dentine sensitivity is defined as short , sharp pain from exposed dentine in response to thermal , tactile , osmotic , or chemical stimuli that can not be ascribed to any other dental defect or disease .
clinically the presence of dentine sensitivity creates challenges for both patients and dental practitioners ; in addition to causing patient discomfort , dentine sensitivity can complicate the provision of restorative treatment and the treatment of periodontal tissues [ 3 , 4 ] .
dentine sensitivity is related to the exposure of dentine tubules resulting from either loss of tooth enamel or loss of periodontal tissue ( gingival recession ) [ 5 , 6 ] . for dentine sensitivity to be incited , dentine tubules must be open at the dentine surface as well as remaining patent to the dental pulp [ 6 , 7 ] . at the microscopic level dentine sensitivity
is currently still described by the hydrodynamic theory proposed by brannstrom [ 8 , 9 ] , which states that following dentine exposure to stimuli such as physical touch , temperature alteration , sweet liquid , and acidic liquid , changes in intratubular fluid movement and intratubular pressure occur .
these changes can cause activation of intratubular nerve fibres of pulpal origin ; this nerve excitation causes pain to be experienced [ 8 , 10 ] . to manage dentine sensitivity products
have been developed for at home use functioning primarily through two modalities : suppressing the excitability of intratubular nerve fibres or reducing the patency of exposed dentine tubules [ 2 , 11 , 12 ] .
when acting to suppress the excitability of nerve fibres within dentine tubules , the most common approach is to elevate the extracellular potassium concentration within the dental pulp .
this can be achieved through a patient applying a dentifrice containing potassium ions in a relatively soluble form to exposed dentine .
movement of potassium ions through patent dentinal tubules will act to raise the response threshold of pulpal nociceptors and reduce their ability to fire when provoked [ 13 , 14 ] .
when considering the effectiveness of agents that reduce dentine sensitivity through reduction in the patency of exposed dentine tubules , the most successful outcomes have been achieved through application of fluoride based gels to dentine [ 2 , 11 , 12 ] .
it is well documented that application of dentifrices containing stannous fluoride [ 1517 ] and sodium fluoride [ 1821 ] can promote the deposition of mineral precipitates within open dentinal tubules , thereby reducing fluid flow in dentine tubules following exposure to stimuli .
a dentifrice containing functionalised tricalcium phosphate and 950  ppm sodium fluoride has been shown to enable dentine tubule occlusion in vitro ; application of this dentifrice to extracted bovine teeth following ph cycling was observed to reduce both dentine tubule opening and tubule diameter in comparison to pretreatment levels .
this finding is notable , as not only there is limited data demonstrating that 1000  ppmf dentifrices can effectively reduce dentine sensitivity , but the availability of such a dentifrice could lower the cost and complexity of  at home  treatment undertaken by a patient suffering from dentine sensitivity , a dentifrice containing 1000  ppmf being appropriate for the control of dental caries in low caries risk patients [ 23 , 24 ] .
importantly , while assessment of the anticaries benefits of this dentifrice has been reported , no assessment of the efficacy of this dentifrice to reduce dentine sensitivity in vivo has taken place .
the aim of the present study was therefore to assess the effectiveness of a dentifrice containing 950  ppm fluoride and functionalised tricalcium phosphate ( ftcp ) in the reduction of dentine sensitivity in vivo .
the null hypothesis was that the effectiveness of the dentifrice containing ftcp and 950  ppm fluoride would not be different to the effectiveness of a dentifrice containing 1000  ppm fluoride in reducing dentine sensitivity .
the present study was a single centre , parallel group , blinded ( subjects and examiners ) randomised controlled clinical trial conducted at the westmead centre for oral health , westmead hospital , sydney , australia .
ethical approval from the western sydney local health network human research ethics committee was obtained prior to commencement of the study ( sac2010/11/4.6 ( 3223 ) hrec/10/wmead/202 ) and the trial was registered with the australia new zealand clinical trials register .
