Biomedical subjects

M van de Ruit

Publications and source records attributed to M van de Ruit.

10 recordsLinked to original sources

PubMed1994

Structural requirements for bisphosphonate actions in vitro.

We investigated the structural requirements for the binding of bisphosphonates to bone mineral and the relation between their affinity for bone and their effects on bone resorption in vitro. For this we used fetal mouse long bones in culture and bisphosphonates with variable R1 and R2 structures. In addition, we studied the effect of structural differences in the incorporation of calcium into bone. We found that bisphosphonates containing a hydroxyl group in the R1 position have the highest affinity for bone mineral. This was related to their capacity to inhibit the incorporation of calcium into long bones but not to their antiresorptive potency. The latter was primarily determined by R2. Furthermore, the effect of bisphosphonates on calcification, but not on resorption of bone explants, was mainly determined by the mode of addition. The continuous presence of bisphosphonate during culture inhibited calcification even at very low concentrations, but short incubation of the bones with relatively high concentrations had no effect. This is probably a result of differences in the availability of the compound to the process of calcification. Because, in vivo, the more potent bisphosphonates inhibit resorption without adversely affecting mineralization of the skeleton and they disappear rapidly from the circulation after administration, we suggest that cultures of bone explants incubated with bisphosphonates for short times rather than cultures in which the drugs are continuously present provide more accurate information about the in vivo effect of these compounds on both resorption and calcification.

Animals↗

PubMed1994

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.

Animals↗

PubMed1993

Leukemia inhibitory factor inhibits osteoclastic resorption, growth, mineralization, and alkaline phosphatase activity in fetal mouse metacarpal bones in culture.

Leukemia inhibitory factor (LIF) has been reported to affect bone metabolism, but results are variable. We examined the effect of mouse recombinant LIF on osteoclastic resorption in fetal bone explants representing different stages of osteoclast development. In cultures of 17-day-old fetal mouse metacarpals in which only osteoclast progenitors and precursors are present, resorption (measured as 45Ca release) was significantly inhibited to 29.2% and to 96.6% in the presence of LIF 100 and 1000 U/ml, respectively. Histologic examination of the explants treated with 1000 U/ml of LIF confirmed the biochemical findings and showed that osteoclast progenitors and precursors remained in the periosteum and did not invade the mineralized matrix. In metacarpals of older fetuses (18- and 19-day-old) in which the mineralized cartilage has been invaded by mature osteoclasts, the inhibition of resorption by LIF (1000 U/ml) was 87.9 and 74.7%, respectively, the latter being significantly less than the inhibition observed in 17-day-old metacarpal cultures. The inhibitory effect of LIF was absent during concurrent administration of PTH or 1,25-(OH)2D3 and could be reversed by PTH. In addition, LIF was found to inhibit growth, mineralization, and alkaline phosphatase activity in metacarpals independently of osteoclastic resorption. These results suggest that LIF affects the development rather than the activity of osteoclasts, probably through an effect on the osteogenic cells. LIF may be an important endogenous regulator of bone metabolism.

Acid Phosphatase↗

PubMed1993

Quantification of adherent and nonadherent cells cultured in 96-well plates using the supravital stain neutral red.

In this study we present a rapid, simple, sensitive, inexpensive, and environment-friendly assay for determination of the number of adherent or nonadherent cells cultured in 96-well plates using the supravital stain neutral red. We describe a validation of the method and demonstrate its application to study the effects of hormones (i.e., parathyroid hormone) and cytokines (i.e., tumor necrosis factor-alpha) on the growth of primary cultures of adherent osteoblast-like cells. In addition we show that this method can also be applied to conveniently determine proliferation of cells which grow in suspension, like the CTLL-2 and B-9 cells, which are widely used to measure IL-2 and IL-6 bioactivity, respectively. In these bioassays the changes in optical density induced by IL-2 and IL-6 measured with the neutral red assay are directly comparable with the relative changes measured with the [3H]thymidine incorporation assay. For all types of cells tested, the optical density at 550 nm was directly proportional to the number of cells. The assay, which can be used for different purposes, is an excellent alternative to already existing methods. It is not only easy to perform but also very reproducible, making it ideal for screening of large numbers of samples. Therefore this assay offers a reliable and flexible tool to determine both stimulatory and inhibitory effects of hormones, cytokines, and drugs on cell growth or to study the effects on cell viability.

Animals↗

PubMed1992

The effect of tissue type plasminogen activator (tPA) on osteoclastic resorption in embryonic mouse long bone explants: a possible role for the growth factor domain of tPA.

