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INTRODUCTION
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].
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