Hydroxyapatite Discs Research Articles

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both alpha5 and alphav-containing integrin heterodimers, whereas only alphav integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of alpha2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.

In vitro evaluation of the effect of nicotine, cotinine, and caffeine on oral microorganisms

The aim of this in vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of some oral bacterial species. It also evaluated the ability of these bacteria to metabolize those substances. Single-species biofilms of Streptococcus gordonii, Porphyromonas gingivalis, or Fusobacterium nucleatum and dual-species biofilms of S. gordonii -- F. nucleatum and F. nucleatum -- P. gingivalis were grown on hydroxyapatite discs. Seven species were studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces naeslundii, and the species mentioned above. The viability of planktonic cells and biofilms was analyzed by susceptibility tests and time-kill assays, respectively, against different concentrations of nicotine, cotinine, and caffeine. High-performance liquid chromatography was performed to quantify nicotine, cotinine, and caffeine concentrations in the culture media after the assays. Susceptibility tests and viability assays showed that nicotine, cotinine, and caffeine cannot reduce or stimulate bacterial growth. High-performance liquid chromatography results showed that nicotine, cotinine, and caffeine concentrations were not altered after bacteria exposure. These findings indicate that nicotine, cotinine, and caffeine, in the concentrations used, cannot affect significantly the growth of these oral bacterial strains. Moreover, these species do not seem to metabolize these substances.

Effects of Er:YAG Laser Irradiation on Biofilm-forming Bacteria Associated with Endodontic Pathogens In Vitro

With the development of dental laser delivery systems that can enter into the root canals, it is possible to use Er:YAG lasers to remove the residual biofilm associated with infected root canals. We examined their effects against biofilms made of Actinomyces naeslundii, Enterococcus faecalis, Lactobacillus casei, Propionibacterium acnes, Fusobacterium nucleatum, Porphyromonas gingivalis, or Prevotella nigrescens in vitro. After Er:YAG laser irradiation with energy densities ranging between 0.38–0.98 J/cm 2, the biofilm samples on hydroxyapatite disks were quantitatively and morphologically evaluated. The Er:YAG laser was effective against biofilms of 6 of the bacterial species examined, with the exception of those formed by L. casei. After irradiation, the numbers of viable cells in the biofilms were significantly decreased, whereas atrophic changes in bacterial cells and reductions in biofilm cell density were seen morphologically. Er: YAG lasers might be suitable for clinical application as a suppressive and removal device of biofilms in endodontic treatments.

Carbonate substituted hydroxyapatite: Resorption by osteoclasts modifies the osteoblastic response

Carbonate substitution within the hydroxyapatite (HA) lattice improves osteoconduction, although the mechanism by which this occurs is unclear. Discs of dense, sintered, phase-pure HA and AB-type carbonate substituted hydroxyapatite (CHA) were cultured for 21 days with human CD14+ cells in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor (NF)-kappaB (sRANKL), during which time osteoclasts developed and resorbed the ceramic surface. Discs were then seeded with human osteoblasts (HOBs), and proliferation and collagen synthesis were measured. On some discs, the conditioned proteinaceous layer left behind by the osteoclasts was preserved. Proliferation of HOBs was increased on resorbed compared to control (unresorbed) surfaces on both materials, provided this osteoclast-conditioned layer was left intact. Collagen synthesis by HOBs was increased on previously resorbed surfaces compared to unresorbed surfaces. This effect was seen on both materials but was seen at an earlier time point on CHA. The results suggest that osteoclasts can condition synthetic bioceramic surfaces and alter the responses of osteoblasts that subsequently populate them. Carbonate substitution may enhance osteoconduction indirectly via effects on enhanced bioresorption.

