Cell Number Ratio Research Articles

A study on the small scale precipitation structure over Germany using the radar network of the German Weather Service

Radar composite images of the German Weather Service's (DWD) radar network are used to investigate the spatial and temporal structure of convective rain areas in postfrontal situations. Thirty-nine days were selected and the respective postfrontal convective rain fields were analysed based on over 3000 radar images, yielding altogether approximately 140000 individual rain areas. Basic parameters of the latter within the convective area such as number of rain areas, occupied area and number of reflectivity peaks contained within one single rain area were determined. Then their diurnal variation and spatial distribution within Germany were investigated. The study focuses on single cells (rain areas with one reflectivity peak) and clusters (rain areas with two or more reflectivity peaks), with the latter being defined as consisting of several interconnected convection cells. Number and area of rain areas follow the expected, typical convective diurnal cycle with a maximum around noon. The number ratio of single cells and clusters, however, remains almost constant over the day. This surprising result may be explained as an equilibrium state between growing single cells, the development of single cells into clusters and decaying of single cells and clusters. Similar results are found for the area covered by single cells or clusters. While single cells dominate in terms of number, clusters provide most of the precipitating area. Single convective cells and small clusters preferably occur over mountain ranges but also along the North Sea coast. Large clusters with more than 9 reflectivity maxima are almost exclusively found along the North Sea coast in an area of about 200 km width.

Testing the Immunosuppressive Effects of Cyclophosphamide in the Popliteal Lymph Node Assay in the Modification of Graft-Vs-Host Reaction (PLNA-GvHR) in the Rat

We tested the hypothesis that popliteal lymph node assay (PLNA) in the modification of graft vs host reaction (GvHR) enabled to assess immunosuppressive potential of xenobiotics. We conducted PLNA in GvHR modification under two experimental conditions using cyclophosphamide (CY). In the first experiment average lymph node weight index (IW, weight ratio of popliteal lymph node of hind footpad injected with parental lymphocytes to lymph node of footpad injected with vehicle) in rats administered intraperitoneally with CY simultaneously with, or 3 days before local GvHR induction was 84% and 42%, respectively less than average IW in external control animals injected with parental lymphocytes into one footpad and vehicle into contralateral footpad. Average lymph node lymphocyte index (IL, ratio of cell number in the lymph node undergoing GvHR to control lymph node) in the tested animals was 92% and 86%, respectively less than in controls. In the second experiment in rats injected subcutaneously into one footpad with parental lymphocytes suspended in solution of CY at the concentrations of 4.6 mM or 18.6 mM and with parental lymphocytes into contralateral footpad, average IW was 88%and 92%, respectively less than average IW in the external control animals injected with parental lymphocytes into both footpads (average IL was 84 and 91%, respectively less than in controls). Our preliminary experiments showed a strong inhibitory effect of CY in both PLNA-GvHR models used and we believe that both models may be of value in designing future protocols aiming at prediction of immunosuppressive potential of chemicals.

Carbon-carbon composite bearing materials in hip arthroplasty: analysis of wear and biological response to wear debris.

Ultra-high molecular weight polyethylene wear particles have been implicated as the major cause of osteolysis, implant loosening and late aseptic failure in total hip arthroplasties in vivo. This study initially screened 22 carbon-carbon composite materials as alternatives for UHMWPE in joint bearings. New bearing materials should satisfy certain criteria--they should have good wear properties that at least match UHMWPE, and produce wear particles with low levels of cytotoxic and osteolytic activity. Initial screening was based on wear resistance determined in short-term tribological pin-on-plate tests. Three materials (HMU-PP(s), HMU-RC-P(s), and SMS-RC-P(s)) which had superior wear resistance were selected for long-term testing. All materials had very low wear factors and SMS-RC-P(s), which had a wear factor of 0.08 +/- 0.56 x 10(-7) mm3/Nm, was selected for the subsequent biological testing and particle size analysis. SMS-RC-P(s) showed good biocompatibility in bulk material form and also the wear particles had low cytotoxicity for L929 fibroblasts in culture compared to metal wear particles. Wear debris size analysis by transmission electron microscopy showed that the particles were very small, with the vast majority being under 100 nm in size, similar to metal wear particles. The potential osteolytic effect of SMS-RC-P(s) wear particles was investigated by culturing particles with human peripheral blood mononuclear cells and measuring TNFalpha production. SMS-RC-P(s) did not significantly stimulate TNFalpha production at a particle volume to cell number ratio of 80:1, indicating that the debris had a low osteolytic potential. The results of this study suggest that carbon-carbon composites, particularly those composed of PAN-based fibers may be important biomaterials in the development of next generation bearing surfaces for use in total joint replacements that have very low wear rates and reduced osteolytic and cytotoxic potential.

