several structural and chemical changes occurring in cementum as a result of periodontal disease prevent the reattachment of periodontal ligament pdl fibroblasts which is essential for periodontal wound healing .
one of the goals of periodontal therapy is the predictable regeneration of the periodontium in areas previously affected by periodontal disease .
scaling and root planing alone are not able to fully eliminate the etiological contaminants and they produce a compact smear layer covering the instrumented surface , which inhibits periodontal tissue reattachment .
thus , various root biomodification agents like citric acid , ethylenediaminetetraacetic acid ( edta ) , and tetracycline are used due to their potential for removing smear layer and exposing the underlying radicular collagen fibrils .
fluorosis is one of the major public health problems affecting the rural indian population which is dependent on ground water . in india ,
it is now an established fact that fluoride ingestion over a period of time can affect the structure and function of cells , tissues , organs , and systems resulting in a variety of clinical manifestations .
the various hard tissue effects of fluorosis are hypomineralization of enamel , dentin , hypercementosis , recession of alveolar crest , root resorption , and hypermineralization , while the soft tissue changes include inhibition of type i collagen synthesis , degree of cross linking , fibroblast growth inhibition , lethal effects on fibroblasts , and morphologic changes .
the effect of fluorosis on periodontal health and disease is rarely discussed in the literature and a few reports on this issue are not consistent .
authors have reported no association between periodontal parameters and fluorosis , increased periodontal scores , and reduced periodontal scores .
further , significant differences in response to various nonsurgical therapeutic modalities exist between fluorosed versus nonfluorosed roots .
very little has been addressed about this issue in literature . considering the above studies on the effects of fluoride on connective tissue and the prevalence of endemic fluorosis in and around davangere district ,
so , the objective of the present in vitro study was to study the attachment of fluorosed fibroblasts to the fluorosed and nonfluorosed root fragments .
freshly extracted fluorosed teeth were obtained from the department of oral surgery , college of dental sciences , davangere , karnataka , india and were used according to a protocol that satisfied the ethical standards of rajiv gandhi university of health sciences , karnataka , india .
freshly extracted fluorosed premolar teeth due to orthodontic reasonsperiodontally healthy teeth with dental fluorosis , which was determined by the clinical examination and history of the subjects hailing from natural high water fluoride areas in and around davangere ( fluoride concentration 1.5 - 3 ppm)periodontally healthy teeth without dental fluorosisperiodontally diseased fluorosed and nonfluorosed teeth with > 7 mm pocket depth pd and > 6 mm attachment loss indicated for extraction . 
 
freshly extracted fluorosed premolar teeth due to orthodontic reasons periodontally healthy teeth with dental fluorosis , which was determined by the clinical examination and history of the subjects hailing from natural high water fluoride areas in and around davangere ( fluoride concentration 1.5 - 3 ppm ) periodontally healthy teeth without dental fluorosis periodontally diseased fluorosed and nonfluorosed teeth with > 7 mm pocket depth pd and > 6 mm attachment loss indicated for extraction . 

teeth extracted due to root cariesteeth with traumatic extractionteeth with proximal caries extending to the cementumteeth with fillings extending beyond cementoenamel junction ( cej)teeth with intrinsic stains caused by other reasons such as porphyria , erythroblastosis fetalis , tetracycline therapy , etc . 
 
teeth extracted due to root caries teeth with traumatic extraction teeth with proximal caries extending to the cementum teeth with fillings extending beyond cementoenamel junction ( cej ) teeth with intrinsic stains caused by other reasons such as porphyria , erythroblastosis fetalis , tetracycline therapy , etc . immediately after extraction , blood , saliva , and soft tissue debris were lightly scrubbed with a sterile scrub and rinsed with sterile normal saline solution ( 0.9% ) .
four specimens were obtained from each tooth root , which was cut with a sterile , double - sided diamond disk operated in an air motor ( kavo l - motor 181 dbn , joinville , brazil ) and contra - angled hand piece ( kavo 2068 fgn , joinville , brazil ) running at low speed with sterile water coolant .
the coronal section was performed 1 mm below the cej and the apical section 4 mm from the root apex .
