In vitro osteoclast-like and osteoblast cells' response to electrospun calcium phosphate biphasic candidate scaffolds for bone tissue engineering.

Successful long term bone replacement and repair remain a challenge today. Nanotechnology has made it possible to alter materials' characteristics and therefore possibly improve on the material itself. In this study, biphasic hydroxyapatite/ß-tricalcium phosphate nanobioceramic scaffolds were prepared by the electrospinning technique in order to mimic the extracellular matrix. Scaffolds were characterised by scanning electron microscopy (SEM) and attenuated total reflectance-fourier transform infrared. Osteoblasts as well as monocytes that were differentiated into osteoclast-like cells, were cultured separately on the biphasic bioceramic scaffolds for up to 6 days and the proliferation, adhesion and cellular response were determined using lactate dehydrogenase cytotoxicity assay, nucleus and cytoskeleton dynamics, analysis of the cell cycle progression, measurement of the mitochondrial membrane potential and the detection of phosphatidylserine expression. SEM analysis of the biphasic bioceramic scaffolds revealed nanofibers spun in a mesh-like scaffold. Results indicate that the biphasic bioceramic electrospun scaffolds are biocompatible and have no significant negative effects on either osteoblasts or osteoclast-like cells in vitro.

Short-term fatty acid effects on adipocyte glucose uptake: mechanistic insights.

The modulation of insulin sensitivity in visceral fat tissue could be important in the treatment of Type 2 diabetes mellitus. Selected fatty acids may impact on insulin-stimulated and basal glucose uptake in adipocytes, thus isolated rat epididymal adipocytes were exposed to 100 microM oleic, arachidonic, eicosapentaenoic, docosahexaenoic or stearic acids and insulin (15 nM) or vehicle for 30 min. Glucose uptake was quantified by measuring uptake of 3H-deoxyglucose/mg adipocyte protein/min. Where appropriate, inhibitors were included to elucidate the mechanisms involved. In this model, insulin stimulated glucose uptake with 62+/-7%. All fatty acids tested, except for stearic acid, depressed insulin-stimulated glucose uptake by an average of 33+/-4.2%. On the other hand, all fatty acids tested except stearic and arachidonic acids, stimulated basal glucose uptake with an average of 34+/-8.1%. Inhibitor studies showed the involvement of prostaglandins, lipoxins, protein kinase C and tyrosine kinase in these processes.

The Brody effect induced by premature ventricular complexes in the ovine heart.

The QRS response of the electrocardiogram to bleeding has been a source of interest to the physiologist for more than a century. Studies in the dog, cat and chicken have shown a reduction in QRS amplitude in response to bleeding. This effect has been explained by the so-called Brody effect, in which the intraventricular mass of blood acts as a conducting medium, augmenting radial conduction, thus resulting in the subsequent reduction in QRS amplitude in conditions where the intraventricular mass of blood is reduced. The aim of this study was to evaluate whether the Brody effect will be present in the ovine heart and, furthermore, to evaluate if the right and left ventricles will demonstrate the same QRS change if the Brody effect is indeed present. This study clearly demonstrated that the Brody effect is present in the ovine heart. Furthermore, two unique aspects emanating from this study are firstly the fact that this is the first study to show that premature ventricular complexes are able to induce the Brody effect and, secondly that there is a very clear difference in the response of the right and the left ventricles when the Brody effect is induced in the ovine heart.

A pulsed DC electric field affects P2-purinergic receptor functions by altering the ATP levels in in vitro and in vivo systems.

Recently it was shown that extracellular ATP, acting through purinergic receptors, has many physiological functions, including opening of Ca(2+)-ion channels, activation and mediation of signal transduction mechanisms as well as activation of the pain sensation. Since electrical stimulation is also known to affect many signal transduction processes as well as the alleviation of pain, we hypothesized that electric stimulation may affect the extracellular release of ATP. We investigated the effects of a small DC electric field (10(1)--10(2) V m(-1) range and with frequencies below 150 Hz) on the release of ATP in vitro (HeLa cells), and on the levels of ATP in vivo (the plasma of healthy volunteers). In HeLa cells ATP release was increased 50 fold, while the total amount of ATP in the cells was increased by 163%. In the plasma a significant decrease (P<0.05) in ATP concentration was seen after electrical stimulation, in all the volunteers. The small DC electric field also affected the cAMP signal transduction system in vitro (HeLa cells and human lymphocytes) and in vivo (human plasma). Decreased levels of cAMP (P<0.05) were seen in HeLa cells and increased levels of cAMP (P<0.05) in isolated human lymphocytes. The cAMP levels in the plasma of the electrically treated volunteers were lower than control values. These results show that the frequency, waveform and signal strength of the applied electric field are suitable for effecting measurable changes on signal transduction in vitro and in vivo.

Activation of signal-transduction mechanisms may underlie the therapeutic effects of an applied electric field.

