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PubMed1988

Mechanical and bone ingrowth properties of a polymer-coated, porous, synthetic, coralline hydroxyapatite bone-graft material.

CHAG, that is, porous hydroxyapatite hydrothermally converted from the calcium carbonate exoskeleton of a coral (genus Goniopora), has been shown to be effective as a scaffold for bone ingrowth. The large pores in the material, however, resulted in low compressive strengths. Compressive testing was performed to assess the changes in mechanical properties by coating the internal surfaces of CHAG with DL-PLA. Plugs of CHAG with thick (3:1 chloroform to DL-PLA by weight), medium (10:1), and thin (30:1) coatings as well as uncoated CHAG were then implanted transcortically in the proximal third of the diaphysis of rabbit tibiae to assess the in vivo response. The mechanical tests demonstrated significantly improved compressive strength, stiffness, and energy absorption for coated specimens compared with uncoated specimens. Coated specimens were not significantly different from canine tibial cancellous bone in strength and stiffness although they achieved only 36% of the energy absorption capacity. Specimens from rabbit tibiae were harvested at 3, 12, and 24 weeks for interface shear strength determination and contralaterally for histological and histomorphometric assessment. At 12 weeks, uncoated CHAG plugs developed an average ultimate interface shear stress of 26.7 MPa compared with 17 MPa for specimens with 30:1 coatings and 8 MPa for specimens with 10:1 and 3:1 coatings. At 24 weeks, there were no significant differences in shear stress between any of the specimens. Histomorphometric assessments showed that the ratio of area fraction of new bone to area fraction of new bone and void space increased from 68-70% for specimens with 3:1 and 10:1 coatings at 3 weeks to 85.5-89.5% at 24 weeks. In comparison, uncoated and 30:1 specimens had area fraction ratios of about 82% at 3 weeks and 93% at 24 weeks. Histologic sections demonstrated direct apposition of new bone to both the coating and the hydroxyapatite as well as degradation of the coating.

Animals↗
PubMed1975

Model system for studying colonization and growth of bacteria on a hydroxyapatite surface.

A model system for the study of bacterial colonization and growth on a hydroxyapatite (HT) surface is described. Hydroxyapatite was crystallized over the surface of porous glass beads. Chemical analysis of the product showed that the ratio of Ca2+/P042- was indistinguishable from that of commercial HT powder. X-ray diffraction analysis supported the conclusion that the product was HT. A system employing [14C]polyethylene glycol, which selectively adsorbs to the glass surface of the beads, was developed to determine the amount of glass surface covered by HT. Over 90% of the glass surface could be covered by our method. The product, HT beads, consisted of approximately 20% (dry weight) HT. The HT beads possess several properties which make them potentially useful for studying microbial adherence, growth, and interactions. These include: (i) chemical similarity to the tooth surface, (ii) large surface area, and (iii) high density. We also describe a method for direct measurement of the microbial mass of cells growing on beads. The method entails immobilizing a sample on a membrane filter (Millipore), staining it with amido black dye, and eluting the dye for spectrophotometric measurement. Streptococcus mutans served as the test organism. For free-growing bacteria the values measured with the filter assay were directly proportional to cell number, with a value of 1 mug of "protein" corresponding to about 1.5 X 10(6) colony-forming units, determined by viable count. For bacteria colonizing the beads, 1 mug of protein corresponded to about 2 X 10(7) colony-forming units on the beads during logarithmic growth. As the culture approached stationary phase, the efficiency of the assay decreased. These data indicate that multiple random samples, taken at a given time, are representative of the entire culture.

Bacteriological Techniques↗
PubMed1979

Inhibition of adsorption of Streptococcus mutans strains to saliva-treated hydroxyapatite by galactose and certain amines.

Adsorption of all of eight strains of Streptococcus mutans to saliva-treated hydroxyapatite (S-HA) surfaces was inhibited by galactose and melibiose, but not by other neutral sugars tested. This observation supports the hypothesis that lectin-like components participate in the attachment of these streptococci to salivary glycoproteins on saliva-treated hydroxyapatite surfaces. Adsorption of all strains was also inhibited by iodoacetate and spermine; other amines tested reduced adsorption of some strains, but not others.

