Calcium carbonate concretions: cyclic occurrence in the hamster vagina.

Three crystalline forms of calcium carbonate were identified in washings of the hamster vagina. Spherical concretions of vaterite and hexagonal concretions of calcite predominate on days 3 and 4 of the 4-day estrous cycle. Dumbbell-like concretions of aragonite predominate during pregnancy and pseudopregnancy. Each polymorph is associated with an acid-insoluble matrix. Concretions disappear after ovariectomy and reappear during daily injections of estrogen and progesterone.

Creation of "amyloid" fibrils from Bence Jones proteins in vitro.

"Amyloid" fibrils have been created from some human Bence Jones proteins by proteolytic digestion under physiologic conditions. These fibrils with an antiparallel, beta-pleated sheet conformation consist of only a portion of the variable region of the immunoglobulin light polypeptide chain and share the physical properties of amyloid fibrils. The relation between amyloidosis and immunoglobulins is thus more firmly established and a pathogenetic mechanism for amyloid fibril formation is suggested.

Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain.

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.

Collagen crosslinks and mineral crystallinity in bone of patients with osteogenesis imperfecta.

In cortical bone samples from patients with osteogenesis imperfecta (OI), the concentrations of hydroxypyridinium cross-linking amino acids in collagen were measured by reversed-phase HPLC and the x-axis crystallinity of the apatite mineral phase was determined by x-ray diffraction. Bone samples from three patients with type I, nine patients with type III, and eight patients with type IV OI were analyzed and compared with human bone from nine controls. The concentration of the two chemical forms of the mature collagen crosslinking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and the ratio HP/LP were found to be alike in bone collagen of OI patients and healthy controls. However, the c-axis crystallinity of the apatite was found to be reduced in the type III and IV OI patients compared with controls. Regression analysis of crosslink concentrations and c-axis crystallinity in OI bones did not show any correlation. Therefore, collagen molecules deposited in the extracellular matrix of OI bone appear to fulfill the structural requirements for the action of the enzyme lysyl oxidase, such that a normal concentration of intermolecular crosslinks is formed compared with healthy bone. Consequently, crosslink formation and apatite crystal growth seem to be regulated independently in OI bone.

Dental composites based on hybrid and surface-modified amorphous calcium phosphates.

The objectives of this study were to prepare hybrid and surface-modified amorphous calcium phosphates (ACPs) as fillers for mineral-releasing dental composites, and determine whether the mechanical strength of the composites could be improved without decreasing their remineralization potential. ACP was hybridized with tetraethoxysilane or zirconyl chloride and surface-treated with 3-methacryloxypropoxytrimethoxy silane (MPTMS) or zirconyl dimethacrylate (ZrDMA). Composites fabricated with unmodified ACP (u-ACP), hybrid or surface-modified ACP filler and photo-activated Bis-GMA, TEGDMA and 2-hydroxyethyl methacrylate (HEMA) (BTH resin), Bis-GMA, TEGDMA, HEMA and MPTMS (BTHS resin) or Bis-GMA, TEGDMA, HEMA and ZrDMA (BTHZ resin) were tested for their remineralizing potential and biaxial flexure strength (BFS). Ion releases from all composites were significantly above the minimum necessary for reprecipitation of apatite. The BFS of unfilled polymers was not adversely affected by immersion in saline solutions. The BFS of BTH and BTHS composites deteriorated upon soaking. However, BTHZ composites were practically unaffected by exposure to saline solutions. Filler hybridization resulted in a modest, but significant, improvement in the BFS (up to 24%) of BTHZ composites. Heterogeneous distribution of the ACP on disk surfaces was detected by the FTIR microspectroscopy analyses. This might have been caused by uncontrolled aggregation of ACP particles that appeared to hinder interfacial filler/resin interactions and diminish the mechanical strength of composites.

Bladder calcifications after photodynamic therapy: analysis of a rare complication.

OBJECTIVES: We analyzed bladder calcifications occurring after photodynamic therapy administered for the treatment of superficial bladder cancer, a finding not previously reported after this treatment. METHODS: Bladder biopsies from 20 patients undergoing photodynamic therapy were evaluated. Bladder calcifications were identified in 2 patients and analyzed for composition. RESULTS: One patient had diffuse microcrystalline deposition in two biopsies composed of calcium oxalate monohydrate A. A second patient had a focal stone at a healing biopsy site composed of monoclinic calcium hydrogen phosphate dihydrate (brushite) (66%), calcium oxalate (25%), hydroxyapatite (6%), and protein (3%). CONCLUSIONS: Rare calcium oxalate and brushite calcifications were identified after photodynamic therapy and presumed to occur because of tissue injury associated with treatment.

