RESUMEN

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Asunto(s)

Biopelículas/efectos de los fármacos , Fosfatos de Calcio/antagonistas & inhibidores , Metacrilatos/química , Polietilenglicoles/química , Biopelículas/crecimiento & desarrollo , Fosfatos de Calcio/metabolismo , Esmalte Dental/efectos de los fármacos , Esmalte Dental/metabolismo , Humanos , Metacrilatos/farmacología , Polietilenglicoles/farmacología , Distribución Aleatoria , Saliva/efectos de los fármacos , Saliva/metabolismo , Streptococcus gordonii/efectos de los fármacos , Streptococcus gordonii/fisiología , Difracción de Rayos X

RESUMEN

Despite the significant advances in cardiac surgery, heart valve replacement still faces a dilemma. While mechanical valves offer lifelong durability they also commit patients to anticoagulation treatment for the rest of their life. On the other hand, bioprosthetic valves have superior hemodynamic performance but durability of the bioprosthesis limits their use to the elderly, with early onset calcification being the primary cause of biomaterial breakdown. Considering that bioprosthetic valves are not reliant upon anticoagulation, there has been much focus on measures to overcome their issues with durability. Firstly, the calcification process has been studied and factors such as young patient age, use of glutaraldehyde fixative, the presence of phospholipids along with cell debris in the valve tissue and mechanical stress have been identified to influence tissue mineralization. Therefore different calcification reduction strategies are being sought: new fixatives have been developed and tested and post-treatments have been added to tissue processing. This review presents the pathophysiology of tissue valve calcification and focuses on the multiple approaches developed to prevent bioprosthetic heart valve calcification, as well as on their general outcomes and translation to clinical applications.

Asunto(s)

Bioprótesis , Calcinosis/prevención & control , Válvulas Cardíacas/cirugía , Animales , Calcinosis/cirugía , Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/metabolismo , Humanos , Solventes/química , Tensoactivos/química

RESUMEN

BACKGROUND AND OBJECTIVE: Dental calculus occurs as a consequence of supersaturation of saliva with respect to calcium phosphates. This mineralization of dental plaque can be delayed by the presence of crystallization inhibitors, such as pyrophosphate or bisphosphonates. Phytate inhibits brushite and hydroxyapatite crystallization and has the potential to prevent dental calculi formation. The aim of the present study was to examine the effects of phytate and zinc, administered in a mouthwash solution, to prevent the formation of dental calculus. MATERIAL AND METHODS: Healthy dental plaque-forming volunteers (n = 25) took part in a randomized, double-blind, three-period crossover clinical study to assess the efficacy of a phytate-containing mouthwash in relation to control and placebo effects. Subjects rinsed their mouths for 1 min, twice each day, with 20 mL of the test solution, without ingestion. Mouthwash efficacy was assessed through quantification of the amounts of calcium, phosphorus and magnesium present in the residues obtained by dental cleaning, performed by a single trained examiner. RESULTS: A good correlation was found among total calcium, magnesium and phosphorus in calcified dental plaque residues, indicating that any of these variables is adequate for evaluating the reduction of plaque crystallization as calcium phosphate. A statistically significant decrease in total calcium, magnesium and phosphorus was found in the phytate-treatment period compared with control and placebo periods, demonstrating the efficacy of the proposed treatment in reducing dental calculus formation. CONCLUSION: The high efficacy of phytate in reducing dental calculus formation suggests that this substance may be an effective treatment for preventing the development of calculus deposits.