longitudinal buccolingual and mesiodistal sections were performed to expose the pulpal wall and to obtain four specimens ( 2  2 mm ; 2 mm thick ) from each root , so that specimens from the same tooth were subjected to each type of treatment . to avoid contamination from the pulp ,
the pulpal wall was separated from the remaining outer portion of root dentin by a bur at low speed parallel to the longitudinal axis of the root .
group 1 : fluorosed healthy controls ( fh , n = 5 ) group 2 : fluorosed diseased controls ( fd , n = 3 ) group 3 : nonfluorosed healthy controls ( nfh , n = 5 ) group 4 : nonfluorosed diseased controls ( nfd , n = 3 ) group 5 : fluorosed teeth treated with scaling and root planing srp ( fd - srp , n = 10 ) group 6 : fluorosed teeth treated with srp + edta ( fd - srp + e , n = 10 ) group 7 : nonfluorosed teeth treated with srp ( nfd - srp , n = 10 ) group 8 : nonfluorosed teeth treated with srp + edta ( nfd - srp + e , n = 10 ) . prior to sectioning , the experimental specimens were scaled and root planed using # 11 and # 12 gracey curette to remove any organic deposits and/or debris . each root fragment was root planed using a total of 20 - 30 strokes . prior to next root fragment treatment , sharpening of the curette was done . experimental root fragments for root biomodification ( groups 6 and 8) were subjected to edta treatment using a neutral ph , 24% edta gel .
the edta gel was burnished on the outer surface of root fragme nts with a small cotton pellet for 2 min .
human periodontal ligament cell culture was obtained from a fluorosed healthy premolar surgically extracted due to orthodontic treatment in an atraumatic manner .
after extraction , the tooth was rinsed with and placed in minimum essential medium ( mem ) with antibiotic supplement .
the mid third portion of the periodontal membrane was collected carefully by scraping with a surgical scalpel and transferred to the 96-well microtitre plate containing 200 l of dulbecco 's minimum eagle 's medium ( dmem ) supplemented with sodium pyruvate ( 0.l g/1 ) , l - glutamine ( 1.16
g / l ) , streptomycin sulfate ( 100 mg/1 ) , penicillin ( 100,000 /1 ) , and 10% fetal bovine serum ( fbs ; complete dmem ) per well .
cells were grown at 37c in a humidified atmosphere containing 5% co2 and 95% air .
satisfactory attachment of fibroblasts to the culture flasks was obtained in 24 - 48 h. the monolayers of cells , about 2 - 3  10 cells / mm , were formed in 2 - 3 weeks .
the media were changed and the sub - culturing was done after the cells reached confluence .
fibroblast - like cells that were growing from tissue biopsies were identified , incubated , and fed every 3 days until confluence .
cells growing in a monolayer could be sub - cultured by physical means like shaking , magnetic stirring or by chemical methods like using proteolytic enzymes ( trypsin ) and chelating agents ( edta ) .
fibroblast sub - cultures were prepared from the primary cultures by removing the spent medium , washing the tissue fragments several times with sterile phosphated buffered saline ( pbs ) , and dissociating the fibroblasts from the outgrowth by treatment with 0.5 - 0.8 ml of trypsin and edta .
the cells that were treated with trypsin and edta were incubated at 37c for 2 min , after which the cells were seen dissociating from the culture flasks .
they were dislodged from the flasks by gentle tapping and the enzyme activity was stopped by adding 5 - 8 ml dmem containing 10% fbs .
the sub - cultures were observed under inverted microscope to confirm their viability before incubation of fibroblasts ( labomed tcm 400 # 7126000 inverted research microscope , biosciences ) . using 96-well culture plates ,
the fluorosed periodontal fibroblasts were seeded at 10,000 cells / well in growth media containing dmem supplemented with 10% fbs , 100 mg streptomycin / ml , 100 units penicillin / ml , and 1.16 mg l - glutamine / ml and allowed to attach and grow at 37c for 24 h. the fluorosed fibroblasts were incubated and the cells from the third passage were chosen . treated root specimens were placed in the culture plates , so that the specimens were covered by the cell suspension and incubated for 3 days . at the end of this period , the cells on samples were rinsed with pbs and fixed with 4% gluteraldehyde .