The influence of fluoride on the seeded growth of apatite from stable supersaturated solutions at pH 7.4.

Previous studies have shown that fluoride affected the formation of apatite in spontaneous precipitations from unstable calcium phosphate solutions partly through its effect on the stability of such nonapatitic precursors as octacalcium phosphate, Ca8H2(PO4)6.5 H2O. The present study shows that fluoride had a comparable effect on the seeded growth of apatite from stable solutions when the degree of solution supersaturation favored the transient development of octacalcium phosphate precursors on the seed surface.

Enamel apatite: chemistry, structure and properties.

The crystal chemistry of enamel apatite is probably the best understood of all the biological apatites. It is an imperfect apatite, low in calcium and hydroxide ions, but rich in substitutional impurities. A few impurities, like fluoride, are beneficial but most appear to disrupt the lattice structure. In this paper, the composition and basic structural features of enamel apatite are described, with particular emphasis on what we presently know, and do not know, about the role impurities play in establishing the fine details of this structure.

The hydrolysis of anhydrous dicalcium phosphate into hydroxyapatite.

Hydrolytic conversion of dicalcium phosphate anhydrous (DCPA; CaHPO4) to non-stoichiometric hydroxyapatite (HAP; Ca10-x(HPO4)x(PO4)6-x(OH)2-x) was investigated under controlled-solution conditions for identification of solution factors which influence the nucleation, growth, and crystal morphology of the apatitic product phase. The hydrolysis experiments (1 mmol to 7.5 mmol CaHPO4/250 mL) were carried out under CO2-free N2 at fixed pH in the presence of 25-100 mmol/L CaCl2 by standard pH-stat techniques at 37 degrees C. The pH values of the experimental solutions ranged from 6.35 to 9.0. The crystal size and shape of the HAP product were established by x-ray diffraction, and by scanning and transmission electron microscopy. Generally, hydrolysis times and the size of the HAP crystals attained upon completion of the conversion were inversely related to pH. However, crystal size decreased sharply below pH 6.5. Crystal morphology was also strongly affected by pH, with the most equi-dimensionally-shaped crystals developing at pH 7.0. Although the HAP first appeared on the surface of the DCPA, the amount of DCPA used did not affect the final HAP crystal size or the time required for the hydrolysis to be completed. These data suggest that DCPA initiated HAP formation but that nucleation density (i.e., number of nuclei/unit area DCPA surface) and subsequent growth events were controlled by solution factors.

The influence of fluoride on apatite formation from unstable supersaturated solutions at pH 7.4.

The present study was undertaken to examine the effect of fluoride on the formation of apatite in aqeous calcium phosphate suspensions prepared by spontaneous precipitation at pH 7.4. The most notable finding was that fluoride greatly curtailed or eliminated the appearance of octacalcium phosphate-like precursor phases in these preparations.

The effect of supersaturation on apatite crystal formation in aqueous solutions at physiologic pH and temperature.

The importance of supersaturation in the dynamics of apatite precipitation from aqueous solutions is well-established. To determine whether this parameter has a comparable impact on the concomitant development of the textural properties of this phase, such as crystal size and shape, we investigated mineral accretion in synthetic solutions seeded with 0.67 g/L apatite over a range of supersaturations at pH 7.4 and 37 degrees C. A dual specific-ion electrode-controlled titration method was used to maintain the seeded reactions under the following solution conditions: 1.0 to 1.8 mmol/L Ca2+, 0.67 to 1.2 mmol/L total phosphate (PO4), Ca/PO4 (initial) = 1.5, 143 mmol/L KNO3, and 10 mmol/L HEPES. Samples were collected for chemical and textural analyses when the seed apatite was reduced by new accretions to 1/2, 1/4, 1/8, 1/16, and 1/32 of the total solids in suspension. All new accretions were found to be apatitic. At the lowest supersaturation, accretion occurred primarily by growth of the seed crystals. However, at the highest supersaturation examined, the crystals at the end of the experiments were actually smaller, on average, than the original seeds, even though the total mass increased 32-fold. The results suggest that proliferation of new crystals supplanted growth of the seed crystals as supersaturation was increased. The results also suggest that differences in tissue fluid supersaturation may contribute to the large disparity in dimensions between dentin and enamel apatite crystals.

Inhibition of seeded growth of enamel apatite crystals by amelogenin and enamelin proteins in vitro.

