smear layer is an amorphous , granulated , and irregular layer covering the root surface when observed under scanning electron microscope ( sem ) . it is constituted of inorganic ( calcium , phosphate ) , and organic material ( odontoblastic process , bacteria , and blood cells ) and bacterial products ( endo and exotoxins ) resulting in hypermineralization of root surface which may function as a physical barrier to the growth of a connective tissue attachment to the root surface . scaling and root planing eliminate the mineralized debris present on periodontally involved root surfaces , but promotes the formation of this thin residous smear layer that might delay the adhesion of new fibroblasts and connective tissue . conditioning of root surfaces by topical application of acid solutions had been introduced as a periodontal regenerative procedure . root biomodifiers have been found to be effective in eliminating adhered organic and inorganic material and retained toxins from the altered root surface , furthermore , they have shown to expose dentin collagen and cementum bound proteins , resulting in a root surface which is conducive in promoting periodontal regeneration . a number of agents have been proposed for the root conditioning including tetracycline hydrochloride ( ttc hcl ) , citric acid ( ca ) , and ethylenediaminetetraacetic acid ( edta ) . the concept of acid demineralization in periodontal therapy was first introduced in the 1800s as a substitute for scaling and calculus removal . ca alters the external characteristics of root surfaces , removes the smear layer after scaling and root planing , demineralizes the treated surface leaving a  mat - like  collagen surface with exposed dentinal tubules , and eliminates bacterial endotoxins from pathologically altered cementum surfaces . tetracycline hcl is an effective antibiotic which is absorbed onto the root surface and slowly released in its active form ( substantivity ) . it acts as calcium chelator , resulting in demineralization of the root surface and removal of the smear layer . furthermore , it is having a potent anti - inflammatory and anti - collagenase activity . ethylenediaminetetraacetic acid is a decalcifying agent operating at a neutral ph by chelating the divalent cations . it preserves the vitality of the remaining periodontal cells close to the root surface , and also has the advantage of biocompatibility ( ph = 7.0 ) as compared to other root conditioning agents . biopure mtad ( dentsply , tulsa dental , usa . ) has been used as an antibacterial root canal irrigant . it stands for a mixture of ttc isomer ( doxycycline ) , acid - ca , detergent - tween80 . it has the ability to remove the smear layer and also exert a potent antimicrobial activity . in the present study , mixture of tetracycline , acid , and detergent ( mtad ) has been used as a novel approach for root biomodification . on the basis of above facts , the present in vitro study was aimed to evaluate and compare novel root canal irrigant and other root biomodifiers for smear layer removal on periodontally involved human teeth . the study sample consisted of 40 freshly extracted single - rooted , periodontally compromised teeth with hopeless prognosis and caries free root surfaces . the teeth with wasting diseases , tooth fracture , endodonticaly treated , and prosthodonticaly restored were excluded from the study . written informed consent was taken prior to extraction from all the patients and ethical committee clearance from the institution was obtained . following extraction samples samples were obtained from the cervical third of the root by making two parallel grooves with a cylindrical bur under copious saline irrigation . the first groove was positioned horizontally at cementoenamel junction and second groove made parallel and 4 mm apical in relation to the first . the diseased root surfaces of all teeth were scaled with an ultrasonic scaler and thoroughly planed with # 12 , 34 gracey curettes ( hu - freidy ) to remove all the diseased cementum . with the help of diamond disc under copious irrigation , the two samples are obtained first by transverse sectioning the root from the grooves and secondly by sectioning the sample longitudinally into two from the middle . total 80 samples were randomly divided into 4 groups : 
