the use of nanoparticles to polish tooth surfaces show the advantage of attaining faster polishing and smoother surface of teeth . it is a known observation in the modem semiconductor industry ; as discussed by lu , z ., n . p . ryde , et al . ( 2005 ) in “ particle adhesion studies relevant to chemical mechanical polishing ” langmuir 21 ( 22 ): 9866 - 72 hereby incorporated by reference ; that in general the surfaces polished with nanosize abrasive particles get noticeably smoother . however , as known in the same chemical mechanical polishing ( cmp ) methods , it is not guaranteed that one gets smoother surface just taking finer abrasive particles . cmp companies have spent millions of dollars to insure that property . here we disclose the use of polishing slurry that leads to nanometer - scale roughness of polished tooth surfaces . furthermore , we disclose that bacteria adhered to smoother surfaces can be more easily removed than from rough surfaces , thereby preventing them from formation of plaques and ultimately keeping bacteria away from tooth surfaces . this is not an obvious result because bacteria attaches to the tooth surface via biochemical means , and therefore can simply ignore the said difference in roughness before and after the polishing . in general , the forces of interaction of an organic substance with flat surfaces are comparatively weaker than with rough corrugated surfaces because of the increased area of the rough surface compared to the flat one . in the case of bacterial attachment , it may not necessarily be that simple . for example , bacteria could still strongly attach even to a rather smooth surface , so the difference in the adhesion could be negligible when we consider the nanoscale roughness . we show that the addition of nanoparticles makes a toothpaste or a polishing slurry that utilizes such abrasives helpful to remove the bacteria , and consequentially , protect teeth against damaging by cariogenic bacteria . while particles can be of any biocompatible nature ( ceria , gold , silica , diamond , alumina , titania , and so on ), various shapes , and the sizes from 0 . 001 to 0 . 1 microns , as an example , we demonstrate the use an abrasive with 60 nm spherical silica particles . the atomic force microscope ( afm ) is a relatively novel method of measurement as discussed by binnig , g ., c . f . quate , et al . ( 1986 ) in “ atomic force microscope .” phys . rev . lett . 56 ( 9 ): 930 - 933 , hereby incorporated by reference . this technique is based on the detection of forces acting between a sharp tip , the afm probe , and sample surface , for example , tooth surface . fig1 illustrates the schematics of the afm setup . the afm probe is attached to a flexible cantilever . a laser beam is reflected from the cantilever and detected by a photodiode . thus , any vertical displacements of the cantilever are detected by the photodiode . when scanning , the probe is brought to a contact , engaged with the surface of interest . scanning over the surface , the afm system records the deflection of the cantilever with sub - nanometer precision . the deflection signal ( or any derivatives of the deflection ) is recorded digitally , and can be visualized on a computer in real - time . the afm technique is much more than just simply the microscopy described above . one can think about the afm probe as a microscopic “ finger ” with a nanosize apex . here we use the afm probe as a sort of a soft brush to remove bacteria from the tooth surface while imaging the tooth surface in contact mode . this method will be described in more detail below . here we use a nanoscope ™ dimension 3100 afm ( veeco / di instruments , inc ., santa barbara , calif .). a standard cantilever holder cell for operation in liquids is employed . roughness measurements are done with the built - in afm software ( version used 5 . 12 , release 4 ). all scanning measurements are performed on the tooth surface immersed in deionized water . a v - shaped standard wide 200 μm afm cantilevers ( veeco , santa barbara , calif .) were used throughout our study . the contact mode is used for afm imaging . the scan speed of 2 - 4 hz is used while imaging in contact mode , 15 hz is used for the raster mode used to monitor removal of the bacteria . when using the rastering mode , the scan angle is set to 90 degrees to provide homogeneous lateral force ( friction force ) responsible for the bacteria removal . streptococcus mutans ( wards - lyophilized bacteria 85 - 1957 ) are received as freeze - dried . it is regenerated by dissolving in 5 ml of culture medium ( tryptic soy broth ) for a minute , and then , placing tooth sample in the growth medium overnight . about 5 ml of the bacterial solution ( bacteria and culture medium ) is centrifuged for about 5 min at 3000 rev / min and the supernatant is discarded carefully to retain the pellet of bacteria at the bottom of the test tube . the pellet is then resuspended in 3 ml of deionized water to wash away the culture medium and acid produced by the bacteria . in culture medium , the optimum ph for growth of a bacterium must be considered and hence sodium acetate buffer is used to maintain the ph of the medium depending on bacterial waste products ( acids ) that accumulate during growth . most bacteria exhibit a relatively narrow range of ph over which they grow . also they grow over a temperature range of about 37 degrees . the bacteria is again centrifuged and the pellet is resuspended in a solution of 15 % glucose and sodium acetate ph 5 . 