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Calcium Modulates Interactions between Bacteria and Hydroxyapatite

¹ Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145, 4000, San Miguel de Tucumán, Tucumán, Argentina;
² Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET);
³ Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán;
⁴ Unidad de Actividad Química, Centro Atómico Constituyentes, Comisión Nacional de Energia Atómica;
⁵ Instituto de Tecnologia Jorge Sabato, Universidad Nacional de General San Martin; and
⁶ Escuela de Posgrado, Universidad National de General San Martin

^* corresponding author, mapella{at}cerela.org.ar

	ABSTRACT

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Bacterial adhesion onto hydroxyapatite is known to depend on the surface properties of both the biomaterial and the bacterial strain, but less is known about the influence of the composition of the aqueous medium. Here, the adhesion of Streptococcus mutans and 3 different Lactobacilli on powdered hydroxyapatite was shown to change with Ca²⁺ concentration. The effect depends on the surface properties of each strain. Adhesion of Lactobacillus fermentum and salivarius (and of Streptococcus mutans at low Ca²⁺) was enhanced with increasing Ca²⁺ concentration. Lactobacillus casei was efficiently removed by adhesion on hydroxyapatite, even without Ca²⁺ addition, and the effect of this ion was only marginal. The results are interpreted in terms of Ca²⁺-mediated adhesion, and relative to the hydrophobic properties of each strain and the electrical properties of the bacterial and solid surfaces (electrophoretic mobility).

KEY WORDS: hydroxyapatite • bacteria • surface properties • interactions

	INTRODUCTION

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Hydroxyapatite (HAP) constitutes more than 90% of dental enamel (de Aza and de Aza, 2004). Upon immersion in oral fluid, synthetic HAP (or any other bare inorganic biomaterial) rapidly develops a proteinaceous film (acquired pellicle), on which bacteria and epithelial cells adhere. All surface properties of the solid are drastically changed by the presence of this film, which defines the net surface charge and the nature of surface chemical groups. Adherent bacteria form a biofilm by adsorption, adhesion, colonization, and invasion of the solid covered by the acquired pellicle (Busscher et al., 1995). Some components of saliva may also promote bacterial aggregation (Weerkamp and McBride, 1980).

This complex picture does not preclude a need for understanding of the behavior predicted by simple models. The Deryaguin-Landau-Verwey-Overbeek (DLVO) theory (Verwey and Overbeek, 1948) provides a simple description of the interaction between biomaterials and bacteria by taking into account electrostatic and London dispersion interactions. The former, defined by surface charges, may be attractive or repulsive; the latter, due to correlations between molecular dipoles, are always attractive. Accordingly, van Loosdrecht et al.(1987) showed experimentally that both charge and hydrophobicity affect the degree of bacterial adhesion on polymers. The ionic strength and dielectric constant of the medium influence both electrostatic and dispersion interactions; in addition to these generic medium effects (Weerkamp et al., 1988), specific ionic effects by fluoride and acetate (Eifert et al., 1984) have also been described.

This paper reports the results of an experimental study of the interaction of HAP with bacterial cells in simple aqueous solution and in human saliva, focused on the effect of Ca²⁺. HAP particles, smaller than bacteria, but able to form aggregates of larger size, were used in our effort to understand the interplay between bacterial co-aggregation and hetero-coagulation of bacteria with particles. Electrophoretic mobilities of bacteria and particles provided information on the operating electrical charges. We used hydrophobicity measurements to explore the influence of hydration forces on the interaction. Three oral cavity strains and a more hydrophilic strain, from cheese, were studied. The interaction of particles and bacteria was also observed by optical and electron microscopy. We used the results to explore the role played by Ca²⁺ in the surface phenomena that define bacteria-HAP adhesion.

	MATERIALS & METHODS

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Powdered hydroxyapatite (Ca/P molar ratio 1.70) was composed of aggregates (ca. 10-µm diameter) of well-crystallized nanoparticles (average size, 100 x 10 x 5 nm) with a Brunauer-Emmett-Teller surface area of 23.5 m² g^–1. Fourier Transform Infrared spectra showed the presence of small quantities of carbonate (weak band at 1460 cm^–1). [For further details on the properties of the hydroxyapatite, see García Rodenas et al.(2005).]

The strains were Streptococcus mutans ATCC25175, Lactobacillus salivarius ATCC11741, and Lactobacillus fermentum ATCC14932 from the oral cavity, and Lactobacillus casei ATCC393 from cheese. The behavior of hydrophilic bacteria was explored with this latter strain representative of micro-organisms entering the oral cavity with food intake.

