Effect of tourmaline on acid production of streptococcus mutans.Note: *p<0.05 was statistically significant as determined by the Student’s t-test for the mean values different from the control group.
Solid freeform fabrication (SFF), also termed as layered manufacturing, produces parts directly from a computer model without part-specific tooling and human intervention (Sachlos et al., 2003; Leong et al., 2003; Kim et al., 2009; Dwivedi and Kovacevic, 2004; Cai et al., 2003; Tian et al., 2002; Guo et al., 2002; Cawley, 1999; Calvert et al., 1998; Alimardani and Toyserkani, 2008; Alemohammad et al., 2007; Li, 2005; Bryant et al., 2003). It has great potential to fabricate functionally graded materials (FGMs).
Through selective slurry extrusion (SSE) based technique of SFF, Xu et al. successfully fabricated multi-material dental crown (Xu et al., 2005) and further proposed a novel approach (as shown in Figure 1), termed as equal distance offset (EDO), to representing and process planning for SFF of functionally graded materials so as to meet the requirement of modelling and fabricating 3D complex shaped FGM objects (Xu and Shaw, 2005).
In EDO (Xu and Shaw, 2005), a neutral arbitrary 3D CAD model is adaptively sliced into a series of 2D layers. Within each layer, 2D material gradients are designed and represented via dividing the 2D shape into several sub-regions enclosed by iso-composition contours. If needed, the material composition gradient within each of the sub-regions can be further specified by applying the equal distance offset algorithm to each sub-region. Using this approach, an arbitrary-shaped 3D FGM object with linear or non-linear composition gradients can be represented and fabricated via suitable SFF machines. The process planning for SFF of FGM objects is shown in Figure 2.
In recent years, the inkjet colour printing based SFF technology has been of great interests in tissue engineering (Calvert et al., 2007; Xu et al., 2006; Saunders et al., 2008; Sanchez et al., 2008; Roth, 2004; Hasenbank et al., 2008; Cui and Boland, 2009), as it can fabricate 3D complex shaped graded material with smooth gradients (Wang and Shaw, 2006).
Human teeth have some very good properties such as high hardness and wearability, good heat insulation, high strength, etc. These properties are related to its graded structure. Figure 3 (Tooth anatomy, (2011).
A normal tooth can be divided into three parts: the crown, the neck and the root. The crown is the part of the tooth that is visible above the gum (gingiva). The neck is the region of the tooth that is at the gum line, between the root and the crown. The root is the region of the tooth that is below the gum. Some teeth have only one root, for example, incisors and canine (eye) teeth, whereas molars and premolars have 4 roots per tooth.
The crown of each tooth has a coating of enamel, which protects the underlying dentine. Enamel is the hardest substance in the human body, harder even than bone. It gains its hardness from tightly packed rows of calcium and phosphorus crystals within a protein matrix structure. Once the enamel has been formed during tooth development, there is little turnover of its minerals during life. Mature enamel is not considered to be a ‘living’ tissue.
The major component of the inside of the tooth is dentine. This substance is slightly softer than enamel, with a structure more like bone. It is elastic and compressible in contrast to the brittle nature of enamel. Dentine is sensitive. It contains tiny tubules throughout its structure that connect with the central nerve of the tooth within the pulp. Dentine is a ‘live’ tissue.
Below the gum, the dentine of the root is covered with a thin layer of cementum, rather than enamel. Cementum is a hard bone-like substance onto which the periodontal membrane attaches. This membrane bonds the root of the tooth to the bone of the jaw. It contains elastic fibres to allow some movement of the tooth within its bony socket.
The pulp forms the central chamber of the tooth. The pulp is made of soft tissue and contains blood vessels to supply nutrients to the tooth, and nerves to enable the tooth to sense heat and cold. It also contains small lymph vessels which carry white blood cells to the tooth to help fight bacteria.
The extension of the pulp within the root of the tooth is called the root canal. The root canal connects with the surrounding tissue via the opening at the tip of the root. This is an opening in the cementum through which the tooth’s nerve supply and blood supply enter the pulp from the surrounding tissue.
