We have studied the structure of lysozyme layers adsorbed at the silica−water interface using specular neutron reflection. The effect of pH on the adsorbed lysozyme layer was examined by manipulating the pH in two different cycles at two constant lysozyme concentrations of 0.03 and 1.0 g dm-3; the first cycle was started at pH = 4 followed by pH = 7 and then 8, before returning to 4; the second cycle was started at pH = 7 followed by a decrease to 4 and then back to 7. The neutron reflectivity profiles showed no hysteresis in either adsorbed amount or structure. There was less adsorption at pH = 4 than at pH = 7 for both lysozyme bulk concentrations. No variation of the reflectivity with time was found at the experimental resolution of about 5 min per measurement. The lysozyme structure at the interface at pH = 4 and pH = 7 was determined from reflectivity profiles at different isotopic compositions of the water. The thickness of the adsorbed layer at the lower concentration of 0.03 g dm-3 was found to be 30 ± 2 Å, suggesting sideways-on adsorption of the ellipsoidally shaped protein. At the higher concentration of 1.0 g dm-3 the thickness of the layer was found to be 60 ± 2 Å, suggesting bilayer adsorption with side-on orientation in each layer. These observations disagree with literature results from surface force and ellipsometric measurements which suggest that a side-on monolayer of 30 Å thickness is formed at dilute bulk concentrations, which switches to end-on adsorption of 45 Å thickness as the bulk concentration increases, eventually reaching a bilayer of 90 Å thickness when the bulk lysozyme concentration is further increased. The neutron measurements indicate that the adsorbed amount and the orientation of the globular protein are determined by the electrostatic repulsion between the lysozyme molecules within the layer.

In a previous study of BSA adsorption onto the hydrophilic silica/water interface using neutron reflection, we examined the concentration dependence of the surface excess of BSA at a pH close to its isoelectric point (IP). The surface excess was found to reach a plateau at a very low bulk protein concentration, suggesting a high affinity of BSA for the oxide surface. This work has now been extended to an investigation of the structure and composition of the BSA layer above and below its IP. It is found that adsorption of BSA is strongly dependent on pH, although the protein concentration has little influence on the surface excess at pH 3 and 7. Changing the pH from the IP substantially reduces the surface excess. The structure of the adsorbed layers below a bulk BSA concentration of 0.5 g dm-3 can be fitted to a single uniform layer distribution over all pH conditions studied, which suggests that there is no significant denaturation. Denaturation generally leads to a more fragmented peptide distribution and a nonuniform density distribution normal to the surface. The thicknesses of the layers below 0.5 g dm-3 were all smaller than the dimension of the short axis of the globular solution structure for BSA, indicating that the molecules are adsorbed sideways-on with their long axes parallel to the solid surface and that adsorption onto the hydrophilic surface results in some structural deformation. The reversibility of BSA adsorption at the hydrophilic silica/water interface was also examined directly. Adsorption was found to be irreversible with respect to changes in BSA concentration but reversible with respect to solution pH at low BSA concentrations only.

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 microm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.

Poly(dimethylsiloxane) (PDMS) elastomers are widely used in biological laboratories to produce prototype micro-bioreactors. The need was identified for a simple modification of the PDMS surface to prevent protein adsorption and cell attachment in certain areas and to increase the level of cell attachment where desirable. A study has been undertaken into the effect on protein adsorption and cell attachment of adsorbed surfactants and sodium hydroxide (NaOH) modification of the surface. The proteins are adsorbed from Dubecco's Modified Eagles Medium (DMEM) supplemented with foetal calf serum, HEPES, antibiotics, and ascorbic acid, as will be used to maintain the cells in the micro-bioreactor. Protein adsorption has been quantified by X-ray photoelectron spectroscopy (XPS) and the viable cell attachment by MTT assay. Protein and cell configuration were studied by atomic force microscopy (AFM) and light microscopy (LM) respectively. A positive correlation is observed between the level of protein adsorption and cell attachment. The Pluronic  F68 surfactant treatment exhibited the greatest reduction in protein adsorption and cell attachment, followed by the Pluronic  F127, with Tween 20 being the least effective. The NaOH treatment showed a substantial increase in both protein adsorption and cell attachment.

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

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In this paper, we establish a general mean value result of arithmetic functions over short intervals with the Selberg-Delange method. As an application, we generalize the Deshouillers-Dress-Tenenbaum's arcsin law on divisors to the short interval case.