A mass separation of chondrocytes from cartilage tissue utilizing an automatic crushing device

Porcine articular cartilage tissue was crushed using Multi-Beads-Shocker^™ and digested with trypsin and collagenase type II. Chondrocyte cell yield was maximized by automatic crushing for 10 s, which was 4 times that by manual dicing with a surgical blade and might be useful for the automation of the cell processing.     

Deposition of DLC film with adhesive W-DLC layer on stainless steel and its tribological properties

Tribological properties of a diamond-like carbon (DLC) coating with an adhesive tungsten-containing DLC (W-DLC) layer were investigated. The coatings were deposited onto AISI316L steel substrates and Si wafers using plasma enhanced chemical vapor deposition and tungsten co-sputtering of the metal target. Methane and argon gases were used as the precursor of the coatings. In this study, three types of coatings were evaluated: DLC/W-DLC on AISI316L (DLC-1), DLC/W-DLC on Si wafer (DLC-2), and DLC on Si wafer (DLC-3). The structural characterizations were performed by transmission electron microscopy and tapping mode atomic force microscopy. At the boundary between the W-DLC layer and the AISI316L substrate, microscopic decohesion or delamination was not observed. The surface roughness of the DLC-1 coating was greater than that of the DLC-2 coating. This feature was derived from the surface roughness of the initial surface of the AISI316L substrate. Friction tests were performed using a rotation-type ball-on-flat configuration tribometer. The observed friction of the DLC-1 coating was unstable compared with the DLC-2 or DLC-3 coatings. This was due to wear debris which had risen to the friction surface resulting in unstable friction on the DLC-1 coating. During the friction studies, the top DLC layer was removed from the adhesive W-DLC layer because the adhesive strength at this part was not enough. In order to achieve the low and stable friction of the DLC coating with the W-DLC layer on AISI316L, it is necessary to improve the smoothness of the surface and the adhesion between the DLC coating and the W-DLC layer.     

Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid

Abstract

Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS) with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-)). For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR)) with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR) although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR) both in air and in PBS(-). A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR) in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR). The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.     

Effect of Oxide Film on the Friction of 52100 Steel in Vacuum

To solve the anomalous behaviors of friction coefficient in reciprocated slidings of 52100 steel in ultrahigh vacuum, studies were made if the wear track, friction level, and number of traverses for a 52100 steel test ball sliding on a 52100 steel flat over a pressure range of 760 to 3 × 10^?9 torr. Plots of the friction coefficient versus number of traverses show a maximum (f = 1.0–1.2) followed by a drop (f = 0.5–0.6). Both the friction force-time curves and microscopic friction area change from smooth to rough at the point of the drop in friction coefficient. The shape of the friction plot is ascribed to a build-up and rupture of the oxide film that forms on the surface of the 52100 steel specimen in atmosphere and is affected by applied load, direction of finishing marks and pressure level of the environment. Further, above a critical pressure of 10^?6 torr the drop was not experienced. It was also found that higher friction does not always mean that the sliding surfaces are clear or more oxide-free.     

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.     

Effects of excess K+ on carbachol-induced contractions in the guinea-pig tracheal muscle

