Biomedical subjects

W Beertsen

Publications and source records attributed to W Beertsen.

At least 55 records · Page 3Linked to original sources

PubMed1996

Cell-bound and extracellular matrix-associated alkaline phosphatase activity in rat periodontal ligament. Experimental Oral Biology Group.

In previous studies it was noted that alkaline phosphatase (ALP) activity in periodontal ligament does not only seem to be related to cells but may also be associated with the extracellular matrix. In an attempt to clarify this we studied the distribution of the enzyme at the electron microscopic level. In addition, ALP-activity was assessed biochemically following extraction of the ligament with (i) agents dissolving the membrane or splitting the phosphatidylinositol anchor (Triton X-100 or phosphatidylinositol-phospholipase C, respectively), and (ii) a matrix-degrading enzyme cocktail (collagenase, hyaluronidase and elastase). Histochemical observations revealed (a) a heterogeneous distribution of ALP-activity, with highest activity adjacent to the alveolar bone and (b) two pools of activity; one bound to cells and one associated with the collagenous extracellular matrix. In line with this were the biochemical data indicating that approximately 10% of the enzyme activity was firmly bound to the extracellular matrix and 90% to plasma membranes. Isoelectric focusing did not reveal differences between the two fractions, both samples yielding a single broad band corresponding with an isoelectric point of about 4.4.

Alkaline Phosphatase↗

PubMed1996

Cytokine-induced endogenous procollagenase stored in the extracellular matrix of soft connective tissue results in a burst of collagen breakdown following its activation.

Numerous data strongly suggest the involvement of cytokines and the matrix metalloproteinase collagenase (MMP-1) in the pathogenesis of periodontitis. Recently, we have demonstrated that, upon culturing under the influence of IL-1 alpha + EGF, a large amount of inactive procollagenase (MMP-1) is stored in the extracellular matrix of periosteal tissue. We now show that this endogenous reservoir of proenzyme can be operative after activation with plasmin and is able to induce a rapid and almost complete breakdown of the collagenous extracellular matrix. The level of collagen degradation following activation showed a strong correlation with the amount of proenzyme that was incorporated in the tissue. The highest level of degradation (70% of the total amount of collagenous proteins) was found with the IL-1 alpha + EGF-treated explants, followed by those treated with IL-1 alpha alone (35%). Explants cultured with EGF or in the absence of cytokines, containing only small amounts of procollagenase, showed little collagen breakdown following plasmin activation (7%). Inhibition of metalloproteinases by EDTA, or blockage of plasmin by PMSF, prevented the degradation in all explants irrespective of the amount of proenzyme present in the tissue. Our findings demonstrate that endogenous proenzyme stored in a native connective tissue matrix can be activated at a later time interval which results in a massive breakdown of the tissue. This study shows a possible pathway of collagenase-induced breakdown without recent de novo synthesis of the enzyme. Such a sequence may be operative in chronic inflammatory diseases, such as periodontitis, where production of procollagenase under the influence of cytokines spans a longer time period, whereas breakdown is often characterized by a cyclic behaviour.

Analysis of Variance↗

PubMed1996

[Dental research in the Netherlands: Ph.D theses 1956-1994].

The number of PhD-theses by Dutch scientists shows a continuous increase over the last four decades, resulting in about 20 theses per year during the early nineties. They cover a wide range of subjects in all major disciplines of dental research and nowadays are usually written in English (in 80% of the cases). A substantial proportion (23%) of all dental theses comes from scientists not trained as dentist but in other disciplines like biochemistry and psychology. So far, the drastic decline in the number of Dental Schools in the Netherlands during the eighties has had little influence on the overall outcome of PhD-theses. The international impact of Dutch dental research seems to increase over the years as shown by the declining fraction of Dutch dental scientists who are never cited in the international dental literature.

Academic Dissertations as Topic↗

PubMed1995

Mineralization of alkaline phosphatase-complexed collagen implants in the rat in relation to serum inorganic phosphate.