Comparison of hydroxyapatite and dental enamel for testing shear bond strengths

Abstract Aims To investigate the feasibility of using artificial hydroxyapatite as a future biomimetic laboratory substitute for human enamel in orthodontic bond strength testing by comparing the shear bond strengths and nature of failure of brackets bonded to samples of hydroxyapatite and enamel. Method One hundred and fifty hydroxyapatite discs were prepared by compression at 20 tons and fired in a furnace at 1300°C. One hundred and five enamel samples were prepared from the buccal and palatal/lingual surfaces of healthy premolars extracted for orthodontic purposes. Orthodontic brackets were bonded to each sample and these were subjected to shear bond strength testing using a custom-made jig mounted in an Instron Universal Testing Machine. The force value at bond failure was obtained, together with the nature of failure which was assessed using the Adhesive Remnant Index. Results The mean shear bond strength for the enamel samples was 16.62 MPa (95 per cent CI: 15.26, 17.98) and for the hydroxyapatite samples 20.83 MPa (95 per cent CI: 19.68, 21.98). The difference between the two samples was statistically significant (p < 0.001). When the nature of failure was assessed with the ARI Index, 83 per cent of the enamel samples scored 2 or 3, while 49 per cent of the hydroxyapatite samples scored 0 or 1. Conclusions Hydroxyapatite was an effective biomimetic substrate for bond strength testing with a mean shear bond strength value (20.83 MPa) at the upper end of the normal range attributed to enamel (15–20 MPa). Although the difference between the shear bond strengths for hydroxyapatite and enamel was statistically significant, hydroxyapatite could be used as an alternative to enamel for comparative laboratory studies until a closer alternative is found. This would eliminate the need for extracted teeth to be collected. However, it should be used with caution for quantitative studies where true bond strengths are to be investigated.

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials

Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.

The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm 2 decreased in the order HA > PMMA > SL ( p < 0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens ( p < 0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.

Influences of starch and sucrose on Streptococcus mutans biofilms

The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms. S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose. Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05). The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH− groups and F− ions. The aim of this experimental investigation was to use the embryonal mouse fibroblast cell line NIH-3T3 for comparative study of basal cytotoxicity of fluoridated biomaterials FHA and FA discs. Hydroxyapatite (HA) disc, high-density polyethylene as negative control and polyvinyl chloride (PVC) containing organotin stabilizer as positive control were used as standard biomaterials. The appropriateness of the use of NIH-3T3 cells and their sensitivity for tested biomaterials were evaluated on the basis of five cytotoxic end points: cell proliferation, cell morphology, lactate dehydrogenase (LDH) released, protein and DNA cell content. The basal cytotoxicity of FHA, FA and HA discs was measured by direct contact method. FHA composite, FA and HA demonstrated in cell line NIH-3T3 nearly similar basal cytotoxicity increasing with the time of treatment. After 72 h of biomaterials treatment, about 25% inhibition of cell number, unchanged morphology of dividing cells, 6.31–0.16% increase of released LDH, about 10% inhibition of cell protein content and about 20% inhibition of DNA content was found. On the other hand, from the growth rates it resulted that NIH-3T3 cells, affected by tested biomaterials, divided about 20% slowlier than the control (untreated cells). Using the linear regression analysis we found out that deviations in measurements of cytotoxicity by four methods were as follows: less than 10% for cell number, protein and DNA content methods and 12.4% for released LDH method. Based on a good correlation of the cytotoxicity of biomaterials obtained from all end points we could conclude that fibroblast NIH-3T3 cell line was appropriate for measuring the basal cytoxicity of tested biomaterials.