the cell attachment onto the cemental surface of the treated root fragments of all the groups at 72-h period was observed in a stereomicroscope ( magnus model msz 
for the cell counting , all root fragments were observed under stereomicroscope at a same work distance 20 mm and the same magnification 100 .
the software overlaid a grid of evenly spaced horizontal and vertical lines ( adobe photoshop , version cs3 ) .
this method allowed for counting on the surface of each root fragment at four extremities of the specimen and at central area . using these standardized conditions ,
since the data were in cells , non - parametric tests were used for intra - group ( kruskal  wallis test ) and inter - group ( mann  whitney test ) comparisons .
for all the tests , a p value of 0.05 or less was considered for statistical significance .

 freshly extracted fluorosed premolar teeth due to orthodontic reasonsperiodontally healthy teeth with dental fluorosis , which was determined by the clinical examination and history of the subjects hailing from natural high water fluoride areas in and around davangere ( fluoride concentration 1.5 - 3 ppm)periodontally healthy teeth without dental fluorosisperiodontally diseased fluorosed and nonfluorosed teeth with > 7 mm pocket depth pd and > 6 mm attachment loss indicated for extraction . 
 
freshly extracted fluorosed premolar teeth due to orthodontic reasons periodontally healthy teeth with dental fluorosis , which was determined by the clinical examination and history of the subjects hailing from natural high water fluoride areas in and around davangere ( fluoride concentration 1.5 - 3 ppm ) periodontally healthy teeth without dental fluorosis periodontally diseased fluorosed and nonfluorosed teeth with > 7 mm pocket depth pd and > 6 mm attachment loss indicated for extraction .

 teeth extracted due to root cariesteeth with traumatic extractionteeth with proximal caries extending to the cementumteeth with fillings extending beyond cementoenamel junction ( cej)teeth with intrinsic stains caused by other reasons such as porphyria , erythroblastosis fetalis , tetracycline therapy , etc . 
 
teeth extracted due to root caries teeth with traumatic extraction teeth with proximal caries extending to the cementum teeth with fillings extending beyond cementoenamel junction ( cej ) teeth with intrinsic stains caused by other reasons such as porphyria , erythroblastosis fetalis , tetracycline therapy , etc .
immediately after extraction , blood , saliva , and soft tissue debris were lightly scrubbed with a sterile scrub and rinsed with sterile normal saline solution ( 0.9% ) .
four specimens were obtained from each tooth root , which was cut with a sterile , double - sided diamond disk operated in an air motor ( kavo l - motor 181 dbn , joinville , brazil ) and contra - angled hand piece ( kavo 2068 fgn , joinville , brazil ) running at low speed with sterile water coolant .
the coronal section was performed 1 mm below the cej and the apical section 4 mm from the root apex .
longitudinal buccolingual and mesiodistal sections were performed to expose the pulpal wall and to obtain four specimens ( 2  2 mm ; 2 mm thick ) from each root , so that specimens from the same tooth were subjected to each type of treatment . to avoid contamination from the pulp ,
the pulpal wall was separated from the remaining outer portion of root dentin by a bur at low speed parallel to the longitudinal axis of the root .
group 1 : fluorosed healthy controls ( fh , n = 5 ) group 2 : fluorosed diseased controls ( fd , n = 3 ) group 3 : nonfluorosed healthy controls ( nfh , n = 5 ) group 4 : nonfluorosed diseased controls ( nfd , n = 3 ) group 5 : fluorosed teeth treated with scaling and root planing srp ( fd - srp , n = 10 ) group 6 : fluorosed teeth treated with srp + edta ( fd - srp + e , n = 10 ) group 7 : nonfluorosed teeth treated with srp ( nfd - srp , n = 10 ) group 8 : nonfluorosed teeth treated with srp + edta ( nfd - srp + e , n = 10 ) . prior to sectioning , the experimental specimens were scaled and root planed using # 11 and # 12 gracey curette to remove any organic deposits and/or debris .
experimental root fragments for root biomodification ( groups 6 and 8) were subjected to edta treatment using a neutral ph , 24% edta gel .
