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  • 1
    Electronic Resource
    Electronic Resource
    Proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63) cultured on previously used titanium surfaces (1996)
    Copenhagen : Munksgaard International Publishers
    Clinical oral implants research 7 (1996), S. 0 
    Details
    ISSN: 1600-0501
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: This study compared osteoblast proliferation, differentiation, and protein synthesis on new and used titanium (Ti) disks to test the hypothesis that cleaning and resterilization of previously used Ti disks does not alter cell response to a particular surface. Ti disks of varying roughness were prepared by one of five different treatment regimens. Standard tissue culture plastic was used as a control. Human osteoblast-like cells (MG63) were cultured on the Ti disks and cell proliferation, cell differentiation, RNA synthesis and matrix production (collagen and noncollagen protein; proteoglycans) measured. After their first use, the disks were cleaned, re-sterilized by autoclaving. and MG63 cells cultured on them as before. At confluence, the same parameters were measured and cell behaviour on new and used disks compared. When confluent cultures of cells on plastic were compared to those cultured on new Ti surfaces, cell number was reduced on the roughest surfaces and equivalent to plastic on the other surfaces. Cell number was further reduced when disks with the roughest surfaces were re-used; no differences in cell number could be discerned after cleaning and re-sterilization. Cell proliferation was inversely related to surface roughness and was less than seen on tissue culture plastic. Re-use of the Ti disks resulted in no change in cell proliferation rate. Alkaline phosphatase specific activity in isolated cells was lowest on the rougher surfaces; no differences between new and used disks were observed. Similarly, enzyme activity in the cell layer was decreased in cultures grown on rougher surfaces, with no effect of prior disk use being noted. RNA synthesis was decreased with respect to plastic in cultures on smoother surfaces and increased on rougher surfaces; prior disk use did not alter RNA synthesis. Collagen production by the cells was decreased on smoother surfaces, but was comparable to tissue culture plastic when grown on rougher surfaces. Non-collagen protein production was unaffected by culture surface and whether or not the disk had been previously used. Proteoglycan synthesis by cells was decreased on all surfaces studied and comparable on both new and used disks. The results of this study indicate that Ti implant surfaces are unaffected by cleaning and resterilization, although rougher surfaces may require more extensive cleaning than smoother ones. This suggests the possibility that implants, in the same patient, could be safely reused. In vivo studies in animals, however, need to be performed before clinical application can be considered.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1034/j.1600-0501.1996.070104.x
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  • 2
    Electronic Resource
    Electronic Resource
    Markers of primary mineralization are correlated with bone-bonding ability of titanium or stainless steel in vivo (1995)
    Copenhagen : Munksgaard International Publishers
    Clinical oral implants research 6 (1995), S. 0 
    Details
    ISSN: 1600-0501
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Critical events in the adaptation of osseous tissues to implant materials involve initial calcification of the newly synthesized bone. Previous studies indicated that bone-bonding but not nonbonding glass ceramics increase the matrix vesicle number, thereby compensating for delayed maturation of the extracellular organelles. The present study assessed whether this was also true for metal implants commonly used in orthopaedics and oral medicine. Bone-bonding titanium (Ti) or nonbonding stainless steel (SS) implants were placed in the right tibias of Sabra rats following ablation of the marrow. At 3, 6, 14, and 21 days postinjury, newly formed endosteal bone in the treated and contralateral limbs was removed and matrix vesicle-enriched membranes isolated. Alkaline phosphatase and phospholipase A2 specific activities and phosphatidylserine (PS) content were determined and compared with those of a nonsurgical control group. Results show that matrix vesicle alkaline phosphatase and phospholipase A2 activity and PS content was increased in the Ti-implanted limbs at 6 (peak). 14, and 21 days, although at levels less than observed in normal ealing. Alkaline phosphatase activity remained elevated throughout the healing period. In contrast, these parameters were markedly inhibited in the SS-implanted limbs with respect to Ti or to normal healing. Both implants altered the systemic response associated with marrow ablation. but in an implant-specific manner. The results support the hypothesis that cells adjacent to bone-bonding materials can compensate for negative effects on primary mineralization during osteogenesis, whereas cells adjacent to nonbonding materials either do not compensate or are further depressed. The data support the use of the rat marrow ablation model as a tool for rapid, initial assessment of biomaterials in bone.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1034/j.1600-0501.1995.060101.x
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  • 3
    Electronic Resource
    Electronic Resource