The influence of molecular weight, crosslinking and counterface roughness on TNF-alpha production by macrophages in response to ultra high molecular weight polyethylene particles

The response of murine macrophages to clinically relevant polyethylene wear particles generated from different polyethylenes at various time points and volumetric doses in vitro was evaluated. Clinically relevant ultra high molecular weight polyethylene (UHMWPE) wear debris was generated in vitro in a lubricant of RPMI 1640 supplemented with 25% (v/v) foetal calf serum using a multi-directional pin-on-plate wear rig under sterile conditions. Wear debris was cultured with C3H murine peritoneal macrophages at various particle volume (μm 3): cell number ratios. The secretion of TNF-α was determined by ELISA. Initially the effect of molecular weight of UHMWPE was considered. Higher molecular weight GUR415HP was shown to have a lower wear rate than the lower molecular weight GUR1120, however a greater volume of the wear debris produced by the high molecular weight GUR415HP was in the 0.1–1.0 μm size range. Wear debris from GUR415HP produced significant levels of TNF-α at a concentration of 1 μm 3/cell while at least 10 μm 3/cell of GUR1120 wear debris per cell was needed to produce significant levels of TNF-α. Secondly the effects of crosslinking GUR1050 was examined when worn against a scratched counterface. The wear rate of the material was shown to decrease as the level of crosslinking increased. However the materials crosslinked with 5 and 10 Mrad of gamma irradiation produced higher percentages of 0.1–1.0 μm size wear particles than the non-crosslinked material. While the crosslinked material was able to stimulate cells to produce significantly elevated TNF-α levels at a particle concentration of just 0.1 μm 3/cell only concentrations of 10 μm 3/cell and above of the non-crosslinked wear debris were stimulatory. When the counterface was changed from scratched to smooth the wear rate for all three GUR1050 materials was further reduced. For the first time nanometre size wear particles were observed from polyethylene which reduced the percentage mass of debris in the 0.1–1.0 μm size range. For all three materials on the smooth counterface only concentrations of 50 μm 3/cell and above were stimulatory. This study has demonstrated that molecular weight, crosslinking and counterface roughness are important factors in determining the biological activity of polyethylene.

A hybrid continuum/particle approach for modeling subsonic, rarefied gas flows

A hybrid approach that combines a continuum approach solving the Navier–Stokes equations and a particle method called the information preservation (IP) method is implemented to simulate subsonic, rarefied gas flows accurately and efficiently. The coupling between the continuum and particle approaches relies on a continuum/particle interface, which is investigated in detail. The IP method preserves information at the macroscopic level that allows the continuum approach to directly use the information from the particle region. In order to correctly generate particles from the continuum region, two strategies are proposed. One strategy adopts a Marshak-type condition, which requires many particles in a cell to control the flux fluctuation due to the microscopic motion of particles. In the second strategy, reservoir cells and buffer cells are used, and this is the approach adopted in our general hybrid scheme. The location of the interface is determined using a continuum breakdown parameter. Studies show that the continuum breakdown parameter suggested by Garcia et al. can be used to determine the location of the interface, but the implementation of the interface also affects the location of the interface. Simulation of a flow over a flat plate shows the performance of the hybrid approach and reveals the effects of the cutoff value to a continuum breakdown parameter. This approach is also applied to study the aerodynamics of a micro-scale airfoil, which is very difficult for a single method. The hybrid approach generally spends less computational time than the IP method for rarefied gas flows, and the numerical performance of the hybrid approach depends on the number ratio of continuum cells to particle cells.