 group a : biopure mtad [ figure 1]group b : tetracycline hcl ( 50mg / ml)group c : citric acid ( ph 1.0)group d : ethylene diaminetetraacetic acid ( 15% ) . group a : biopure mtad [ figure 1 ] group b : tetracycline hcl ( 50mg / ml ) group c : citric acid ( ph 1.0 ) group d : ethylene diaminetetraacetic acid ( 15% ) . these agents were freshly prepared every time and were applied by  active burnishing  on the curetted root surfaces for 3 min . the cotton pellets are changed every 30 seconds to ensure consistent solution application . following treatment , all samples were dehydrated in a graded series of ethanol ( 1090% ) for 30 min each and finally in 100% acetone for 30 min more . the samples were dried under the lamp and then mounted on the aluminum stubs and inserted in sc7640 sputter coater machine for gold / palladium coating on specimens . all the specimens were examined in a polaron - sem ( leo-430 ) at a magnification of 3500 and photomicrographs were evaluated to ascertain the extent of root biomodification by removal of smear layer , patent dentinal tubules in relation to the total number of dentinal tubules . the photomicrographs were distributed to three calibrated , trained blind examiners to determine the degree of smear layer removal according to the root surface modification index ( sampaio 's index ) . the scores are as follows : root surface without smear layer , with dentinal tubules completely opened ; no evidence of smear layer in the dentinal tubule gaps . root surface without smear layer , with dentinal tubules completely opened ; evidence of smear layer in the dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; evidence of dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; no evidence of dentinal tubule gaps . root surface covered with smear layer , with irregular aspect and presence of grooves and/or scattered debris . the number of totally patent smear free dentinal tubules out of the total number of dentinal tubules and percentage patency was calculated . the sampaio 's index , patent dentinal tubules and percentage patency of four groups ( mtad , ttc hcl , ca , and edta ) were compared by analysis of variance ( anova ) and the significance of mean difference between the groups was done by tukey 's post - hoc test after ascertaining normality by shapiro - wilk test and homogeneity of variances by levene 's test . a two - sided (  = 2 ) p < 0.05 was considered statistically significant . samples were obtained from the cervical third of the root by making two parallel grooves with a cylindrical bur under copious saline irrigation . the first groove was positioned horizontally at cementoenamel junction and second groove made parallel and 4 mm apical in relation to the first . the diseased root surfaces of all teeth were scaled with an ultrasonic scaler and thoroughly planed with # 12 , 34 gracey curettes ( hu - freidy ) to remove all the diseased cementum . with the help of diamond disc under copious irrigation , the two samples are obtained first by transverse sectioning the root from the grooves and secondly by sectioning the sample longitudinally into two from the middle . total 80 samples were randomly divided into 4 groups : 
 group a : biopure mtad [ figure 1]group b : tetracycline hcl ( 50mg / ml)group c : citric acid ( ph 1.0)group d : ethylene diaminetetraacetic acid ( 15% ) . 
  group a : biopure mtad [ figure 1 ] group b : tetracycline hcl ( 50mg / ml ) group c : citric acid ( ph 1.0 ) group d : ethylene diaminetetraacetic acid ( 15% ) . these agents were freshly prepared every time and were applied by  active burnishing  on the curetted root surfaces for 3 min . the cotton pellets are changed every 30 seconds to ensure consistent solution application . following treatment , following the chemical treatment , all samples were dehydrated in a graded series of ethanol ( 1090% ) for 30 min each and finally in 100% acetone for 30 min more . the samples were dried under the lamp and then mounted on the aluminum stubs and inserted in sc7640 sputter coater machine for gold / palladium coating on specimens . all the specimens were examined in a polaron - sem ( leo-430 ) at a magnification of 3500 and photomicrographs were evaluated to ascertain the extent of root biomodification by removal of smear layer , patent dentinal tubules in relation to the total number of dentinal tubules . the photomicrographs were distributed to three calibrated , trained blind examiners to determine the degree of smear layer removal according to the root surface modification index ( sampaio 's index ) . the scores are as follows : root surface without smear layer , with dentinal tubules completely opened ; no evidence of smear layer in the dentinal tubule gaps . root surface without smear layer , with dentinal tubules completely opened ; evidence of smear layer in the dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; evidence of dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; no evidence of dentinal tubule gaps . root surface covered with smear layer , with irregular aspect and presence of grooves and/or scattered debris . the number of totally patent smear free dentinal tubules out of the total number of dentinal tubules and percentage patency was calculated . the sampaio 's index , patent dentinal tubules and percentage patency of four groups ( mtad , ttc hcl , ca , and edta ) were compared by analysis of variance ( anova ) and the significance of mean difference between the groups was done by tukey 's post - hoc test after ascertaining normality by shapiro - wilk test and homogeneity of variances by