Adsorption↗
PubMed1980

Attachment of oral Cytophaga species to hydroxyapatite-containing surfaces.

Model systems simulating the cementum portion of teeth were used to characterize the attachment process by which certain species of oral Cytophaga initiate the colonization of the tooth root surface in vitro. The adsorption of these bacteria to spheroidal hydroxyapatite beads and mechanically powdered root material followed Langmuir isotherm kinetics. From such data, the number of binding sites per 20 mg of substrate and the affinity constants were evaluated for two strains of Cytophaga sp. Resting cells of the two strains tested adhered relatively tenaciously to hydroxyapatite beads in numbers similar to those observed with cells of Streptococcus sanguis. Attachment of bacteria to the substrates was partially inhibited by (i) coating the substrates with human serum or saliva, (ii) pretreating cell suspensions with proteinase K or phospholipase C or D, or (iii) exposing the cells to temperatures greater than 60 degrees C for 15 min. Treating resting cell suspensions with pronase, neuraminidase, phospholipase A2, or 0.1 M ethylenediaminetetraacetic acid had no effect on the attachment process.

Absorption↗
PubMed1981

Differences in the adsorptive behavior of human strains of Actinomyces viscosus and Actinomyces naeslundii to saliva-treated hydroxyapatite surfaces.

Human strains of Actinomyces viscosus and A. naeslundii differ in the time of their appearance and in their patterns of colonization in the mouth. Strains of these organisms were found to differ in their abilities to adsorb to saliva-treated hydroxyapatite (S-HA) surfaces, thought to mimic the teeth, and these differences parallel their patterns of colonizing the dentition. Thus, strains of A. viscosus tended to adsorb in higher numbers to hydroxyapatite (HA) treated with saliva of older children and adults than with saliva of younger children (ages 6 to 11). These salivary changes may account for the increased frequency with which this organism can be isolated from the mouths of children as they grow older. In contrast, strains of A. naeslundii and Streptococcus mutans did not show a preference for attaching to either type of S-HA. Strains of A. viscosus also generally adsorbed in higher numbers than A. naeslundii to HA treated with adult saliva; this may explain why higher proportions of A. viscosus are usually recoverable from the teeth of adults, even though A. naeslundii is generally present in higher proportions in saliva. Significant variation was noted between strains and between saliva samples collected from different donors. The differences in adsorptive behavior of strains of these species suggests that they are binding to different receptors in the salivary glycoprotein coating on HA surfaces. Adsorption of A. naeslundii ATCC 12104 was enhanced when S-HA was pretreated with neuraminidase, but this had little effect upon the adsorption of other Actinomyces strains tested. Adsorption of strain ATCC 12104 to S-HA was also strongly inhibited by fructose and sucrose and weakly inhibited by glucose, maltose, galactose, and lactose. However, other strains of A. naeslundii tested were affected less, or not at all, by these sugars. Adsorption of two strains of A. viscosus was not affected by any of the sugars or amines tested.

Actinomyces↗
PubMed1982

Enzymatic modification of bacterial receptors on saliva-treated hydroxyapatite surfaces.

Certain properties of experimental pellicles formed by the adsorption of salivary components on hydroxyapatite surfaces change over time. To determine whether enzymes likely to be present in the oral environment could induce such changes, pellicles were treated with saliva which had been incubated for 18 h at 35 degrees C to promote the elaboration of microbial enzymes. This treatment markedly reduced the numbers of Streptococcus mutans MT3 and JBP and S. sanguis FC-1 and C5 cells which attached, but it had little or no effect on the attachment of S. mitis RE7, Actinomyces viscosus LY7 and CK-8, Bacteroides gingivalis 381, or B. melaninogenicus subsp. intermedius 581. Heating the incubated saliva at 60 degrees C for 30 min partially reduced its pellicle-modifying activity, whereas heating at 80 degrees C for 30 min or 100 degrees C for 15 min completely eliminated such activity. This indicated that the saliva contained heat-labile substances, presumably enzymes, which could affect the pellicle receptors involved in the attachment of S. mutans and S. sanguis. Treatment of saliva-treated hydroxyapatite with commercially obtained enzyme preparations also affected bacterial attachment. Thus, treatment with galactose oxidase reduced the numbers of the S. mutans strains which attached, whereas treatment with neuraminidase reduced the adsorption of S. sanguis FC-1 but not that of S. sanguis C5. Treatment with beta-glucosidase preparations derived from almonds significantly reduced the attachment of all of the streptococcal strains studied, but, when subjected to isoelectric fractionation, the adherence-inhibiting activity did not correlate directly with beta-glucosidase activity. Treatment of the pellicles with trypsin or eight other glycosidases did not affect streptococcal attachment. Exposure of the enzymatically modified pellicles to fresh saliva did not restore the streptococcal receptors. Collectively, the data suggest that some bacterial receptors in the pellicle coating of teeth can be modified by enzymes likely to be present in the oral environment, and these interactions may affect oral bacterial ecology.