All strains were stored at –20°C in skim milk (9% w/v) with yeast extract (0.5% w/v) and subcultivated by 3 successive transfers in LAPTg (Raibaud et al., 1961) broth for S. mutans and L. salivarius, and MRS broth for L. fermentum and L. casei. They were grown aerobically in the same media at 37°C for 24 hrs. Active cultures of each strain were centrifuged (5000 g, 10 min), and the pellets were suspended in an adequate volume of 0.1% peptone water to yield ca. 10⁸ CFU mL^–1. We determined bacterial counts by plating in the agar medium after incubation at 37°C for 48 hrs.

All other reagents were analytical grade.

We prepared samples for microscopic observations in an optical Karl Zeiss Axilab (immersion objective 100X) by mixing 100 µL bacterial culture (10⁸ CFU mL^–1) with 200 µL HAP suspension (1.25 g L^–1) containing ^–2 mol L^–1), at either KNO₃ or CaCl₂ (10 neutral pH. After 15 min, a 200-µL aliquot was placed on a layer of agar gel for microscopic observation. Ancillary observations were performed in a Philips 515 scanning electron microscope, with de-aggregated (sonicated) HAP samples.

Electrophoretic mobilities were measured in a Rank Brothers apparatus (Cambridge, UK) at various Ca(NO₃)₂ concentrations. Constant ionic strength (I = 0.01 mol L^–1 ) was obtained by KNO₃ addition. Before measurement, HAP samples were conditioned for 3 days. Bacterial mobilities were determined in the stationary phase. Measurements were also performed in natural saliva, with and without added Ca²⁺.

We determined hydrophobicity by measuring the partition between water and toluene, hexadecane, or p-xylene (Rosenberg et al., 1980). Bacterial cultures were incubated for 16 hrs, rinsed 3x with physiological solution, and re-suspended until the absorbance at 560 nm was 0.5–0.6. The culture (3 mL) and the organic solvent (1 mL) were mixed and vigorously shaken for 90 sec. After phase separation (15 min), the absorbance of the aqueous fraction was measured. Surface hydrophobicity percentage was calculated as HB% = 100 (1 - A/A₀), where A₀ and A are the absorbances before and after extraction.

For adsorption measurements, HAP was suspended in sterile natural saliva diluted (volume ratio 1:1) with aqueous solutions containing various amounts of Ca(NO₃)₂ and KNO₃, with I = 0.02 mol L^–1 maintained. [Saliva was collected unstimulated from a healthy volunteer individual. No IRB approval of study protocol was necessary.] Saliva was clarified by centrifugation at 20,000 g for 20 min at 4°C and sterilized by filtration. Equal volumes of the HAP suspension (10 mg mL^–1) and bacterial culture (ca. 10⁸ CFU) were mixed and left in contact for 2 hrs. Sedimentation of the solid containing HAP and adhered bacteria was achieved by centrifugation at 500 rpm for 1 min. The supernatant with non-adhered bacteria was removed; the solid was re-suspended in 0.1% peptone water and gently vortex-shaken.

S. mutans and Lactobacillus cells adsorbed to the HAP surface were counted by plate count in the agar media; the plates were incubated at 37°C for 48 hrs.

	RESULTS

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Mobilities of the bacterial strains in aqueous KNO ₃ solutions were generally negative in the whole spanned pH and Ca²⁺ concentration ranges (Figs. 1A-1D). The mobility sign (and hence of the surface net charge) was due to carboxylate and other ionized weak acidic groups of the bacterial surface (Cann, 1978; Rose et al., 1993, 1994). The addition of Ca²⁺ made the mobility less negative. Only S. mutans showed charge reversal at the highest Ca²⁺ concentration and lowest pH.

View larger version (10K): [in this window] [in a new window]   Figure 1. Electrophoretic mobility vs. pH profiles of Streptococcus mutans (A), Lactobacillus casei (B), Lactobacillus salivarius (C), Lactobacillus fermentum (D), and HAP (E) in 0.01 mol L–1 KNO3. [Ca 2+] = 0 (O), 5 x 10–4 (), 1 x 10–3 (), and 1 x 10–2 () mol/L. Also shown in (A) and (C), measurements in the presence of natural saliva: [Ca2+] = 1 x 10–2 mol L–1 (); [Ca2+] = 0 (). Vertical bars indicate SD of triplicate experiments.