    Underlying mechanisms at the bone-surface interface during regeneration (1997)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of periodontal research 32 (1997), S. 0 
    Details
    ISSN: 1600-0765
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The goal of regenerative therapy around teeth and implants is to create a suitable environment in which the natural biological potential for functional regeneration of periodontal ligament and/or bone can be maximized. In order for the regenerative process to be successful, the following factors must be addressed: prevention of acute inflammation from bacteria, mechanical stability of the wound, creation and maintenance of blood clot-filled space, isolation of the regenerative space from undesirable competing tissue types, and the creation of a desirable surface chemistry, energy, roughness and microtopography that can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation and, therefore, the regeneration process. This paper will review how surface characteristics (chemistry and roughness) can affect cell response and local factor production. To evaluate the effect of surface chemistry on cell proliferation and differentiation costochondral chondrocytes were grown on standard tissue culture plastic dishes sputter-coated with different materials. The results indicate that surface materials can elicit differential responses in cell metabolism and phenotypic expression in vitro. In a second study, the effect of varying titanium surface roughnesses on osteoblast-like cell behavior was examined. Surface roughness was found to alter osteoblast proliferation, differentiation and matrix production in vitro. In addition, production of PGE2 and TGFβ by these cells was also shown to increase with increasing surface roughness, indicating that substrate surface roughness also affects cytokine and growth factor production. The role of surface roughness in determining cellular response was further explored by comparing the response of osteoblasts grown on new and previously used surfaces. The results of these latter studies showed that cell proliferation, expression of differentiation markers and overall matrix production are not altered when cells are grown on used vs. virgin surfaces. This suggests the possibility that implants may be re-used, especially in the same patient, if they are appropriately treated. In this context, it should also be noted that rougher titanium surfaces may require more extensive cleaning procedures. From a global perspective, these studies provide some insight into how bone regeneration can be optimized in the presence of an implant or tooth root residing at the site of a bony defect. Since the new bone being produced, during regeneration, grows from a distal area toward the implant or tooth root surface, it is hypothesized that the osteoblasts growing on the surface of the implant may produce local factors that can affect the bone healing process distally. In short, it appears that the surface characteristics of an implant, particularly roughness, may direct tissue healing and, therefore, subsequent implant success in sites of regeneration by modulating osteoblast phenotypic expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1600-0765.1997.tb01399.x
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  • 4
    Electronic Resource
    Electronic Resource
    Evidence for the Involvement of Carbonic Anhydrase and Urease in Calcium Carbonate Formation in the Gravity-Sensing Organ of Aplysia californica (1997)
    Springer
    Calcified tissue international 61 (1997), S. 247 -255 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Calcium carbonate —Aplysia californica— Statoconia — Urease — Carbonic anhydrase.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900330
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  • 5
    Electronic Resource
    Electronic Resource
    Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of β-Glycerophosphate and ascorbic acid (1994)
    Springer
    Calcified tissue international 54 (1994), S. 399-408 
    Details
    ISSN: 1432-0827
    Keywords: Matrix vesicles ; Osteoblast-like cells ; Metalloproteinases ; Ascorbic acid ; β-Glycerophosphate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 μg/ml), β-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or β-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00305527
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  • 6
    Electronic Resource
    Electronic Resource
    24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix (1997)
    Springer
    Calcified tissue international 61 (1997), S. 313-321 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Chondrocyte cultures — 24,25-(OH)2D3— Matrix vesicles — Protein kinase C — Phospholipase A2— Monensin — Quinacrine — Nongenomic.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)2D3 [14]. In the present study, we examined whether the effect of 24,25-(OH)2D3 on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10−9–10−7 M 24,25-(OH)2D3. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)2D3-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)2D3-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)2D3-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)2D3 on PKC involved phospholipase A2 (PA2). Quinacrine (a PA2 inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)2D3, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA2 plays a role in the nongenomic regulation of PKC by 24,25-(OH)2D3. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)2D3 modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)2D3 regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900341