Food Habits of Two-Year-Old Chinese Mitten Crab (Eriocheir sinensis) Stocked in Lake Bao'an, China

ABSTRACT Gastric mills of 362 specimens of two-year-old Chinese mitten crab (Eriocheir sinensis), which contained recognizable food items, from Lake Bao'an, China were examined. The food items were macrophytes, algae, arthropods, oligochaetes, fish, protozoa, rotifers, gastropods, and detritus, and the percent frequencies of occurrence (FO) for these items were 87.3%, 82.0%, 48.2%, 28.2%, 28.7%, 0.3%, 0.6%, 0.3% and 88.7%, respectively. Unidentified animal tissue was often observed and had a FO of 46.1%. In total, FO of plants (macrophytes + algae) was 87.7% and of animals was 89.8%. However, 5.8% of the gastric mills contained only animals, 5.3% had only macrophytes, and 0.3% contained only algae. There was no significant difference (p>0.05) in food habits between male and female crabs. The ratio of cell number of macrophytes to algae was about 156:1.

Sarpogrelate HCl, a selective 5-HT 2A antagonist, retards the progression of atherosclerosis through a novel mechanism

Although sarpogrelate HCl is widely used for the prevention of arterial thrombosis, its effect on atherosclerosis is unknown. Accordingly, we here investigated the effects of sarpogrelate HCl on a rabbit model of atherosclerosis. Male rabbits were fed a 0.5% cholesterol diet (HCD) (Gp 1), HCD with vitamin E (Gp 2), HCD with vitamin E and sarpogrelate (Gp 3), or HCD with sarpogrelate alone (Gp 4) for 8 weeks. The atherosclerotic area was decreased by feeding of vitamin E and sarpogrelate (16.9±2.0% in Gp 1 vs. 8.2±2.0% in Gp 3). Tone-related basal NO release was higher in Gps 3 and 4. Acetylcholine-induced relaxation tended to be improved in Gp 3. The amount of eNOS mRNA was increased in Gp 4, and aortic cyclic GMP concentration showed the same tendency. O 2 − release tended to be decreased in Gps 2 and 3. The matrix metalloproteinase-1 (MMP-1)-positive area was decreased, and the percentage ratio of cell numbers of smooth muscle cells/macrophages in the plaque was increased in Gp 3. The results demonstrated that sarpogrelate HCl retards the progression of atherosclerosis in rabbits, and that this effect is enhanced by concomitant administration of vitamin E. Although upregulation of eNOS may play a role as one of the underlying mechanisms, our results suggest that an additional mechanism—possibly involving the antiproliferative effects of sarpogrelate HCl on smooth muscle cells and macrophages—may also play an important role.

Perinatal Neurosteroid Levels Influence GABAergic Interneuron Localization in Adult Rat Prefrontal Cortex