S. mutans’ mobility in saliva (Fig. 1A) was similar to that observed in 1 x 10^–3 mol L^–1 Ca²⁺, regardless of the Ca²⁺ added to the saliva (0 or 1 x 10^–2 mol/L). The quenching of the Ca²⁺ influence is attributed to the presence of this ion in the bacterial culture. L. salivarius’ mobility was not quenched in the same way; the results (Fig. 1C) suggest a Ca²⁺ level in the culture of ca. 5 x 10^–3 mol L^–1.

HAP mobility in water was negative in the absence of added Ca²⁺ in the range 5.5 < pH < 9.5. Bell-shaped, positive values were measured in the presence of high Ca²⁺ (Fig. 1E).

L. casei was hydrophilic, whereas S. mutans and L. salivarius were essentially hydrophobic (Fig. 2). The measurements of L. fermentum hydrophobicity in hexadecane deviated considerably from the values in the other 2 solvents. Sweet et al.(1987) have reported that hexadecane does not always produce accurate or reproducible results; hence, the abnormally low value measured in hexadecane was discarded. Other effects, such as cell lysis induced by xylene, are not supported by our data.

View larger version (19K): [in this window] [in a new window]   Figure 2. Bacterial hydrophobicities in hexadecane (), p-xylene (), and toluene (). Bars represent the average of at least 6 measurements, with SD = ± 3%.

View larger version (119K): [in this window] [in a new window]   Figure 3. Bacteria-hydroxyapatite interaction. (A-C) Optical photomicrographs (bar = 50 µm) of suspensions containing HAP and L. salivarius (A,B) or L. casei (C). In (A), no Ca2+ was added; in (B) and (C), [Ca2+] = 1 x 10–2 mol L–1. (D) Scanning electron microscope micrograph of suspensions containing L. salivarius and hydroxyapatite with [Ca2+] = 1 x 10–2 mol L–1 (bar = 1 µm).

View larger version (4K): [in this window] [in a new window]   Figure 4. Removal percentage of bacteria by centrifuged hydroxyapatite precipitate (see text) as a function of log [Ca2+]: (A) Lactobacillus salivarius () and Lactobacillus fermentum (); (B) Streptococcus mutans; (C) Lactobacillus casei. Vertical bars indicate SD of triplicate experiments.

1. Removal of the 2 highly hydrophobic rods (L. fermentum and L. salivarius) increased with increasing Ca²⁺ concentration (Fig. 4A).
   
2. The same trend, but at high Ca²⁺ concentration, was observed for the hydrophobic coccus S. mutans (Fig. 4B).
   
3. The highly hydrophilic rod from cheese (L. casei) was efficiently removed even without added Ca²⁺, and the effect of addition was low or negligible (Fig. 4C).
   
4. At high Ca²⁺, the adsorption of all strains reached values higher than 40%, and the difference between L. casei and the other strains largely disappeared.
   

	DISCUSSION

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The electrophoretic mobility, µ, is frequently used to calculate the -potential, the electrical potential at the slipping plane. This plane separates the fluid that moves together with the particle from the outer volume in which counter-ions move in the opposite direction; it is usually assumed to coincide with the onset of the diffuse region of the double layer (outer Helmholtz plane, OHP). is determined by the charge density on the particle surface and by the extent of charge neutralization within the OHP. Although, in simple cases, is proportional to (Hunter, 1981), in the case of bacteria, their electrical conductivity may affect the relation between µ and (van Loosdrecht et al., 1987). Although Sonohara et al.(1995) have derived an equation that calculates potentials from experimental mobility data, with inclusion of the effect of ionic penetrability, it is safer to discuss the charge of the bacteria using raw mobility data (van Loosdrecht et al., 1987).

The mobility/pH profiles for hydroxyapatite in water containing Ca²⁺ have been interpreted in terms of a simple surface complexation model, involving adsorption/desorption of Ca²⁺ and H⁺, together with the protolytic equilibria of dissolved phosphate. [Surface complexation models assume that surface charge develops through chemical complexation reactions involving surface species and dissolved ions, and that these interactions can be written as mass-law equilibria. This subject has been extensively treated in the literature; see, e.g., Stumm and Morgan (1996) and Blesa et al.(1993). For further details, see García Rodenas et al.(2005).] Many factors may influence the mobility of enamel, especially the presence of organic matter; thus, very diverse values of the isoelectric point (pH value at which = 0) have been reported (Kambara et al., 1978).

Bacterial mobility does not change much with pH in the range 6 < pH < 10.5, because the ionization degree of surface groups is roughly constant. In contrast, Ca²⁺ adsorption affects the mobility appreciably. Positive values in L. casei indicate strong chemisorption.