The effect of enamel matrix proteins on the seeded growth of enamel apatite crystals was studied in stable supersaturated solutions at pH 7.4 and 37 degrees C. Of the two major protein classes in the enamel matrix, the enamelins were considerably more effective than the amelogenins in retarding seeded growth. However, the amelogenin species that did show significant inhibitory activity are those known to be lost first from the enamel matrix during the rapid mineralization stage of enamel maturation.

Setting reactions and compressive strengths of calcium phosphate cements.

Setting reactions and compressive strengths of a self-hardening calcium phosphate cement (CPC) were investigated. The CPC consists of tetracalcium phosphate (TTCP) and anhydrous dicalcium phosphate (DCPA). The cement specimens were prepared by mixing 0.7 g of the powder (TTCP 72.9 wt% + DCPA 27.1 wt%) with 0.175 mL of the liquid (25 mmol/L H3PO4 and 1.32 mmol/L sodium fluoride). The specimens were removed from the molds at pre-determined time intervals after being mixed, and their compressive strengths were measured. Immediately afterward, the fractured specimens were rapidly frozen in ethanol (-80 degrees C), lyophilized, and examined by powder x-ray diffraction and scanning electron microscopy (SEM). The results showed that (1) hydroxyapatite was the only reaction product; (2) the reaction was nearly completed within four h, during which both the reaction product and compressive strength increased linearly with time, resulting in a strong correlation between the two; and (3) fully set CPC consisted primarily of small rod-like crystals and some platy crystals.

Quantitative assessment of the efficacy of amorphous calcium phosphate/methacrylate composites in remineralizing caries-like lesions artificially produced in bovine enamel.

Recent studies show that methacrylate-based composites with amorphous calcium phosphate (ACP) as a filler can release supersaturating levels of calcium and phosphate ions in proportions favorable for apatite formation. These findings suggest that such composites could be effectively used as coatings for remineralizing teeth damaged by tooth decay. To examine this hypothesis, we tested composites in vitro for their efficacy to remineralize artificially formed caries-like lesions in extracted bovine incisors. Single 120-microns-thick sagittal tooth sections were placed in holders that exposed only the carious enamel surface. The exposed surfaces were coated with a 1-mm- to 1.5-mm-thick layer of the composite containing, by mass, 40% apatite, silica, or P2O7(-4)-stabilized ACP and 60% photoactivated resin comprised of Bis-GMA, TEGDMA, HEMA, and ZrM. The photocured composite-coated sections were immersed either in a remineralizing solution for 4 weeks at 37 degrees C (static model) or cyclically immersed in demineralizing (0.5 h) and remineralizing solutions (11.5 h) for 2 weeks (dynamic model). Quantitative digital image analysis of matched 102 microns x 220 microns areas from contact microradiographs taken of the sections before and after immersion showed that lesions coated with ACP-filled composites fractionally recovered 71% +/- 33% of their lost mineral compared with 14% +/- 13% for apatite controls in the static model and 38% +/- 16% compared with -6% +/- 24% in the dynamic model. The results suggest that sealants based on ACP-filled methacrylate composites have the potential to remineralize carious enamel lesions.

Simultaneous caries induction and calculus formation in rats.

Weanling specific pathogen-free Osborne-Mendel rats were fed a high-calcium, high-phosphorus diet with various levels of sucrose and inoculated with Streptococcus sobrinus strain 6715-13WT and Actinomyces viscosus strain OMZ-105 in order to determine whether calculus and caries could develop simultaneously. Rats consumed diets designated RC-16-5, RC-16-25, or RC-16-50 which partially replaced the corn starch component with progressively higher levels of sucrose, thus, to 5, 25, or 50% sucrose. In general, bacterial recoveries of A. viscosus declined with higher sucrose content of the diet, but a pattern of recovery for S. sobrinus was less clear with respect to dietary sucrose. S. sobrinus, however, was recovered at higher percentages from the tooth surface flora at the later two of three sampling dates. Most calculus--identified by the brittle quality, staining characteristics, and apatitic x-ray diffraction patterns of tooth surface deposits--was formed on the maxillary molars, and most carious lesions occurred on mandibular molars. While there was minimal association of the calculus score with the amount of sucrose in the diet, calculus scores increased greatly from 23 to 43 days after infectious challenge. Caries scores, of both fissure and smooth surfaces, by contrast, increased in a dose-response fashion with increasing dietary sucrose and with time. It is thus possible to induce calculus formation and caries simultaneously in specific pathogen-free Osborne-Mendel rats consuming a high-calcium and -phosphorus diet conducive to calculus formation and containing sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved properties of amorphous calcium phosphate fillers in remineralizing resin composites.