the edta gel was burnished on the outer surface of root fragme nts with a small cotton pellet for 2 min .
human periodontal ligament cell culture was obtained from a fluorosed healthy premolar surgically extracted due to orthodontic treatment in an atraumatic manner .
after extraction , the tooth was rinsed with and placed in minimum essential medium ( mem ) with antibiotic supplement .
the mid third portion of the periodontal membrane was collected carefully by scraping with a surgical scalpel and transferred to the 96-well microtitre plate containing 200 l of dulbecco 's minimum eagle 's medium ( dmem ) supplemented with sodium pyruvate ( 0.l g/1 ) , l - glutamine ( 1.16
g / l ) , streptomycin sulfate ( 100 mg/1 ) , penicillin ( 100,000 /1 ) , and 10% fetal bovine serum ( fbs ; complete dmem ) per well .
cells were grown at 37c in a humidified atmosphere containing 5% co2 and 95% air .
satisfactory attachment of fibroblasts to the culture flasks was obtained in 24 - 48 h. the monolayers of cells , about 2 - 3  10 cells / mm , were formed in 2 - 3 weeks .
the media were changed and the sub - culturing was done after the cells reached confluence .
fibroblast - like cells that were growing from tissue biopsies were identified , incubated , and fed every 3 days until confluence .
cells growing in a monolayer could be sub - cultured by physical means like shaking , magnetic stirring or by chemical methods like using proteolytic enzymes ( trypsin ) and chelating agents ( edta ) .
fibroblast sub - cultures were prepared from the primary cultures by removing the spent medium , washing the tissue fragments several times with sterile phosphated buffered saline ( pbs ) , and dissociating the fibroblasts from the outgrowth by treatment with 0.5 - 0.8 ml of trypsin and edta .
the cells that were treated with trypsin and edta were incubated at 37c for 2 min , after which the cells were seen dissociating from the culture flasks .
they were dislodged from the flasks by gentle tapping and the enzyme activity was stopped by adding 5 - 8 ml dmem containing 10% fbs .
the sub - cultures were observed under inverted microscope to confirm their viability before incubation of fibroblasts ( labomed tcm 400 # 7126000 inverted research microscope , biosciences ) .
using 96-well culture plates , the fluorosed periodontal fibroblasts were seeded at 10,000 cells / well in growth media containing dmem supplemented with 10% fbs , 100 mg streptomycin / ml , 100 units penicillin / ml , and 1.16 mg l - glutamine / ml and allowed to attach and grow at 37c for 24 h. the fluorosed fibroblasts were incubated and the cells from the third passage were chosen . treated root specimens were placed in the culture plates , so that the specimens were covered by the cell suspension and incubated for 3 days . at the end of this period ,
the cell attachment onto the cemental surface of the treated root fragments of all the groups at 72-h period was observed in a stereomicroscope ( magnus model msz 
for the cell counting , all root fragments were observed under stereomicroscope at a same work distance 20 mm and the same magnification 100 .
the software overlaid a grid of evenly spaced horizontal and vertical lines ( adobe photoshop , version cs3 ) .
this method allowed for counting on the surface of each root fragment at four extremities of the specimen and at central area . using these standardized conditions , cell counts of pdl fibroblasts
results were presented as mean  standard deviation for quantitative data . since the data were in cells , non - parametric tests were used for intra - group ( kruskal  wallis test ) and inter - group ( mann  whitney test ) comparisons .
for all the tests , a p value of 0.05 or less was considered for statistical significance .
a total of 56 root fragments from both fluorosed and nonfluorosed , healthy and diseased teeth were included in this in vitro study to evaluate the effect of scaling and root planing and edta root biomodification on fluorosed and nonfluorosed root fragments .
the attachment of fluorosed fibroblasts to the root surface was assessed by stereomicroscope and counted using grid applied through software .
the results of the study are interpreted in figure 1 . in the nonfluorosed group ,
significantly greater attachment was found in diseased ( p = 0.036 ) and scaled root fragments ( p = 0.008 ) as compared to healthy root fragments .
