Suspension culture of differentiated rat heart myocytes on non-adhesive surfaces

Cardiac myocytes isolated from adult rat ventricles have been maintained in a stable, differentiated state for prolonged periods by the use of suspension culture on hydrophobic tissue culture inserts or agarose-coated plates. The success of this procedure depends on the use of low-serum media to prevent myocyte-myocyte interaction and proliferation of any residual endothelial cells. Myocytes cultured in this manner retain many of their structural characteristics, suggesting that maintenance of their elongated irregular shape is not dependent on interaction with extracellular matrix. They also exclude trypan blue, can be vitally stained by the uptake and reduction of the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], synthesize myosin and when returned to adhesive surfaces are capable of attachment and attendant dedifferentiation. Stability of the myocytes in suspension permits their use in co-culture experiments; specifically, myocytes separated from endothelial cells by the hydrophobic membrane of the tissue culture insert stimulated proliferation of the latter cells, suggesting this to be a useful system for studying myocyte-endothelial cell interaction.     

Spatially controlled cell engineering on biodegradable polymer surfaces

Controlling receptor-mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1-3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin-mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6-8). This provides a mechanism for mimicking natural cell-to-matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron-scale pattern features (9-14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron-scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide-poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.     

Biomimetic peptides that engage specific integrin-dependent signaling pathways and bind to calcium phosphate surfaces

Many important matrix proteins involved in bone remodeling contain separate domains that orient the protein on hydroxyapatite and interact with target cell receptors, respectively. We have designed two synthetic peptides that mimic the dual activities of these large, complex proteins by binding to calcium phosphate minerals and by engaging integrin-dependent signaling pathways in osteoblasts. The addition of either PGRGDS from osteopontin or PDGEA from collagen type I to the HAP-binding domain of statherin (N15 domain) did not alter its alpha-helical structure or diminish its affinity for hydroxyapatite. Immobilized N15-PGRGDS bound MC3T3-E1 osteoblasts predominantly via the alpha v beta 3 integrin and induced focal adhesion kinase (FAK) phosphorylation at comparable levels to immobilized osteopontin. Immobilized N15-PDGEA bound MC3T3-E1 osteoblasts predominantly through the alpha 2 beta 1 integrin and induced similar levels of FAK phosphorylation. Although both peptides induced FAK phosphorylation with similar time courses, only the N15-PDGEA peptide induced ERK1/2 phosphorylation, showing that these peptides are also capable of engaging integrin-specific signaling pathways. This peptide system can be used to study adhesion-dependent control of signaling in the context of the relevant biomineral surface and may also be useful in biomaterial and tissue engineering applications.     

工程陶瓷的磨削钻孔及表面形成机理

结合工程陶瓷的加工特性,采用特定组份的铜基结合剂烧结金刚石钻头对其进行孔加工试验研究.结合剂体积分数为48%Cu、30%Co、6%Ni、5%WC、5%Ti、4%Sn、2%Cr.根据烧结金刚石钻头的磨削加工特点,导出了唇面单颗金刚石磨粒的平均载荷的计算公式;在此基础上,结合陶瓷磨削表面的扫描电镜观察,分析了金刚石钻头磨削钻孔的陶瓷材料去除机理.结果表明,在试验条件下,陶瓷材料虽然以脆性断裂去除为主,但也有部分材料发生塑性变形去除.     

Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.     

Increasing the filtration rate of phospho-gypsum using surfactant

Phospho-gypsum is a by-product of processing phosphate rock for phosphoric acid production by acidulation with sulfuric acid. The size distribution of phosphor-gypsum is a major factor for the economics of the process as it greatly affects the filterability of the acid. Surface active agents proved to be very effective additive for growth of gypsum crystals. Two phosphate concentrates, one from Egypt (New Valley) and the other from USA (South Florida) were tested for phosphoric acid production with modification of gypsum crystals using non-ionic surfactant (CMR-100) containing a mixture of C₆–C₂₂ sorbitan esters. The studies were carried out using a semi-continuous laboratory-scale unit simulating the dihydrate process conditions.The mean diameter of the phospho-gypsum crystals increases in the presence of surfactant for both phosphate concentrates. The surfactant leads to decreasing the viscosity and modification of gypsum crystals through decreasing the fine size fractions and attainment of large and uniform crystal shape. The mean diameter of New Valley phosphor-gypsum increases from about 30.0 μm to 36.6 μm while the mean diameter of South Florida phospho-gypsum increases from about 30.3 μm to about 38.4 μm. On the other hand, the average surface area of both New Valley and South Florida phosphor-gypsum were decreased from 4461 and 8069 cm²/g without surfactant to 3284 and 3995 cm²/g with surfactant, respectively. In addition, the surfactant leads to an increase in P₂O₅ recovery of 1–2%, which consequently improves plant productivity.     