OBJECTIVES: The rationale for this study was based on the hypothesis that the mechanical strength of methacrylate composites containing the bioactive filler, amorphous calcium phosphate, can be enhanced by synthesizing this filler in the presence of glass-forming agents. Specifically, this study was conducted to prepare composites with zirconia- and silica-modified amorphous calcium phosphate fillers, and to determine whether the remineralization potential from the release of calcium and phosphate ions and the mechanical properties of the corresponding methacrylate composites were enhanced. METHODS: The modified amorphous calcium phosphates were synthesized at pH 10.5 by mixing 800 mmol/L Ca(NO3)2 solutions and either 250 mmol/L zirconylchloride (ZrOCl2) or 4.4 mol/L tetraethoxysilane (TEOS) solutions with solutions containing 525 mmol/L Na2HPO4 and 11 mmol/L Na4P2O7. After washing and drying, the amorphous calcium phosphates were mixed with visible light-activated resins and photopolymerized to form composite disks that were then examined for their ability to release Ca2+ and total ionic phosphate (PO4(3-) + HPO4(2-) + H2PO4-, hereafter indicated as PO4) by immersion in HEPES-buffered (pH 7.4) saline at 37 degrees C. Solution ion concentrations were compared at regular intervals up to 265 h. Biaxial flexural strengths of the composites before and after immersion were compared, and significant differences were established by Student's test (p < 0.05). RESULTS: Both ZrOCl2- and TEOS-modified amorphous calcium phosphate composite disks released Ca2+ and PO4 ions at sustained levels requisite for remineralization to occur. The transformation of amorphous calcium phosphate into hydroxyapatite within the composites was also retarded, particularly in the case of amorphous calcium phosphate modified with ZrOCl2. Biaxial flexure strength values of composite disks showed that TEOS- and ZrOCl2-amorphous calcium phosphate-filled composites increased in strength by 33% and 21% before immersion and by 25% and 27% after immersion, respectively, compared to unmodified amorphous calcium phosphate composites (controls). All strength increases except TEOS after immersion were significant (p < 0.05). SIGNIFICANCE: Properly modified amorphous calcium phosphate fillers can be used to prepare bioactive composites with enhanced mechanical properties for more demanding dental applications without compromising their remineralizing potential.

Mechanical properties of bioactive amorphous calcium phosphate/methacrylate composites.

OBJECTIVES: The aim of this study was to determine whether amorphous calcium phosphate (ACP)-containing composites, which have the ability to release mineralizing levels of Ca and PO4 ions, have appropriate mechanical properties for use as base and lining materials. METHODS: Composites of pyrophosphate-stabilized ACP particulates (mass fraction of 40%) and photo-activated methacrylate resins (mass fraction of 60%) were tested for biaxial flexure strength (BFS), diametral tensile strength (DTS), and compressive strength (CS). Hydroxyapatite (HAP; mass fraction of 40%), and micro-sized glass (mass fraction of 50%) composites as well as a commercial visible light curable base/liner were also tested. The significance between mean values was determined by Student-Newman-Keuls multiple comparisons (p < 0.05). RESULTS: BFS of dry and wet (24 h at 37 degrees C in water) ACP composites (60.3 and 62.0 MPa, respectively) were significantly lower than those of the comparison materials (79.2-109.3 MPa). CS values were likewise lower (62.9 MPa dry and 67.6 MPa wet vs 80.6-196.8 MPa) except for the wet base/liner (58.5 MPa). DTS of the dry ACP composite (21.8 MPa) was comparable with that of the HAP (22.8 MPa) and glass (25.5 MPa) composites, but lower than that of the base/liner (36.2 MPa). DTS decreased significantly when the ACP composite was wet (17.8 MPa). SIGNIFICANCE: These results suggest that the remineralizing ACP polymeric composites, although mechanically weaker in some respects than other polymeric composites, have properties suitable for use as base and lining materials.

Behavior of a calcium phosphate cement in simulated blood plasma in vitro.