Actinomyces↗
PubMed1985

Actinobacillus actinomycetemcomitans strains Y4 and N27 adhere to hydroxyapatite by distinctive mechanisms.

Actinobacillus actinomycetemcomitans strains Y4 and N27 absorb to spheroidal hydroxyapatite in roughly the same numbers per milligram of substrate and with the same tenacity as two previously tested Cytophaga species. Although the two strains of A. actinomycetemcomitans exhibited similar affinities and number of binding sites for SHA, their response to enzyme treatment and heating were very different. The capacity of strain Y4 to attach to spheroidal hydroxyapatite was diminished by treatment with proteases and phospholipases and was unaffected by neuraminidase, while strain N27 was unaffected by proteases and phospholipases and lost its binding capabilities when treated with neuraminidase.

Actinobacillus↗
PubMed1985

Surface properties of Streptococcus sanguis FW213 mutants nonadherent to saliva-coated hydroxyapatite.

Seventeen mutants of Streptococcus sanguis FW213 nonadherent to saliva-coated spheroidal hydroxyapatite were isolated after mutagenesis with ethyl methanesulfonate, nitrosoguanidine, nitrous acid, hydroxylamine, or 2-aminopurine. Enrichment for nonadherent mutants was accomplished by successive adsorptions of the adherent strains to saliva-coated hydroxyapatite. After enrichment, variant colonial morphology on tryptic agar was used as a screening technique for selection of nonadherent mutants, with loss of colonial opacity frequently associated with loss of adherence ability. These mutants were further characterized for additional surface properties, including twitching motility, saliva-induced aggregation, coaggregation with Actinomyces species, surface hydrophobicity, and presence of fimbriae. Results from these assays indicated that the nonadherent mutants fell into six phenotypic groups. A correlation between the loss of adherence ability, a decrease in cell fimbriation, and a decrease in surface hydrophobicity is apparent.

Adhesiveness↗
PubMed1985

Antibodies that bind to fimbriae block adhesion of Streptococcus sanguis to saliva-coated hydroxyapatite.

Antibodies raised against a fimbriated, adhesive strain of Streptococcus sanguis (FW213) were found to block the adhesion of this organism to saliva-coated hydroxyapatite. Antibodies were made specific for adhesion antigens by adsorption with isogenic, nonadhesive mutants (for rabbit polyclonal adsorbed antibody) or selection based on nonreactivity with two nonadhesive mutants (for monoclonal antibody). Rabbit antibody raised against isogenic, nonfimbriated nonadhesive mutants served as a control for antibodies present, but not related to fimbriation. Adsorbed antibody and monoclonal antibody were shown to be specific for fimbriae (antigen 1), since both antibodies could be seen by immune electron microscopy to bind 3.6-nm fimbriae, reacted only with the fimbriated parent and not the mutants in a whole bacterial cell enzyme-linked immunosorbent assay, and could immunoprecipitate fimbriae from fimbrial extracts of FW213. Antibodies isolated from preimmune and mutant sera did not react with fimbriae in any of the above assays. Only adsorbed antibody and monoclonal antibody were capable of blocking the adhesion of FW213 to saliva-coated hydroxyapatite. Adsorbed antibody, purified to immunoglobulin G (IgG), was an effective inhibitor of adhesion without causing interfering cellular aggregation. Monoclonal IgG, papain-cleaved to Fab fragments to prohibit cell-to-cell cross-linking, was also a potent inhibitor of S. sanguis FW213 adhesion. Both IgG from mutant sera and Fab fragments from normal mouse IgG could not be shown to block adhesion. These data further support the hypothesis that S. sanguis fimbriae are involved in adhesion.

