Biomedical subjects

J Klein-Nulend

Publications and source records attributed to J Klein-Nulend.

60 records · Page 4Linked to original sources

PubMed1990

Evidence that adenosine 3',5'-monophosphate mediates hormonal stimulation of prostaglandin production in cultured mouse parietal bones.

The present investigation was undertaken to examine the possible role of cAMP in PTH-stimulated prostaglandin (PG) production in organ cultures of neonatal mouse parietal bones. Cultures were treated with PTH, forskolin, isobutylmethylxanthine (IBMX), and 8-bromo-cAMP (8BrcAMP). We found that similar concentrations of PTH stimulate cAMP formation and increase PG production in this culture system. Forskolin, a direct activator of adenylate cyclase, was also a potent stimulator of cAMP and PG production. The effect was dose dependent, with a maximum at 10(-5) M. The time courses for PTH- and forskolin-stimulated PG production were similar, and there was a close and similar correlation between cAMP production at 15 min and PGE2 production at 6 h for both agents. An increase in PG production was also observed when IBMX, which elevates cAMP levels in cells by inhibiting cAMP phosphodiesterase, or the cAMP analog 8BrcAMP was added to the cultures. In addition, IBMX enhanced the PGE2 responses to PTH, forskolin, and 8BrcAMP. These findings indicate that stimulation of PG production by PTH may be mediated by cAMP.

1-Methyl-3-isobutylxanthine↗

PubMed1989

Inhibition by 17 beta-estradiol of PTH stimulated resorption and prostaglandin production in cultured neonatal mouse calvariae.

Previous attempts to show a direct effect of physiological concentrations of 17 beta-estradiol (beta E2) on bone in vitro have been unsuccessful. We describe a culture system using neonatal mouse calvariae in which beta E2 in the range 1 pM to 1 nM inhibited parathyroid hormone (PTH) stimulated prostaglandin E2 (PGE2) release by 50 to 70% in the presence and absence of cortisol. In addition, beta E2 reduced medium calcium concentration and release of previously incorporated 45Ca by 10 and 20%, respectively, in PTH stimulated cultures. Indomethacin did not block beta E2 effects on resorption. 17 alpha-Estradiol (alpha E2) reduced PTH stimulated 45Ca release but not PGE2 release. Thus, beta E2 has direct effects on bone consistent with its known effects to decrease bone resorption in vivo.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

Effects of two inhibitors of anion transport on bone resorption in organ culture.

The present investigation was undertaken to examine the effects of 4-acetamido-4'-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), known amino-reactive and selective inhibitors of anion exchange across the plasma membrane, on bone resorption in organ cultures of fetal rat long bones. Cultures were treated with SITS and DIDS under control unstimulated conditions or with PTH. Both SITS and DIDS were found to be potent inhibitors of 45Ca release from previously labeled fetal rat long bones. Both control resorption and the response to PTH were inhibited in a dose-related fashion. SITS and DIDS also inhibited the incorporation of [3H]thymidine in bone. The effects on resorption and [3H]thymidine incorporation were reversible when the drugs were withdrawn. These findings indicate that SITS and DIDS are potent inhibitors of bone resorption which may act by blocking the anion exchange, Cl-/HCO3-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro.

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.

Animals↗

PubMed1987

Increased bone formation and decreased bone resorption in fetal mouse calvaria as a result of intermittent compressive force in vitro.

We have shown earlier that hypertrophic chondrocytes of growth plate cartilage in vitro react to an intermittent compressive force (ICF) of physiological magnitude by an increased calcification of the matrix. In this communication, we report the influence of ICF on bone metabolism, i.e., osteoblastic and osteoclastic activity, using fetal mouse calvaria in vitro. Seventeen-day-old calvaria were cultured for 5 days under control conditions (atmospheric pressure), or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). Osteoblastic activity was monitored by measuring alkaline phosphatase (AP) activity and 45Ca incorporation into the bone mineral. Osteoclastic resorption of the mineral phase was monitored by measuring the release of 45Ca from prelabeled bone rudiments. In addition, the total mineral content (Ca and Pi) of the calvaria was determined. Exposure to ICF resulted in a significant increase in bone formation, indicated by an enhanced alkaline phosphatase activity and increased incorporation of 45Ca, as well as a decreased bone resorption. The combined effects led to a net increase in mineral content per calvarium of some 16%. We conclude that both osteoblasts and osteoclasts are affected by intermittent compressive force. Osteoblasts are stimulated, and osteoclasts are inhibited in their activity and/or growth. The effect of ICF on osteoblasts is comparable with the effect on fetal growth plate chondrocytes; both cell types respond to ICF by an increase in calcium-phosphate mineral deposition in the matrix. The lower bone resorption may be a direct effect of ICF on osteoclasts, but it is also possible that osteoblasts play an intermediate role.

Alkaline Phosphatase↗

PubMed1986

Increased calcification of growth plate cartilage as a result of compressive force in vitro.

The influence of intermittent compressive force (ICF) and of continuous compressive force (CCF) on calcification of growth plate cartilage was investigated, using organ cultures of fetal mouse cartilaginous long bone rudiments. Sixteen-day-old metatarsal rudiments, still consisting of uncalcified cartilage, were isolated and cultured for 5 days. Initial calcification of hypertrophic cartilage occurred under control conditions (atmospheric pressure), and under the influence of ICF or CCF by intermittently or continuously compressing the gas phase above the culture medium. Calcification was monitored by means of 45Ca and 32P incorporation into calcium-phosphate mineral and by morphometric methods. Both ICF and CCF increased cartilage calcification, but ICF was about twice as effective as CCF. Killed rudiments did not calcify during the culture period, nor did ICF or CCF increase the incorporation of label. The effects of ICF and CCF on calcification could not be mimicked by increasing the PO2 and PCO2 levels in the gas phase. The length of the central zone of calcified cartilage was significantly increased by ICF and CCF. We conclude that hypertrophic chondrocytes respond directly to ICF and CCF by an increased deposition of calcium-phosphate mineral in the matrix. Discontinuous mechanical stimulation evokes a higher cellular response than does continuous stimulation.

Animals↗