Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.     

A VIP antagonist distinguishes VIP receptors on spinal cord cells and lymphocytes

Vasoactive intestinal peptide (VIP) is a neuropeptide which also interacts with cells of the immune system. The paucity of specific VIP receptor antagonists has hampered studies of possible receptor heterogeneity and of VIP function. To aid in achieving these goals, a new VIP antagonist, a hybrid between neurotensin and VIP, has been synthesized. This peptide interacted with VIP receptors on spinal cord cells with an affinity 10-fold greater than VIP itself. In contrast, 1000-fold higher concentrations of the antagonist were required to displace labeled VIP from its receptor on lymphoid cells as compared to VIP itself, suggesting VIP receptor heterogeneity between immune and spinal cord cells.     

Modulators of growth in primary culture of rat proximal tubular cells II

Summary: The present studies assessed the effects of manipulating extracellular sodium (Na) concentration and Na transport on cellular hypertrophy and hyperplasia in primary culture of rat proximal tubular cells. A concentration-dependent effect on thymidine incorporation and protein content was observed with cell culture media Na concentration of 130, 140 and 150 mmol/L. This effect was independent of osmolality (matched with mannitol) and no stimulatory effect occurred if choline was substituted for Na. Cells derived from sham-operated (Sx) animals exposed to a higher media concentration of Na (150 vs 140 mmol/L) had both stimulated thymidine incorporation to 186.8 ± 35.41% (P<0.05) and enhanced cell protein content to 134.7 ± 135% (P<0.05). This effect was more pronounced in cell cultures derived from unilaterally nephrectomized (Nx) animals, being 212.8 ± 31.5% (P<0.01) for thymidine incorporation (P<0.05 vs cells from sham-operated animals grown in high Na media) and 114.4 ± 3.2% (P<0.001) for protein content (P=0.11 vs sham-operated cells grown in similar conditions). the addition of 10^?4 mmol/L ethylisopropyl amiloride hydrochloride (EIPA) to Nx cells in a normal or high Na concentration media resulted in a decrease in cellular protein content to 82.6 ± 6.8% (P<0.05) and 85.5 ± 0.2% (P<0.0001) compared to respective controls. 10^?4 mol/L EIPA in media supplemented with insulin-like growth factor (IGF-1) blocked the proliferative response normally seen in response to this growth factor from 156.6 ± 13.7 to 27.5 ± 3.1% (P<0.0001) compared to control. However, the presence of EIPA did not abrogate the hypertrophic response elicited by IGF-1 (cell protein content 128.1 ± 13.1% of control with IGF-1 vs 124.9 ± 12.5 with IGF-1 and EIPA; P= n.s.). Addition of 10^?4 mol/L EIPA to 10% serum derived from either Sx or Nx animals blocked the growth response to the sera, limiting the cellular protein content to 76.6 ± 5.5% (P<0.0001) and 89.7 ± 4.4% (P<0.0001) and thymidine incorporation to quiescent levels of 0.2 ± 0.1% (P<0.0001) and 0.4 ± 0.1% (P<0.0001) compared to respective controls. In summary, rat renal proximal tubular cell growth is influenced by Na concentrations in the cell culture environment and inhibited in the presence of EIPA. This supports a role for altered epithelial transport in the cellular growth response to a number of stimuli.     

Region-specific constitutive gene expression in the adult porcine meniscus.

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.     

Macrophage-like cells from explant cultures of rat sciatic nerve produce apolipoprotein E

Apolipoprotein E is synthesized and secreted by degenerating peripheral nerve, but the role of resident endoneurial cells in this process is not clear. To exclude the involvement of nonresident cells, we examined the cellular source of endoneurial apolipoprotein E in explant cultures of rat sciatic nerve. The cellular outgrowth from these explant cultures released apolipoprotein E into the culture medium. The cellular outgrowth contained fibroblasts, Schwann cells, and a population of cells with many phenotypic characteristics of macrophages, including the production of apolipoprotein E. No other cell type in the cultures appeared to contribute to this production. These data suggest that apolipoprotein E is produced by resident endoneurial cells in explant cultures and that these cells are macrophages.     