1. In smooth muscles isolated from the guinea-pig trachea, the effects of dihydropyridines, nifedipine and nicardipine on contractions produced by carbachol (Cch) were studied in normal (6 mM) and excess K+ concentration (60 mM). The tonic contraction produced by 1 microM Cch was highly dependent on the external Ca2+ concentration ([Ca2+]0) and was not significantly affected by cyclopiazonic acid or thapsigargin, Ca2+ uptake inhibitor. 2. [Ca2+]0-tension curves were steeper in the presence of 1 microM Cch (the Hill coefficient: 2.5) than in the presence of 60 mM K+ (Hill coefficient: 1.6) and their ED50 of Ca2+ was 0.16 and 0.39 mM, respectively. An increase of K+ to 60 mM in the presence of 1 microM Cch shifted the curve to the left roughly in parallel (ED50: 0.12 mM, Hill coefficient: 2.3). 3. [Ca2+]0-tension curve in the presence of 1 microM Cch was shifted to the right in parallel by nifedipine (1 microM). This was markedly potentiated by 60 mM K+ (the increase in ED50 of Ca2+ being 3 times at 6 mM and 15 times at 60 mM K+). No tension was evoked by Ca2+ up to 2.5 mM in 60 mM K+ solution containing 1 microM nifedipine but no Cch. 4. In the absence of nifedipine, Cch-induced contractions were potentiated by 60 mM K+, whereas in the presence of nifedipine, Cch-induced contractions were markedly inhibited by 60 mM K+. These mechanical changes were accompanied by an increase or a decrease in intracellular Ca2+. 5. A hypothesis is presented to explain the results which suggests that the kinetics of Ca2+ influx though a single type of pathway is modulated by membrane potential and receptor activation and that the susceptibility of the pathway to dihydropyridine blockade is closely related to the Ca2+ influx kinetics with receptor activation reducing and membrane depolarization increasing the susceptibility.     

Surgical significance of supernumerary parathyroid glands in renal hyperparathyroidism

In secondary hyperparathyroidism (2HPT) fundamentally all parathyroid glands, including supernumerary glands, become hyperplastic, and stimulation of parathyroid glands continues after parathyroidectomy (PTx). Therefore supernumerary glands have special significance during surgery for 2HPT, whether persistent or recurrent HPT. In the present study 570 patients underwent initial total PTx with a forearm autograft. The frequency, type, location, histopathology, and clinical significance of the supernumerary glands were evaluated. At the initial operation 90 supernumerary glands were removed from 82 to 570 patients (14.4%); 12 patients (2.1%) required extirpation of supernumerary glands for persistent/recurrent HPT. Altogether 104 supernumerary glands were identified at operation in 94 of the 570 patients (16.5%). Among these 104 glands, 25 (24.0%) were of the rudimentary, or split, type and 79 (76.0%) of the proper type. Supernumerary glands were most frequently identified in the thymic tongue (53/104, 51.0%); 32 (60.4%) of these 53 glands were identified only microscopically. In 6 of the 570 cases (1.1%), reoperation was required for persistent HPT due to supernumerary glands located in the mediastinum, and 6 patients underwent neck reexploration for recurrence. Histopathologically, 61 of 104 (58.7%) supernumerary glands, including 36 glands recognized only microscopically, showed diffuse hyperplasia, and 43 (41.3%) displayed nodular hyperplasia. Residual small supernumerary glands with diffuse hyperplasia have the potential to be transformed to nodular hyperplasia during long-term hemodialysis. Therefore all parathyroid glands including supernumerary glands should, if possible, be removed at the initial operation. Routine removal of the thymic tongue and careful examination of the regions surrounding the lower poles of the thyroid, especially on the left side, are important steps in the surgical treatment.     

Structure and expression of the human SM22alpha gene, assignment of the gene to chromosome 11, and repression of the promoter activity by cytosine DNA methylation

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.     

Leaching and cytotoxicity of formaldehyde and methyl methacrylate from acrylic resin denture base materials

Acrylic resin dentures have the potential to elicit irritation, inflammation, and an allergic response of the oral mucosa. Studies of substances leachable from acrylic resins, their cytotoxicity to cultured cells, and means of reducing their leaching were systematically conducted. Under in vivo and in vitro conditions, formaldehyde and methyl methacrylate were significantly leached into human saliva and saliva-substitute buffer, especially from autopolymerized resins. Both leachable substances showed cytotoxic potentials in the range of their leaching concentrations. Formaldehyde was cytotoxic at lower concentrations than methyl methacrylate. Preleaching in water reduced subsequent leaching of both formaldehyde and methyl methacrylate, and the amount of reduction depended on an increase in the preleaching temperatures. Immersion of acrylic resin dentures in hot water (50 degrees C) before insertion is recommended, especially for autopolymerized resins used either for rebasing or as denture base materials, to minimize the risk of adverse reactions in patients who wear acrylic resin dentures.