The present study was designed to determine the relationship between mineralization of collagenous matrices and serum levels of calcium and inorganic phosphate. Collagen slices were prepared from bovine dentin or cortical bone and complexed with varying amounts of intestinal alkaline phosphatase (ALP). The enzyme was added to induce de novo mineralization. The ALP-complexed slices were implanted subcutaneously over the skull and in the dorsolateral aspect of the abdominal wall in female Wistar rats of various ages (5-, 10-, 20-, or 35-week-old) and in young male rats fed on a low-P diet. After 1-4 weeks, the implants were removed and analyzed for calcium and phosphate content. In addition, serum levels of calcium and phosphate (total and inorganic) were determined. It was shown that the highest mineral influx occurred in the younger rats (which were also highest in serum P(i)), whereas almost no mineral uptake occurred in the older ones. Also in rats fed on a low-P diet (which were low in serum P(i), a strongly decreased mineral influx was noted. In all animal groups a positive correlation was found between the degree of mineralization and serum P(i). No distinct relationship was found between serum Ca/organic phosphate levels and mineral influx in the implants. In vitro incubation of ALP-collagen conjugates in serum from younger and older rats confirmed our view that serum P(i), besides local levels of ALP, is important in de novo mineral deposition. For accretion of mineral in partially remineralized collagenous carriers, ALP activity was not required.

Aging↗

PubMed1995

Type VI collagen is phagocytosed by fibroblasts and digested in the lysosomal apparatus: involvement of collagenase, serine proteinases and lysosomal enzymes.

Type VI collagen is present in most connective tissues, where it is considered to play a crucial role in the attachment of cells to the extracellular matrix and/or in the three-dimensional organization of the collagen meshwork. Although some information is available on its formation, the mechanisms involved in its degradation are not understood. Here, we present evidence for lysosomal digestion of type VI collagen by fibroblasts of periosteal explants. In the lysosomal apparatus of these cells, broad-banded filamentous aggregates characterized by 100-nm periodicity were found, which proved to consist of type VI collagen as indicated by their stainability with anti-type VI collagen antibodies. By interfering with synthesis (ascorbate or alpha, alpha-dipyridyl), intracellular translocation of collagen-containing vesicles (colchicine) as well as phagocytosis (cytochalasin B), it was shown that the intracellular broad-banded type VI collagen represented phagocytosed material. In the presence of acidotropic agents (NH4Cl and methylamine) the amount of intracellular type VI collagen increased significantly (5- to 10-fold), suggesting that a rise of pH in the endosomal/lysosomal apparatus causes inhibition of its degradation. By using a variety of proteinase inhibitors, it was found that inhibition of collagenase (when used in combination with NH4Cl), or inhibition of cysteine proteinases (both with and without NH4Cl), resulted in an increased amount of intracellular type VI collagen, whereas inhibition of serine proteinases significantly lowered the level of intracellular type VI collagen. The data presented are the first to indicate a pathway by which type VI collagen degradation may occur: fibroblasts phagocytose type VI collagen and subsequently digest this collagen in their lysosomal apparatus. Degradation depends on the activity of several enzymes, among them collagenase and serine proteinases, probably exerting their activity in the extracellular space just before the actual internalization. After uptake, digestion involves pH-sensitive lysosomal enzymes, including those belonging to the class of cysteine proteinases.

Animals↗

PubMed1995

Alkaline phosphatase activity in the periodontal ligament and gingiva of the rat molar: its relation to cementum formation.

Alkaline phosphatase (ALP) is a glycoprotein thought to be involved in processes leading to mineral formation in tissues like bone and cementum. In the rat molar periodontium, several regions are associated with the formation of cementum (periodontal ligament, inner part of the gingiva), whereas other areas are not (e.g., the outer part of the gingiva just beneath the outer oral epithelium). In an attempt to establish how the spatial distribution of ALP activity relates to cementum formation, we assessed the activity of the enzyme quantitatively in the periodontium of the rat maxillary molars, by using the indoxyl-tetrazolium salt method. It appeared that the distribution of enzyme activity in the ligament was heterogeneous, indicating local variations in the phosphate household. Highest activity was found in areas related to mineralization, adjacent to the alveolar bone and cementum. Enzyme activity was higher adjacent to cellular cementum than to acellular cementum. With respect to acellular cementum, a highly significant positive correlation was found between ALP activity and cementum thickness, which indicates a close relationship between local phosphate production and cementum formation rate. An interesting observation in the connective tissue of the gingiva mesial to the first molar was a sharp demarcation between an ALP-positive inner part, adjacent to the tooth, and an ALP-negative outer part, underneath the outer oral epithelium. In the interdental gingiva, the entire connective tissue proved positive for the enzyme, suggesting that this region consists of the combined inner gingival parts of two adjacent teeth.

Alkaline Phosphatase↗

PubMed1995

Cytokines modulate phagocytosis and intracellular digestion of collagen fibrils by fibroblasts in rabbit periosteal explants. Inverse effects on procollagenase production and collagen phagocytosis.

