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Biomedical subjects

W Beertsen

Publications and source records attributed to W Beertsen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Collagen breakdown in soft connective tissue explants is associated with the level of active gelatinase A (MMP-2) but not with collagenase.

Recent data suggest that gelatinase A (matrix metalloproteinase-2, MMP-2) plays an important role in the degradation of collagen of soft connective tissues. In an attempt to investigate its participation in more detail we assessed the digestion of collagen in cultured rabbit periosteal explants and compared this with the level of active MMP-2 and collagenases. The data demonstrated that both collagen degradation and MMP activity increased with time. Conditioned medium obtained from explants cultured for 72 h showed that the level of active MMP-2 correlated with collagen degradation (r = 0.80, d.f. = 23, P < 0.0001). Such a relationship was not found with collagenase activity (r = -0.08, d.f. = 21, NS). The possible involvement of MMP-2 in collagen degradation was investigated further by incubating explants with selective gelatinase inhibitors (CT1166, CT1399 and CT1746). In the presence of these compounds breakdown of collagen was almost completely abolished (approximately 80%). Finally we assessed whether periosteal fibroblasts had the capacity to degrade collagen type I that conferred resistance to collagenase activity. Breakdown of this collagen did not differ from degradation of normal collagen. Taken together, our data provide support for the view that MMP-2 plays a crucial role in collagen degradation of soft connective tissue.

Animals↗
PubMed1999

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone.

Data in the literature suggest that site-specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.

Amides↗
PubMed1999

Alkaline phosphatase activity in human periodontal ligament: age effect and relation to cementum growth rate.

Recently, a relationship was demonstrated between the thickness of the cementum layer in rat molars and the activity of alkaline phosphatase (ALP) in the adjoining periodontal ligament (PDL). It was the aim of the present study to investigate whether such a relationship also exists in the periodontium of man. Healthy deciduous and permanent teeth free from periodontitis were obtained from 74 patients, varying in age from 3 to 78 yr, and their PDL dissected from the middle one-third of the roots. ALP activity was measured in PDL extracts and expressed per hydroxyproline content. It was shown that ALP activity was relatively high in children. After puberty its concentration decreased to level off at about half the concentration found in the younger age groups. The activity of the enzyme in the PDL correlated positively with the yearly cementum thickness increment as calculated from data published previously.

Adolescent↗
PubMed1999

Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation.

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.

Alkaline Phosphatase↗
PubMed1999

Blood circulation as source for osteopontin in acellular extrinsic fiber cementum and other mineralizing tissues.

Osteopontin (OPN) is one of the major non-collagenous proteins in root cementum and other mineralized tissues. Although most of this mineral-seeking protein is thought to be produced by local tissue cells, some of it might enter the mineralizing matrix from the blood. To test this hypothesis, we followed the distribution of a single dose of purified porcine or rat 125I-labeled OPN injected i.v. in rats, in mineralizing and non-mineralizing tissues and in subcutaneously implanted collagenous implants. The animals were killed 30 or 48 hrs after injection. Tissues (calvaria, tibia, lower and upper jaws) were harvested and processed for radioautography and biochemical analysis. Tissues as well as calcifying collagenous implants proved to have taken up radiolabel. In EDTA extracts of long bones, the majority of the radiolabel was demonstrated to be associated with intact OPN. The iodinated protein was also found in the acellular extrinsic fiber cementum (acellular cementum) layer investing the continuously growing incisors, in laminae limitantes, cement lines, and in forming bone near the mineralization front. Further, the label was present in the circumpulpal dentin of the incisors, and some of it appeared to have been incorporated into developing enamel. It is concluded that OPN in acellular cementum and other mineralizing tissues may-at least partially-originate from sources outside the direct environment following its transportation via serum.

Animals↗
PubMed1998

Involvement of V-ATPases in the digestion of soft connective tissue collagen.

The contribution of vacuolar H+-ATPases (V-ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum). Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase inhibitors, bafilomycin A1 and folimycin, decreased overall collagen degradation by 40 and 50% after 24 and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in extracellular and intracellular collagen degradation in soft connective tissue.

Animals↗
PubMed1998

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

Animals↗
PubMed1998

Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue.