Biochemical engineering of neural cell surfaces by the synthetic N-propanoyl-substituted neuraminic acid precursor

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration, and pathogenesis of diseases. During times of intense plasticity within the nervous system, such as development and regeneration, sialylation of neural cells is distinct from the time of its maintenance. In this study, a synthetic precursor of neuraminic acid, N-propanoylmannosamine (N-propanoyl neuraminic acid precursor (P-NAP)), is applied to the culture medium of oligodendrocyte progenitor cells, microglia, astrocytes, and neurons from neonatal rat brains to alter sialylation of glycoconjugates within these cells. P-NAP is metabolized and incorporated as N-propanoyl neuraminic acid into glycoproteins of the cell membrane. P-NAP stimulates the proliferation of astrocytes and microglia but not of oligodendrocyte progenitor in vitro. However, P-NAP increases the number of oligodendrocyte progenitor cells expressing the early oligodendroglial surface marker A2B5 epitope. In the presence of P-NAP, cerebellar neurons (but not astrocytes) in microexplant cultures start to express the oligodendroglial progenitor marker A2B5 epitope, which is normally undetectable on these cells. The controls, which were performed in the absence of any additive or in the presence of the physiological precursor of neuraminic acid, N-acetylmannosamine, did not show any increase in A2B5 expression.     

Biomimetic peptide surfaces that regulate adhesion, spreading, cytoskeletal organization, and mineralization of the matrix deposited by osteoblast-like cells

In an effort to regulate mammalian cell behavior in contact with solid material surfaces, we have functionalized surfaces with different ratios of both the putative cell binding (-Arg-Gly-Asp-) domain and a consensus heparan-binding domain. The peptide sequences -Arg-Gly-Asp- (-RGD-) and -Phe-His-Arg-Arg-Ile-Lys-Ala- (-FHRRIKA-) or mixtures of the two in the ratios of 75:25 (mimetic peptide surface I), 25:75 (mimetic peptide surface II), and 50:50 (mimetic peptide surface III) were immobilized on model surfaces using a heterobifunctional cross-linker to link the peptide(s) to amine-functionalized quartz surfaces. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry, thickness of the overlayers, and surface density of immobilized peptides ( approximately 4-6 pmol/cm2). The degree of rat calvaria osteoblast-like cell spreading, focal contact formation, cytoskeletal organization, proliferation, and mineralization of the extracellular matrix (ECM) on model biomaterial surfaces was examined. Mimetic peptide surface II (MPS II) and MPS III supported the highest degree of cell spreading (p < 0.05), following 4 h of incubation, compared to MPS I, homogeneous -RGD-, and homogeneous -FHRRIKA- grafted surfaces. Furthermore, MPS I, MPS II, MPS III, and homogeneous -RGD- surfaces promoted the formation of focal contacts and stress fibers by attached bone cells. The strength of bone cell detachment following 30 min of incubation was significantly higher (p < 0.05) on MPS II surfaces compared to homogeneous -RGD- and -FHRRIKA-. However, the degree of cell proliferation on the peptide surfaces were not significantly different from each other (p > 0.1). Following 24 d in culture, the areas of mineralized ECM formed on MPS II and MPS III surfaces were significantly (p < 0.05) larger than those of other surfaces. These results demonstrate that utilizing peptide sequences incorporating both cell- and heparin-adhesive motifs can enhance the degree of cell surface interactions and influence the long-term formation of mineralized ECM in vitro.     

Patterned delivery of immunoglobulins to surfaces using microfluidic networks

Microfluidic networks (microFNs) were used to pattern biomolecules with high resolution on a variety of substrates (gold, glass, or polystyrene). Elastomeric microFNs localized chemical reactions between the biomolecules and the surface, requiring only microliters of reagent to cover square millimeter-sized areas. The networks were designed to ensure stability and filling of the microFN and allowed a homogeneous distribution and robust attachment of material to the substrate along the conduits in the microFN. Immunoglobulins patterned on substrates by means of microFNs remained strictly confined to areas enclosed by the network with submicron resolution and were viable for subsequent use in assays. The approach is simple and general enough to suggest a practical way to incorporate biological material on technological substrates.     

Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase

Tenascin is a large oligomeric glycoprotein of the extracellular matrix. Its location is innervated muscle tissues. We investigated immunohistologically, using two monoclonal antibodies (mah) against Tenascin, biopsied denervated human muscle of children and adults. Tenascin was present in the interstitial space among denervated muscle fibres. Accumulation of Tenascin in denervated adult muscle tissue was frequent, accumulation in denervated muscle tissue of children was sparse and weak. The two antibodies reacted correspondingly. Tenascin was not only found in the vicinity of atrophic muscle fibres, but also close to normally sized fibres, suggesting an early stage of denervation even before reduction of muscle fibre volume.     

Concentration of hepatitis A and B virus antigens using water-soluble polymers

A possibility of using reversibly sedimented polymeric system for the concentration of hepatitis A and B virus antigen has been demonstrated. As polymeric systems, tetrasole-containing polyelectrolytes and interpolymeric complex polymethacryl acid-poly-1-vinylpyrrolidone were used, capable of sedimenting in acid medium and dissolving in media with the neutral pH.     

Reversible polymers formed from self-complementary monomers using quadruple hydrogen bonding

Units of 2-ureido-4-pyrimidone that dimerize strongly in a self-complementary array of four cooperative hydrogen bonds were used as the associating end group in reversible self-assembling polymer systems. The unidirectional design of the binding sites prevents uncontrolled multidirectional association or gelation. Linear polymers and reversible networks were formed from monomers with two and three binding sites, respectively. The thermal and environmental control over lifetime and bond strength makes many properties, such as viscosity, chain length, and composition, tunable in a way not accessible to traditional polymers. Hence, polymer networks with thermodynamically controlled architectures can be formed, for use in, for example, coatings and hot melts, where a reversible, strongly temperature-dependent rheology is highly advantageous.     

Shape from stereo: a systematic approach using quadratic surfaces

Postnatal development of the neuro- and viscerocranium with special reference to the maxillodental structures was studied morphometrically by analyzing computer tomograms and radiograms of human and monkey heads of different age groups. The following parameters were used: the prognathic angle, the clivus angle, the palate-incisivus angle, the interincisival angle and the viscerocranial quotient. In the newborn primates including man, all parameters measured were relatively similar; postnatally, however, characteristic differences in the growth pattern between man and monkey were developing. In monkey, a marked prognathic growth of the viscerocranium was found associated with characteristic positional changes of the frontal teeth, whereas the growth of the neurocranium was retarded. Here, unlike the human, a flattening of the skull base was observed. In contrast, the human skull showed no major proportional changes during its postnatal development compared with the original spherical skull form of the newborn.     

Effects of surfactant distribution and ventilation strategies on efficacy of exogenous surfactant

The effects of both surfactant distribution patterns and ventilation strategies utilized after surfactant administration were assessed in lung-injured adult rabbits. Animals received 50 mg/kg surfactant via intratracheal instillation in volumes of either 4 or 2 ml/kg. A subset of animals from each treatment group was euthanized for evaluation of the exogenous surfactant distribution. The remaining animals were randomized into one of three ventilatory groups: group 1 [tidal volume (VT) of 10 ml/kg with 5 cmH2O positive end-expiratory pressure (PEEP)]; group 2 (VT of 5 ml/kg with 5 cmH2O PEEP); or group 3 (VT of 5 ml/kg with 9 cmH2O PEEP). Animals were ventilated and monitored for 3 h. Distribution of the surfactant was more uniform when it was delivered in the 4 ml/kg volume. When the distribution of surfactant was less uniform, arterial PO2 values were greater in groups 2 and 3 compared with group 1. Oxygenation differences among the different ventilation strategies were less marked in animals with the more uniform distribution pattern of surfactant (4 ml/kg). In both surfactant treatment groups, a high mortality was observed with the ventilation strategy used for group 3. We conclude that the distribution of exogenous surfactant affects the response to different ventilatory strategies in this model of acute lung injury.