Degradation of fibrillar collagen may occur in the extracellular space by enzymes, such as the metalloproteinase collagenase, or in the lysosomal apparatus of fibroblasts following phagocytosis. As the mechanisms involved in the regulation of the latter process are unknown, we investigated possible modulating effects of the cytokines epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta) on both collagen phagocytosis and the release of collagenase in an in vitro model employing periosteal tissue explants. The data demonstrated that the level of intracellular collagen digestion could be influenced by cytokines: IL-1 alpha inhibited and TGF-beta enhanced phagocytosis of fibrillar collagen by periosteal fibroblasts, whereas the cytokines had an opposite effect on the release of procollagenase. In combination, IL-1 alpha and TGF-beta proved to have an antagonizing effect on either parameter. PDGF and EGF had no effect on phagocytosis or collagenase release. The level of phagocytosed collagen correlated positively with the actual breakdown of collagen as assessed by the release of hydroxyproline but negatively with the level of released procollagenase. Our findings demonstrated that cytokines are able to modulate both the phagocytosis of collagen fibrils by fibroblasts and their subsequent intracellular breakdown, as well as the release of procollagenase, an enzyme considered crucial for extracellular collagenolysis. Moreover, our data show a negative correlation between these two parameters. It is concluded that IL-1 alpha, EGF and TGF-beta may be important in modulating the contribution of the intracellular and extracellular route of collagen breakdown.

Animals↗

PubMed1995

Cytokines modulate contraction of periosteal explants from rabbit calvariae.

Periosteal explants obtained from rabbit calvariae are introduced as a model system to study contraction of soft connective tissue. Culturing of these explants resulted in a substantial time-dependent decrease of the surface area which coincided with a similar decrease in volume. A 75% reduction of the initial explant surface area was observed after a culture period of 72h in the presence of 10% serum. Also in the absence of serum contraction of explants was found, indicating that serum components were not essential. Following devitalization of the explants no contraction did occur. A strong inhibition of contraction was found following interference with the formation of microfilaments, microtubules or intracellular cyclic-AMP levels by using cytochalasin B, colchicine or dibutyryl-cAMP, respectively. These data indicated that viable cells and an intact cytoskeleton were a prerequisite for contraction to occur. A number of cytokines (EGF, aFGF, bFGF, IGF-1, PDGF, TGF-beta and IL-1alpha) was tested for their ability to influence contraction. IL-1alpha was shown to inhibit contraction from the 48h culture period on. Anti-IL-1alpha-serum completely abolished this effect. The IL-1alpha-inducible inhibition of contraction was also partially blocked by indomethacin. TGF-beta enhanced contraction dose-dependently during the 24-48h culture period, whereas TGF-1 and IL-1alpha, added to the cultures in combination, proved to antagonize each other. The other growth factors did neither influence contraction not the IL-1alpha-induced inhibition of contraction.

Actin Cytoskeleton↗

PubMed1995

[Dental research in the Netherlands: an international orientation].

Analysis of dental scientific literature (section Dentistry & Odontology of the Science Citation Index) has revealed that the majority of the contributions to dental science come from the USA, the UK and Japan. Also Dutch scientists showed up as active contributors, especially when viewed against the distribution of IADR-members and dental practitioners over the world. The scientific output in the Netherlands is evenly spread over the various dental subdisciplines, although some emphasis is seen on the fields of cariology and social dentistry.

Dentistry↗

PubMed1994

Identification of new rat dentin proteoglycans utilizing C18 chromatography.

Although only one small PG has been identified in dentin until now, a preliminary investigation has shown indications of the presence of several new proteoglycans (PGs) in rat incisor dentin. The aim of the present investigation was to isolate and characterize these PGs, which were labeled with 35S to facilitate the analysis. C18 chromatography resolved five dentin PGs. Based on SDS-polyacrylamide gel electrophoresis, their size varied from 100 to 400 kDa. The core proteins of the first four PGs appeared as 25, 40, 70, and 115 kDa bands. They stained turquoise with Stains All but did not stain with Coomassie Brilliant Blue. The core protein of the fifth PG appeared at about 45 kDa. This core protein stained with Coomassie Brilliant Blue but not with Stains All. In all PGs, the glycosaminoglycans consisted mainly of chondroitin 4-sulfate. To investigate their incorporation into predentin (young dentin that is not yet mineralized) and dentin, rat dentin PGs were pulse-labeled by injecting rats with [35S]sulfate at 5, 28, 55, or 177 h before killing the animals. Radiolabeling of predentin PGs was highest after 5 h and decreased rapidly (76%) over the next 50 h. In dentin PGs, a large percentage (34%) of the final quantity of incorporated 35S (at 177 h) was already present at 5 h. C18 chromatography of dentin PGs for each of the four time intervals showed similar 35S distribution patterns representing all five PGs, whereas the predentin appeared to contain mainly the fifth PG. This study demonstrates the existence of several apparently novel PGs in dentin that can be resolved by the use of a new method. These PGs were found in mineralized dentin and are thought to be rapidly transported toward the mineralization front. Part of the predentin PGs, on the other hand, seems to be lost as mineralization proceeds.