Unfortunately, dental caries is one of the most important problems in human oral diseases (Namba et al., 1982). It is known that streptococcus mutans play one of most important roles in dental caries (Jessica et al., 2007). During metabolizing carbohydrates, streptococcus mutans produces organic acid, which can induce the demineralization of tooth surface and results in dental caries (Ooshima et al., 2000).
Some literatures disclosed that the dental plaque pH after a sucrose rinse can decrease to 4.5 or even 4 (Hefferren et al., 1981; Thylstrup et al., 1986). A pH of higher than 6 is considered to be the safe area, a plaque pH of 6.0~5.5 is the potentially cariogenic area, and pH of 5.5~4 is the cariogenic or dangerous area for cavity formation. Therefore, the maintenance of a higher pH value in the plaque is very important for the anticaries.
The fluoride has been used for about five decades in caries prevention. However, it is difficult to control its quantity to a proper level (Nakajo et al., 2008); excessive fluoride is harmful to human body and insufficient fluoride will not take effect for anticaries action. In recent years much attention has been paid to developing fluoride-free techniques that can prevent human teeth from caries(Scherp et al., 1971; Allakera and Ian Douglas, 2009).
Some natural products were reported to be candidates of new anticariogenic substances (Shouji et al., 2000; Matsumoto et al., 1999). However, there is not scientific evidence about the effect of natural tourmaline on the streptococcus mutans.
Tourmaline is a kind of electropolar mineral belonging to the trigonal space group of R3m, whose general chemical formula can be written as XY3Z6Si6O18(BO3)3W4, where X is Na+, Ca2+, K+, or vacancy; Y is Mg2+, Fe2+, Mn2+, Al3+, Fe3+, Mn3+, Cr3+, Ti4+ or Li+; Z is Al3+, V3+, Cr3+, or Mg2+; and W is OH-, F-, or O2- (Castañeda et al., 2006). Its Crystal structure of tourmaline is shown in Figure 4 (Fuchs et al., 1998).
The most important feature among the electric properties of the tourmaline is the possession of spontaneous and permanent poles, which would produce an electric dipole, especially in a small powder with a diameter of several microns or less (Jin et al., 2003). Therefore, a strong electric field exists on the surface of a tourmaline powder (Nakamura et al., 1994; Zhu et al., 2008). The electric field effect of tourmaline powders can influence the redox potential of water and regulated the pH value of solution (Xia et al., 2006). The maintenance of pH value is very important for the growth and metabolism of bacteria (Esgalhado et al., 1995).
Therefore, the graded composite materials were fabricated with nano-tourmaline and nano-hydroxyapatite powders by direct inkjet colour printing for the study of inhibiting function on streptococcus mutans. Moreover, the mechanism by which tourmaline inhibits the growth and acid production of streptococcus mutans was also explained from the view of pH value.
2. Printing setup
The printer consists of a printing table and two piezoelectric XJ126 monochromatic print head (Xaarjet Ltd., Cambridge, UK). There are a total of 134 nozzles viewable on the nozzle plate. The active nozzles are numbered 1 to 126, with nozzle 1 referenced to the datum features on the printhead (as shown in Figure 5), linking a ME1+ continuous ink supply system (Epson, Japan) for the different inks.
A PC104 Programmable Multi-Axis Controller (Delta Tau Ltd. California, USA) for the sliding table and print head movement, and a XaarJet HPC for print head operation were incorporated. The control program was XUSB application in windows XP. The XJ126 print head is a 126 nozzle, piezoelectric drop-on-demand array of print width 17.14 mm and nozzle diameter 50 μm. The sliding table moved at 50 mm/s for printing. The gap between the nozzle plate and the printing surface was maintained at 1 mm by Z displacement. The graded colour pattern was written using Adobe Photoshop software and converted to monochrome binary image files, in device-independent bitmap format, for printing pattern on the substrate. The processing flow of an image is shown in Figure 6.
