism involved in aging process of immortalized human keratinocyte (HaCaT) and primary human epidermis keratinocyte of adults (HEKa) irradiated by ultraviolet B(UVB). METHODS: HEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-beta-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase (hTERT). RESULT: Strong positive SA-beta-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from (1.81 +/-0.25)% to (4.43 +/-0.28)% and necrosis rate increased from (0.05 +/-0.01)% to (0.10 +/-0.03)% in HaCaT cell, but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast, apoptosis rate of in HEKa cells significantly increased from (0.65 +/-0.05)% to (59.53 +/-2.35)%, and the necrosis rate in HEKa cells also reached (3.89 +/-0.24)%(P<0.05). Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines, intracellular level of SOD decreased and MDA increased remarkably after UVB exposure, and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression. CONCLUSION: The stress-induced premature senescence (SIPS) in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress, not related to the telomerase expression.

Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin.

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.

Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine.

The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process ofgelation. The findings should contribute to a better understanding of polymer-cell interactions, which could be crucial in possible medical treatments. Alginate-yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50-600 microm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell-alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5x10(8) and 1x10(9) cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.

Kinetics of degradation by immobilized cells with ultrasonic irradiation.

Linear alkylbenzene sulphonate (LAS) decompositions by immobilized cells with ultrasonic irradiation were investigated at the optimized condition in order to gain insight into the kinetics of the decomposition process. Firstly, by analyzing the decomposition process of LAS theoretically, showed the kinetic model of suspending cells and immobilized cells both followed the MONOD model (namely micro=micromaxs/(ks+s) during wastewater treatment, then discussed the kinetics model of LAS degradation by immobilized cells with ultrasonic irradiation at the presupposition conditions, and then the two unknown parameters ([See text]) in the gained model were researched at the condition of laboratory. Moreover, experiments have been done to validate the parameters ([See text]) in the kinetics equation, the result shows the valid kinetics equation of LAS degradation is at the LAS concentration of 30-80 mg/L:

Apparatus for exposing cell membranes to rapid temperature transients.

We seek to determine whether cell membranes contain sensors that trigger a downstream response to temperature excursions. To do this, we have developed a novel apparatus for exposing a cell membrane to an extremely rapid temperature excursion in the nanosecond range. Cells are plated on a gold surface that is back-heated by a pulsed laser and cooled by conduction of heat into the glass substrate and the liquid medium. Analysis using the heat diffusion equation shows that the greatest temperature rise is localized within a region tens of nanometres thick, suitable for specifically heating a cell membrane without heating the remainder of a cell. We refer to this device as a nanosecond hotplate.

Structure of dried cellular alginate matrix containing fillers provides extra protection for microorganisms against UVC radiation.

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a approximately 1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.