the scaled + edta treated root fragments showed significantly lesser attachment . in the fluorosed group ,
all the groups showed similar fibroblast attachment with no attachment to fluorosed scaled and edta - treated root fragments . on comparison of fluorosed and nonfluorosed groups
, there was no significant difference between healthy , diseased scaled , and edta - treated root fragments .
there is a possibility that the fluorosed root cementum may be altered in mineralization status ( hypomineralization ) similar to fluorosed enamel and dentin , which has not been studied so far .
the morphometric analysis of fibroblasts exposed to fluoride ions revealed altered morphology and decrease in area and spherical volume as compared to normal fibroblasts .
changes of fibroblast shape ( circumference and diameter ) that were found might suggest folding of their surface area .
cell morphology can be regarded as an indicator for the affinity of cells to differently treated root surfaces .
although flat cells are firmly attached to the substratum by means of numerous attachment extensions and lamellopodia , round cells can be considered poorly attached .
the cell morphology and root surface alterations play an important role in cell - to - root interaction . in the current study ,
considering the possible changes in fluorosed specimens ( fibroblasts and root cementum ) , a first attempt was made to study and compare the fluorosed fibroblast attachment ( fl - fa ) to nonfluorosed and fluorosed root fragments after srp and srp + edta treatment that are known to help and enhance fibroblast attachment .
the results are discussed as follows : in the nonfluorosed group , significantly greater attachment was found in the untreated diseased ( p = 0.036 ) and srp - treated nonfluorosed diseased groups ( p = 0.008 ) as compared to healthy group , similar to the observations of fardal et al . who used an in vitro model to assess initial attachment of human gingival fibroblasts to six periodontally diseased root surfaces .
two purposes of acid demineralization are collagen exposure and removal of smear layer produced due to instrumentation . as fibroblast adhesion and locomotion
is facilitated by collagen , the collagen exposure favors fibroblast adhesion and the smear layer prevents it .
the delicate balance of these two factors is vital to fibroblast attachment and both the factors determine the extent of fibroblast attachment . in the present study
, significantly lesser attachment was found in srp + acid treated nonfluorosed diseased root fragments which could be due to inadequate removal of smear layer as suggested by various authors and overdemineralization of the tooth surface .
contrary to the above studies which report an inadequate removal of smear layer by edta , an in vitro study and an in vivo animal study by blomlof et al . reported effective removal of smear layer using edta .
few authors reported attachment of significantly greater number of fibroblasts to specimens treated with root biomodification . the in vitro and in vivo effects of edta root biomodification are not consistent in the literature .
though there is no conclusive evidence regarding the benefits of root conditioning , it is being widely practiced in clinical management .
the scaled + edta - treated fluorosed root fragments did not reveal fibroblast attachment which could be due to incomplete removal of the smear layer and overdemineralization of the root surface .
there is a possibility that 24% edta was deleterious to supposedly hypomineralized fluorosed root surface as suggested by two studies .
( 2009 ) observed the incomplete removal of smear layer from edta - treated fluorosed root , wider dentin tubules , and peritubular areas in fluorosed dentin , which are the evidences of hypomineralization .
another study conducted by dhingra and vandana showed more amount of root surface melting in the fluorosed group ( 73.33% ) than in the nonfluorosed roots ( 66.69% ) after er : yag application , which was probably due to hypomineralized fluorosed root .
no similar studies exist in the literature as it is the first of its kind done in the literature .
the comparison of fluorosed fibroblast attachment to fluorosed and nonfluorosed , healthy , diseased , and diseased treated root fragments was the main objective of this study .
the composition of the root cementum will influence the fibroblast attachment which occurs as a part of physiologic turnover .
the tendency for a greater cell attachment to non - demineralized cementum may be due to the higher collagen content of cementum .
type i collagen and its degradation products are known to be chemotactic stimulants for polymorphonuclear cells pmns , macrophages , and fibroblasts .
the need of the hour is to explore the chemical composition of fluorosed cementum . in the fluorosed and nonfluorosed diseased groups ,
different root surface qualities are reported to influence fibroblast functions , for example , endotoxins from periodontal pathogens may penetrate the root surface and have been implicated in inhibiting fibroblast proliferation , synthesis , and attachment .