Reduction of VSC and salivary bacteria by a multibenefit mouthrinse

To evaluate the effectiveness of a multibenefit mouthrinse containing 0.05% cetylpyridinium chloride (CPC) and 0.025% sodium fluoride in reducing volatile sulfur compound (VSC) levels and total cultivable salivary bacteria, at both 4 h and overnight. In vitro analysis of efficacy was performed using saliva-coated hydroxyapatite disc substrates first treated with the mouthrinse, then exposed to whole human saliva, followed by overnight incubation in air-tight vials. Headspace VSC was quantified by gas chromatography (GC). A clinical evaluation was conducted with 14 subjects using a crossover design. After a seven-day washout period, baseline clinical measurement of VSC was performed by GC analysis of mouth air sampled in the morning prior to eating, drinking or performing any oral hygiene. A 10 mL saline rinse was used to sample and enumerate cultivable salivary bacterial levels via serial dilution and plating. Subjects were instructed to use the treatment rinse twice daily in combination with a controlled brushing regimen. After one week the subjects returned in the morning prior to eating, drinking or performing oral hygiene to provide samples of overnight mouth air and salivary bacteria. The subjects were then immediately rinsed with the test product, and provided additional mouth air and saliva rinse samples 4 h later. A multibenefit rinse containing 0.05% CPC and 0.025% sodium fluoride was found to reduce VSC in vitro by 52%. The rinse also demonstrated a significant clinical reduction in breath VSC (p < 0.05) of 55.8% at 4 h and 23.4% overnight relative to baseline VSC levels. At both time points, the multibenefit rinse was more effective than the control; this difference was statistically significant at the overnight time point (p < 0.05). Total cultivable salivary bacteria levels were also reduced significantly (p < 0.05) at 4 h and overnight by this mouthrinse compared to baseline levels and the control. A multibenefit mouthrinse was shown to reduce in vitro VSC levels via headspace analysis and clinically at the 4 h and overnight time points. A significant reduction in total cultivable salivary bacteria was also observed at all time points, supporting the VSC data.

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.

Electrovector effect on bone-like apatite crystal growth on Inside pores of polarized porous hydroxyapatite ceramics in simulated body fluid

Porous hydroxyapatite (HA) disks and blocks with interconnecting pores (pore size≅150 μm, interconnecting window size≅30 μm, and porosity≅75%) were polarized and bone-like apatite formations on the pore surfaces were investigated by immersion test using simulated body fluid (SBF) to estimate the electrovector effect in porous bodies. Thermally stimulated depolarization current (TSDC) measurements were used to estimate the stored charges of polarized porous HA disks and blocks as 11 and 7 μC cm-2, respectively, demonstrating that both the porous disks and blocks were successfully polarized. After immersion in SBF for 14 d, bone-like apatite formations within the pores were observed in the polarized and non-polarized HA disks and blocks. Based on scanning electron microscopic (SEM) images of the pores located around the center of the disks and the blocks (inside-pores), formation of bone-like apatite was accelerated in the polarized HA compared with that in the non-polarized HA. Although the weight of the polarized and non-polarized porous HA increased linearly with immersion time regardless of shape, the rate of weight change of the polarized HA was greater than that of the non-polarized HA. It was demonstrated that the electric charges on the inside-pore surface of porous HA have a positive effect on the formation of bone-like apatite crystals.

Mechanical behaviour of porous hydroxyapatite

The aim of the study was to investigate the role of microstructure and porosity on the mechanical behaviour of sintered hydroxyapatite. Hydroxyapatite disks with four different porosities were used in this investigation. With a nanoindentation system, elastic modulus, hardness, contact stress–strain relationship, energy absorption and indentation creep behaviour were investigated. The elastic modulus and hardness of hydroxyapatite exhibited an exponential relationship (e − bP ) with the porosity P, which is similar to Rice’s finding with the minimum solid area model. High porosity samples showed more substantial inelastic behaviour, including higher energy absorption, no linear elastic region in the contact stress–strain curve and some indentation creep behaviour. We conclude that porous microstructure endows hydroxyapatite with inelastic deformation properties, which are important in a material for bone substitution usage.

Adsorption behavior of statherin and a statherin peptide onto hydroxyapatite and silica surfaces by in situ ellipsometry

The salivary protein statherin is known to adsorb selectively onto hydroxyapatite (HA), which constitutes the main mineral of the tooth enamel. This adsorption is believed to be crucial for its function as an inhibitor of primary (spontaneous) and secondary (crystal growth) precipitation of calcium phosphate salts present in saliva. A fragment corresponding to the first 21 N-terminus amino acids of statherin (StN21) was previously found to reduce the rate of demineralization of HA. Therefore, the interfacial properties of this peptide and statherin onto silica, hydrophobized silica and HA discs was studied by in situ ellipsometry. Their reversibility induced by dilution and elutability induced by buffer and sodium dodecyl sulfate (SDS) was also determined. The results revealed that statherin adsorbed at a greater extent onto the HA as compared to StN21, suggesting that the hydrogen bonding between the uncharged polar residues at the C-terminal region of statherin and HA contributes to its adsorption. However, on both silica surfaces the peptide adsorption appeared to proceed in a similar way. Onto the hydrophobized silica the adsorption of both peptides was suggested to occur either via multilayer formation or adsorption of aggregates from solution, while onto the hydrophilic silica adsorption of peptide aggregates from solution was the suggested mechanism. Further, both peptides were observed to be strongly adsorbed onto HA, even after SDS treatment, in comparison to the layers adsorbed onto hydrophobized silica. Both peptide layers were found to be weakly adsorbed onto the hydrophilic silica surface as they were totally removed by buffer dilution.