Animals↗

PubMed1994

Mineralization of alkaline phosphatase-complexed collagenous implants in the rat: relation with age, sex, and site of implantation.

This study was designed to determine the effects of age, sex, and site of implantation on the extent of alkaline phosphatase-complexed collagen sheets mineralization in the animal body. Collagen sheets were prepared from bovine dentin and cortical bone and complexed with varying amounts of intestinal alkaline phosphatase (ALP). Controls were without enzyme or with heat-inactivated enzyme. Sheets were implanted subcutaneously over the skull and in the dorsolateral abdominal wall in 5- or 20-week-old male and female Wistar rats. After 2-3 weeks the implants were removed and analyzed for phosphate and calcium content. Our findings have shown that alkaline phosphatase-induced mineralization of collagenous implants is influenced to a considerable extent by age, sex, and site of implantation. Highest mineral influx was seen in the younger males. Implants in younger females and older males contained less mineral, whereas those installed in the older females were almost free of calcium phosphate deposits. Dentinal implants in the skull region contained more mineral than those in the abdominal wall.

Abdominal Muscles↗

PubMed1994

Bound phosphoproteins enhance mineralization of alkaline phosphatase-collagen complexes in vivo.

Phosphoproteins (PP) covalently bound to a collagenous matrix have been reported to promote its mineralization in vitro. It was the aim of the present study to determine whether PP also enhance the mineralization of collagen in vivo. To this end, collagen slices were prepared from demineralized bovine cortical bone. Following immobilization of rat dentin phosphoprotein (PP) to the slices, bovine intestinal alkaline phosphatase (ALP) was bound according to the SATA-MHS coupling method. Controls were without enzyme. The slices were implanted into skin pockets prepared over the skull of female Wistar rats (6-10 weeks old). After 3-31 days the implants were removed and analyzed for calcium and phosphate content. It was shown that slices of PP-treated bone collagen mineralized more rapidly and to a greater extent than bone collagen slices without PP. Controls remained free of mineral. It is concluded that mineralization of collagenous matrices, induced by alkaline phosphatase, is enhanced by bound phosphoproteins following implantation in subcutaneous connective tissue.

Alkaline Phosphatase↗

PubMed1994

Ankylosis of the mouse molar after systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP).

The proposed homeostasis between fibroblasts in the periodontal ligament and bone cells lining the inner aspect of the alveolar wall is thought to be responsible for maintaining the width of the periodontal ligament space. In the present study, we attempted to interfere with this cellular balance by systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). The maxillary left molars of mice were extracted. They then received a daily subcutaneous injection of HEBP (10 mg P/kg b.w.) for periods of 10, 30 or 50 days. A 2nd group of animals received HEBP without molar extraction. Control groups were injected with Ringer's solution. The animals were sacrificed after the last injection and the mandibles processed for light and electron microscopy. HEBP treatment caused a significant decrease of the width of the periodontal ligament space which was influenced by time. Ankylosis started to occur after 30 days, predominantly in the interradicular areas and was more common around unopposed teeth. At 50 days, signs of resorption were seen along the acellular cementum layer. During HEBP-administration, extensive deposition of newly formed cementum matrix was observed along the cellular cementum but this activity was not seen along the acellular cementum. It is concluded that HEBP interferes with the mechanisms that mediate preservation of the periodontal ligament width.

Alveolar Process↗

PubMed1994

Repair processes in the periodontium following dentoalveolar ankylosis: the effect of masticatory function.