Neurosteroids are a class of steroids synthesized de novo in the brain, several of which are potent modulators of GABA(A) receptor function. In developing brain GABA(A) receptor, stimulation plays a trophic role. Cortical levels of the GABAergic neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) vary dramatically across development; during the second week of life, elevated levels of 3alpha,5alpha-THP are associated with decreased GABA(A) receptor function. To determine whether alteration of endogenous 3alpha,5alpha-THP levels during development alters GABAergic interneurons in prefrontal cortex (PFC) at maturity, rat pups were exposed to 3alpha,5alpha-THP (10 mg/kg) on postnatal day 1 (P1), P2, and P5. On P80, frontal cortex tissue was assayed for GABAergic cell localization (parvalbumin and calbindin immunoreactivity), agonist-dependent [(3)H] dizocilpine (MK-801) binding to NMDA receptors in cortical homogenates, muscimol-mediated (36)Cl(-) influx into synaptoneurosomes, and 3alpha,5alpha-THP levels. The localization of parvalbumin-labeled cells was markedly altered; the ratio of cell number in the deep layers (V-VI) versus superficial layers (I-III) of adult PFC increased twofold in animals exposed to 3alpha,5alpha-THP on P1 or P5. Relative microtubule-associated protein-2 and calbindin immunoreactivity were not altered by perinatal 3alpha,5alpha-THP administration. Agonist-dependent [(3)H]MK-801 binding was decreased in PFC but not parietal cortex homogenates, whereas muscimol-mediated (36)Cl(-) influx and 3alpha,5alpha-THP levels were unchanged in frontal cortex of adult males exposed to 3alpha,5alpha-THP on P5. These data are consistent with a change in the distribution of a subset of interneurons in response to neurosteroid exposure and suggest that GABAergic neurosteroids are critical for normal development of GABAergic systems in the PFC.

Cytokine secretion in patients with acute promyelocytic leukemia after treatment with all-trans retinoic acid

Objective: To detect the modulation of cytokines production by acute promyelocytic leukemia (APL) cells before or after exposure to all-trans retinoic acid (ATRA). Methods: Diagnoses were performed according to the FAB cytological classification criteria and cytogenetic criteria. Bone marrow or blood samples from APL patients were collected in heparinized microfuge tube. Primary APL cells were separated and purified by traditional Ficoll-Hypaque density centrifugation and enriched after adherence to plastic surfaces. IL-1β, IL-6, IL-8, TNFα and G-CSF levels in the supernatants of cultured leukemia cells were estimated by ELISA method. NBT method was used to detect the differentiation of APL cells at the same time. Results: 96 h after exposure to ATRA at 10−6 M in vitro or 60 mg/day in vivo, APL cells showed a significant increase of IL-1β (P<0.05) and G-CSF (P<0.05) production, and a significant decrease of IL-6 (P<0.05) and IL-8 (P<0.05), however, there was no obvious variation of TNFα. On the other hand, the proliferation of APL cells in vitro was statistically correlated to the IL-1β secretion or G-CSF secretion. And the cell number ratio in patients with detectable IL-1β or G-CSF was higher than that without detectable IL-1β or G-CSF. Conclusion: IL-1β and G-CSF secretion may play an important role in the proliferation of APL cells after exposure to ATRA.

Serum from patients anesthetized with opiates less effective in the support of chondrocyte growth in vitro.

Risk of viral and/or prion disease transmission associated with the use of fetal bovine serum in clinical cell culture has led to the increasing use of autologous human serum in tissue engineering. A relatively large volume of blood is needed and so, to decrease patient discomfort, we have investigated the feasibility of taking blood when the patient is anesthetized. Two serum samples were prepared from each of 22 patients: (1). from the awake patient (PRE) and (2). from the patient 5 min after induction of general anesthesia (PER). The sera were compared for their ability to support the in vitro proliferation of primary human chondrocytes, determined by cell counting. The effects of anesthetic agents on the PER/PRE cell number ratio were established by analysis of variance and stepwise multilinear regression analysis. The PER sample supported higher growth in 2 of 22 patients, equivalent growth in another 11, and significantly lower growth in the remaining 8. Only the opiate analgesics (fentanyl [Sublimaze], alfentanyl [Rapifen], and diamorphine) had a significant and inhibitory effect on chondrocyte proliferation. It is suggested that opiate analgesics be avoided when blood is taken to support the in vitro growth of human cells.

Effects of clinically relevant alumina ceramic wear particles on TNF- α production by human peripheral blood mononuclear phagocytes

The recent introduction of microseparation of the components of ceramic-on-ceramic hip prostheses during hip simulations has produced clinically relevant wear rates, wear patterns and wear particles. This provided an opportunity to determine the response of primary human peripheral blood mononuclear cells to clinically relevant alumina ceramic wear particles in vitro. Alumina ceramic wear particles were generated in a hip joint simulator under microseparation conditions. The particles showed a bi-modal size distribution with nanometer sized (5– 20 nm ) and larger particles (0.2– >10 μm ). The particles were cultured with human peripheral blood mononuclear cells obtained from six different donors at particle volume to cell number ratios of 1, 10, 100 and 500 μm 3 . After 24 h incubation the viability of the cells and the levels of TNF- α were determined. The response to the microseparation wear particles was compared to that of commercially available alumina powder with a uniform morphology and mean size of 0.5 μm . All six Donors PBMNC produced significantly elevated levels of TNF- α when stimulated with 100 μm 3 of the alumina powder per cell. Volumetric concentrations of 10 and 1.0 μm 3 per cell failed to stimulate a significant response by the cells from any of the six donors. Three of the six Donors PBMNC secreted significantly elevated levels of TNF- α when stimulated with 100 μm 3 of the microseparation wear particles, whereas the other three failed to respond to the wear debris at this concentration. All of the Donors PBMNC produced significantly elevated levels of TNF- α when stimulated with 500 μm 3 of the microseparation wear particles per cell. Thus, a greater volume of the microseparation wear particles was required to activate the PBMNC than the alumina powder. This was probably due to the microseparation wear particles having fewer particles in the critical size range (0.1– 1 μm ) for macrophage activation compared to the alumina powder. It can be concluded that alumina ceramic wear particles generated under microseparation conditions are capable of inducing osteolytic cytokine production by human mononuclear phagocytes. However, the volumetric concentration of the particles needed to generate this response is extremely high and given the low wear rates ( <4 mm 3 per million cycles) of ceramic-on-ceramic bearings, even under severe microseparation conditions, it is unlikely that this concentration threshold will be achieved in vivo.

Comparison of the response of human peripheral blood mononuclear cells to challenge with particles of three bone cements in vitro

This study compared the effects of different sizes of three clinically relevant endotoxin free bone cement particles on primary human macrophage TNF-α production in vitro. The bone cements used were CMW original, CMW1RO and Palacos R. The cement wear debris was generated aseptically and then sequentially filtered to produce the size ranges 0.1– 1 μm , 0.1– 10 μm , 1– 10 μm and >10 μm . The debris was cultured with human peripheral blood mononuclear cells at particle volume (μm 3) per cell ratios of 100:1, 10:1 and 1:1. TNF-α production was determined by ELISA and cell viability by MTT conversion. CMW1RO particles induced increased TNF-α production by PBMNCs when tested in the size range 0.1– 1 μm , and also to a lesser degree in the sizes 0.1– 10 μm and 1– 10 μm at the particle volume (μm 3) to cell number ratios of 100:1 and 10:1. The increase in TNF-α production induced by Palacos R debris was only observed with the particle size ranges less than 10 μm at the ratio of 100:1. This study demonstrated that bone cement particles are capable of inducing raised TNF-α production in vitro. This is dependent upon cement particle size, volume and cement particle type, with cement particles containing radio-opaque additives being the most active.

Comparison of the cytotoxicity of clinically relevant cobalt–chromium and alumina ceramic wear particles in vitro