Effect of chlorhexidine/thymol and fluoride varnishes on dental biofilm formation in vitro

This study evaluated the effect of chlorhexidine/thymol (CHX/T) and fluoride (F) varnishes on biofilm formation in vitro. Hydroxyapatite discs coated with varnish were first immersed in saline for 0, 3, 7 or 14 d, then immersed in pasteurized saliva. The discs were incubated for 20 h with a bacterial suspension containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, and Veillonella dispar. Uncoated discs were used as controls. Growth of bacteria on the discs was evaluated by culture and by scanning electron microscopy (SEM). Bacterial vitality was examined by fluorescence staining. In the CHX/T-treated group, bacterial accumulation was delayed, and the total number of bacteria was significantly lower than in the controls. In the F-treated group, the total number of bacteria did not differ from the control, although the number of S. oralis was lower. Bacterial vitality in the CHX/T and F groups did not differ from that in the controls. The total number of bacteria on the CHX/T-treated discs immersed in saline was significantly higher than that on the non-immersed discs. Biofilm development was inhibited by the CHX/T varnish but not by the F varnish. The effect of the CHX/T varnish decreased following the immersion of discs in saline.

Characterization of Bone Cell Behaviors on Collagen Grafted Hydroxyapatite Surfaces

In order to enhance bone cell adhesion on hydroxyapatite (HA), collagen was used as a surface-grafting material on HA substrates because the collagen is a major constituent of connective tissues and has been regarded as one of the most excellent coating materials for bone bonding. First, HA disks (10mmΦ x 1mm) were prepared and then collagen was immobilbized on the HA surface using an 3-APTES coupling agent on HA disk surfaces. MC3T3-E1 osteoblasts were seeded on the collagen-grafted and non-grated HA disks and cultured in a Dulbecco’s modified eagle medium containing 10% fetal bovine serum for 4 hrs to evaluate the cell adhesion on the HA samples. The osteoblasts on the collagen-grafted sample were more spread than those on the non-grafted sample. It is believed that collagen-grafted HA surface provides suitable sites for cell attaching due to the high biocompatibility of collagen

Fluoropolymers as low-surface-energy tooth coatings for oral care

A range of low-surface-energy fluoropolymers has been synthesised and their effectiveness as dental-care coatings for plaque, stain and erosion prevention has been evaluated using a series of oral care models employing pressed discs of calcium hydroxyapatite or sections of human teeth. Since the blocking of dentinal tubules is a key mechanistic strategy in the treatment of dentine hypersensitivity, the capability of these non-permanent fluoropolymer coatings to occlude the pore structure of human dentine and to reduce the outward flow of simulated dentinal fluid has also been investigated. Several of the fluoropolymer coatings have been found to inhibit bacterial adhesion but no correlation has been established between anti-adhesion efficacy and fluorine content or surface energy. All the fluoropolymers have been seen to reduce stain uptake by pellicle-coated HA discs, with homopolymers being considerably more effective than copolymers. Some fluoropolymer coatings have also been shown to inhibit the acid demineralisation of hydroxyapatite discs and to reduce dentine permeability. Coatings of the 2:1 copolymer of 1 H,1 H,2 H,2 H-perfluorodecyl acrylate and 2-hydroxyethyl acrylate are most promising, exhibiting significant anti-adhesion and anti-erosion efficacy and reducing dentine permeability to a level that is comparable with that achieved with the standard treatment employed in commercial anti-sensitivity formulations.