Asunto(s)

Antiinfecciosos Locales/uso terapéutico , Cálculos Dentales/prevención & control , Antisépticos Bucales/uso terapéutico , Ácido Fítico/uso terapéutico , Triclosán/uso terapéutico , Adolescente , Adulto , Anciano , Calcio/análisis , Fosfatos de Calcio/antagonistas & inhibidores , Estudios Cruzados , Cristalización , Placa Dental/química , Método Doble Ciego , Durapatita/antagonistas & inhibidores , Femenino , Humanos , Magnesio/análisis , Masculino , Persona de Mediana Edad , Fósforo/análisis , Placebos , Adulto Joven , Zinc/uso terapéutico

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/química , Osteopontina/farmacología , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Precipitación Química , Cromatografía Líquida de Alta Presión , Femenino , Leche , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo

RESUMEN

Calcification is the most frequent cause of the clinical failure of bovine pericardium bioprosthetic valves, preventing their widespread application for surgical treatment. The aim of this study was to minimize calcific degeneration in bovine pericardium by using a chelating agent, ethylenediaminetetraacetic acid (EDTA). Freshly excised bovine pericardium was dissected free from adhering fat tissue and cut into 1-cm(2) pieces that were rinsed in phosphate-buffered saline solution (PBS) and transferred into 4 degrees C PBS containing 1% glutaraldehyde (GA) for initial fixation, then allocated into two groups. Group I received the same treatment in a fresh solution for 5 more days. Group II underwent an additional fixation step in PBS solution (pH 7.4, 37 degrees C) containing 11% EDTA for a period of 48 h (30 ml/g tissue) and was then transferred into freshly prepared PBS + 1% GA solution at 37 degrees C for another 3 days. To investigate the calcification rate, pericardial patches were inserted into the dorsal pouches of 25 male Wistar rats for 21 days. Calcium levels were measured with an atomic absorption spectrophotometer and examined histo-pathologically. The calcium content of EDTA-treated pericardium (Group II), 21 +/- 3.8 microg/mg, was significantly lower than that of Group I, 43.3 +/- 9.2 microg/mg. Assessment of the degree of calcification in the histological sections generally agreed well with the results of the chemical analyses. Calcium deposition in Group I samples were found to be solid mineral depositions, whereas in the Group II pericardial samples, only smaller traces of calcium were found. Calcific degeneration in bovine pericardium can be reduced by using chelates such as EDTA.

Asunto(s)

Calcinosis/prevención & control , Quelantes/farmacología , Ácido Edético/farmacología , Pericardio/efectos de los fármacos , Animales , Bioprótesis , Fosfatos de Calcio/antagonistas & inhibidores , Bovinos , Masculino , Ratas , Ratas Wistar

RESUMEN

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9-24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , alfa-Fetoproteínas/fisiología , Animales , Calcio/metabolismo , Fosfatos de Calcio/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ácido Etidrónico/farmacología , Concentración de Iones de Hidrógeno , Cinética , Masculino , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Factor Plaquetario 4/metabolismo , Unión Proteica , Radioinmunoensayo , Ratas , Temperatura , Factores de Tiempo , alfa-Fetoproteínas/metabolismo

RESUMEN

The present studies show that the previously reported ability of fetuin to inhibit the precipitation of hydroxyapatite from supersaturated solutions of calcium and phosphate in vitro is accompanied by the formation of the fetuin-mineral complex, a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein that was initially discovered in the serum of rats treated with etidronate and that appears to play a critical role in inhibiting calcification in vivo. Rat serum potently inhibited the precipitation of calcium phosphate mineral when the concentration of calcium and phosphate were increased by 10 mm each, and the modified serum was incubated at 37 degrees C for 9 days; in the absence of serum, precipitation occurred in seconds. Large amounts of the fetuin-mineral complex were generated in the first 3 h of this incubation and remained throughout the 9-day incubation. Purified bovine fetuin inhibited the precipitation of mineral for over 14 days in a solution containing 5 mM calcium and phosphate at pH 7.4 at 22 degrees C, whereas precipitation occurred in minutes without fetuin. There was a biphasic drop in ionic calcium in the fetuin solution, however, from 5 to 3 mM in the first hour and from 3 to 0.9 mM between 20 and 24 h; these changes in ionic calcium are due to the formation of complexes of calcium, phosphate, and fetuin. The complex found at 24 h to 14 days is identical to the fetuin-mineral complex found in the serum of etidronate-treated rats, whereas the complex found between 1 and 20 h is less stable.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , alfa-Fetoproteínas/fisiología , Animales , Calcio/metabolismo , Fosfatos de Calcio/metabolismo , Bovinos , Centrifugación , Cromatografía en Gel , Ácido Etidrónico/farmacología , Concentración de Iones de Hidrógeno , Masculino , Fosfatos/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Temperatura , Factores de Tiempo , alfa-Fetoproteínas/metabolismo