subjects included within the study were from the pool of patients eligible to receive treatment at the westmead centre for oral health . from this patient pool , during an initial  off waiting list  examination ,
eight hundred and fifty individuals indicated they suffered from dentine hypersensitivity and were willing to participate in the study .
of these 850 individuals a total of 71 subjects were recruited for the study having met the strict inclusion criteria . all subjects which participated in the study consented to participation prior to the inclusion . as part of the consent process
, patients were made aware of adverse effects of dentifrices , namely , abrasion and staining of hard and soft tissues and the detrimental sequelae of excessive fluoride ingestion . to be considered for this study ,
subjects were required to meet all of the following inclusion criteria : being aged between 18 and 70 years;demonstrating good general health with no history of chronic illness;possession of at least 2 teeth with an exposed root surface which are responsive on probing ( 50  g force ) or a 1  s duration cold air blast ( 70  psi ) ; these teeth were not to exhibit diagnosed caries , defective restorations , or signs of fracture on initial assessment ; these teeth were not to have had dental restorations , periodontal surgery , or orthodontics that has resulted in postoperative pain in the immediate past 3 months;a willingness to read , understand , and sign the consent form;a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion .
being aged between 18 and 70 years ; demonstrating good general health with no history of chronic illness ; possession of at least 2 teeth with an exposed root surface which are responsive on probing ( 50  g force ) or a 1  s duration cold air blast ( 70  psi ) ; these teeth were not to exhibit diagnosed caries , defective restorations , or signs of fracture on initial assessment ; these teeth were not to have had dental restorations , periodontal surgery , or orthodontics that has resulted in postoperative pain in the immediate past 3 months ; a willingness to read , understand , and sign the consent form ; a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion .
a subject was excluded from participation in the study if any of the following conditions applied : use of a desensitising agent in the 3 months prior to the study;undertaking regular medical treatment involving anti - inflammatory or analgesic use;being pregnant or nursing;exhibiting a known allergy to any ingredients in the examined dentifrices;suffering from conditions which could increase the level of acid within the oral cavity : bulimia , gastric reflux disease;excessive dietary exposure to acids ; lemons  2 times per day , raw tomatoes  2 times per day , acidic drinks  1 litre per day ( sports drinks , energy drinks , or fruit juice ) , wine  3 standard glasses per day;an inability to read the oral hygiene instructions provided to each participant . use of a desensitising agent in the 3 months prior to the study ; undertaking regular medical treatment involving anti - inflammatory or analgesic use ; being pregnant or nursing ; exhibiting a known allergy to any ingredients in the examined dentifrices ; suffering from conditions which could increase the level of acid within the oral cavity : bulimia , gastric reflux disease ; excessive dietary exposure to acids ; lemons  2 times per day , raw tomatoes  2 times per day , acidic drinks  1 litre per day ( sports drinks , energy drinks , or fruit juice ) , wine  3 standard glasses per day ; an inability to read the oral hygiene instructions provided to each participant . allocation of subjects to four study groups was randomised through use of a computer algorithm to limit the impact of age , gender , diet , and current level of oral hygiene on the study results .
group a. this group brushed teeth twice daily with a dentifrice containing 1000  ppm fluoride ions ( mfp ) : colgate cavity protection ( colgate - palmolive , new york , ny , usa , ) .
group b. this group brushed teeth twice daily with a dentifrice containing 1000  ppm fluoride ions ( naf ) + 19300  ppm potassium ions ( kno3 ) : sensodyne total care ( glaxosmithkline , sydney , nsw , australia ) .
group c. this group brushed teeth twice daily with a dentifrice containing ftcp and 950  ppm fluoride ions ( naf ) : clinpro tooth crme ( 3 m espe , st .
group d. this group brushed teeth twice daily with a dentifrice containing ftcp and 950  ppm fluoride ions ( naf ) : clinpro tooth crme ( 3 m espe , st .