It has recently been shown that administration of the drug 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) perturbs the homeostasis in the periodontal ligament resulting in an osteoid-mediated ankylosis between the alveolar wall and the root surface. In the present study, the events after discontinuing HEBP administration were investigated and the effect of function on repair of the periodontal ligament was evaluated. In mice, the maxillary left molars were extracted. They then received a daily subcutaneous injection of HEBP (10 mg P/kg b.w.) for a period of 50 days, were killed 24 h, 14, 28, 56 and 112 days after the last injection and their mandibles processed for light microscopy. HEBP administration caused a significant decrease of the width of the periodontal ligament space with localized ankylosis. In the period after discontinuing HEBP treatment, the newly-formed bone did first mineralize and was then partly resorbed resulting in the disappearance of the ankylotic areas. Furthermore, root resorption was seen. Finally, the periodontal ligament regained its normal architecture and width and a new layer of cementum was formed. Functional teeth showed more root resorption than hypofunctional ones and a more rapid repair of the periodontal ligament. It is concluded that in the period after HEBP treatment the periodontal ligament regains its normal width by a repair process including extensive root resorption, that seems to accelerate this process.

Alveolar Process↗

PubMed1994

Formation of afibrillar acellular cementum-like layers induced by alkaline phosphatase activity from periodontal ligament explants maintained in vitro.

Fibroblasts of the periodontal ligament, by their alkaline phosphatase (ALP) activity, are considered to play a role in the formation of acellular cementum. As a means of exploring this hypothesis, periodontal ligament explants from rat incisors were cultured in direct contact with bovine dentin slices in the presence of 10 mmol/L beta-glycerophosphate. Periosteal and pericardial tissue explants were maintained under similar conditions. After two weeks, the slices were harvested and processed for electron microscopic examination. Controls included periodontal ligament explants to which the ALP-inhibitor levamisole was added. The results suggest that only ALP-positive cultures from periodontal ligament and periosteum form mineralized layers along the dentin. After demineralization, layers consisted of fine filamentous or granular material of moderate electron-density and resembled afibrillar acellular cementum. Our findings support the hypothesis that periodontal ligament fibroblasts, by means of their ALP activity, play a pivotal role in the formation of acellular cementum.

Alkaline Phosphatase↗

PubMed1994

Immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme. The effect of the cytokines IL-1 alpha and EGF.

The effect of interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1 alpha in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells. Explants cultured in the presence of IL-1 alpha alone contained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, non-cultured periostea contained no detectable enzyme. Extraction of collagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total activatable enzyme synergistically increased (10-fold) under the influence of IL-1 alpha and EGF, whereas IL-1 alpha alone showed a 4-fold enhancement compared to control or EGF-incubated explants. (3) The latent fraction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1 alpha + EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase, on the other hand, appeared to be present in a relatively high concentration in explants cultured without cytokines (2.45 mU/explant versus 0.36 mU/explant in IL-1 alpha + EGF-treated explants).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The prevalence and distribution of rests of Malassez in the mouse molar and their possible role in repair and maintenance of the periodontal ligament.

This study sought to determine the prevalence and distribution of these rests under normal conditions and after giving 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), a drug that interferes with homeostasis in the periodontal ligament. Normally the rests of Malassez were equally distributed on all root aspects with the exception of the mesial of the mesial root of the first mandibular molar. With increasing age their number decreased. A similar distribution was seen in the animals treated with the bisphosphonate, in which there was a severe reduction in width of the periodontal ligament, at several sites resulting in ankylosis. After discontinuing HEBP the normal width of the periodontal ligament space was restored. Repair was characterized by root resorption, elimination of ankylosis, cementum formation and regeneration of ligament. These processes seemed to have no connection with the prevalence and distribution of rests of Malassez. It is concluded that rests of Malassez are unlikely to play an important part in the homeostasis of periodontal ligament and do not seem to be a prerequisite for its repair and maintenance.

Animals↗

PubMed1993

Mineralization of dentinal collagen sheets complexed with alkaline phosphatase and integration with newly formed bone following subperiosteal implantation over osseous defects in rat calvaria.

We addressed the question to what extent alkaline phosphatase (ALP) can induce mineralization of a collagenous matrix implanted subperiosteally, and how the graft interacts with the underlying bone. Bovine intestinal ALP was bound to sheets of guanidine-extracted, demineralized bovine dentin by using the crosslinking agent 1-ethyl-3(3-dimethylaminopropyl)carbodiimide.HCl. The complexes (with active enzyme) and control grafts (no enzyme) were implanted over osseous defects in opposite halves of rat calvaria. After time intervals varying from 3-12 weeks, the calvaria were processed for light and electron microscopic examination and histomorphometric analysis. The ALP-containing sheets (but not their controls) rapidly accumulated mineral crystals. As the complexes mineralized, osteoblasts appeared and formed a layer of bone in direct contact with the grafted material. The results indicate that ALP induced the deposition of mineral crystals, and strongly suggest that it is this mineral component which influenced the formation of bone.

Alkaline Phosphatase↗