RESUMEN

This study quantitatively compares the inhibition of calcium phosphate (CaP) precipitation by the salivary acidic proline-rich proteins (PRPs) statherin and histatin-1. Saliva and CaCl2 in 125 mM imidazole buffer (pH 7.0) were incubated with potassium phosphate and a hydroxyapatite (HAP) suspension, for 30 min at 25 degrees C, then filtered through nitrocellulose. The calcium (Ca) concentration in the filtrate was measured by atomic absorption spectrophotometry, then deducted from that in the initial solution to determine the amount of CaP precipitation after 30 min. The values of the inhibitory activities on CaP precipitation relative to crude parotid saliva were 4.7, 4.9, 6.9, and 65.8 for histatin-1, large PRPs, small PRPs, and statherin, respectively.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Fosfopéptidos/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Adulto , Femenino , Histatinas , Humanos , Concentración de Iones de Hidrógeno , Cinética , Masculino , Glándula Parótida/metabolismo , Fosfopéptidos/aislamiento & purificación , Saliva/fisiología , Proteínas y Péptidos Salivales/aislamiento & purificación

RESUMEN

OBJECTIVE: To examine the effect of basic calcium phosphate (BCP) crystals on expression of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in human fibroblasts. METHOD: Using a semi-quantitative reverse transcription-polymerase chain reaction method and phosphocitrate (PC), a specific inhibitor of the biological effects of BCP crystals, we examined the effects of BCP on the steady state transcript levels of metalloproteinase (MMP)-1, -3, -9 and -13 and TIMP-1 and -2 in human fibroblasts. DNA primers against elongation factor were used as internal controls. RNAs isolated from human fibroblasts treated with BCP crystals (50 microg/ml) in the presence or absence of PC (10(-3) M) were used as templates, and RNA from untreated control cultures and cultures treated with Interleukin-1-beta (IL-1beta) were used as negative and positive controls, respectively. RESULTS: We observed increases in MMP-1, -3, -9 and -13 transcripts by BCP crystals. BCP crystal down-regulated TIMP-1 and -2 over untreated controls. Western blot analysis confirmed that BCP crystals down-regulate the synthesis of TIMP-1 and -2. While IL-1beta up-regulated MMP-1, -3, -9 and -13, it had no significant effect on expression of either TIMP. In all cases, PC specifically reversed the differential regulation of MMPs and TIMPs by BCP crystals but had no effect on IL-1beta induction of MMP expression. CONCLUSION: The ability of BCP to induce the synthesis of degradative MMPs while down-regulating the synthesis of the naturally occurring counterpart TIMPs may explain the changes consistent with a role of BCP crystal in the pathogenesis of degenerative changes in osteoarthritis. The ability of PC to reverse both degradative effects of BCP crystal suggests that PC can be a potential therapeutic agent for BCP crystal deposition diseases.