paul , mn , usa ) and a directed  pea sized  topical application of clinpro tooth crme onto sensitive teeth before sleeping without rinsing . at no time during the trial were subjects made aware of what test group they belonged to or which product they were using ; all packaging was discarded prior to distribution and toothpaste tubes were wrapped in generic sticky white labelling . at the study commencement ,
all subjects were provided with a new toothbrush and dental floss and were given the same oral hygiene instructions verbally and as a take home pamphlet .
subjects were also provided with a two - minute timer in a bid to maintain strict and consistent adherence to the prescribed two - minute brushing time twice daily .
patients were assessed at three time points during the study : baseline , 6 weeks , and 10 weeks .
the clinical assessment was completed by three senior dental officers at westmead centre for oral health to limit the effect of clinician variation on study results .
standardisation was also maintained through each clinician recalibrating the process of stimulus provision to sensitive surfaces at the commencement of each assessment day .
examiners remained blinded to test groups to which participants belonged at all examination points throughout the study .
at each assessment , each sensitive tooth surface was exposed to three different stimuli that were applied directly onto the identified sensitive tooth .
the three stimuli included an air blast , tactile stimulation , and application of a hypertonic solution .
the identified tooth surface was exposed to air delivered from a standard dental unit triplex syringe from an operating distance of approximately 1  cm for a period of 1  s at an operating temperature of 21c ( 5c ) .
a pressure gauge was mounted to the dental unit and calibrated prior to each assessment day to ensure air was delivered at a standard pressure of 70  psi . 

the identified tooth surface was stroked for 3  s using a standard dental explorer probe that was held perpendicular to the surface using a force of 50  g. 
 hypertonic solution .
a 70% hypertonic sucrose solution was applied to the identified tooth surface for 3  s. the solution was at room temperature at the time of application . following application of each stimulus patients
rated the pain / sensitivity experienced using an 11-point numbered pain rating scale ( nrs-11 ; figure 1 ) .
these scores were recorded on clinical test forms . following the 6-week examination patients returned the study dentifrice
wash - out  period to elapse before reexamination at 10 weeks to quantify any prolonged actions of each test dentifrice ; 6-week usage of a dentifrice has been shown to provide sufficient time to allow maximum benefit of a desensitising product .
patient compliance in the use of the study dentifrice was established at the 6-week examination .
patients were asked to return products which were issued and each tube was weighed to calculate the total amount of toothpaste used over the 6-week period . for each test group
the mean pain score for each stimulus modality ( air blast , tactile , and hypertonic solution ) was calculated by patient and by tooth at each assessment point ( baseline , 6 weeks , and 10 weeks ) . additionally , the mean pain scores for each test group for each stimulus were combined and divided by 3 to give a total combined modalities sensitivity ( cms ) score at each assessment point .
the percentage change relative to baseline in mean pain score for each stimulus and the percentage change in cms relative to baseline were calculated for each test group .
ancova ( with baseline as the covariate ) with a tukey hsd post hoc test was used for between group comparisons .
of the 850 individuals from the westmead centre for oral health patient pool who indicated suffering from dentine sensitivity , 80 individuals satisfied the inclusion criteria .
these 80 individuals were randomly allocated to the four study groups ( a  d ) .
the study population exhibited a mean age of 40.9 and a range of 1767 years of age .
seventy - one of the 80 subjects completed the 10-week clinical study and complied with the protocol given .
of the participants who completed the study , there were 54 females and 17 males .
no adverse soft tissue or hard tissue effects were observed by the assessing clinicians during the study ; however one participant withdrew from the study reporting an allergic reaction to colgate cavity protection .
the 9 patients that did not complete the study did so as they no longer wanted to attend the recall appointments on the basis of convenience .
at 6 weeks ( end of the treatment phase ) all groups showed a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 12% reduction , sensodyne total care a 19% reduction , clinpro tooth crme ( brushing only ) a 45% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 43% reduction .
the reduction in evaporative sensitivity scores from base line of both clinpro tooth crme groups at 6 weeks was significantly greater ( p  0.05 , 95%  ci ) than the reduction in evaporative sensitivity scores of both the positive control group ( sensodyne total care ) and the negative control group ( colgate cavity protection ) .