0 ( the buffer to maintain the ph of the culture medium ). this bacterial solution is applied on tooth surface to form films . to grow the bacterial films on the tooth surface , the tooth samples are then immersed in bacterial suspension overnight in water bath ( american optical model # 406015 ) at 37 ° c . the sample is rinsed with di water from the sides to remove excess of bacterial colonies from the sample . the tooth samples used for the research are milk teeth . the surfaces are not treated with any chemicals for our study . the milk tooth is cut into four pieces using a cutter and each piece is fixed to a glass slide with water insoluble glue ( loctite e - 30cl hysol epoxy adhesive ) with their enamel facing up . the glue is allowed to cure overnight . the abrasive slurry used here is prepared from 60 nm colloidal silica particles in aqueous medium of neutral ph ( is adjusted with acetic acid ( fisher scientific )). the particles are used from a commercial slurry ( klebosol , rodel ild 1300 ). the polishing is done with a polyurethane polishing pad ( rodel ic 1400 ) attached to a polishing machine ( lecco corporation gp - 10 , grinder and polisher ). a load of 5 g is attached to the end of glass slide where the tooth is glued . the sample to be polished is held gently on the rotating polishing pad . there is a constant flow of slurry . after about 20 sec the tooth is viewed under an optical microscope to check for the amount of polish . in order to image the tooth surface under afm the tooth is removed from the glass surface and glued to a petri dish ( 35 × 10 mm ). the tooth sample is ready to be immersed in bacterial suspension . the tooth samples are immersed in bacterial suspension overnight in water bath ( american optical model # 406015 ) at 37 ° c . the sample is rinsed with di water from the sides to remove excess of bacterial colonies from the sample . the polishing paste used for the study is the commercially available crest ® cavity protection regular paste ( proctor & amp ; gamble , inc .) and a specialized paste nupro ® with fluoride ( orange — fine grit as well as mint — medium grit ( dentsply caulk , inc .) used by dentists for cleaning of tooth surface . the abrasives used in crest ® tooth paste are of the order of micrometers whereas those in the nupro ® paste range from 1 - 177 μm depending on whether it is fine or coarse grit . the tooth paste crest ® as well as nupro ® ( 5 g ) are dissolved in 10 ml of di water and stirred for 5 min . the tooth samples are polished as mentioned above , but the slurry is replaced with the solution of the crest ® and nupro ® pastes , respectively . the sample is observed under microscope for desired polish surface and is engaged for 2 min . aqueous solution of methylene blue dye ( biological grade , sigma - aldrich , inc .) is used to visualize bacteria adhered to the tooth surface . bacterial film as formed on tooth surface is stained with a few drops of methylene blue for about 2 min and then rinsed with water to wash away excess dye from tooth surfaces . such a staining allows optical differentiating between bacteria and dental surface , which stays white while bacteria become bluish . the polishing is done by using a polyurethane pad ( ic1400 , rodel , inc .) attached to a polishing machine ( lecco corporation gp - 10 , grinder and polisher ). a load of 5 gm is attached to the top of one end of glass slide where the tooth is glued , see fig2 . the other side of glass slide is gently held by fingers . a constant flow of slurry of nanoparticles is supplied . after about 20 sec of polishing , the tooth is checked under an optical microscope to estimate the degree of polishing . similar procedure but with longer polishing times of about 1 to 2 min is used when using commercially available crest ® cavity protection regular paste ( procter and gamble , inc . ), and a specialized polishing paste nupro ® with fluoride ( fine grit , dentsply caulk ) used by dentist for cleaning of tooth enamel . the longer times in the latter case are used to attain approximately the same removal of the enamel during the polishing ( as is qualitatively estimated by optical microscopy ). in order to image the tooth surface under afm the tooth sample is placed in a petri dish . we compare roughness of virgin tooth surface and the surface of the same tooth ( split into four pieces ) when polished with , tooth paste , polishing paste , and nanoparticle slurry . optical images of these four surfaces are illustrated in fig3 . fig4 a - d shows representative afm images of the surfaces shown in fig3 a - d . as one can see from these images , the slurry polished tooth exhibited a smoother surface than the tooth paste polished surface . one can see that the surfaces polished with crest ® and nupro ® look even rougher at micron scale than the virgin tooth . this is not very surprising as both toothpastes