Successful treatment of various medical complaints with an applied electric field has been reported over the years. The identities of the cellular mechanisms that are influenced by this type of treatment and facilitate the positive effects, remain elusive. A study of many in vitro and in vivo reports revealed that the beneficial effects can be attributed to the activation of membrane proteins, and specifically proteins involved in signal-transduction mechanisms. Not only may the proteins be affected but it is now well established that enhanced Ca(2+)influx, observed to follow electric stimulation of cells, also contributes to many calcium-dependent cellular processes which can be linked to the therapeutic effects discussed in this paper. An hypothesis of the physical changes caused by an applied, relatively small (10(3)to 10(4)V m(-1)range), electric field with low to moderate frequency (below 150 Hz), is postulated.

Calcium, gamma-linolenic acid and eicosapentaenoic acid supplementation in senile osteoporosis.

Recent animal work suggests that gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) enhance calcium absorption, reduce excretion and increase calcium deposition in bone. A pilot study was set up to test the interactions between calcium and GLA + EPA in humans. Sixty-five women (mean age 79.5), taking a background diet low in calcium, were randomly assigned to GLA + EPA or coconut oil placebo capsules; in addition, all received 600 mg/day calcium as the carbonate. Markers of bone formation/degradation and bone mineral density (BMD) were measured at baseline, 6, 12 and 18 months. Twenty-one patients were continued on treatment for a second period of 18 months, after which BMD (36 months) was measured. At 18 months, osteocalcin and deoxypyridinoline levels fell significantly in both groups, indicating a decrease in bone turnover, whereas bone specific alkaline phosphatase rose indicating beneficial effects of calcium given to all the patients. Lumbar and femoral BMD, in contrast, showed different effects in the two groups. Over the first 18 months, lumbar spine density remained the same in the treatment group, but decreased 3.2% in the placebo group. Femoral bone density increased 1.3% in the treatment group, but decreased 2.1% in the placebo group. During the second period of 18 months with all patients now on active treatment, lumbar spine density increased 3.1% in patients who remained on active treatment, and 2.3% in patients who switched from placebo to active treatment; femoral BMD in the latter group showed an increase of 4.7%. This pilot controlled study suggests that GLA and EPA have beneficial effects on bone in this group of elderly patients, and that they are safe to administer for prolonged periods of time.

The mammalian metabolite, 2-methoxyestradiol, affects P53 levels and apoptosis induction in transformed cells but not in normal cells.

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.

Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells.

The effects of arachidonic acid (AA) and gamma-linolenic acid (GLA) on cell cycle progression and apoptosis induction, using flow cytometry, were compared on normal human skin fibroblasts, strain HSF43 with wild type (wt) p53, large T antigen transformed HSF43 cells (line E8T4) with non functional p53, and on two lymphoblast cell lines, TK6 with wt p53 and WTK1 with mutant p53. AA and GLA caused similar dose (50, 75 and 100 microg/ml AA and GLA) and time dependent (24 and 48 h) induction of apoptosis in each cell line. The degrees of the response of the four cell lines were, however, different. The normal HSF43 cells were most resistant against apoptosis induction and the WTK1 cells most susceptible. The apoptosis induction appeared to be independent of functional p53. Cell cycle progression was also similarly affected by AA and GLA in the two cell types. In the fibroblast type cells (HSF43 and E8T4) S- and G2/M-phase arrests were evident after 48 h exposure to AA and GLA, and in the lymphoblast cell lines (TK6 and WTK1) the cells were arrested in the G1-phase.

The possible role of sodium-potassium adenosine triphosphatase in preterm labour.

An unacceptably high incidence of preterm labour is seen in the black and coloured communities of South Africa. This hypothesis proposes that sodium-potassium adenosine triphosphatase activity plays an important role in preterm labour. The impaired activity of the sodium pump leads to increased cytosolic calcium levels, which may trigger contraction of myometrial smooth-muscle cells, resulting in preterm labour.

[Peroxisomal diseases--a survey]. / Siektes van peroksisome--'n oorsig.

Peroxisomes are ubiquitous cytoplasmic structures in mammalian tissues. The metabolic functions of these organelles include synthesis of plasmalogens and other ether lipids, beta-oxidation, especially of very long-chain fatty acids (VLCFAs, > C22) and their derivatives, inactivation of hydrogen peroxide by peroxisomal catalase and involvement in several other metabolic pathways, e.g. gluconeogenesis, catabolism of purines and polyamines and detoxification of ethanol. Peroxisomal diseases which may arise from genetic faults in the biogenesis of the organelle or aberrant targeting of one or more proteins to the peroxisome, are divided into three groups based on the extent of loss of peroxisomal functions. Prototype of the first group is the cerebro-hepato-renal syndrome of Zellweger (ZS) which shows generalised loss of peroxisomal functions and absence of demonstrable mature peroxisomes in the liver. Other syndromes which are briefly discussed include neonatal adrenoleukodystrophy (NALD) and infantile Refsum syndrome (IRS) which may be regarded as milder variants of ZS, and diseases caused by loss of a limited number of peroxisomal functions (rhizomelic chondrodysplasia punctate). However, the group of peroxisomal diseases with the highest incidence are those syndromes where only a single peroxisomal function is impaired. The most common peroxisomal disease, X-linked adrenoleukodystrophy (XALD) belongs to this group. XALD develops as a result of an isolated defect of peroxisomal acyl-CoA synthetase with resultant accumulation of VLCFAs, especially C26:0. Primary hyperoxaluria type 1 is caused by deficient activity of peroxisomal alanine: glyoxylate aminotransferase due to aberrant targeting of this enzyme to mitochondria and not peroxisomes, a unique example of a genetic enzyme trafficking defect. The primary diagnosis of these syndromes is usually based on clinical findings and measurement of accumulated or depleted metabolites in the body e.g. VLCFAs, bile acid intermediates, phytanic acid, pipecolic acid and plasmalogens. Therapy includes dietary adjustments e.g. supplementation with oleic acid derivatives to normalise elevated VLCFAs in XALD. Treatment with hypolipidaemic drugs and certain peroxisomal substrates which induce proliferation of mature peroxisomes offers promise in the therapy of these debilitating and often fatal diseases.