Osteoblasts produce proteolytic enzymes and their production is regulated by osteotropic agents. It has been suggested that these proteases play a role in bone resorption by removing the superficial collagenous layer from the bone matrix and indirectly inducing migration of osteoclast precursors towards the bone matrix. We examined the effect of the plasminogen activator tPA on osteoclastic resorption using 17-day-old mouse embryonic long bone explants representing different stages of osteoclast development, that is, radii containing already mature osteoclasts and metacarpals containing no mature osteoclasts but only osteoclast precursors/progenitors which are still confined to the periosteum. Tissue type PA stimulated osteoclastic resorption (measured as 45Ca-release) in 17-day-old fetal metacarpals but not in radii of the same animal. Blocking the enzymatic activity of tPA did not inhibit its effect on osteoclastic resorption. Plasmin, the direct product of PA enzymatic activity, did not induce osteoclastic resorption. However, a tPA-mutant missing the growth-factor-like domain of the molecule, failed to stimulate 45Ca-release from the metacarpals. In addition, in both systems tPA and transforming growth factor alpha had similar effects on osteoclastic resorption. The finding that tPA stimulated 45Ca-release only in the metacarpals suggests that tPA has an effect on osteoclast formation rather than on the activity of already mature osteoclasts. Under the experimental conditions used this effect seems to be mediated by the growth factor domain of tPA rather than by the enzymatic activity of the molecule.

Amino Acid Chloromethyl Ketones↗

PubMed1984

Automated determination of heparin with chromogenic substrates.

Spectrophotometric heparin assays which are based on the catalytic effect of heparin on either the inactivation of thrombin or that of factor Xa by antithrombin III, were adapted for use in a laboratory batch analyzer. Optimal conditions were determined for assays using the chromogenic substrates Chromozym-Th and S-2238 with thrombin, and S-2222 with factor Xa. Inactivation of the clotting enzyme by antithrombin III was stopped by addition of chromogenic substrate. Assays thus obtained appeared to be applicable in a wider range of heparin concentrations and were less dependent on plasma antithrombin III concentration that known manual spectrophotometric methods. The best results were obtained with the methods based on thrombin inactivation and applying a logarithmic reference curve.

Blood Coagulation Tests↗

PubMed1984

Automated spectrophotometric heparin assays. Comparison of methods.

Three automated spectrophotometric heparin assays were investigated. The day-to-day reproducibilities in routine laboratory use were compared with two commercial manual kits for heparin determination. Regression analysis of the activated partial thromboplastin time (APTT) on results of any of the heparin assays shows that the heparin concentration cannot be deduced from the APTT values found in patients receiving heparin. The automated heparin assays that employ thrombin and Chromozym-Th or S-2238 were found to be most suitable for routine heparin determination. Heparin concentrations obtained from assays based on factor Xa inactivation were not significantly different from those employing thrombin (p less than 0.01), but revealed a wider standard deviation. The relationship between APTT and heparin level found was not related to the plasma antithrombin III concentration. The extra antithrombin III that is added in the assays had to be freed of heparin neutralising activity to obtain reliable estimates of the heparin concentration in the low range (0-200 U/l).

Chromogenic Compounds↗

PubMed1984

Determination of low molecular weight heparin in clinical laboratory.

The applicability was investigated of automated spectrophotometric heparin assays and three clotting assays for determination of two low molecular weight (LMW) heparin fractions: Org 10172 and DxN10 and two infractionated commercially available heparins. The relative activity of the two commercially available heparins was similar in the anti-Xa assay, in the anti-IIa assay and in 3 clotting assays. The LMW heparins showed markedly different relative activity in all 5 assays. The activities of those heparin preparations relative to the standard heparin were compared in the 5 assays, but standardization against a standard heparin preparation appeared impossible. Methods of heparin determination can be used to monitor treatment with a heparin preparation only if the same preparation is used as a reference substance.

Chondroitin Sulfates↗

PubMed1984

Heparin neutralization during collection and processing of blood inhibited by pyridoxal 5'-phosphate.

Spectrophotometric heparin assays are expected to be independent on clotting factors, either activated or nonactivated, but could be sensitive to heparin neutralization during blood collection. It was shown that more than 200 U of heparin/l plasma can completely be neutralized during blood processing. Because the heparin neutralization is not constant but dependent on the sample as well as on the type of sample handling, one cannot beforehand compensate and correct the results for possible heparin neutralization. We tested the use of pyridoxal 5'-phosphate (PLP) to prevent heparin neutralization. As the use of PLP in the citrate tube decreased the heparin neutralization to a negligible effect, PLP-citrate tubes are to be preferred for all plasma heparin determinations.

Antithrombin III↗

PubMed1982

Interindividual variation in relationships between plasma heparin concentration and the results of five heparin assays.

In order to determine their value for estimating the heparin concentration in plasma, we established the relationship between test result and heparin concentration in plasma from various individuals, for five assays used with heparin treatment. Only assays which can be carried out routinely in clinical laboratories were considered. The thrombin time and the whole blood recalcification time give pointless and ambiguous information respectively, concerning the heparin level. The activated partial thromboplastin time with and without heparin neutralisation give only a rough estimate. The spectrophotometric method using a chromogenic substrate gives the best information. The latter can be improved by using a non-linear (parabolic) equation for the calculation of the reference curve. Current heparin therapy, controlled with the aid of a clotting assay, may result in plasma heparin concentrations that vary widely from one patient to another.

Blood Coagulation Tests↗