3. Materials and methods
Tourmaline powders were from the Inner Mongolia Autonomous Region of China, whose chemical compositions were analyzed by EDAX Phoenix energy dispersion spectroscope (EDS) and given in the mass ratio as follows: Al2O3, 34.98%; B2O3, 10.94%; K2O, 0.036%; Na2O, 0.91%; CaO, 2.534%; MgO, 0.2%; SiO2, 34.6%; Fe2O3, 15.8%. The preparation of nano-hydroxyapatite powders was reported in Ref. (Shih et al., 2004). The streptococcus mutans was purchased from Tianjin Medical University, China. Brain heart infusion (BHI) broth, mitis salivarius agar (MSA) and phenol red broth were purchased from Hangzhou Tianhe Microorganism Reagent Co., Ltd., China. Other reagents were analytical.
These tourmaline and hydroxyapatite inks with ethyl alcohol as the carrier and oleic acid as the dispersant had a solid loading of 15 and 20 wt%, respectively. After mixing the powders in ethyl alcohol, the suspensions were kept in the ultrasonicator for 1 h. Then, sedimentation experiments for more than 24 h confirmed that this was indeed the case. To provide the visual appreciation of the graded materials fabricated, the tourmaline suspension was mixed with 20 wt% of the commercial XaarJet magenta ink, whereas the hydroxyapatite suspension was mixed with 20 wt% of the XaarJet cyan ink. The streptococcus mutans can be cultured on the graded materials.
The compositional profiles were determined using a scanning electron microscope (Philips XL30, Amsterdam, Holland) equipped with energy dispersive spectrometry (EDS). The X-ray diffraction (XRD) analysis was performed on a Philips-X’Pert TMD diffractometer with Cu Kα radiation (λ= 0.154056 nm). The patterns were scanned over the 2θ angular range 10~60° at a scan rate of 0.03° 2θ s-1.
The growth of streptococcus mutans was examined at 37 ℃ in 0.95 ml of BHI broth containing 1% glucose and various concentrations of tourmaline. These tubes were inoculated with 0.1 ml of an overnight culture grown in the BHI broth, and incubated at 37 ℃ for 1 day. The optical density (OD) of cells was measured spectrophotometrically at 600 nm.
The filter-sterilized tourmaline was added to 0.95 ml of the phenol red broth containing 1% glucose, which was then inoculated with 0.05 ml of the seed culture of streptococcus mutans. The cultures were incubated at 37 ℃ for 1 day, and the pH of the cultures was determined using a precise pH meter (PHS-3B, Shanghai Precision & Scientific Instrument Co., Ltd., China).
Fifteen grams of tourmaline were added with 100 ml of bacterial suspensions. The initial pH of the medium was changed between 5.0 and 11.0, whereas the temperature was constant at 37 ℃. The pH of the bacterial suspension at different time was measured with precise pH meter mentioned above.
Ten replicates were made for each test. Data were analyzed using the statistical package for social sciences (SPSS). The data were expressed by the mean ± S.D. Differences between means of the experimental and control groups were evaluated by the Student’s t-test. The level of significance for statistical analyses was 0.05.
4. Results and discussion
XRD patterns of the tourmaline powder is shown in Figure 7. From Figure 7, we can see that the main crystal structure of the tourmaline powder does not change apparently and no other phase of tourmaline can be found before and after printing, indicating that the crystal structures of tourmaline powders were not changed. Through calculation, the crystal volume had no change either.
Graded materials were printed using 10 overprinted layers at a printer defined resolution of 185 dot-per-inch (dpi). As shown in Figure 8(a), the tourmaline in the two graded materials of dimensions 10 mm×10 mm increases with a linear gradient from the left to the right. And in Figure 8(b), the tourmaline in the elliptical two graded object increases with a linear gradient from the center to the edges. These compositional profiles of graded materials match that of the design very well.
The streptococcus mutans was cultured on the graded materials in Figure 8(a). The compositional profiles of the graded materials before and after culturing streptococcus mutans determined by using an energy-dispersive spectrometer are shown in Figure 9.