Asunto(s)

Fosfatos de Calcio/farmacología , Fibroblastos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Western Blotting/métodos , Fosfatos de Calcio/antagonistas & inhibidores , Células Cultivadas , Citratos/farmacología , Colagenasas/genética , Colagenasas/metabolismo , Humanos , Interleucina-1/farmacología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Modelos Biológicos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética

RESUMEN

Several individual species of dental plaque bacteria have the ability to initiate the precipitation of calcium phosphate minerals in vitro; other plaque components have been shown to inhibit mineralisation. We have examined subjects' overall plaque mineralisation promoter and inhibitor properties, and have attempted to correlate them with supragingival calculus development over 6 months. Three-day-old plaque was collected from 22 adult subjects at the start and end of the study. To detect promoter activity, the plaque was placed in a suspension of brushite, the liquid phase of which was supersaturated with respect to hydroxyapatite. The extent of mineralisation was determined by the rise in phosphate concentration over 4 days. To detect inhibitor activity, plaque was placed in a similar suspension, which also contained hydroxyapatite. Promoter activity was compared with that hydroxyapatite, and inhibitor activity was compared with polyaspartate. The subjects' teeth were scaled at the start of the study, and calculus deposition was measured at the end using the Volpe Manhold method. Most plaque samples showed some promoter or inhibitor activity, or both, but no significant correlation existed between these activities and a subject's development of calculus. A significant inverse correlation existed between plaque mineralisation promoter activity and its inhibitor activity at the start of the study. Our results suggest that the nucleating and mineralisation inhibitory properties of young plaque will probably not be a useful target for a practical preventive methodology for supragingival calculus.

Asunto(s)

Fosfatos de Calcio/metabolismo , Cálculos Dentales/metabolismo , Placa Dental/metabolismo , Adulto , Anciano , Fosfatos de Calcio/agonistas , Fosfatos de Calcio/antagonistas & inhibidores , Cristalización , Cálculos Dentales/química , Cálculos Dentales/etiología , Placa Dental/química , Placa Dental/complicaciones , Depresión Química , Femenino , Humanos , Hidroxiapatitas/metabolismo , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Estimulación Química

RESUMEN

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP: MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin: ACP: MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP: MMP-9 and fibrin: ACP: MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5-2.5 microM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 microM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 microM) by higher phosphate (500 microM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 microM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP: MMP-9 complexes (50-100 microM). Neither POP nor PCP had any effect on preformed fibrin: ACP: MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin: ACP: MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/farmacología , Difosfatos/farmacología , Difosfonatos/farmacología , Fibrina/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Unión Competitiva/efectos de los fármacos , Humanos , Sustancias Macromoleculares , Inhibidores de la Metaloproteinasa de la Matriz , Unión Proteica/efectos de los fármacos

RESUMEN

Supersaturation with respect to calcium salts (oxalate and phosphate) is the driving force leading to crystalluria and nephrolithiasis. High-molecular-weight urinary inhibitors are recently described molecules capable of altering the process of kidney stone formation. By inhibition of crystal nucleation, growth and aggregation and by inhibition of crystal interaction with tubular cells, these proteins efficiently prevent stone formation and retention in the urinary tract. But in spite of considerable efforts, characterization of these proteins is still under way. Besides the pathophysiology of risk factors for calcium salts supersaturation such as idiopathic hypercalciuria or hyperoxaluria, the renal involvement of protein inhibitors is the most exciting field in the comprehensive approach of nephrolithiasis, a disease that affects up to 10% of people in Western countries.

Asunto(s)

Oxalato de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/antagonistas & inhibidores , Cálculos Renales/prevención & control , Animales , Oxalato de Calcio/metabolismo , Fosfatos de Calcio/metabolismo , Cristalización , Humanos , Nefrocalcinosis/prevención & control , Proteínas/fisiología

RESUMEN

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

Asunto(s)