OBJECTIVES: The purpose of this study was to gain a better understanding of the integration of calcium phosphate cement (CPC) implants in biological tissue. METHODS: An in vitro continuous flow system was employed to examine the protracted behavior of disc-shaped specimens of this bioactive material under sustained physiological-like solution conditions. Weight measurement, diameteral tensile strength measurement (DTS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and powder x-ray diffraction (XRD) were used to characterize the CPC samples as a function of immersion time. RESULTS: When CPC was immersed in simulated blood plasma in which the Ca (2.5 mmol/L) and inorganic phosphate (1.0 mmol/L) levels were kept constant, both the weight and DTS of the specimens steadily increased to about 1.5 times their original values over a period of 20 wk. SEM observations showed new precipitate formations in intimate contact with the original CPC surface. FTIR and XRD analyses revealed that the precipitate was a B-type carbonate hydroxyapatite (OHAp), the type of OHAp observed in bone and dentin. On the other hand, the interior of CPC discs did not show an increase in either bulk density or OHAp content. Thus, the increases in weight and DTS are attributable to the OHAp precipitation on the CPC surface. SIGNIFICANCE: The results suggest that under in vivo conditions, CPC implants would not dissolve in physiological fluids. OHAp coatings may form on the implants, which may enhance bonding of implants to bone by mechanically strengthening the interface between them.

The effect of short chain fatty acid administration on hepatic glucose, phosphate, magnesium and calcium metabolism.

Intra peritoneal administration of the short chain fatty acids, acetate, propionate and butyrate, in amounts calculated to reach 20 mM in total body water were given to fed and 48 hour starved male Wistar rats. One half hour after administration, the livers were freeze-clamped and the hepatic contents of various intermediary metabolites were measured. The liver content of total glycolytic intermediates was elevated by short chain fatty acids. In fed animals, the portion of glycolysis from fructose 1,6-bisphosphate (FBP) to PEP was elevated 2 to 4 fold. In 48 hour starved animals, where gluconeogenesis is active, the portion of the gluconeogenetic pathway from FBP to glucose was elevated 1.5 to 3.5 fold with the exception of the butyrate treated animals where blood glucose was not elevated. The metabolites of the hexose-monophosphate pathway that were measured, namely 6-phosphogluconate, ribulose 5-phosphate and xylose 5-phosphate were increased in both fed and starved animals. The free cytoplasmic [NAD+]/[NADH], [NADP+]/[NADPH], and [epsilon ATP]/[epsilon ADP] X [epsilon Pi] ratios were all decreased in both fed and starved animals after short chain fatty acid administration. The liver content of calcium increased 1.2 to 2 fold in fed animals and 2 to 3 fold in starved animals while total liver magnesium was either unchanged or increased only 1.2 times. The liver pyrophosphate (PPi) content increased a minimum of 10 fold in fed animals and over 100 fold in starved animals. In all cases no PPi could be detected in vivo by 31P NMR even though in the starved rats the PPi levels approached those of ATP. The liver content of inorganic Pi increased 1.3 to 1.5 fold in fed animals and 1.5 to 2 fold in starved animals. The total "rapidly metabolizing" Pi pool, that includes adenine and guanine nucleotides, glycolytic and shunt intermediates, Pi and PPi increased 1.3 times in fed animals (from 13.8 mumole/g fresh weight) and 1.5 to 1.7 fold in starved animals (from 15.7 mumol/g fresh weight). The total phosphate taken up from blood and entering the rapidly turning over pool of liver phosphate ranged between 4 and 12 mumols/g of liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Amorphous Calcium Phosphate-Based Bioactive Polymeric Composites for Mineralized Tissue Regeneration.

Amorphous calcium phosphate (ACP), a postulated precursor in the formation of biological hydroxyapatite, has been evaluated as a filler phase in bioactive polymeric composites that utilize dental monomers to form the matrix phase on polymerization. In addition to excellent biocompatibility, these composites provided sustained release of calcium and phosphate ions into simulated saliva milieus. In an effort to enhance the physicochemical and mechanical properties and extend the utility of remineralizing ACP composites to a greater variety of dental applications, we have focused on: a) hybridizing ACP by introducing silica and/or zirconia, b) assessing the efficacy of potential coupling agents, c) investigating the effects of chemical structure and compositional variation of the resin matrices on the mechanical strength and ion-releasing properties of the composites, and d) improving the intrinsic adhesiveness of composites by using bifunctional monomers with an affinity for tooth structure in resin formulations. Si- and Zr-modified ACPs along with several monomer systems are found useful in formulating composites with improved mechanical and remineralizing properties. Structure-property studies have proven helpful in advancing our understanding of the remineralizing behavior of these bioactive composites. It is expected that this knowledge base will direct future research and lead to clinically valuable products, especially therapeutic materials appropriate for the healing or even regeneration of defective teeth and bone structures.