As shown in Figure 9(a), before culturing streptococcus mutans, the graded compositional profiles vary in a linear distribution and match the designed composition. After cultured on the graded materials, the streptococcus mutans shows a reverse distribution to that of tourmaline (Figure 9(b)). To further investigate the effect of tourmaline on streptococcus mutans, the bacteria were exposed to 10, 30, 70, 100, 150, and 200 mg/ml of tourmaline culture. Figure 10 shows the effect of tourmaline on the growth of streptococcus mutans.
The bacteria were inoculated to BHI broth with each concentration of tourmaline and anaerobically incubated for 1 day at 37℃. The optical density of A600 was read by a spectrophotometer. From Figure 10, it can be seen that the tourmaline has an antibacterial activity against streptococcus mutans in a dose dependent manner, and exhibits significant inhibition at concentrations higher than 30 mg/ml compared to the control group (p < 0.05).
Streptococcus mutans grow in plaque, and releases various organic acids during metabolizing carbohydrates. The organic acids demineralise tooth surfaces and initiate the dental caries. The effect of tourmaline on acid production of streptococcus mutans is shown in Table 1.
|Control||10||5.14 ± 0.023|
|10||10||5.15 ± 0.028|
|30||10||5.90 ± 0.072*|
|70||10||6.87 ± 0.018*|
|100||10||7.97 ± 0.017*|
|150||10||8.50 ± 0.202*|
|200||10||8.52 ± 0.130*|
The decrease of pH was significantly inhibited in the presence of tourmaline (30–200 mg/ml) compared to the control group. Except antibacterial activity of tourmaline, the reason may be that the strong electric field of tourmaline would affect the water molecule in the culture and produce plenty of negative ions, which can neutralize the organic acid in the culture.
The comparison of Figure 8 and Table 1 clearly shows that the antibacterial performance of tourmaline has the similar tendency of increasing pH value of the bacterial suspensions. This result gives the support to the suggestion given by Dashper and Reynolds (Dashper and Reynolds, 2000) that streptococcus mutans exhibit significantly lower maximum culture OD at pH 7.1 compared with at pH 6.0 and 6.3.
The pH value is very important for the growth and metabolism of bacteria (Xia et al., 2006; Matsumoto et al., 1999). In what follows, we will give the mechanism by which tourmaline acts on the inhibition of streptococcus mutans from the view of pH value.
Figure 11 shows the effect of tourmaline on the different pH values of the bacterial suspensions.
In Figure 11, 15 grams of tourmaline were added with 100 ml of bacterial suspensions. The initial pH of the medium was changed between 5.0 and 11.0, whereas the temperature was constant at 37℃.
It can be seen that the pH value was shifted toward 8.5 irrespective of whether it was acidic or alkaline. The increase rate of pH value in acidic solution was than the decrease rate in basic solution. Surface adsorption and ion exchange adsorption of H+ helped the increase rate of pH value in acidic solution more than the decrease rate in basic solution.
The electrode characteristics of tourmaline particles influenced the redox potential of water and regulated the pH value of solution to about 8.5, which was consistent with the results of Yoshitake et al. (Yoshitake et al., 1996), Zhan et al. (Zhan et al., 2006) and Xia et al. (Xia et al., 2006). The mechanism of which is that the streptococcus mutans was remained in the culture of pH at about 8.5 self-adjustment induced by tourmaline. The maintenance of pH value was very important for the growth and metabolism of bacteria. This culture inhibits growth and acid production of streptococcus mutans.
In this chapter, the graded biomaterial can be fabricated by inkjet printing. Moreover, to the best of our knowledge, this study is the first to use self-adjustment pH value property of tourmaline for inhibiting the growth and acid production of streptococcus mutans. As the results show, tourmaline can inhibit the growth and acid production of streptococcus mutans, and exhibits significant inhibition at concentrations 30~200 mg/ml, resulting from pH at about 8.5 self-adjustment induced by tourmaline. Therefore, this material has an inhibitory effect on streptococcus mutans, and can be used as a promising biomaterial for preventing human teeth from caries.
The authors would like to thank the “211” Project of Hebei University of Technology, and the Natural Science Foundation of Hebei Province under Grant Numbers: E2004000052 and E2006000039, and the National Natural Science Foundation of China under Grant Number: 60973079, for the financial support.