levene 's test . a two - sided (  = 2 ) p < 0.05 was considered statistically significant . root surface without smear layer , with dentinal tubules completely opened ; no evidence of smear layer in the dentinal tubule gaps . root surface without smear layer , with dentinal tubules completely opened ; evidence of smear layer in the dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; evidence of dentinal tubule gaps . root surface covered with smear layer , with uniform aspect ; no evidence of dentinal tubule gaps . root surface covered with smear layer , with irregular aspect and presence of grooves and/or scattered debris . the number of totally patent smear free dentinal tubules out of the total number of dentinal tubules and percentage patency was calculated . the sampaio 's index , patent dentinal tubules and percentage patency of four groups ( mtad , ttc hcl , ca , and edta ) were compared by analysis of variance ( anova ) and the significance of mean difference between the groups was done by tukey 's post - hoc test after ascertaining normality by shapiro - wilk test and homogeneity of variances by levene 's test . a two - sided (  = 2 ) the sampaio 's index scores of group a , b , c , d ranged from 12 , 24 , 45 , and 13 respectively [ figure 2a  d ] , with mean  sd 1.67  0.49 , 3.27  0.59 , 4.20  0.41 and 2.33  0.62 , respectively [ table 1 ] . comparing the mean scores of four groups , anova revealed significantly different sampaio 's index scores ( f = 64.09 , p < 0.001 ) , patent dentinal tubule scores ( f = 140.42 , p < 0.001 ) and mean percentage patency scores of four groups ( f = 141.70 , p < 0.001 ) . scanning electron microscope photomicrographs of ( a ) mtad , ( b ) ethylenediaminetetraacetic acid , ( c ) tetracycline hydrochloride , ( d ) citric acid sampaio 's index , patent dentinal tubules , and percentage patency ( meansd ) of four experimental groups further , tukey test [ table 2 ] revealed significantly different and lower mean sampaio 's index , mean patent dentinal tubule and percentage patency scores of mtad group as compared to ttc hcl ( p < 0.001 ) , ca ( p < 0.001 ) and edta ( p = 0.006 ) groups . similarly , the mean sampaio 's index , patent dentinal tubule and percentage patency scores of edta group also lowered significantly as compared to both ttc hcl ( p < 0.001 ) and ca ( p < 0.001 ) . further , the mean sampaio 's index scores of ttc hcl group was also found significantly different and lower as compared to ca ( p < 0.001 ) . however , like patent dentinal tubules ( p = 0.974 ) , the mean percentage patency scores also not differed between ttc hcl and ca ( p = 0.427 ) that is , found to be statistically the same . significance ( p ) of mean difference of sampaio 's index , patent dentinal tubules , percentage patency between four experimental groups one of the objectives of periodontal therapy is the restoration of the lost periodontium and conversion of the periodontitis  affected root surface into a substrate which is biocompatible for epithelial and connective tissue cell adherence and attachment . methods to achieve this objective include scaling and root planing , and use of demineralizing agents . scaling and root planing leads to the formation of smear layer which inhibits the growth of a connective tissue attachment to the root surface . demineralizing agents have shown to expose the dentinal collagen , widening the orifices of dentinal tubules , remove cementum bound proteins and retained toxins from altered root surface . thus , the root surface smear layer removal with chemicals was carried out during periodontal therapy to enhance regeneration of the lost periodontal attachment apparatus . many chemical agents have been proposed as root conditioning agents , some of them are ca , ttc hcl group , edta . the other agents used are carbon dioxide laser , neodymium : yttrium , aluminum , garnet laser , and erbium : yttrium , aluminum , garnet laser and many more . as previously established in a systematic review by mariotti ( 2003 ) and american academy of pediatrics , position paper ( 2005 ) , ttc hcl , ca , and edta remove the smear layer but do not provide clinical benefit to the patient during regenerative procedures . as biopure mtad ( dentsply ltd . , usa . ) is used as an efficacious root canal irrigant for smear layer removal , so in the present study , we have utilized this novel agent as a root biomodifier for smear layer removal on periodontally involved human teeth . ca removes the inorganic materials and tween-80 reduces the surface tension and benefits the diffusion of acids into the root canal irregularities and dentinal tubules . ( 2003 ) demonstrated that mtad is an effective solution for the removal of the smear layer ; it also did not significantly change the structure of the dentinal tubules . ttc hcl , 50 mg / ml concentration was used according to previous studies in which ishi et al . ( 2000 ) inferred that ttc hcl concentrations