The relation between hydrophobicity and mobility is a matter of debate. Pelletier et al.(1997) reported no correlation, whereas van der Mei et al.(1988) described a shift in the isoelectric point of a series of S. salivarius HB mutant strains that parallels the surface ratio (lipoteichoic)/(teichoic) acids. Both hydrophobicity and mobility measurements are subject to large intrinsic errors, and a detailed analysis of a possible correlation is not warranted. The lower mobility of the hydrophilic strain reported here, however, is beyond doubt.

Hydrophilic interfaces result from high surface density of ionizable groups, and the very low mobilities of L. casei must therefore be due to charge neutralization by the strong chemisorption of counter-ions, an effect that may run parallel to ionic conductivity effects (van Loosdrecht et al., 1987). Ca²⁺ is probably already present in high concentrations in the corresponding cultures. The decreased influence of Ca²⁺ addition on the mobility in saliva supports this assertion.

Cann (1978) and Rose et al.(1993) have proposed that bacterial adhesion in saliva takes place through irreversible, long-range interactions mediated by polymers, followed by reversible adhesion between anionic groups, mediated by calcium binding. In the oral cavity, tooth enamel covered by the acquired pellicle exposes negatively charged groups to the fluid, and calcium ions provide the natural chemical bridges with the bacterial cells. In filtered sterile saliva, with no acquired pellicle, the surface calcium ions of enamel provide the bridges. In a medium containing HAP particles, bacteria may adhere to the solid and may also co-aggregate. It has been reported that biofilms formed 15 min after inoculation on hydroxyapatite disks consist mainly of single, non-aggregated cells. At longer times, S. oralis SK248 (OMZ 607) easily forms spheroidal or ovoid micro-colonies; cylindrical cells, such as those of A. naeslundii OMZ 745, also give rise to spheroidal structures on the HAP substrate, with ease (Guggenheim et al., 2001). The present study was carried out under very different conditions, and involved the interaction of bacteria with smaller hydroxyapatite particles aggregated in clusters similar in size to the bacteria. Both co-aggregation and adhesion took place under these conditions, the former leading to larger aggregates at low Ca²⁺ concentrations.

The effect of exogenous Ca²⁺ on adhesion may now be explained. The availability of Ca²⁺, at low concentrations, determined the affinity of L. fermentum, L. salivarius, and S. mutans for HAP. The behavior of L. casei was diverse, suggesting that a solution layer rich in Ca²⁺ already surrounded this strain. These effects were obvious in spite of the possible Ca²⁺ sequestering effect of saliva components. Adhesion of bacteria and solid particles took place via linking Ca²⁺ in all cases; indeed, at the pH of saliva at low Ca²⁺ levels, electrostatic repulsion must operate against simple bacteria-substrate hetero-coagulation. Ca²⁺ not only gives rise to charge reversal, but also provides the bridges to link, through functional groups, HAP and bacterial surfaces. At high Ca²⁺, bacterial co-aggregation may also be favored, and a decrease in the extent of hetero-coagulation results. This conclusion applies both to L. casei and S. mutans; this result was not achieved by L. fermentum and L. salivarius.

The small HAP particles had a marked tendency toward forming large clusters, but the nature of the interaction with bacteria was similar regardless of the size of the cluster. Hetero-coagulation was obvious, but adhesion of small HAP particles onto the larger bacteria was also seen by SEM in sonicated media.

Yoshida et al.(1998) described both tightly and loosely adherent bacteria. Although our study did not explore this issue, it is probable that the affinity is not constant, since Ca²⁺ concentration varies, and the solid removes more bacteria at higher Ca²⁺, including less tightly adherent bacteria.

The interaction between bacterial and HAP surfaces depends not only on the nature and number of available anchoring functional groups, but also on the medium calcium ions that bind the functional groups of the bacteria and the biomaterial. Adhesion would be favored by high calcium concentrations, which in turn may originate from acid attack on HAP at low pH values. Thus, lactic acid formation in the early stages of caries attack on enamel may indirectly generate conditions favorable for further bacterial adhesion. Bacterial co-aggregation may compete with hetero-coagulation for hydrophobic strains at lower solid/bacteria ratios, especially in low Ca²⁺ media.

	ACKNOWLEDGMENTS

 
This work was supported by CONICET (PIP 02679 and Fellowship to SCV), CIUNT (Project G305), ANPCyT (PICT 06-06631), and CNEA. The authors gratefully thank Prof. R. Rodriguez Clemente for supplying the hydroxyapatite sample, and C. Barrionuevo for editing the manuscript.

Received November 2, 2005; Last revision September 8, 2006; Accepted September 25, 2006

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