the hypermineralization of diseased root is reported to act as a barrier to fibroblast attachment . at this juncture , it is important to elucidate the various structural and chemical changes in fluorosed cementum induced by periodontal disease such as hypermineralization , endotoxins , etc . in the srp group ,
the configuration and composition of scaled root surfaces are important factors capable of affecting the generation of new connective tissue attachment to root surfaces on which the attachment has been lost due to periodontitis .
recently , it was shown that extracts of cementum affect the synthetic activities of fibroblasts and that they promote fibroblast attachment .
these observations indicate that the cementum may play a role in periodontal connective tissue formation by regulating the activities of cells which are necessary for the synthesis of matrix constituents .
there is an absolute need to study the composition of fluorosed cementum which regulates fibroblast attachment and functional activities .
various authors have utilized gingival fibroblasts from human origin , and continuous mouse fibroblast cell lines ( l-929 ) , v79 fibroblasts , and human gingival fibroblast - fmm1 cell line have also been used for the same purpose . but
it is relatively free of tissue remnants and shows minimal alteration of cell morphology and function when cultured . as the human periodontal fibroblasts can be obtained easily , the use of nonoral and nonhuman cells should be avoided as the extrapolation of the reports is not encouraging from the clinical point of view .
the current in vitro study was conducted using human periodontal fibroblasts derived from dental roots from endemic fluorosis area , which were cultured and used for fibroblast attachment .
usage of histologic or scanning electron microscopy ( sem ) analysis can be considered for better analysis of the attachment .
overall , srp proves to be a standard requirement for a healthy attachment to occur both in fluorosed and nonfluorosed roots .
also , 24% edta does not seem to be a promising agent for root biomodification in fluorosed roots in a given concentration and time of treatment .
further studies are required to assess fluorosed cementum composition and mineralization status both during healthy and diseased status , to ascertain the possible morphologic alteration of fluorosed fibroblasts obtained from an endemic area .
 background : fluorosis causes mineralization changes in the tooth and can lead to morphologic alterations of fibroblasts . to understand the effect of fluorosis on periodontal healing , the initial step during healing , such as fibroblast attachment to the root surface , needs to be evaluated . 
 hence , the objective of the present study was to study the attachment of fluorosed fibroblasts on the fluorosed and nonfluorosed root fragments.materials and methods : a total of 56 fluorosed and nonfluorosed , periodontally healthy and diseased tooth roots were obtained and allotted to eight groups : fluorosed and nonfluorosed healthy controls ( fh and nfh , respectively ) , fluorosed and nonfluorosed diseased controls ( fd and nfd , respectively ) , fluorosed and nonfluorosed roots treated with scaling and root planing ( fd + s and nfd + s , respectively ) , and similar groups treated with scaling and root planing and 24% ethylenediaminetetraacetic acid ( edta ) gel application for 2 min ( fd + se and nfd + se , respectively ) . after the respective treatment , the root fragments were incubated in the human periodontal ligament fibroblast cells obtained and cultured from freshly extracted healthy human fluorosed premolar tooth root.results:in the nonfluorosed roots category , greater attachment was found in the untreated nonfluorosed diseased ( p = 0.036 ) and srp - treated nonfluorosed diseased groups ( p = 0.008 ) as compared to the nonfluorosed healthy group . while in the fluorosed roots category , no significant difference was observed in fl - fa ( p > 0.05 ) within the group . 
 however , no attachment was observed in edta - treated fluorosed root fragments . when fluorosed groups were compared to nonfluorosed groups 
 , no significant changes were noted between the groups.conclusion:srp proves to be a standard requirement for fibroblast attachment to occur both in fluorosed and nonfluorosed roots . 
 although there was no significant difference in attachment between srp and srp + edta among fluorosed roots , edta does not seem to be a promising agent for root biomodification in fluorosed roots in a given concentration and time of treatment . 
INTRODUCTION
MATERIALS AND METHODS
Inclusion criteria
Exclusion criteria
Procedural steps
Fibroblast culture
Fibroblast sub-culture
Incubation of treated root specimens in fluorosed fibroblast culture
Cell attachment
Cell counting
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION