to the resin block surfaces (μg/mm2) was greater than that for the other salivary proteins (Figure 2).
3.2. Adhesion of S. mutans (Quantitative Adherence of Radiolabeled Bacteria)
The values for disintegrations per minute (dpm) were significantly (p < 0.05) lower for all resin blocks soaked in any of the five kinds of salivary protein than for those for the unsoaked blocks (control). No significant difference in dpm was obtained with respect to filler content rate, though the dpm value for Mucin 1 and Lysozyme tended to decrease with the increasing S-PRG filler content rate. In addition, the dpm value for Mucin 1 was lower than that for the other four kinds of salivary protein, but not significantly so (Figure 3).
Figure 1. Mean values for adsorption of the 5 kinds of salivary protein fluid to different S-PRG resin blocks after different time of incubation. (a) Mucin; (b) Lactoferrin; (c) IgA; (d) Cystain C; (e) Lysozyme.
Figure 2. Amount of salivary proteins adsorbed per 1 mm2 area of resin block surface.
Figure 3. Quantitative adherence of radiolabeled S. mutans (No significant difference was found between specimens having the same characters. MUC: Mucin 1, LAC: Lactoferrin, CYS: Cystatin C, LYZ: Lysozyme).
3.3. SEM Observation
Adhesion of S. mutans was found on the surfaces of all resin blocks. However, reduced adhesion was observed for the resin blocks that had been incubated with any of the salivary proteins (Figures 4(a)-(f)). Figure 4(b) shows the SEM of S. mutans that had adhered to S-PRG resin coated with Mucin 1. Little adhesion found with S. mutans regardless of the content rate of the S-PRG filler. Moreover, the number of S. mutans that had adhered to the Lysozyme-coated resin tended to decrease with an increase in S-PRG filler content rate.
The current study found that the amount of the salivary protein adsorption varied according to the content of S-PRG filler. Therefore the null hypothesis was rejected. The surface properties of the material used for caries treatment are always influenced by the saliva in the mouth. More than 300 kinds of bacteria exist in the human mouth, and a biofilm is formed on the teeth as a dental plaque  . Therefore, it is necessary to know the relationships among the restoration materials, saliva and the plaque in studying about dental materials   .
The characteristics of the resin composite containing an S-PRG filler, are that salivary proteins are more readily adsorbed than resin composites without S-PRG filler, and the amount of plaque formation is low    . In our past in vitro study, electrophoresis analysis (SDS-PAGE) revealed that a resin composite containing the S-PRG filler has the property of adsorbing salivary proteins of 25 kDa or less, whereas the S-PRG filler alone can adsorb salivary proteins of up to 14 kDa  . Moreover, the main proteins detected in vivo on the surface of a resin composite containing the S-PRG filler as determined in an immunity SEM method were Cystatin C and Lysozyme  . Therefore, we considered the possibility of plaque formation could be influenced by salivary protein in the mouth that is adsorbed on the surface of a resin composite containing S-PRG filler. In our in vitro study, the amount of adsorption of various salivary proteins was measured by using three kinds of resin composite, each having a different content of the S-PRG filler. We also examined the relationship between bacteria adhesion to the resin composite and the salivary protein covering it.
The absorbance (OD280) of the salivary protein fluid decreased with increasing incubation time for each of the five kinds of salivary protein tested. Regarding the amounts of each salivary protein that adsorbed to the resin block (μg/mm2) regardless of the content of the S-PRG filler, we calculated that Mucin 1 gave the greatest adsorption, followed by Lysozyme. That is, there was almost a correlation between the amounts for various salivary proteins and simulated human whole saliva in this study. Moreover, the amount of adsorption of the Lysozyme increased as the content of the S-PRG filler was increased. The means of interaction between Lysozyme and hydroxyapatite in the mouth is thought to be an electrostatic attraction; because the tooth surface becomes negatively charged when Ca2+ is discharged and Lysozyme, which is positively charged, is then attracted to the teeth  . On the other hand, the resin composite containing
Figure 4. SEM of S. mutans adhered to S-PRG resin coated with 5 kinds of salivary protein fluid. (a) Control: non-coated with the salivary protein; (b) Mucin; (c) Lactoferrin; (d) IgA; (e) Cystain C; (f) Lysozyme.
S-PRG filler discharges various inorganic ions (Al3+, Sr2+, Na+, ,) in the solution such as saliva, and the amount of discharge is proportional to the content of the S-PRG filler. Therefore, as the ionization potential is high in the case of the resin composite with a high content of the S-PRG filler, the positively charged Lysozyme may unite with the part of resin composite containing the negatively charged S-PRG filler.
We examined bacterial adhesion by using radiolabeled S. mutans and by SEM observation. Though the bacteria adhered to all the resin blocks coated with any of the salivary proteins, their adherence was less than that in the control group. Moreover, the resin composites coated with Mucin 1 showed less adhesion than those coated with the other salivary proteins. Adhesion of the radiolabeled bacteria to the Lysozyme-coated composite increased with an increase in the S-PRG filler content, and the number of adherent S. mutans seen by SEM observation tended to decrease with the increase in this content. These two experimental results approximated one another, indicating that the amount of adsorption and the kind of the salivary protein influenced the adhesion by S. mutans.
The initial conditioning salivary coat plays an important role in bacterial adhesion to a restorative surface  . However, it must be pointed out that although the acquired pellicle itself is free of bacteria  , it is the starting point for microbial colonization on oral hard surfaces, whereby the salivary pellicle acts as a receptor for the initial adhesion of bacteria. Indeed, the formation of oral biofilms on hard surfaces is a complex process that begins with salivary pellicle formation and pellicle adsorption to the surface, and then progresses to passive transport of bacteria to the pellicle surface, followed by irreversible adhesion and multiplication of the attached organisms  .
Among the five kinds of salivary proteins tested in this study, S. mutans adhesion to resin composites coated with any of these salivary proteins was low. In addition, in the case of Lysozyme there was somewhat of a correlation between the increases in bacterial adhesion with increasing S-PRG filler content. Many anti-bacterial proteins are found in human whole saliva, and Lysozyme is classified as a non-immunoglobulin protein  . Moreover, adsorbed Lysozyme is effective in preventing Streptococcus from adhering to the hydroxyapatite covered by saliva  . Our finding suggest that the S-PRG filler used in a dental restoration material exposed to endogenous Lysozyme, which is an anti-fungoid salivary protein that is selectively adsorbed, is very profitable to prevent secondary caries.
The adherence of S. mutans was low when the resin blocks were soaked in any of the five kinds of salivary proteins tested regardless of the content of the S-PRG filler. In addition, the amount of adsorption of Lysozyme to the composite increased with increasing content of the S-PRG filler. Moreover, the adhesion of S. mutans to the surface of a resin block coated with Lysozyme seemed to become lower with an increase in the content of the S-PRG filler, though no significant difference was found.
The authors would like to thank Shofu Inc. for providing the materials needed.