The degradation of soft connective tissue collagen is considered to depend on the activity of various proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases and cysteine proteinases. In the present study, we investigated the contribution of these enzymes to this process. Using a general inhibitor of MMPs (SC44463), collagen degradation was strongly inhibited, by about 40% after 24 h and up to 80% after 72 h of culturing. Blockage of cysteine proteinase activity (with leupeptin or E-64) reduced breakdown at these time intervals by 50% and 20%, respectively. Given the abundant presence of gelatinases--in particular gelatinase A (MMP-2)--in the tissue, the effect of an inhibitor selective for gelatinases (CT1166) was studied. Gelatinase inhibition resulted in a dose-dependent decrease of collagen breakdown up to 90% after 48 h. The ability of gelatinase A to degrade collagens was demonstrated by the induction of breakdown in devitalized explants by addition of activated gelatinase A, or by activation of endogenous enzyme with 4-aminophenylmercuric acetate. This latter effect was not found with plasmin, an activator of MMPs other than gelatinase A. Finally, the relevance of gelatinase A to the in vivo degradation of soft connective tissue collagen was implicated by the significant correlation found between its activity and the collagen turnover rates of four soft connective tissues (tooth pulp, periodontal ligament, molar gingiva and skin). We conclude that collagen degradation in soft connective tissue is mediated by MMPs and to a lesser extent by cysteine proteinases. Our data are the first to attach a key role to gelatinase A in this process.

Animals↗
PubMed1998

Formation of reparative acellular extrinsic fiber cementum in relation to implant materials installed in rat periodontium.

The aim of the present study was to determine the nature of the cells associated with the formation of reparative acellular extrinsic fiber cementum (AEFC). Calcifying collagen membranes, hydroxyapatite particles and/or non-resorbable ePTFE membranes were implanted in lesions made in the periodontium of the rat mandibular incisor. The incisor was prevented from erupting further, and the animals were killed after 1-8 wk. In the first week, next to cells with a fibroblastic phenotype, epithelial cells (ECs) migrating out of the reduced enamel epithelium were among the cells colonizing the wounds. From 2 wk on, the ECs showed regressive changes and disappeared. Along mineralized implant surfaces, a basophilic layer enriched in osteopontin (but without detectable amelogenin) was deposited. After 3 wk (when ECs were no longer present) the healing tissue transformed into a well-organized PDL-like tissue, and AEFC started to develop. Along the non-mineralizing ePTFE membranes, AEFC did not form except in regions where small calcified bodies were present. It is concluded that reparative AEFC layers are formed only along calcified surfaces. The cells associated with this reparative activity are periodontal ligament cells with a fibroblastic phenotype.

Animals↗
PubMed1998

EGF and IL-1 alpha modulate the release of collagenase, gelatinase and TIMP-1 as well as the release of calcium by rabbit calvarial bone explants.

Matrix metalloproteinases (MMPs), among which is collagenase (MMP-1), are likely to be involved in various steps of the bone resorption process. As both production of these enzymes and bone resorption appear to be mediated by cytokines, we investigated the effects of two cytokines, IL-1 alpha and EGF, on the release of collagenase, gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1 and calcium by rabbit calvariae. It was found that all these parameters increased under the influence of these cytokines. The release of calcium--used as a parameter of bone resorption--was highest in the combined presence of the cytokines. Although the absolute and relative enhancement by a combination of IL-1 alpha and EGF was most pronounced for collagenase (7-fold), both gelatinase A (5-fold) and gelatinase B (1.5-fold) had increased simultaneously. Calvariae produced a high level of MMP inhibitor (TIMP-1), especially under the influence of the cytokines; periosteum released little inhibitor. It is concluded that IL-1 alpha and EGF are likely to play a modulating role in the process of bone resorption.

Analysis of Variance↗
PubMed1998

Type VI collagen is associated with microfibrils and oxytalan fibers in the extracellular matrix of periodontium, mesenterium and periosteum.

Type VI collagen was immunolocalized in several soft connective tissues at the light and electron microscopic level. Positive labeling was found in all tissues examined, periodontal ligament, gingiva, mesenterium and periosteum. The labeled structures could be divided into 2 categories: microfibrils intermingling with collagen fibrils, and those that formed bundles (oxytalan fibres and elastin-associated microfibrils). Control sections incubated with antibody preabsorbed to purified type VI collagen, or with non-immune antibody, proved to be negative. Our observations indicate that the structural organization of type VI collagen varies from small microfibrillar structures associated with the collagen and elastin fibre systems to highly ordered parallel arrays of oxytalan bundles.

Animals↗
PubMed1998

Cysteine proteinases and matrix metalloproteinases play distinct roles in the subosteoclastic resorption zone.