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  • 7
    Electronic Resource
    Electronic Resource
    Purification, Amino Acid Sequence, and cDNA Sequence of a Novel Calcium-Precipitating Proteolipid Involved in Calcification of Corynebacterium matruchotii (1998)
    Springer
    Calcified tissue international 62 (1998), S. 350-358 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Microbial calcification — Proteolipid — Dental calculus — Amino acid sequence — cDNA nucleotide sequence.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. It produces membrane-associated proteolipid capable of inducing hydroxyapatite formation in vitro. This proteolipid was purified from chloroform:methanol extracts by chromatography on Sephadex LH-20 and migrated on SDS-polyacrylamide gel electrophoresis at 6-9 kDa. Removal of covalently attached acyl moieties by methanolic KOH decreased its molecular mass to approximately 5.5 kDa. The amino acid sequence of the apoproteolipid indicated a peptide of 50 amino acids, a calculated molecular weight of 5354 Da, and an isoelectric point of 4.28. Sequence analysis revealed an 8 amino acid sequence with homology to human phosphoprotein phosphatase 2A as well as several potential acylation sites and one phosphorylation site. The purified proteolipid induced calcium precipitation in vitro. Deacylation of the proteolipid by hydroxylamine treatment resulted in 〉50% loss of calcium-precipitating activity, suggesting that covalently attached lipids are required. Degenerate oligonucleotide primers, based on the amino acid sequence, were used to amplify the gene for the 5.5 kDa proteolipid from total chromosomal DNA of C. matruchotii by PCR. A 166 bp cDNA was isolated and sequenced, confirming the amino acid sequence of the proteolipid. Thus, we have sequenced a unique bacterial proteolipid that is involved in the formation of dental calculus by precipitating Ca2+ and possibly in transport of inorganic phosphate, necessary for hydroxyapatite formation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900443
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  • 8
    Electronic Resource
    Electronic Resource
    TGFβ1 Regulates 25-Hydroxyvitamin D3 1α- and 24-Hydroxylase Activity in Cultured Growth Plate Chondrocytes in a Maturation-Dependent Manner (1999)
    Springer
    Calcified tissue international 64 (1999), S. 50-56 
    Details
    ISSN: 1432-0827
    Keywords: Key words: TGFβ1 — Chondrocytes — 1α-Hydroxylase — 24-Hydroxylase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Chondrocytes metabolize 25-(OH)D3 to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)2D3 and 24,25-(OH)2D3. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1α-hydroxylase and 24R-hydroxylase, and their regulation by TGFβ1. Basal 1α- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1α- and 24R-hydroxylase activities determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFβ1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 released into the conditioned media by resting zone chondrocyte cultures in response to TGFβ1 were measured. In culture, all three cell types were found to contain 1α- and 24R-hydroxylase activities. Basal 1α-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFβ1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFβ1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1α-hydroxylase activity by 20–30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [3H]-24,25-(OH)2D3 by resting zone cells increased with TGFβ1 treatment, while [3H]-1,25-(OH)2D3 production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1,25-(OH)2D3 and 24,25-(OH)2D3 from 25-(OH)D3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFβ1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900578
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  • 9
    Electronic Resource
    Electronic Resource
    Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica (1995)
    Springer
    Journal of comparative physiology 177 (1995), S. 415-425 
    Details
    ISSN: 1432-1351
    Keywords: Aplysia ; Carbonic anhydrase ; Statoconia ; Organ culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17°C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 × 10−4 M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00187478
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  • 10
    Electronic Resource
    Electronic Resource
    Ascorbic acid stimulates the resorption of canine articular cartilage induced by a factor derived from activated rabbit macrophages (1985)
    Springer
    Rheumatology international 5 (1985), S. 103-107 
    Details
    ISSN: 1437-160X
    Keywords: Macrophage-derived factor ; Cartilage resorption ; Ascorbic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Articular cartilage explants from the knees of mongrel dogs release 5–10% of their proteoglycan content spontaneously when cultured for 4 days in serum-free modified Bigger's medium. A factor synthesized and secreted by lipopolysaccharide-stimulated rabbit macrophages can stimulate this release of proteoglycan by 2 to 3-fold. The release of proteoglycan in response to macrophage factor is maximal in the presence of 1.5–50 μg/ml l-ascorbic acid. In the absence of ascorbate, or with high levels of ascorbate (150 μg/ml), the effect of the factor is diminished by 50%. d-isoascorbate, reduced glutathione, or dithiothreitol cannot substitute for l-ascorbate in producing this effect, while dehydroascorbate can.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00541328
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