there was no significant difference ( p  0.05 , 95%  ci ) in the reduction of evaporative sensitivity scores from baseline when comparing the sensodyne total care and colgate cavity protection groups at the end of the 6-week treatment phase ( tables 1 and 2 and figure 2 ) . at 10 weeks ( four weeks after cessation of treatment )
, all four groups demonstrated a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 54% reduction .
the reduction in evaporative sensitivity scores at 10 weeks for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p  0.05 , 95%  ci ) than the reduction in evaporative sensitivity demonstrated by the negative control colgate cavity protection .
there was no significant difference ( p  0.05 , 95%  ci ) in the reduction in evaporative sensitivity scores at 10 weeks from baseline when comparing groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) . at 6 weeks ( end of the treatment phase )
all groups showed a reduction in tactile sensitivity from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 39% reduction , clinpro tooth crme ( brushing only ) a 44% reduction , and clinpro tooth crme ( brushing + topical application ) a 62% reduction . at 6 weeks the only group to show a significant reduction ( p  0.05 , 95%  ci ) in tactile sensitivity scores in comparison to the negative control group ( colgate total protection ) was clinpro tooth crme ( brushing + topical application ) .
there were no other comparisons that displayed significantly different sensitivity scores ; both clinpro tooth crme groups were not significantly different ( p  0.05 , 95%  ci ) from the positive control group , sensodyne total care ( tables 1 and 2 and figure 3 ) . at 10 weeks ( four weeks after cessation of treatment ) , not all groups showed a reduction in tactile sensitivity from baseline , with colgate cavity protection showing a 6% increase in sensitivity .
however , sensodyne total care showed a 37% reduction in tactile sensitivity , clinpro tooth crme ( brushing only ) a 14% reduction , and clinpro tooth crme ( brushing + topical application ) a 64% reduction from baseline . a significant reduction ( p  0.05 , 95%  ci ) in tactile sensitivity at 10 weeks from baseline for clinpro tooth crme ( brushing + topical application ) and sensodyne total care in comparison to colgate cavity protection was observed .
no significant difference ( p  0.05 , 95%  ci ) in the reduction in tactile sensitivity scores from baseline to 10 weeks was observed between clinpro tooth crme ( brushing only ) and sensodyne total care .
additionally , no significant difference ( p  0.05 , 95%  ci ) in tactile sensitivity score reduction at 10 weeks was observed between clinpro tooth crme ( brushing only ) and colgate cavity protection ( tables 1 and 2 and figure 3 ) . at 6 weeks
all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 32% reduction , sensodyne total care a 41% reduction , clinpro tooth crme ( brushing only ) a 40% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 61% reduction . at 6 weeks the difference in % reduction from baseline was not significant ( p  0.05 , 95%  ci ) for any of the four groups ( tables 1 and 2 and figure 4 ) . at 10 weeks
, all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 42% reduction , sensodyne total care a 56% reduction , clinpro tooth crme ( brushing only ) a 22% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 66% reduction .
the only groups to exhibit a significant difference ( p  0.05 , 95%  ci ) in hypertonic sensitivity reduction at 10 weeks compared to baseline were sensodyne total care and clinpro tooth crme ( brushing + topical application ) .
there was no significant difference ( p  0.05 , 95%  ci ) in hypertonic sensitivity reduction at 10 weeks between the groups using colgate cavity protection , sensodyne total care , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 4 ) . at 6 weeks ( end of the treatment phase )
all groups exhibited a reduction in the combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 30% reduction , clinpro tooth crme ( brushing only ) a 42% , and clinpro tooth crme ( brushing + topical application ) showing a 52% reduction . at the end of the 6-week treatment phase , both clinpro tooth crme study groups showed a significant reduction ( p  0.05 , 95%  ci ) in cms when compared to the negative control group using colgate cavity protection .