Concern over polyethylene wear particle induced aseptic loosening of metal-on-polyethylene hip prostheses has led to renewed interest in alternative materials such as metal-on-metal and alumina ceramic-on-alumina ceramic for total hip replacement. This study compared the effects of clinically relevant cobalt–chromium and alumina ceramic wear particles on the viability of U937 histiocytes and L929 fibroblasts in vitro. Clinically relevant cobalt–chromium wear particles were generated using a flat pin-on-plate tribometer. The mean size of the clinically relevant metal particles was 29.5±6.3 nm (range 5– 200 nm ). Clinically relevant alumina ceramic particles were generated in the Leeds MkII anatomical hip simulator from a Mittelmieier prosthesis using micro-separation motion. This produced particles with a bimodal size distribution. The majority (98%) of the clinically relevant alumina ceramic wear debris was 5– 20 nm in size. The cytotoxicity of the clinically relevant wear particles was compared to commercially available cobalt–chromium (9.87 μm±5.67) and alumina ceramic (0.503±0.19 μm) particles. The effects of the particles on the cells over a 5 day period at different particle volume (μm 3) to cell number ratios were tested and viability determined using ATP-Lite TM. Clinically relevant cobalt–chromium particles 50 and 5 μm 3 per cell reduced the viability of U937 cells by 97% and 42% and reduced the viability of L929 cells by 95% and 73%, respectively. At 50 μm 3 per cell, the clinically relevant ceramic particles reduced U937 cell viability by 18%. None of the other concentrations of the clinically relevant particles were toxic. The commercial cobalt–chromium and alumina particles did not affect the viability of either the U937 histiocytes or the L929 fibroblasts. Thus at equivalent particle volumes the clinically relevant cobalt–chromium particles were more toxic then the alumina ceramic particles. This study has emphasised the fact that the nature, size and volume of particles are important in assessing biological effects of wear debris on cells in vitro.

Testicular fine needle aspiration cytology from a stallion with testicular degeneration after external genitalia trauma

A seven-year-old Quarter Horse had a serious external genitalia trauma with severe swelling of ventrum, penis, prepuce and scrotum after falling over a fence. Appropriated treatment was rapidly started after clinical examination. During recovery period, the spermatogenesis assess by semen evaluation was not possible due to stallion's inability to ejaculate. Therefore, for testicular evaluation fine needle aspiration cytology (FNAC) was performed. The first FNAC showed a deviation of germ cell line towards immature cells, mainly by primary spermatocytes (59.5%) with very few late spermatids and spermatozoa (2.5% each), and an increased Sertoli cells/germ cells ratio (478/100), which characterized testicular degeneration. One month after the first FNAC, the second exam presented a drastic decrease of Sertoli cells/germ cells ratio (7/100) and marked increase of mature cell number, specially by early and late spermatids (50% and 24.5%, respectively). In this case, the results of both FNAC could demonstrate a partial recovery of spermatogenesis activity. Two months later, the stallion had mated two mares successfully and they became pregnant. In conclusion, the adequate treatment allowed a complete recovery of the stallion's reproductive function, and since semen collection was impossible during treatment, testicular FNAC showed to be an efficient diagnostic method for evaluating acute damage in the spermatogenesis.

Morphological and histochemical characteristics of mast cells and the content of in-tissue histamine in various pathological parathyroids: do mast cells participate in hormone secretion in human parathyroids?

The possibility of the participation of mast cells in human parathyroid hormone secretion was studied with regard to the frequency, distribution, and sub-types of mast cells and the content of in-tissue histamine, a chemical mediator in mast-cell granules, in human parathyroids with various pathological conditions. The above factors were compared between those of a 'normal' parathyroid group and those of 'pathological' parathyroids associated with adenoma and hyperplasia. Specimens were scanned for the mean value of the mast cell number per field of microscopic view and for the ratio of the mast cell number in glandular parenchymal tissue to that in interstitial tissue. The activated state of the mast cells was examined through classifying the mast cells into two sub-types, mucosal mast cells and connective-tissue mast cells. The high-performance liquid chromatography (HPLC) method was used for assay of in-tissue histamine. The frequency of mast cells showed no difference between the groups, whereas the distribution of mast cells, showed a distinct difference. The occurrence rate of mast cells in glandular parenchymal tissue in the 'pathological' group presented an increase as compared with that in the 'normal' group. Furthermore, the occurrence rate of mucosal mast cells in an activated state also showed an increase. This suggests that mast cells are likely to participate in parathyroid hormone secretion. The histamine-content in the 'normal' group was significantly larger than that in the 'pathological' group, which was a different outcome from that observed in mast cells from the results of light microscopy. This may require taking into consideration the difference in the histamine content of the mast cells themselves between that of mucosal mast cells and connective-tissue mast cells.