Direct determination of saturable and non-saturable calcium uptake in the developing rat duodenum.

Significant changes in saturable and non-saturable intestinal calcium uptake processes occur during development of the rat. A modified 'free floating disc' assay was used to measure total, active and passive duodenal calcium uptake on matching duodenal segments from male Sprague Dawley rats aged 14 to 56 days of age. The results were compared and found to give patterns similar to data published for the in situ ligated loop technique.

Supplemented gamma-linolenic acid and eicosapentaenoic acid influence bone status in young male rats: effects on free urinary collagen crosslinks, total urinary hydroxyproline, and bone calcium content.

The effect of different ratios of the prostaglandin precursors gamma-linolenic (GLA) and eicosapentaenoic (EPA) acids on bone status in growing rats measured as a function of free urinary pyridinium crosslinks and hydroxyproline levels was investigated. Male Sprague-Dawley rats were weaned onto an essential fatty acid deficient diet and from their fifth week, different groups of rats received a balanced, semisynthetic diet, supplemented with different ratios of GLA:EPA supplied as a mixture of evening primrose oil (EPO) and fish oil (FO). Controls were supplemented with linoleic (LA; sunflower oil) and alpha-linolenic (ALA; linseed oil) acids (3:1) or a commercially available rat chow. Animals were terminated at 84 days and femur length, ash weight, calcium content, free urinary pyridinium crosslinks (Pyd and Dpyd), total hydroxyproline (Hyp), and creatinine levels measured. Free urinary Pyd and Dpyd are good indicators of bone status and they correlated well with Hyp. Pyd and Dpyd excretion were significantly decreased in the higher GLA:EPA dietary groups and correlated well (r = 0.7) with Hyp levels. Concomitantly, bone calcium content increased significantly in the same dietary groups. These results suggest that diet supplementation with relatively high GLA:EPA ratios are more effective in inhibiting bone resorption than LA:ALA.

Calcium transport by isolated brush border and basolateral membrane vesicles: role of essential fatty acid supplementation.

Intestinal calcium transport is important in whole body calcium homeostasis and it is therefore of interest to understand the mechanism of absorption and its regulation by 1;25-dihydroxyvitamin D3 (1,25 (OH)2D3) (vitamin D). Significant changes in lipid composition of membranes have previously been shown in response to vitamin D3 administration. Deficiency in essential fatty acids (EFAs) may influence the vitamin D-dependent calcium absorption in the intestinal tract. The purpose of this study was to investigate the effect of unsaturated fatty acid supplementation on calcium transport. Simultaneous measurements of calcium transport, membrane fluidity and lipid structure have rarely been performed on the same preparation. Intestinal membrane vesicles were prepared using a novel procedure. Vesicles prepared from fish oil and evening primrose oil supplemented animals revealed the highest calcium transport over time as well as the highest degree of unsaturation as compared to those from animals which were unsupplemented or given sunflower or coconut oil. The relative content of polyunsaturated fatty acids in the intestinal membranes may change fluidity, enhance calcium transport and may influence the action of vitamin D3 on calcium absorption.

Induction of osteogenesis.

Bone induction studies on baboons using standard millipore tissue chambers showed that lyophilised bone, bone marrow and thrombocytes (growth factors) are necessary for optimal osteogenesis. The decalcification of bone tissue before transplantation leads to the impairment of new bone growth.

In vitro determination of bone mineral content of the femur neck. Use of computed tomography.

An accurate method for the in vitro measurement of mineral content of femoral bone by means of computed tomography (CT) is presented. The bone mineral content (BMC) of the femoral head in patients with a subcapital fracture was determined and compared with that in patients with osteo-arthritis. There was a satisfactory standard curve and a highly significant relationship between the CT number (Hounsfield number) and a standard bone equivalent reference (K2HPO4) was obtained at two different energies (96 and 125 kV). The calculated error in the calibration procedure was less than 1,5% and the overall error of the method was 8,8%. The femoral BMC of patients in the fracture group was found to be substantially lower (P less than 0.001) than that in osteoarthritis.