clinpro tooth crme when used with an additional topical application also demonstrated a significant reduction ( p  0.05 , 95%  ci ) in dentine sensitivity compared to the positive control , sensodyne total care , after 6 weeks ( tables 1 and 2 and figure 5 ) . at 10 weeks ( 4 weeks after cessation of treatment ) , all groups showed a reduction in combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing an 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) a 54% reduction .
the reduction in cms for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p  0.05 , 95%  ci ) than the reduction in cms from baseline of the negative control colgate cavity protection at four weeks after cessation of treatment .
there was no significant difference ( p  0.05 , 95%  ci ) in cms reduction from baseline to 10 weeks between groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 5 ) .
the ability for fluoride ions sourced from a dentifrice to form fluoride - calcium precipitates that occlude dentine tubules with the consequence of reducing dentine sensitivity is well documented [ 1821 , 27 , 28 ] .
this ability occurs due to the negative electric charge of fluoride ions , which results in their binding with calcium cations .
once bound to calcium cations present within a dentifrice the fluoride ions are rendered incapable of combining with cations present at the tooth surface . to overcome the effect of reduced fluoride ion availability at the tooth surface caused by intradentifrice fluoride - calcium bonding
, dentifrice manufacturers have traditionally acted to increase the concentration of fluoride ions within a dentifrice . in recent times , however , altering the chemical structure of calcium complexes within a dentifrice to reduce the bonding affinity between calcium cations and fluoride anions has been undertaken as an alternative solution to simple fluoride ion concentration increase .
one such example of this has been the development and incorporation of altered calcium phosphate complexes , such as functionalised tricalcium phosphate , which is currently incorporated within clinpro tooth crme . functionalised tricalcium phosphate ( ftcp ) is produced through beta tricalcium phosphate ( tcp ) complexes being milled with sodium lauryl sulfate ( sls ) .
the tcp crystal structure exhibits several reactive sites including calcium - oxygen clusters ( cao3 , cao7 , and cao8 ) and lattice defects .
these reactive sites are available to undergo chemical interaction with anions such as fluoride ions . to reduce the reactivity of these sites within b - tcp complexes
the incorporation of sls within the tcp structure therefore impedes fluoride ions from combining with calcium ions in the dentifrice , so in turn potentially increasing the concentration of both calcium and fluoride to tooth surfaces .
[ 25 , 3234 ] have reported a synergistic relationship between ftcp and fluoride with regard to enamel remineralisation ; enamel that is remineralised through a fluoride / ftcp combination demonstrates significantly greater surface and subsurface rehardening following ph cycling compared to that achieved by fluoride application alone .
notably , the results of the present study suggest that this remineralisation synergy between fluoride and ftcp may also produce a benefit in terms of enhancing dentine tubule occlusion ; the two test groups that utilised clinpro tooth crme ( 950  ppm f ) demonstrated a greater level of sensitivity relief for all stimuli except the hypertonic test at 10 weeks ( group c ) in comparison to the group using colgate total , despite the fact that colgate total contains a greater concentration of fluoride ions ( 1000  ppm f ) .
previous microscopic analysis showing the ability of clinpro tooth crme to reduce the diameter of tubule openings following ph cycling to a greater degree than sensodyne nupro 5000 and topex renew supports this possibility . within the present study a greater reduction in dentine sensitivity
was observed when an additional topical application of clinpro tooth crme was placed to supplement application provided through brushing alone .
this finding is in contrast to reports in the literature stating that there is no evidence to suggest that additional topical application increases the effectiveness of a desensitizing dentifrice .
first an intentional topical application of a dentifrice potentially allows a greater concentration of fluoride ions to be applied to a tooth surface in comparison to that applied through  unintentional  brushing alone .
secondly , through an additional topical application , fluoride ions can remain at a sensitive surface for a longer duration than following brushing and rinsing and thirdly through increasing the duration of fluoride ion presence upon a sensitive surface , the propensity for fluoride migration and tubule occlusion is raised .
the greater success of clinpro tooth crme when applied as an additional topical application rather than brushing alone may also be a result of greater salivary fluoride concentration as a by - product of topical application and no rinsing following application .