are not intended to provide the polishing at nanoscale . fig3 illustrates optical images of ( fig3 a ) virgin tooth , ( fig3 b ) a toothpaste polished tooth , and ( fig3 c ) a dental toothpaste ( nupro ®) polished tooth and ( fig3 d ) slurry polished tooth . fig4 a - d illustrates a series 15 × 15 μm 2 afm images of ( fig4 a ) virgin tooth , ( fig4 b ) a toothpaste ( crest ®) polished tooth , and ( fig4 c ) a dental toothpaste ( nupro ®) polished tooth , and ( fig4 d ) a slurry polished tooth . quantitative analysis of roughness is done for the areas of 5 × 5 μm 2 . this is justified by the size of s . mutans bacteria . we assume that the roughness should be analyzed over the area comparable with the bacterium size to contribute to the bacterial adhesion . this is plausible because it is unlikely that a bacterium would develop adhesion to an area much larger than its size . statistical distributions for the roughness obtained are as illustrated in fig5 a - 5d . each roughness measurement corresponds to a region of 5 × 5 μm 2 extracted from a larger 20 × 20 μm 2 image . in total , 8 images of 20 × 20 μm 2 are recorded , and each image divided into approximately 10 - 16 images of 5 × 5 μm 2 . fig5 a - 5d illustrate a roughness distribution for ( 5 a ) a virgin tooth , ( 5 b ) a tooth paste polished tooth , ( 5 c ) a nupro ® polished tooth , and ( 5 d ) a slurry polished tooth surface . the average roughness and standard deviation ( with respect to the normal distribution ) of the distributions of fig5 a - d are shown in table 1 . one can see that on sub 5 - micron level neither crest ® nor nupro ® pastes improved the roughness . this is in agreement with both optical images of fig3 a - d and afm images of fig4 a - d . the surface polished with the slurry of nanoparticles show an order of magnitude decrease in roughness . in the next section we will discuss if this decrease is significant by the bacterial adhesion . table 1 illustrates average roughness and standard deviation of the tooth surfaces both virgin and polished . to correlate the adhesion of s . mutans bacteria to the tooth surface with surface roughness , we used the raster afm scanning method briefly described in the materials and methods section . this technique is analogous to the action of a single fiber of a soft cleaning brush . the afm probe scans / rasters rather fast over the tooth surface . see fig6 . because we are working in the contact mode , the rastering is done with a constant load force . this helps to monitor removal of the bacteria in a highly controlled way . fig6 illustrates a schematic of rastering mode . during the rastering , the afm probe moves left - right fast , while slowly scanning up and down . the bacteria attached to the surface can be removed by the , action of lateral force of the afm probe . this mimics the action of a single fiber of a soft cleaning brush . after the first scans , weakly attached bacteria are removed by the action of lateral force of the afm probe . after several scans , more tightly attached bacteria are also removed . and finally , the bacteria with strongest adhesion are removed last . while rastering , afm can , in principle , collect the images of the surface with the bacteria on it , although quality is insufficiently low . we used those images only to monitor when we need to stop and make a regular , slow afm scan . fig7 a - c illustrate a series of such images of slurry polished tooth surface with s . mutans adhered , which are taken after certain number of scans . the total scanning period is about 2 minutes , completing approximately a dozen full scans . fig7 a is taken after initial couple of scans . it shows exposed relatively small areas of tooth surface , which initially is covered with a continuous layer of bacteria . fig7 b shows the bacterial adhesion after approximately 1 minute . fig7 c demonstrates the remaining bacteria as at the end of the scan series . one can see that the bacteria are initially removed from the smooth area , fig7 a and stayed longer on the rougher one , fig7 b and 7c . quality of the images is enough to find roughness of those corresponding areas . it is estimated that the roughness of the areas in which the bacteria are removed after the first scans ( shown in fig7 c with stars ) ranges within 1 . 8 - 2 . 4 nm . in contrast , the areas in which bacteria are removed only after the complete series of scans ( shown in fig7 c with squares ) have the roughness of 5 . 0 - 5 . 