MATERIALS AND METHODS

Rectangular shaped dentin specimens were prepared from the bovine incisor roots. The specimens’ surfaces were ground flat and then polished by diamond slurries with particle sizes up to 0.25 μm. Specimens were washed in an ultrasonic bath with deionized water, ethanol and again with deionized water each for 15 min to remove smear plugs and micro-biomes. Scanning was carried out using a non-contact profilometer (NCP) with a red laser light source (VK-X150/X160, Keyence Co., Ltd., Osaka, Japan) and images were saved.

RESULTS AND DISCUSSION

The biofilms that were formed on the dentin specimens in 24 h appeared creamy-white slimy substances on necked eyes and top surface was not smooth characteristically. CLSM 3D images confirmed that showing mountains, valleys and river-like channels running in between (Figure 1, left). In this particular image, the height of the biofilm (or vertical thickness) appeared to be more than 60 µm as color gradation displayed unevenness between the biofilm clusters. Most interestingly, the top-most surface of individual clusters was not essentially smooth; porous appearances were remarkable in between already smoothened parts. CLSM 3D images of 7D biofilm have shown further maturation; in vertical direction in exceeded 240 mm (Figure 1, right). Color gradation clearly indicated that mountains, valleys continued growing - though river-like channels were disappearing and most of the top surface became significantly smooth. Also, porousness could not be detected - instead they appeared as low-pitched rough surfaces at this stage. Deep ditch-like whole could be detected.

Root dentin surface images are shown in Figure 2. The surface was remarkably smooth with open dentinal tubules - open tubule size was very small as obvious in the dentin before biofilm attack (left). After 1D biofilm attack the surface became rougher and tubular orifice became wider which are easily detectable on the image (middle). As biofilm attack continued for 7 days the surface roughened further and tubular orifice widened remarkably as demineralization continued (right). Some notable information with 3D color gradation of the biofilm and dentin demineralization was acquired with this VK-X series of CLSM. The VK-X achieves high-resolution sensing by using a 16-bit photomultiplier as its laser receiving element. Also, it is capable of nanoscale measurements, even from a distance. The non-contact profilometeric image of the unstained biofilm could be taken in less than a minute unlike other biofilm imaging reported so far [11]. Therefore, biofilm maturation can be monitored at any stage without disturbing their growth. Also, the same biofilm specimen can be analyzed using any other method as the partially air dried biofilm remains unharmed. Identification of porous areas was possible due to the penetration and return of CLSM laser through premature or loosely condensed matrix of the biofilms and that would help in determining the time of application of antibacterial agents during in vitro studies. Clear images of normal dentin and demineralized dentin are also helpful for many in vitro experiments; e.g. testing degree of demineralization and remineralization of dental hard tissues [12].

1. Featherstone JDB (2008) Dental caries: A dynamic disease process. Aust Dent J 53: 286-291.
2. Scheie AA, Petersen FC (2004) The biofilm concept: Consequences for future prophylaxis of oral diseases? Crit Rev Oral Biol Med 15: 4-12.
3. Black GV (1886) Gelatine forming micro-organisms. Independent Practitioner 7: 546-548.
4. Loesche WJ (1986) Role of Streptococcus mutans in human dental decay. Microbiol Rev 50: 353-380.
5. Bourbia M, Ma D, Cvitkovitch DG, Santerre JP, Finer Y (2013) Cariogenic bacteria degrade dental resin composites and adhesives. J Dent Res 92: 989-994.
6. Hayati F, Okada A, Kitasako Y, Tagami J, Matin K (2011) An artificial biofilm-induced secondary caries model for in vitro studies. Aust Dent J 56: 40-47.
7. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, et al. (2002) Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci 99: 14434-14439.
8. Li J, Wang W, Wang Y, Zeng AP (2013) Two-dimensional gel-based proteomic of the caries causative bacterium Streptococcus mutans UA159 and insight into the inhibitory effect of carolacton. Proteomics 23-24: 3470-3477.
9. Gyo M, Nikaido T, Okada K, Yamauchi J, Tagami J, et al. (2008) Surface response of fluorine-polymer incorporated resin composites to cariogenic biofilm adherence. Appl Environ Microbiol 74: 1428-1435.
10. Oyanagi T, Tagami J, Matin K (2012) Potentials of mouthwashes in disinfecting cariogenic bacteria and biofilms leading to inhibition of caries. Open Dent J 6: 23-30.
11. Bowen WH, Koo H (2011) Biology of Streptococcus mutans-derived glucosyl transferases: Role in extracellular matrix formation of cariogenic biofilms. Caries Res 45: 69-86.
12. Fernández CE, Tenuta LMA, Del Bel Cury AA, Nóbrega DF, Cury JA (2017) Effect of 5,000 ppm fluoride dentifrice or 1,100 ppm fluoride dentifrice combined with acidulated phosphate fluoride on caries lesion inhibition and repair. Caries Res 51: 179-187.