Fosfatos de Calcio/farmacología , Pirofosfato de Calcio/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citratos/farmacología , Proteínas Quinasas Activadas por Mitógenos , Transducción de Señal , Fosfatos de Calcio/antagonistas & inhibidores , Pirofosfato de Calcio/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , División Celular , Células Cultivadas , Cristalización , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Interleucina-1/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proto-Oncogenes Mas , Transducción de Señal/efectos de los fármacos , Ácidos Tricarboxílicos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Inhibitory effects of a rhubarb (Rhei rhizoma) solution and its fractions on the formation of calcium phosphate precipitates were studied. The rhubarb solution inhibited both the amorphous calcium phosphate (ACP) formation and the rate of hydroxyapatite (HAP) transformation, and extended the induction time. When the solution was fractionated using membrane filters, a filtrate with the molecular weight between 3 and 10 kDa (with 2/3 recovery of polyphenols) was found to be responsible for both the ACP formation and the extension of the induction time. Another filtrate with the molecular weight below 3 kDa (with 1/3 recovery of polyphenols) may be responsible for the inhibition of both the ACP formation and the rate of HAP transformation, and the extension of the induction time. When the extract of rhubarb was fractionated using a Sephadex LH-20 column chromatography, fraction IV greatly inhibited the formation of calcium phosphate precipitates, while fractions I, II and III slightly inhibited that reaction. Our finding suggests that fraction IV may contain useful substance(s) for the prevention of oral calcium phosphate precipitation (calculus formation). However, strong calcium chelating properties would limit the concentration that could be safely employed.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Extractos Vegetales/química , Plantas Medicinales , Rheum/química , Análisis de Varianza , Calcio/química , Fosfatos de Calcio/metabolismo , Quelantes/química , Fraccionamiento Químico , Precipitación Química , Cálculos Dentales/prevención & control , Durapatita/metabolismo , Estadísticas no Paramétricas

RESUMEN

Non-collagenous proteins of dentin and bone have important effects on mineralization which have been studied by various in vitro systems. We developed an in vitro mineralization system using electrophoretic gels as diffusion media of calcium and phosphate ions. Calcium and phosphate ions were diffused naturally or propelled by electric potential. Calcium phosphate was precipitated in the gel, and the precipitation was affected by proteins in the gel which had been separated by electrophoresis. We applied this system to analysis of non-collagenous proteins of dentin. Among the proteins, phosphophoryns promoted calcium phosphate precipitation in the natural-diffusion system. A non-collagenous protein having a molecular mass of 60 kDa inhibited precipitation. The results were different, however, in the electric-diffusion system, in which phosphophoryns had a negative effect. The present system enabled us to compare the effects of plural proteins rapidly, even using unpurified material.

Asunto(s)

Dentina/metabolismo , Fosfoproteínas/metabolismo , Calcificación de Dientes , Animales , Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/química , Fosfatos de Calcio/metabolismo , Bovinos , Precipitación Química , Dentina/química , Difusión , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular/metabolismo , Geles , Fosforilación , Ratas

RESUMEN

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE1 on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE1 inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE1 (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE1-induced increase in intracellular cAMP by at least 60%. The PGE1-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2',5'-dideoxyadenosine (ddA) (10 microM) and ddA reversed the PGE1-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyryl cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase, inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE1 on HF collagenase mRNA levels. PGE1 inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Colagenasas/biosíntesis , AMP Cíclico/metabolismo , Fibroblastos/efectos de los fármacos , Prostaglandinas E/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Alprostadil/farmacología , Bucladesina/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Colagenasas/genética , Cristalización , Didesoxiadenosina/farmacología , Dinoprostona/farmacología , Inducción Enzimática , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathy. BCP crystals induce the secretion of matrix-degrading enzymes such as collagenase. No prophylactic or therapeutic agents are recognized to ameliorate the cartilage damage associated with BCP deposits in joints. As a chondroprotective effect of prostaglandins (PG) has been suggested, we studied the effect of misoprostol, a PGE1 analogue, on BCP crystal-induced mitogenesis and collagenase messenger RNA (mRNA) accumulation in human fibroblasts (HF). Mitogenesis was determined by 3H-thymidine incorporation assays and collagenase mRNA accumulation by Northern blot analysis, in HF stimulated with BCP crystals in the presence or absence of misoprostol. Misoprostol caused concentration-dependent inhibition of BCP crystal-induced mitogenesis. The inhibition of BCP-stimulated mitogenesis was not specific as misoprostol also inhibited the mitogenic response to 10% serum. There was only 50 (+/-5)% inhibition of serum-induced mitogenesis by misoprostol at 500 ng/ml, the concentration that completely inhibited BCP crystal-induced mitogenesis. Misoprostol also inhibited the accumulation of collagenase mRNA in BCP-stimulated HF by 63%. These data suggest that misoprostol may inhibit the synovial proliferation and cartilage degradation that accompany BCP crystal deposition.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Colagenasas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Misoprostol/farmacología , Mitógenos/antagonistas & inhibidores , Northern Blotting , Células Cultivadas , Colagenasas/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , ARN Mensajero/efectos de los fármacos , Líquido Sinovial/metabolismo

