Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: Special emphasis on collagen.

The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.

Cementum and dentin in hypophosphatasia.

Hypophosphatasia (HPP) often leads to premature loss of deciduous teeth, due to disturbed cementum formation. We addressed the question to what extent cementum and dentin are similarly affected. To this end, we compared teeth from children with HPP with those from matched controls and analyzed them microscopically and chemically. It was observed that both acellular and cellular cementum formation was affected. For dentin, however, no differences in mineral content were recorded. To explain the dissimilar effects on cementum and dentin in HPP, we assessed pyrophosphate (an inhibitor of mineralization) and the expression/activity of enzymes related to pyrophosphate metabolism in both the periodontal ligament and the pulp of normal teeth. Expression of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) in pulp proved to be significantly lower than in the periodontal ligament. Also, the activity of NPP1 was less in pulp, as was the concentration of pyrophosphate. Our findings suggest that mineralization of dentin is less likely to be under the influence of the inhibitory action of pyrophosphate than mineralization of cementum.

Phagocytosis of fibronectin and collagens type I, III, and V by human gingival and periodontal ligament fibroblasts in vitro.

BACKGROUND: Electron microscopic studies have suggested that the volume density of collagen-containing vacuoles in fibroblasts is higher in the periodontal ligament (PDL) than in the gingiva. Whether this difference reflects intrinsic differences in phagocytic capacity among the cells in these tissues is not known. METHODS: PDL and gingival fibroblasts were isolated from subjects and cultured under identical conditions in the presence of fluorescent beads coated with collagen type I, III, or V or fibronectin. Control beads were coated with bovine serum albumin or an enamel matrix protein mixture that does not constitute part of the extracellular matrix of PDL and gingiva. After various time intervals (1 to 24 hours), the percentage of cells that had internalized beads was assessed by flow cytometry. Since alkaline phosphatase activity has been suggested to play a role in collagen phagocytosis, the activity of this enzyme was determined for all cell populations. RESULTS: The results demonstrated the following order in the percentage of cells internalizing protein-coated beads: fibronectin > collagen type I > III > V. Internalization of collagen type I-coated beads exceeded that of beads coated with bovine serum albumin or enamel matrix proteins by 6 and 3 times, respectively. No differences were observed in collagen phagocytic activity between PDL and gingival fibroblasts, and no relationship could be demonstrated between collagen phagocytosis and alkaline phosphatase activity. CONCLUSIONS: We conclude that differences in collagen phagocytosis between PDL and gingiva, as observed in vivo, are not likely to be explained in terms of intrinsic phagocytic capacities of these cells.

Perichondrium-wrapped collagenous matrices to induce chondroneogenesis: an in vitro study.

OBJECTIVE: To develop a model for cartilage regeneration in vitro, to be used for cartilage reconstruction in vivo. METHODS: Collagenous matrices were wrapped in a perichondrium layer. The matrices served as carriers to allow migration of cells from the perichondrium into the matrix. Culture conditions stimulated cell growth and proliferation. RESULTS: After 4 weeks of culturing, microscopic examination showed an increase of cell layers around the matrices but also of cells migrated into porous matrices. Immunohistochemical staining of these cultured cells expressed type II collagen intracellularly. CONCLUSIONS: This model seems appropriate to culture mucoperichondrial explants in combination with collagenous matrices. Cells migrate into the pores of the matrix, survive, and synthesize matrix components. Actual formation of cartilage has not been shown to occur. Adding growth factors to this model may influence induction of this activity.

Enamel matrix-derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts.

BACKGROUND: Although it is claimed that enamel matrix-derived proteins (EMP) can be used to promote new attachment formation around periodontally involved teeth, the underlying biological mechanism is not understood. It was the aim of the present study to investigate the effects of EMP on the behavior of human periodontal ligament (HPLF) and gingival fibroblasts (HGF) in vitro, with special focus on their attachment properties, the expression of alkaline phosphatase (ALP) activity, the release of transforming growth factor (TGF)beta1, and their proliferative rate. METHODS: Fibroblast populations were obtained from 10 individuals with a healthy periodontium and cultured in chemically defined medium on culture plates coated with EMP, purified collagen type I, or their respective vehicles. Experiments were performed in the absence of serum for periods up to 48 hours. RESULTS: It was shown that HGF barely attached and spread on EMP-coated substrata, whereas HPLF attached and spread within 24 hours. However, when cultured on purified collagen type I, both cell types showed rapid attachment and spreading. Furthermore, the expression of ALP activity was significantly enhanced under the influence of EMP, especially in HPLF. HPLF and HGF both released significantly higher levels of TGFbeta1 in the presence of EMP. EMP did not influence 3H-thymidine incorporation by HPLF and HGF. CONCLUSIONS: Our results indicate that HPLF and HGF respond differently to EMP. A more rapid attachment of HPLF to this substratum might contribute, during the initial stages of periodontal healing, to selective outgrowth and colonization of exposed root surfaces in vivo.

Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of tissue non-specific alkaline phosphatase activity.

