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W Beertsen

Publications and source records attributed to W Beertsen.

At least 19 recordsLinked to original sources
PubMed1992

Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases.

The site of action of cysteine-proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP-inhibitor trans-epoxy-succinyl-L-leucylamido (4-guanidino)-butane (E-64) and the MMP-inhibitor N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (Cl-1) in an in vitro model system of PTH-stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor-treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor-treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E-64 and Cl-1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.

Animals↗
PubMed1992

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A.

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.

Amino Acid Sequence↗
PubMed1992

Alkaline phosphatase induces the mineralization of sheets of collagen implanted subcutaneously in the rat.

To determine whether alkaline phosphatase (ALP) can cause the mineralization of collagenous matrices in vivo, bovine intestinal ALP was covalently bound to slices of guanidine-extracted demineralized bovine dentin (DDS). The preparations were implanted subcutaneously over the right half of the rat skull. Control slices not treated with the enzyme were implanted over the left half of the skull of the same animals. Specimens were harvested after periods varying from 1 to 4 wk. It was shown that ALP-coupled DDS rapidly accumulated hydroxyapatite crystals. 4 wk after implantation, the content of calcium and phosphate per microgram of hydroxyproline amounted up to 80 and 60%, respectively, of that found in normal bovine dentin. Our observations present direct evidence that ALP may play a crucial role in the induction of hydroxyapatite deposition in collagenous matrices in vivo.

Alkaline Phosphatase↗
PubMed1991

Cell-seeding.

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Animals↗
PubMed1991

Alkaline phosphatase induces the deposition of calcified layers in relation to dentin: an in vitro study to mimic the formation of afibrillar acellular cementum.

An attempt was made to test the hypothesis that alkaline phosphatase, an enzyme which is abundant in periodontal ligament, plays a role in the formation of acellular root cementum. Thin slices of bovine dentin were incubated in Iscove Modified Dulbecco's Medium supplemented with 10% normal rabbit serum and 10 mmol/L beta-glycerophosphate (beta-GP) or folded into pericardial explants. Intestinal bovine alkaline phosphatase (APase), covalently linked to agarose beads, was added to the cultures. In the presence of the enzyme, the dentin slices were covered with thin layers of mineralized material. Such layers were not observed in cultures not provided with APase-beads or beta-GP. They also did not form in relation to demineralized dentin. The layers of calcified material appeared to consist of crystallites embedded in a granular matrix of moderate electron density, which often exhibited the presence of incremental lines and resembled the matrix of afibrillar acellular cementum formed under in vivo conditions. When pericardial explants were interposed between the enzyme-containing beads and the dentin, mineral deposition in relation to the dentin was retarded. This finding lends support to the view that soft connective tissues interfere with the free diffusion of phosphate.

Alkaline Phosphatase↗
PubMed1991

The distribution of magnesium in developing rat incisor dentin.

Previous studies have shown that rat incisor dentin contains a considerable amount of magnesium that is distributed heterogeneously. The cementum-related dentin, especially its incisal portion, is richest in magnesium. It was the purpose of the present study to investigate the changes that occur in the magnesium content during dentin maturation. Cross-sections were prepared from rat incisors at the apical, middle, and incisal levels. By means of an electron microprobe, tracings were made of the Ca-, Mg-, and P- signal frequencies. Comparison of corresponding dentin layers within and between the cross-sections showed that the Mg/P molar ratio was always higher in the cementum-related dentin (CRD) than in the enamel-related dentin (ERD) and increased from the apex toward the incisal edge. Especially in the incisal cross-section, an increase in Mg/P was found from the older (peripheral) toward the younger (central) dentin layers. As the Mg/P ratio varied from 0.07 to 0.33, the Ca/P ratio was found to fluctuate from 1.48 to 1.15. The two ratios appeared to be highly correlated (r = -0.97; p less than 0.001), suggesting that Mg replaces Ca and is bound to phosphate.

Animals↗
PubMed1990

Interleukin 1 increases the production of collagenase but does not influence the phagocytosis of collagen fibrils.

The aim of the present study was to determine whether interleukin 1 alpha (Il-1), a cytokine known to have a stimulatory effect on collagenase production, also influences the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. Mouse long bones and calvariae both with surrounding periosteum were cultured for 24 or 48 hours in media containing varying concentrations of the cytokine. The periostea were subjected to morphometric analysis in order to assess the volume density of phagocytosed collagen fibrils in fibroblasts. The results indicated that neither in calvarial nor in long bone periosteum the uptake and intracellular degradation of collagen by fibroblasts was influenced by Il-1. However, between both tissues the amount of collagen phagocytosed differed considerably. It appeared that within 48 hours periosteal fibroblasts of calvariae ingested at least three times more fibrillar collagen than those of long bone periosteum. This finding suggests intrinsic differences between these connective tissues as to the phagocytic behaviour of the fibroblasts. Analysis of collagenase activity in the media demonstrated that under the influence of Il-1 collagenase release increased about 1.5- to 2-fold, most of the enzyme being in a latent form. The media also proved to contain an inhibitor of collagenase, its production not being affected by Il-1. It is concluded that under the conditions tested Il-1 does not seem to play a role in the regulation of the intracellular pathway of collagen digestion.