between 50 and 125 mg / ml might alter dentin surfaces by removing smear layer and also maximize tubule openings in a short period of time , if repeated applications were performed . another agent used was ca ph 1 was also applied for 3 min as recommended by earlier studies . edta of 15% concentration , ph of 4.35 was used as given by lasho et al . the present study photomicrographs were evaluated using the root surface modification index ( sampaio 's index ) for smear layer removal [ table 2 ] . the lowest score was achieved in group a / mtad which indicates complete smear layer removal and complete opening of dentinal tubules , which was in accordance with the study by mozayeni et al . ( 2009 ) who stated that mtad revealed the presence of more abundant and larger dentinal tubule opening when compared with other agents . the result of mtad was statistically significant with ttc hcl , ca , and edta . however , to date , no in vitro or in vivo studies have been conducted utilizing mtad as a root biomodifier . the patent dentinal tubules and percentage patency intergroup comparison by z - test showed highly statistically significant difference among the groups . mtad had higher statistically significant number of patent and percentage patency of dentinal tubules when compared with ttc hcl ( p = 0.0001 ) , ca ( p < 0.0001 ) and edta ( p = 0.0455 ) . ( 2009 ) who inferred that mtad was more efficacious in removing smear layer and opening dentinal tubules of instrumented root canals because of being a combination agent of doxycycline , ca and a detergent tween-80 which reduces the surface tension and helps in better penetration of these acids into smear layer . zhang et al . ( 2003 ) demonstrated in his study that mtad is less cytotoxic to the surrounding cells and tissues than edta . ethylenediaminetetraacetic acid was statistically significant than ttc hcl and ca , because edta is better in smear layer removal and exposure of dentinal tubules by making the root surfaces more biocompatible according to silverio et al . blomlf and lindskog ( 1995 ) also stated that edta in a neutral ph was more effective to demineralize dentin than the acidic agents like ca , and selectively remove mineral from a dentin surface , exposing a collagenous matrix . ttc hcl when compared with ca ( p = 1.0000 ) did not had any statistically significant difference which was in accordance with the studies done by lafferty et al . ( 1993 ) who demonstrated comparable results of ca and ttc because of their similar acidic ph ( ttc hcl 1.75 ph , ca ph 1 ) . in summary , our results confirm that the root condition agents / root biomodifiers are effective in removing the smear layer and exposing dentinal tubules . mtad being most efficacious followed by edta , ttc hcl , and ca . despite the limitations of the study , further longitudinal in vitro and in vivo studies to establish mtad as a root conditioning agent are warranted . the study has established that the various root biomodifiers : mtad , ttc hcl 50 mg / ml , ca ph 1 , and edta 15% modifies the dentin surface by smear layer removal and exposure of dentinal tubules . hence , the application of mtad as a root biomodifier may have a significant role in periodontal regeneration both in vitro and in vivo .
background : smear layer removal and collagen fiber exposure may improve regeneration which can be accomplished by use of root biomodifiers . 
 these enhance the degree of connective tissue attachment to denuded roots . 
 the objective of this in vitro study was to evaluate and compare novel root canal irrigant and other root biomodifiers for smear layer removal on periodontally involved human teeth.materials and methods : forty human teeth were collected and stored in saline . after scaling and root planing , two samples were obtained from each tooth . 
 thus , a total of 80 dentin blocks were randomly divided into four groups : mixture of tetracycline , acid and detergent ( mtad ) , tetracycline hydrochloride ( ttc hcl ) , citric acid ( ca ) , and ethylenediaminetetraacetic acid ( edta ) . 
 the agents were applied for 3 min by active burnishing . immediately following treatment , 
 the specimens were rinsed , dehydrated , fixed and prepared for scanning electron microscope and were examined at  3500 magnification . 
 sampaio 's index was evaluated by the previously trained blind examiner using photomicrographs . 
 groups were compared using analysis of variance followed by tukey 's post - hoc test.results:mixture of tetracycline , acid , and detergent is most efficacious in removing the smear layer and showed statistically significant dentinal tubules opening , followed by edta , ttc hcl , and ca.conclusion:mixture of tetracycline , acid and detergent and conventional root biomodifiers used in the study alters the dentin surface by smear layer removal and exposure of dentinal tubules . 
 hence , mtad as a root biomodifier may have a significant role in periodontal regeneration .