Twelve hours exposure to inhomogeneous high magnetic field after logarithmic growth phase is sufficient for drastic suppression of Escherichia coli death

When Escherichia coli B was aerobically grown at 43 °C in a medium whose concentration was one-fourth that of the Luria–Bertani (LB) medium supplemented with 1.5 g/l of glutamic acid, drastic cell death was observed after the end of the logarithmic growth phase. However, when the same experiment was conducted under inhomogeneous 5.2–6.1 T magnetic field, cell death was extremely suppressed and the ratio of viable cell number under high magnetic field to that under geomagnetic field reached as much as 100,000. When the magnetic field exposure was restricted to 12 h after the logarithmic growth phase, a similar high degree of suppressive effect on the death was observed. The findings that the amount of sigma S protein encoded by the rpoS gene under the high magnetic field was larger than that under the geomagnetic field, and that the magnetic field effect disappeared when the rpoS gene-deficient strain was cultivated under the high magnetic field, suggest the interaction of magnetic field with a stationary phase specific gene.

Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei.

Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogeneous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56--70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.

A novel three-dimensional tissue equivalent model to study the combined effects of cyclic mechanical strain and wear particles on the osteolytic potential of primary human macrophages in vitro.

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.

Comparison of the response of three human monocytic cell lines to challenge with polyethylene particles of known size and dose.

The response of three human monocytic cell lines (Monomac-1, U937 and THP-1) to challenge with polyethylene particles of known size and dose was evaluated. Particles with a mean size of 0.21, 0.49, 4.3, 7.2, and 88 microm were co-cultured with the cells for 24 hours prior to the assessment of cell viability and production of the pro-inflammatory cytokines, IL-1beta, IL-6 and TNFalpha. Additionally, GM-CSF and prostaglandin E2 were measured in culture supernatants from particle stimulated U937 cells. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 100 : 1, 10 : 1, 1 : 1 and 0.1 : 1. None of the test fractions had any effect on cell viability. Only the response of the U937 cell line was demonstrated to be comparable to that of primary macrophages as determined in a previous study. Furthermore only particle volume to cell number ratios of 10 : 1 or greater consistently stimulated significantly enhanced levels of cytokine secretion with particles within the phagocytosable size range (0.1 to 10 microm) being the most biologically active. No response was observed when U937 macrophages were stimulated with the largest (88 microm) particles at any of the volume ratios tested in this study. These results suggest that the size and volume of polyethylene particles are critical factors in macrophage activation. In addition, the U937 cell line has been shown to be a suitable model for the in vitro study of macrophage-particle interactions.

Comparison of the response of primary human peripheral blood mononuclear phagocytes from different donors to challenge with model polyethylene particles of known size and dose

The response of primary human peripheral blood mononuclear phagocytes to challenge with polyethylene particles of known size and dose was evaluated. Particles with mean sizes of 0.21, 0.49, 4.3, 7.2, and 88 μm were co-cultured with cells for 24 h prior to the assessment of cell viability and production of the osteolytic mediators IL-1 β, IL-6, TNF α, GM-CSF and PGE 2. All particle fractions were evaluated at particle volume (μm 3) to cell number ratios of 10 : 1 and 100 : 1 which were previously identified as being the most biologically active and clinically relevant. The heterogeneity of human individuals was clearly evident both in the profile and the magnitude of the response of the donors evaluated in this study (the response of Donor 5 being 2- to 15-fold lower than that of the other donors). Only the sub-micrometre particles stimulated significantly enhanced cytokine secretion at the ratios tested: mean particle sizes of 0.49 and 0.21 μm being the most biologically active. Macrophages stimulated with particles outside this size range produced considerably lower levels of mediator. These results compared favourably with the results of earlier studies, which demonstrated that particles within the phagocytosable size range (0.1–10 μm) were the most biologically active. These results, therefore, confirm earlier findings and suggest that the size and volume of polyethylene particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life and could provide the basis for a valuable diagnostic tool to identify those patients most at risk of implant loosening.