Animals↗
PubMed1990

The inorganic components of cementum- and enamel-related dentin in the rat incisor.

Recently, we have shown that, in rodent incisors, the crown- and root-analogue dentin (enamel- and cementum-related dentin) show differences in mineralization rates (Beertsen and Niehof, 1986) and composition of the organic matrices (Steinfort et al., 1989). It was the aim of the present study to determine whether these differences were accompanied by differences in the inorganic components. Rat incisors were analyzed by means of hardness measurements, microradiography, and the determination of Ca, Mg, and PO4 content. The outer circumpulpal dentin layer of the enamel-related dentin (ERD) was considerably harder and denser than the comparable layer of the cementum-related dentin (CRD). Concomitantly, a higher Ca and PO4 content was found for the ERD than for the CRD, while the reverse occurred with respect to Mg. From the apical end of the incisor toward the incisal edge, the Ca/PO4 ratio tended to decrease for both ERD and CRD, while the Mg/PO4 ratio increased. All differences appeared to be statistically significant. It is concluded that differences in the non-collagenous organic matrix were accompanied by differences in the inorganic components. More specifically, a relatively high content of highly phosphorylated phosphoproteins (ERD) was associated with a higher Ca and a lower Mg content.

Animals↗
PubMed1990

Formation of acellular root cementum in relation to dental and non-dental hard tissues in the rat.

After the periodontium of the rat was wounded, the formation of acellular extrinsic fiber cementum (AEFC) did not appear to be restricted to the hard dental tissues (pre-existing cementum, dentin, and enamel). Layers resembling AEFC were also deposited along the inner wall of the alveolar bone. At the time of observation (six weeks after being wounded), cells other than fibroblast-like cells could not be distinguished close to the newly formed AEFC-like layers. We suggest that the deposition of this material was related to the activity of periodontal ligament fibroblasts.

Alveolar Process↗
PubMed1990

The possible relationship between the mineralization of dentin and the composition of its organic matrix.

Using the rat incisor as a model, evidence is presented that enamel-related (ERD) and cementum-related (CRD) dentins exhibit differences in composition. Whereas the ERD is rich in highly phosphorylated phosphoproteins, the CRD is rich in lowly phosphorylated phosphoproteins. These differences are accompanied by and possibly related to differences in composition and physical properties of the mineral phase.

Animals↗
PubMed1989

Differences between enamel-related and cementum-related dentin in the rat incisor with special emphasis on the phosphoproteins.

In order to determine whether qualitative and quantitative differences exist between the non-collagenous proteins of crown and root dentin, rat incisors were separated into their enamel- and cementum-related dentin portions (ERD and CRD, respectively). Isolation of the mineral-bound proteins was performed under nondegradative conditions. Analytical procedures included DEAE-chromatography on high pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and determination of phosphate, protein, and hydroxyproline. The results have shown that considerable differences exist among the two dentins with respect to the quantity of the various phosphoproteins. For this group of proteins as a whole, the ERD contains about 2 times the amount of organic phosphate found in the CRD and about 1.4 times the amount of protein. The content of higher phosphorylated phosphoproteins was about 4 times higher in the ERD than in the CRD, whereas the reverse was shown for the lower phosphorylated phosphoproteins. All differences were found to be statistically significant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that while the ERD contains phosphoproteins with an apparent molecular mass of 98 kDa, the CRD contains two classes of phosphoproteins one of 98 and one of 88 kDa. The relevance of the observed differences in phosphoprotein distribution is discussed in relation to their possible role in mineralization.

Aging↗
PubMed1989

The influence of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on dental root resorption in the mouse.

It has been hypothesized that phagocytosis of a layer of mineralized material which is sometimes deposited along the root surface following trauma is an important factor in the initiation of the resorption of teeth. To test this hypothesis, freezing of the mouse incisor periodontium was used as a model where deposition of mineral was prevented by the systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP, 10 mg P/kg b.w.) for time periods varying from 2 to 31 days. The animals were killed either 24 hours or 1 month after the last injection and their mandibles were processed for light microscopy. In the HEBP-treated animals, killed 24 hours after the last injection, no mineralized material had been deposited along the root surface, and very little resorption was seen. Large amounts of bone matrix were deposited in the intraperiodontal space (often causing localized ankylosis). In HEBP-treated animals killed 1 month after the last injection, again no layer of mineralized material along the root surface was seen, but now root resorption had become manifest, occupying up to 40% of the cement surface. This occurred irrespective of the duration of HEBP administration. In the saline-treated animals, extensive resorption of the incisor was noted (up to 90% of its surface). It is concluded that the layer of mineralized material deposited after the periodontium is frozen does not play a decisive part in the onset of root resorption. HEBP treatment slows down root resorption but does not prevent it.

Animals↗
PubMed1989

Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts.

The effect of various metalloproteinase-inhibiting compounds on collagen phagocytosis by fibroblasts was studied in cultured periosteal tissue. Evidence is presented indicating that neither anti-collagenase nor anti-stromelysin interfere with the uptake of collagen fibrils from the extracellular space and their intracellular digestion. Similar results were obtained with tissue inhibitor of metalloproteinases (TIMP). In the presence of the proteinase inhibitor leupeptin, a compound which strongly inhibits the intracellular degradation of phagocytosed collagen, a time-dependent increase in the amount of internalized collagen was found. This increase proved to be similar in explants treated as well as in those not treated with the metalloproteinase-inhibiting compounds. It is concluded that enzymes, such as collagenase and stromelysin, do not play a crucial role in the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. If these enzymes are involved it must be prior to these events. Based on the morphometric data the intralysosomal degradation time of collagen was calculated to be about 30 minutes. A comparison with findings in the literature on collagen metabolism in the periodontal ligament of the rat molar suggests that all collagen degraded may pass through the phagolysome pathway during physiological turnover and remodelling.

Animals↗
PubMed1989

Calcification of dentinal collagen by cultured rabbit periosteum: the role of alkaline phosphatase.

Periostea were dissected from 1-2 weeks old rabbit calvaria and folded around decalcified and extracted bovine dentin matrix slices (DMS). The cocultures were grown in serum-containing medium supplemented with beta-glycerophosphate or other organic phosphate esters. [45Ca]-uptake measurements indicated that the DMS calcified. Initiation of the calcification process was associated with alkaline phosphatase activity and could be prevented by adding the inhibitor L-levamisole to the culture medium. Using [32P]-adenosine-monophosphate as a substrate for phosphatase activity it was demonstrated that very little, if any, phosphate was utilized for the phosphorylation of higher molecular weight substances. The results suggest that over 99% of the phosphate produced was laid down in inorganic form. Further, it was noted that mineral deposition in the DMS was accompanied by the simultaneous inclusion of methylene blue and PAS-positive substances whose nature, origin and function remain to be determined. The results lend support to the theory that alkaline phosphatase is involved in the initiation of calcification processes by raising the local concentration of phosphate ions.

Alkaline Phosphatase↗
PubMed1988

Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption.

To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in an in vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded. It was further noticed that in 12 of 287 osteoclasts, cytoplasmic vacuoles were present containing collagen fibrils that could not be distinguished from those in cartilage or bone. Their intracellular localization was proved by the study of serial sections. Finally, a significant reduction was shown as to the relative surface density of electron-translucent vacuoles; this would seem to suggest reduced endocytic activity of the cells. Our observations support the view that cysteine-proteinases play an important role in osteoclastic bone resorption. It was further noticed that the in vitro effects of leupeptin and E-64 in certain respects resemble ultrastructural features of pycnodysostosis, an osteopetrosislike bone disorder. The data are in line with the hypothesis that this disease is caused by insufficient activity of osteoclastic cysteine-proteinases.

Animals↗
PubMed1987

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde.

The histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used. Continuous monitoring of the formation of the fluorescent reaction product showed that after a certain lag phase, depending on the enzyme activity in the tissue, discrete granules appeared which became increasingly fluorescent with incubation time. Subsequently, recrystallization and redistribution of the final reaction product started to occur. It is concluded that the coupling reaction with 5-nitrosalicylaldehyde is sufficiently fast for a proper localization of proteinase activity and can be used for 'kinetic' analysis of enzyme activity. The method provides indications of relative amounts of cathepsin B activity in different cell types within a tissue section. It appeared from the study on metacarpal bone explants that fibroblasts in perichondrium and periosteum contained a relatively high cathepsin B activity whereas chondrocytes showed a low but distinct activity. This observation suggests that cysteine proteinases are not only involved in collagen degradation by fibroblasts but that they also play a role in the intracellular digestion of collagen by chondrocytes.

Aldehydes↗