Adhesiveness↗
PubMed1987

Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite.

Cell-to-cell interaction (coaggregation) between Propionibacterium acnes PK93 and Streptococcus sanguis DL1 was measured on saliva-coated hydroxyapatite beads (SHA) at bacterial concentrations between 1.3 X 10(6) and 6.7 X 10(8) cells per ml. Four hundredfold more DL1 than PK93 cells adhered to the saliva-coated beads, and the adherence of S. sanguis was proportional to cell input. SHA precoated with 3 X 10(8) DL1 cells bound 75 to 80% of available PK93 cells at all input amounts tested, up to an input of 8 X 10(7) cells. Adherence of PK93 to DL1-coated SHA approached saturation at an input of approximately 10(9) PK93 cells, when 1.5 X 10(8) bound. The coaggregation on SHA occurred either in buffer or saliva and was inhibited by N-acetylgalactosamine and by lactose; the attachment of DL1 to SHA was not inhibited by these sugars. S. sanguis 34 and heat-treated DL1 cells, neither of which form coaggregates with PK93, attached to SHA, but such cells did not bind PK93 cells. The findings of this study indicate that bacteria unable to attach to saliva-coated hydroxyapatite can indeed adhere to such a surface by strong lectin-mediated cell-to-cell interactions with bacteria already attached to the surface.

Acetylgalactosamine↗
PubMed1979

Hydroxyapatite phagocytosis by human polymorphonuclear leucocytes.

Hydroxyapatite crystals were incubated with human polymorphonuclear leucocytes and samples examined by light and electron microscopy after 3, 8, 30, and 120 minutes' incubation. Phagocytosis of crystals occurred at 3 minutes and increased with incubation. Degranulation of neutrophils, loss of cytoplasmic density, and cell necrosis were greatest in cells mixed with apatite and increased with time. Avid in vitro phagocytosis of hydroxyapatite crystals lends further support to the potential of these crystals as causes of human arthritis.

Humans↗
PubMed1982

Enzymatic release of microspheroids containing hydroxyapatite crystals from synovium and of calcium pyrophosphate dihydrate crystals from cartilage.

Incubation of minced, focally calcified, chondromatosis tissue obtained at operation from a patient with Milwaukee shoulder (rotator cuff defect and glenohumeral osteoarthritis associated with synovial fluid, hydroxyapatite crystals in microspheroidal masses, collagenase, and neutral protease) with partially purified mammalian synovial cell collagenase released masses of hydroxyapatite crystals of the same size as those originally found in the patient's synovial fluid. Incubation of mineral articular cartilage obtained from a shoulder joint at arthroplasty for a destructive arthropathy in a patient with generalised calcium pyrophosphate dihydrate (CPPD) crystal deposition with partially purified synovial cell collagenase freed CPPD crystals from their matrix. These data are compatible with a previously postulated mechanism linking microcrystals to destructive arthropathies, that is, crystal endocytosis by synovial cells stimulating collagenase secretion with subsequent enzymatic crystal "strip-mining', releasing additional crystals into the synovial fluid in a self-perpetuating cycle.

Calcium Pyrophosphate↗
PubMed1971

Hydroxyapatite in the pathogenesis of cystic fibrosis.

Submandibular saliva collected from cystic fibrosis patients and control subjects was separated by centrifugation into an insoluble deposit and a clear supernatant. The resulting calcium and phosphorus analyses performed on both fractions warranted a closer investigation as a consistent Ca/P molar ratio of 1.5 was found in the deposit of the cystic fibrosis patients, while no consistent ratio >1.0 was found in the deposit of the control subjects. The expected result, that calcium and phosphorus in the deposit of cystic fibrosis patients is present as the solid phase of hydroxyapatite, was confirmed by a detailed comparison of x-ray powder diffraction patterns of an ashed sample of this deposit and a similarly treated synthetic sample of hydroxyapatite.

Adolescent↗
PubMed1988

Rabbit synoviocyte inositol phospholipid metabolism is stimulated by hydroxyapatite crystals.