子宫内膜体外构建及其应用研究进展

体外构建子宫内膜是利用组织工程学原理,在体外构建结构、形态与功能上与体内子宫内膜相似的三维模拟体。种子细胞分离培养、生物支架材料的合理选择及体外构建方法的优化是体外构建子宫内膜的重要因素。构建的子宫内膜在病理研究、三维培养体系、胚胎发育学及移植等方面具有重要的研究意义。只有更进一步模拟自然子宫的结构,优化构建技术路线和培养条件,才可以再造出真正意义上的组织工程化子宫。     

Detection frequencies and the colony-forming unit recovery of oral treponemes by different cultivation methods

Selected media were compared for primary isolation and detection of oral treponemes from clinical samples. Forty-eight subgingival plaque samples from 45 patients suffering from periodontitis were anaerobically cultivated for 2 weeks at 37°C. Of the 9 media studied, Medium 10 (M10), which was supplemented with 10% rabbit serum and incubated using the plate-in-bottle method, supported the highest colony-forming units of the anaerobes. The treponemal colonies were detected at least on one medium from 83% of the subgingival plaque samples. The new oral spirochete medium in an anaerobic chamber supported the highest detection frequency of the oral treponemes (64% of samples); however, M10 in the plate-in-bottle was found to produce the highest colony-forming unit recovery of the oral treponemes (median 3.6% of the total colony-forming units). This study suggests that M10 in the plate-in-bottle and new oral spirochete medium in the anaerobic chamber are essential in cultivating oral treponemes.     

High Cell-Density Culture System of Hepatocytes Entrapped in a Three-Dimensional Hollow Fiber Module with Collagen Gel

Abstract: A compact three-dimensional (3D) module is needed for hepatocyte culture in order to develop an effective hybrid artificial liver system that can retain hepa-tocellular structure and differentiated functions. We treated the 3D module with collagen gel to entrap rat hepatocytes. This method yielded a high hepatocellular density (2 times 10⁷ cells/ml) over a period of 14 days and maintained the secretion of albumin and ureogenesis at the same level as the control monolayer method. The ammonia removal remained at 43% of the Day 0 value over 8 days of perfusion. Our data show that this approach may be useful for liver support therapy in an ex-tracorporeal circuit.     

Involvement of programmed cell death in preimplantation embryo demise

Fragmentation is frequently observed in animal and human embryosobtained via in-vitro fertilization (IVF), and is known to beassociated with decreased pregnancy rates and poor survivalfollowing cryopreservation. We postulate that embryo fragmentationis a consequence of activated programmed cell death (PCD) andsubsequent apoptosis and discuss evidence of morphological,histological and biochemical features compatible with the occurrenceof PCD in preimplantation embryos. If PCD is an underlying causeof the high incidence of the fragmentation seen in human pre-embryos,it remains to be determined whether this is reflective of thenatural incidence of lethal chromo somes in the human populationor due to the IVF procedure and culture conditions.     

The assessment of in vitro modulation of milk fat globule membrane expression by human breast carcinomas

Although knowledge of functional differentiation and tumour-associated changes of breast carcinomas can be gained by the application of antibodies directed against the milk fat globule membrane, more significant information may be obtained by assessment of the potential of breast carcinomas to modulate their antigenic phenotype. In this study, the extend to which primary tumours can undergo modulation in vitro has been investigated, with consideration of the suitability of organ culture in combination with the immunohistochemical detection of two milk fat globule membrane epitopes, HMFG1 and HMFG2, as methods for detecting this. The preservation of three of the 30 carcinomas assessed, all poorly differentiated, was poor after 3 days of culture. The viability of the other 27 was variable, and was greater in the better differentiated tumours and with the addition of insulin. Expression of the milk fat globule membrane epitopes was generally well maintained. Six of the carcinomas showed a significant change in antigen expression, with this being more frequent in tissues incubated with insulin. Hence, a small group of carcinomas have been identified which appear to have a greater capacity to undergo functional differentiation. Organ culture is considered to be a suitable method for maintaining the tissues in vitro for such evaluation, but the problems encountered in quantifying the immunohistochemical staining, because of antigenic heterogeneity, were such that it is suggested that other approaches be employed.