5 nm . thus , we can conclude that the adhesion of s . mutans to the polished tooth surface is well correlated with the roughness at nanoscale . fig7 a - c illustrate a series of afm images of a slurry polished tooth surface with s . mutans adhered . one can clearly see that bacteria are easier removed from the smooth surfaces than those adhered to the rougher surfaces . the afm is a highly precise technique . however , to get a better idea about how representative the results obtained in the previous sections are , we complement those results with a rather simple qualitative method . we monitor the removal of the bacterium film with a regular optical microscopy . to show the advantage of highly polished surfaces for bacteria removal , we use a soft painting brush ( made of squirrel tail fur ). methylene blue is commonly used in gram stains . here we found that this dye allows us clean differentiation between s . mutans and tooth enamel . a continuous bacterial film is stained in blue as seen in fig8 a . the soft painting brush is gently used to remove this film . this is monitored under bright field reflected optical microscope at certain intervals . the gradual removal of the bacterial film from the surface polished with nanoparticle slurry took a minute , fig8 b and 8c . one can see the polished area as a shiny exposed region . ( t junction of two trenches is used as a landmark to find the same area on the optical microscope after cleaning with the brushes .) but in order to get rid of all the bacterial films , a hard brush is used to get a clean surface as seen from the final image , fig8 d . fig8 a - d illustrate optical images demonstrating removal of bacterial films from the partially polished tooth surface at macroscale ( each image is 1 . 5 mm in width ). dark bluish spots are the bacterial films ( stained with methylene blue dye ). fig8 a illustrates a surface completely covered with the film . fig8 b and 8c illustrate surfaces after some gentle cleaning with a soft brush ( fig8 b preceding fig8 c ). fig8 d illustrates the surface after the hard brush cleaning . the dentin abrasion values of various dental products is outlined in u . s . pat . no . 6 , 280 , 707 (&# 39 ; 707 ), hereby incorporated by reference , assigned to dentsply international , inc . describes the product now known as nupro ®. rda ( radioactive dentin abrasion ) value is measured according to the process described in hefferen , j . dent . res ., vol . 55 , no . 4 , pp . 563 - 573 , hereby incorporated by reference . the prophylaxis paste according to the present invention has abrasion values above those of the dentifrices currently known commercially . the abrasive ratings provided below gives an example of the commercial prophy pastes and their relative dental abrasion ( rda ) values . product having dentin abrasion ( rda ) values include nupro ® paste fine grit , 704 ; nupro ® paste medium grit , 256 ; nupro ® paste coarse grit , 362 ; crest ® regular toothpaste , 81 ; crest ® tartar control toothpaste , 126 ; colgate ® tartar control toothpaste , 112 ; aim ® tartar control toothpaste , 127 ; aquafresh ® tartar control toothpaste , 125 and close - up tartar control toothpaste , 120 as shown in the &# 39 ; 707 patent . the slurry polish of the instant invention has estimated rda values in excess of 750 and may exceed 1000 . the rda value as used in the present description may be determined by any of the standard methods including the grabensteder method or the hefferen method . the hefferen method is preferred because it is recognized by the american dental association . while the rda is the method for evaluation the abrasive nature of normally , toothpaste , another method known as the relative enamel abrasion ( rea ) is normally used for evaluating prophy paste . there is no specific correlation between the recognized rda and rea test methods . according to the present invention , a prophy paste having a rea value of preferably above about 5 is within the scope of the invention . in dentistry there is a concern for the existence of too a high a value of abrasiveness . the method described herein minimizes that because the effects of high abrasiveness can be decreased by adding various chemical elements . a high rda is more effective because it polishes faster . as discussed above , the main advantage / difference of the inventive slurry / paste is that the inventive slurry makes the dental surface very smooth . as discussed above afm is used to measure the roughness of dental surface . the data gathered from such a measurement may be used to develop a measurement of the abrasiveness and effectiveness of various tooth pastes . then illustrative embodiments and modifications thereto described hereinabove are merely exemplary . it is to be understood that other modifications to the illustrative embodiments will readily occur to persons of ordinary skill in the art . all such modifications and variations are deemed to be within the scope and spirit of the present invention as will be defined in the accompanying claims . binnig , g ., c . f . quate , et al . ( 1986 ). “ atomic force microscope .” phys . rev . lett . 56 ( 9 ): 930 - 933 . lu , z ., n . p . ryde , et al . ( 2005 ). “ particle adhesion studies relevant to chemical mechanical polishing .” langmuir 21 ( 22 ): 9866 - 72 .
a method for polishing tooth surface which eases removal of a bacterial film later grown on the polished surface . the method includes the steps of providing a providing a toothpaste or slurry comprising a plurality of inorganic abrasive particles smaller than 100 nm in size , the inorganic abrasive particles comprising one or more of silica , ceria , titania , zirconia , silicon nitrite , and silica carbide , wherein the inorganic abrasive particles are sufficiently hard to be abrasive ; and polishing a tooth surface with the toothpaste or slurry to attain an ultra - smooth tooth surface which can be easily cleaned .