Inhibition of prostaglandin E2 synthesis partially ameliorates some aspects of synovitis, but joint destruction still progresses. Other aspects of phospholipid metabolism may play a role in synovial tissue pathophysiology. Products of phosphatidylinositol metabolism can activate intracellular processes in response to extracellular stimuli. We asked whether this pathway is activated in synoviocytes in monolayer tissue culture by the addition of hydroxyapatite (HA) crystals in medium. These crystals are found in pathological human synovial fluid. These crystals are associated with the secretion of degradative enzymes and with a destructive arthritis in humans. Rabbit synoviocyte cultures, previously incubated with [3H]inositol to label inositol phospholipids, were stimulated with the addition of hydroxyapatite (180 micrograms/ml) to the cultures. There was enhanced intracellular accumulation of [3H]inositol monophosphate (30-100%) after 4 h. This indicated an increased phospholipase C activity. The radioactivity in [3H]inositol bis- and trisphosphates was too low to reliably measure. The use of [32P]Pi allowed detection of these compounds. In the presence of HA, incorporation of [32P]Pi into phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate was increased. In addition, cultures exposed to [32P]Pi during stimulation with HA had an increased content of [32P]inositol monophosphate, bisphosphate, and trisphosphate.

Animals↗
PubMed1989

Structural probe analysis on the attachment of salivary glycoproteins to hydroxyapatite using Fourier-transformed infrared spectroscopy.

The mechanisms of interaction between a salivary glycoprotein and hydroxyapatite has been studied using Fourier-transformed infrared spectroscopy. The spectra of the reacted mixtures showed evidence of change at 1,230-1,250 cm-1 due to the S = O stretching vibrations of the ester sulphate groups and at 775 and 828 cm-1 attributed to sulphate groups in the 6-position of N-acetylgalactosamine. Evidence of amide change in the regions of 1,670 and 1,550 cm-1 is also apparent due to involvement of the polypeptide region. It is proposed that the exposed parts of the protein core are involved in the attachment of the glycoprotein to hydroxyapatite with the main interactive residues being the sulphate groups present on the carbohydrate apo-protein moiety. The results are considered of value in relation to mechanisms involved in the coating of exposed apatite surfaces during the formation of acquired enamel pellicle.

Absorption↗
PubMed1991

Effect of pore topology on the remineralization rate of surface-softened enamel and synthetic hydroxyapatite: an experimental comparison and mathematical model.

Experiments with surface-softened synthetic hydroxyapatite show similar remineralization rates as surface-softened human enamel. The mineral gain with respect to time relates to parameters governing the precipitation of mineral in the specimens. A mathematical model regarding the mineral precipitation on the pore surface is fitted to the experimental values. We obtained comparable precipitation rates for apatite and tooth enamel. The geometry of the pores, however, proved to be different: a mostly tube-like pore structure must be attributed to demineralized enamel, while the synthetic hydroxyapatite specimens remineralize like a ramified, crack-like microstructure of pores.

Chi-Square Distribution↗
PubMed1977

Synchronized crystal dissolution behavior for tooth enamel and synthetic (NBS) hydroxyapatite.

The synchronized cyrstal dissolution hypothesis previously proposed to explain the unusual dissolution behavior of human dental enamel and hydroxyapatite in partially saturated acidic media has been critically examined with dissolution-dialysis transport experiments. The findings are in accord with the hypothesis. A model based upon a variable effective solubility for the hydroxyapatite crystal is proposed.

Chemical Phenomena↗
PubMed1977

Vapor phase adsorption of water on hydroxyapatite.

Water vapor adsorption isotherms were determined gravimetrically on three hydroxyapatite sampel differing in preparation and with surface areas of 70.4, 22.5, and 3.0 M2/gm, respectively. Heats of adsorption for the first layer of water were found to be 13.3, 13.2, and 13.9 kcal/mole on these hydroxypatites. From repeated thermal desorption and adsorption cycles of water, stepwise adsorption was observed which diminished with each outgassing cycle until it disappeared after the fourth cycle. Cross-sectional area of adsorbed water molecule on hydroxyapatite surface was estimated at 11.5 A2. Standard free energies, isosteric heats, changes in enthalpy, and entropy of adsorption of water on HAP samples outgassed at 300 C were determined.

Adsorption↗