RESUMEN

The effects of indapamide (a nonthiazide antihypertensive diuretic) on the growth promoting activity of serum, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) or Ca3(PO4)2 on Balb/C 3T3 cells were studied. Indapamide inhibited the growth promoting activity of serum, but furosemide (a nonthiazide antihypertensive diuretic) had no such inhibitory effect. Indapamide inhibited the growth promoting activity of PDGF, but not that of FGF and Ca3(PO4)2. The present experiments demonstrate that indapamide selectively inhibits the growth promoting activity of PDGF.

Asunto(s)

Indapamida/farmacología , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Células 3T3 , Animales , Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/farmacología , Ratones , Ratones Endogámicos BALB C , Factor de Crecimiento Derivado de Plaquetas/farmacología

RESUMEN

Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Proteínas y Péptidos Salivales/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/aislamiento & purificación , Cromatografía por Intercambio Iónico , Cristalografía , Electroforesis en Gel de Poliacrilamida , Hexosaminas/análisis , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/aislamiento & purificación , Fosfatos/análisis , Fosfoproteínas/análisis , Fosfoproteínas/aislamiento & purificación , Prolina/análisis , Prolina/aislamiento & purificación , Dominios Proteicos Ricos en Prolina , Conejos , Colorantes de Rosanilina , Proteínas y Péptidos Salivales/análisis , Proteínas y Péptidos Salivales/farmacología , Dodecil Sulfato de Sodio

RESUMEN

So that a possible involvement in the mineralization of dental plaque could be investigated, the effects of silicic acid on calcium phosphate precipitation were assessed in vitro. By measuring the decrease in Ca2+ concentration (by means of ion-selective electrodes), we determined both spontaneous precipitation and seeded crystal growth from solutions that contained 1 mmol/L calcium, 7.5 mmol/L phosphate, 50 mmol/L Hepes pH 7.2, and various amounts of silicic acid. Polymerized silicic acid, but not its monomer, was found both to cause a 60% reduction in the lag period that precedes spontaneous precipitation and to enhance the growth rate of seeded hydroxyapatite crystals. Silica suspensions showed effects similar to those of polysilicic acid. In all cases, the precipitated material was found to be hydroxyapatite. Whereas seeded brushite crystals grew slowly without silicic acid, hydroxyapatite was the only mineral detected after crystal growth in the presence of silicic acid. Apparently, polysilicic acid acted as a substrate for hydroxyapatite nucleation, inducing secondary nuclei on both hydroxyapatite and brushite crystals. The finding that polysilicic acid could overcome part of the inhibitory effect of a phosphoprotein on calcium phosphate precipitation gave additional support for the idea that polysilicic acid and silica may promote the formation of dental calculus.

Asunto(s)

Fosfatos de Calcio/antagonistas & inhibidores , Ácido Silícico/farmacología , Dióxido de Silicio/farmacología , Catálisis , Precipitación Química , Cristalización , Durapatita , Hidroxiapatitas , Fosvitina/farmacología