The capacity of the periodontal ligament to alter its structure and mass in response to mechanical loading has long been recognized. However, the mechanism by which periodontal cells can detect physical forces and respond to them is largely unknown. Besides transmission of forces via cell-matrix or cell-cell interactions, the strain-derived flow of interstitial fluid through the periodontal ligament may mechanically activate the periodontal cells, as well as ensure transport of cell signaling molecules, nutrients and waste products. Mechanosensory cells, such as endothelial and bone cells, are reported to respond to a flow of fluid with stimulated prostaglandin E2 (PGE2) and nitric oxide production. Therefore, we examined the PGE2 and nitric oxide response of human periodontal ligament and gingival fibroblasts to pulsating fluid flow and assessed the expression of tissue non-specific alkaline phosphatase activity. Periodontal ligament and gingival fibroblasts were subjected to a pulsating fluid flow (0.7 +/- 0.02 Pa, 5 Hz) for 60 min. PGE2 and nitric oxide concentrations were determined in the conditioned medium after 5, 10, 30 and 60 min of flowing. After fluid flow the cells were cultured for another 60 min without mechanical stress. Periodontal ligament fibroblasts, but not gingival fibroblasts, responded to fluid flow with significantly elevated release of nitric oxide and decreased expression of tissue non-specific alkaline phosphatase activity. In both periodontal ligament and gingival fibroblasts, PGE2 production was significantly increased after 60 min of flowing. Periodontal ligament fibroblasts, but not gingival fibroblasts, produced significantly higher levels of PGE2 during the postflow culture period. We conclude that human periodontal ligament fibroblasts are more responsive to pulsating fluid flow than gingival fibroblasts. The similarity of the early nitric oxide and PGE2 responses to fluid flow in periodontal fibroblasts with bone cells and endothelial cells suggests that these three cell types possess a similar sensor system for fluid shear stress.

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.

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.

[Survey of 50 years of veterinary remedy provision in the Netherlands]. / Overzicht van 50 jaar diergeneesmiddelen-voorziening in Nederland.

This article gives an overview of a half century of veterinary product provision in the Netherlands, and describes the availability of veterinary products from 1945 and onwards. The development of antibiotics, sulfonamides, anthelmintics, endocrine products, vaccines, and other product groups is discussed briefly. The importance of the research carried out by leading pharmaceutical companies in collaboration with the Faculty of Veterinary Medicine Utrecht and other governmental research institutes is stressed. This article also emphasizes the importance of good and responsible product information for veterinarians. The introduction of the veterinary drug law in 1986 has had great consequences for the industry, distributors, and wholesalers as well as for practising veterinarians.

Degradable bisphosphonate-alkaline phosphatase-complexed hydroxyapatite implants in vitro.

Degradable hydroxyapatite (HA) implants complexed with the resorption inhibiting agent bisphosphonate (PCP) and the mineralizing agent alkaline phosphatase (ALP) can theoretically maintain alveolar bone mass directly after extraction of teeth. The present in vitro study investigated the surface properties of PCP-ALP-complexed HA implants in relation to the requirements of implant behavior and action. Adsorbed PCP (pH 3.49) resulted in a flattening and broadening of the phosphate peaks and the formation of carbonate peaks in the HA pattern of the implant indicating a chemical alteration of the HA surface. Adsorption of ALP onto PCP-altered HA surfaces was 26% lower than onto HA implant blank surfaces. PCP-ALP-complexed HA implants released the PCP and ALP steadily and continuously over observation periods of, respectively, 75 and 14 days. During these observation periods, the ceramic grains of the HA implant became smaller and intergrain boundaries became broader. These morphologic characteristics suggested preconditioning of the HA implant surface for future bonding and degradation in vivo. Individual grains were no longer bonded to other grains and detached from the implant which had become rounded in shape. From in vitro mice experiments we found that PCP concentrations between 10(-4) and 10(-3) M resulted in 45Ca-release from the bone HA. Our calculations showed, however, that only a total concentration of 1.4 x 10(-4) M PCP was gradually released over the whole observation period. In another experiment, it appeared that a PCP concentration in solution < 10(-3) M did not reduce ALP activity. It is concluded that release of PCP by the PCP-ALP-complexed implants is maintained at levels in the range to impair osteoclast bone resorption but not high enough to block osteoblast activity. The amount of ALP released can lead to induction of bone formation onto implant surfaces. pH-induced alterations in the microstructure and chemistry of the HA surface allow for controlled degradation of the HA implants in vitro. A PCP-ALP-complexed HA implant acting as temporary scaffolding for alveolar bone growth enhancement, mineralization, and maintenance seems to be a reasonable concept for preservation of the edentulous alveolus.

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.

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.

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.

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.

Effect of bound phosphoproteins and other organic phosphates on alkaline phosphatase-induced mineralization of collagenous matrices in vitro.

The aim of the present study was to determine to what extent the rate at which collagen mineralizes correlates with the amount and nature of bound phosphate groups. Sheets of collagen prepared from demineralized bovine dentin or cortical bone were complexed with various concentrations of phosphoserine [(P)Ser] or rat dentin phosphoproteins (PP; lowly or highly phosphorylated PP, LPP or HPP). Alternatively, phosphate groups were removed from the collagenous carrier material by treatment with phosphatases. Mineralization was achieved by incubation in culture medium supplemented with 45Ca, alkaline phosphatase and 10 mM beta-glycerophosphate. The sheets were monitored for uptake of 45Ca and lag times calculated and plotted against the amount of bound phosphate. It was observed that dephosphorylation of the carrier causes an increase in lag time and that rat PP decreases lag times in a concentration-dependent way. HPP were more effective than LPP. (P)Ser or other small organic P-containing molecules had hardly any influence on lag time. It is concluded that next to the amount of bound phosphate, the nature of phosphorylated substances has considerable influence on the rate of mineralization of a collagenous carrier.

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 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 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.