Chitosan nanoparticles for loading of toothpaste actives and adhesion on tooth analogs

AbstractDelivery and sustained release of toothpaste actives is an important but unexplored area. In this work, chitosan nanoparticles were prepared by a water‐in‐oil emulsion/glutaraldehyde crosslinking method. The typical number average diameter of chitosan and toothpaste active (cetylpyridiniumchloride and NaF) nanoparticles was within the range of 100–500 nm. The particles increased their size at higher pH value. The morphology, adherence, and stability of these nanoparticles were investigated by scanning electron microscopy, transmission electron microscopy, and X‐ray photoelectron spectroscopy. The size of the chitosan/NaF nanoparticles was doubled after they were stored at 4°C for 20 days, and then kept constant till 251 days, the examined time so far. These particles showed good stability in toothpaste lixivium after incubated at 60°C for 30 days too. By contrast, the chitosan/cetylpyridiniumchloride nanoparticles were easy to form floccules in the toothpaste lixivium. The loaded toothpaste actives showed a sustained released behavior for at least 10 h. All the particles could adhere onto the tooth analogs such as hydroxyapatite discs and glass slides in a simulated brushing and rinsing process. In vitro cell culture did not find any cytotoxicity of the as‐prepared chitosan nanoparticles. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Evaluation of Hydroxyl Groups on Hydroxyapatite and Calcium Metaphosphate by Grafting TEOS and APTES

OH groups on hydroxyapatite (HA) and calcium metaphosphate (CMP) were evaluated by grafting tetraethyl orthosilicate (TEOS) and 3-aminopropyltriethyloxysilane (3-APTES) which can bond covalently with OH group of ceramic biomaterials. The prepared HA and CMP disks were soaked in pH 2 and 5 of acidic water and ethanol solution respectively, where pH change of each solution was measured during soaking of samples. After grafting TEOS and 3-APTES on HA and CMP disks, samples were ultrasonically cleaned in distilled water and soaked in pH 5 of ethanol solution, and pH measurement was carried out in the same manner. The pH value of HA and CMP in aqueous solution at pH 2 increased with time continuously, resulting from dissolution of HA and CMP by acidic condition on surface. At pH 5 in aqueous solution, it was the same though the pH increase was smaller. In case of ethanol at pH 5 with HA, though pH value went up slightly, the curve became saturated with time, while there was no change in pH with CMP. After grafting TEOS and 3-APTES, pH values were stabilized with few changes, indicating that there was no direct chemical reaction between the acidic media and the surface of samples due to covalently grafted TEOS and 3-APTES layer. In conclusion, it was confirmed that OH group on the surface of HA was crystallographic and chemical one rather than physically adsorbed one by grafting TEOS or 3-APTES and it will serve an effective binding site for calcium and phosphate ions, or minerals.

An in vitro biofilm model of subgingival plaque

Numerous biofilm models have been described for the study of bacteria associated with the supragingival plaque. However, there are fewer models available for the study of subgingival plaque. The purpose of this study was to develop and validate a model that closely mimicked the composition of the subgingival flora. The model was developed as follows: calcium hydroxyapatite disks were coated overnight with 10% sterile saliva, placed in flat-bottomed tissue culture plates containing trypticase-soy broth, directly inoculated with a small aliquot of dispersed subgingival plaque, incubated anaerobically, and transferred to fresh medium at 48-h intervals until climax (steady-state) biofilms were formed ( approximately 10 days). The model, based on samples from eight periodontitis patients and eight healthy subjects, yielded a multi-species, heterogeneous biofilm, consisting of both gram-positive and gram-negative species, and comprising 15-20 cultivable species associated with the subgingival flora. The species present and their proportions were reflective of the initial cultivable subgingival flora. Comparisons of the initial plaque samples from healthy subjects and the mature biofilms showed 81% similarity in species and 70% similarity in the proportions present. Biofilms formed from samples obtained from periodontally diseased subjects were 69% similar in species and 57% similar in the proportions present. The biofilm model described here closely reproduces the composition of the cultivable subgingival plaque both in the species present and in their relative proportions. Differences existed between biofilms grown from diseased and non-diseased sites with the former being characterized by the presence of periodontal pathogens at microbially significant levels.