Digestion of calvarial bone by osteoclasts depends on the activity of cysteine proteinases and matrix metalloproteinases (MMPs). It is unknown, however, whether these enzymes act simultaneously or in a certain (time) sequence. In the present study, this was investigated by culturing mouse calvarial bone explants for various time intervals in the presence or absence of selective low molecular weight inhibitors of cysteine proteinases (E-64, Z-Phe-Tyr(O-t-Bu)CHN2 or CA074[Me]) and MMPs (CI-1, CT1166, or RP59794). The explants were morphometrically analyzed at the electron microscopic level. All proteinase inhibitors induced large areas of nondigested demineralized bone matrix adjacent to the ruffled border of actively resorbing osteoclasts. The appearance of these areas proved to be time dependent. In the presence of the cysteine proteinase inhibitors, a maximal surface area of demineralized bone was seen between 4 and 8 h of culturing, whereas the metalloproteinase inhibitors had their maximal effect at a later time interval (between 16 and 24 h). Because different inhibitors of each of the two classes of proteolytic enzymes had the same effects, our data strongly suggest that cysteine proteinases attack the bone matrix prior to digestion by MMPs. In line with the view that a sequence may exist were differences in the amount of proteoglycans (shown with the selective dye cuprolinic blue) in the subosteoclastic demineralized areas induced by the inhibitors. In the presence of the cysteine proteinase inhibitor, relatively high levels of cuprolinic blue precipitates were found, whereas this was less following inhibition of metalloproteinases. These data suggested that cysteine proteinases are important for digestion of noncollagenous proteins. We propose the following sequence in the digestion of calvarial bone by osteoclasts: after attachment of the cell to the mineralized surface an area with a low pH is created which results in dissolution of the mineral, then cysteine proteinases, active at such a low pH, digest part of the bone matrix, and finally, when the pH has increased somewhat, MMPs exert their activity.

Amides↗
PubMed1997

Cytokines modulate routes of collagen breakdown. Review with special emphasis on mechanisms of collagen degradation in the periodontium and the burst hypothesis of periodontal disease progression.

In this paper, we review recent work on collagen degradation, 2 main routes of breakdown are described and their relevance during healthy and inflammatory conditions of the periodontium is discussed. Special attention is paid to the possible role of cytokines, in particular interleukin 1 (IL-1) and transforming growth factor beta (TGF-beta), on the modulation of collagen phagocytosis and metalloproteinase production. IL-1 has been shown to have a dual function in collagen digestion. It inhibits the intracellular phagocytic pathway, but at the same time, it strongly promotes extracellular digestion by inducing the release of collagenolytic enzymes like collagenase. TGF-beta has an opposite effect on both pathways and antagonizes IL-1. Collagenase is released in an inactive form, and a considerable fraction of the proenzyme may become incorporated in the extracellular matrix. This reservoir of latent enzyme can be activated (for instance by plasmin), leading to a sudden and extensive breakdown of the collagenous fibre meshwork. It is suggested that this phenomenon may also take place during progressive periodontitis and could explain an episodic nature of collagenolysis, clinically resulting in bursts of attachment loss (burst hypothesis).

Animals↗
PubMed1997

[Dental research in the Netherlands. Citations in publications].

This study examines the citation frequency (via the Science Citation Index) of publications by Dutch dental scientists that have appeared in the international literature since World War II. It was found that most citations were obtained by a relatively small group of researchers, many of them working in the fields of cariology and periodontology. High scores were particularly found among scientists who had their academic training in disciplines other than dentistry. Out of 109 frequently cited papers over 25% was microbiology-oriented and most of them could be characterized as original basic dental research.

Dental Research↗
PubMed1997

[The development of dental research in the Netherlands].

From the acquisition of an academic status (ius promovendi) in 1947 till now, dental research in the Netherlands has reached maturity. An even increasing body of PhD-theses and publications have found their way towards the international scientific dental community. Since the early nineties most research groups have been brought together in the 'Interuniversitaire Onderzoekschool Tandheelkunde' (Netherlands Institute for Dental Sciences), a collaboration of the academic dental institutions in Amsterdam, Nijmegen and Utrecht.

Dental Research↗
PubMed1996

Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling.

Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membrane-bound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding non-collagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor beta and interleukin 1 alpha.

Animals↗
PubMed1996

Loss of attachment in the marginal periodontium of the rat incisor under non-inflammatory conditions. Expression of alkaline phosphatase activity. Experimental Oral Biology Group.

Alkaline phosphatase (ALP) has been suggested to play a role in acellular cementum formation and maintenance of periodontal attachment. In an attempt to determine whether changes in attachment level are associated with altered expression of ALP-activity in the periodontium we induced natural loss of attachment in rats by pinning the lower incisor to the jaw bone. Previous studies have shown that this procedure results in regressive changes in the marginal periodontium without any inflammatory response. Six months after blockage of eruption the attachment level on the experimental (right) side had shifted about 700 microm in the apical direction. On the control (left) side the apical termination of the junctional epithelium had remained stationary with respect to the alveolar crest. Our observations have shown that during the first few weeks of the experiment loss of attachment is accompanied by considerable reduction of ALP-activity in the supracrestal part of the periodontium. At later time intervals, however, no distinct relation was found between apical migration of junctional epithelium and loss of ALP-activity in the supracrestal region, indicating that the two phenomena are not directly related to each other. The domain of the ALP-positive fibroblasts in the supracrestal extension of the periodontal ligament decreased in size and was replaced by ALP-negative connective tissue cells probably coming from the outer gingival domain. Since at all time intervals a distinct demarcation could be observed between the ALP-positive and ALP-negative areas, we interpret our data as indicating that ligament and gingival cells do not mix.

Alkaline Phosphatase↗