this possibility is consistent with studies that have examined the effect of patient activity following dentifrice application on the level of salivary fluoride .
nordstrm and birkhed determined that a topical application of dentifrice to a tooth surface combined with regular brushing resulted in a greater concentration of salivary fluoride than when compared to brushing alone .
sjgren and melin identified that a single post - brushing rinse with fluoridated water decreased fluoride concentration by a factor of two when compared to no rinsing , while rinsing with fluoridated water two times following brushing decreased salivary concentration by a factor of 5 when compared to no rinsing .
the reduction in cms of the negative control group over the study duration can be in part attributed to the placebo effect which is well documented in dentine sensitivity studies [ 7 , 38 , 39 ] .
additionally the hawthorne effect , which describes a positive response to noninterventional treatment , should also be assumed to have had some impact on the study results . improved oral hygiene in patients enrolled in the study
could have also reduced perceived dentine sensitivity across all groups as a reduction in tooth surface plaque facilitated increased dentifrice access to dentine tubules .
these reasons may also account for the antihypersensitivity effects continuing for the month following the cessation of the use of the treatment dentifrices .
an identified limitation of the study was the numbers of patients enrolled in each study group . due to the breadth of the potential patient pool , patients eligible for treatment at westmead centre for oral health ,
it was anticipated that including 40 subjects per group would be an achievable goal over a 3-year study period . as a result of the very strict exclusion criteria , especially the requirement that the use of a desensitising agent in the 3 months prior to the study excluded an individual from being a subject , patient recruitment was extremely difficult .
however despite the smaller numbers than originally forecast per group , the results of the present study provide useful information . not only are the numbers sufficient to demonstrate statistical significance , but when tooth number rather than patient number is used for analysis the subject number exceeds 50 for each group and the statistical outcomes remain the same .
the addition of ftcp to a dentifrice suitable for daily oral hygiene can enhance the ability of dentifrice fluoride to reduce dentine sensitivity . in the present study
twice daily brushing with clinpro tooth crme resulted in a similar reduction in dentine sensitivity to that achieved through brushing with a dentifrice containing kno3 + f ( sensodyne total care ) .
if clinpro tooth crme is used twice daily for brushing in combination with a nightly topical application , it can be more effective in reducing dentine sensitivity than twice daily brushing with sensodyne total care .
 
 background . to assess the clinical efficacy of a dentifrice containing fluoride and functionalised tricalcium phosphate ( ftcp ) in reducing dentine sensitivity . methods . a 10-week parallel blind randomised control trial was conducted . 
 subjects were assigned to one of four groups and instructed to brush twice daily : a : colgate cavity protection ( 1000  ppmf - mfp ) ; b : sensodyne total care ( 1000  ppmf - naf + 19300  ppmk+-kno3 ) ; c : clinpro tooth crme ( 950  ppmf - naf + ftcp ) ; and d : clinpro tooth crme ( brushing + additional topical application ) . 
 seventy - one patients were assessed at baseline , 6 weeks , and 10 weeks for cold , tactile , and hypertonic sensitivity using the nrs-11 pain rating scale . a combined modalities sensitivity score ( cms ) was calculated . results . at 6 weeks , patients reported the following reduction in cms : a ( 20% ) ; b ( 30% ) ; c ( 42% ) ; d ( 52% ) . at 10 weeks , patients reported the following reduction in cms : a ( 18% ) , b ( 40% ) , c ( 24% ) , and d ( 54% ) . 
 the only cms comparisons to show a significant difference ( p < 0.05 ) were between groups a and d ( 6 and 10 weeks ) . 
 conclusions . 
 addition of ftcp to a dentifrice enhances the ability of dentifrice fluoride in reducing dentine sensitivity . 
 using clinpro tooth crme twice daily for brushing can be as effective to reduce dentine sensitivity as twice daily brushing using sensodyne total care 
 . however , additional nightly topical application of ftcp , in addition to twice daily brushing , showed an enhanced reduction in dentine sensitivity . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions