|Year : 2014 | Volume
| Issue : 1 | Page : 29-35
Comparative assessment of fluorosed and nonfluorosed fibroblast attachment on fluorosed and nonfluorosed teeth after scaling and root planning and ethylenediaminetetraacetic acid root biomodification
Kharidi Laxman Vandana1, Neha Girotra1, Nallur Vaman Jayashree1, Kishore Bhat2
1 Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India
2 Department of Microbiology, Maratha Mandal Dental College, Karnataka, India
|Date of Web Publication||18-Aug-2014|
Department of Periodontics, College of Dental Sciences, Davangere - 577 004, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Objectives: Fluorosis causes mineralization changes in the tooth and can lead to morphological alterations of fibroblasts. To evaluate the effect of fluorosis on periodontal healing, the initial step while healing such as, fibroblast attachment onto the root surface requires to be evaluated on the fluorosed and nonfluorosed tooth using nonfluorosed as well as fluorosed fibroblasts originated from the subjects influenced by high-water fluoride. Hence, the objective of the current study was to study and compare the attachment of nonfluorosed and fluorosed fibroblasts on the fluorosed and nonfluorosed root fragments. Materials and Methods: A total of 112 fluorosed and nonfluorosed, periodontally healthy and diseased tooth roots were obtained and allotted to eight groups : f0 luorosed healthy (FH) and non-FH (NFH) controls, fluorosed diseased (FD) and non-FD (NFD) controls, fluorosed and nonfluorosed teeth treated with scaling and root planning (SRP) (FD SRP and NFD SRP) and similar groups treated with SRP and ethylenediaminetetraacetic acid (EDTA) (FD SRP + EDTA and NFD SRP + EDTA) burnishing treatment with 24% EDTA gel for 2 min. After the respective treatment half of the root fragments in each group were incubated in the human periodontal ligament fibroblast cells obtained and cultured from freshly extracted FH and NFH human premolar tooth root. The nonfluorosed fibroblasts are elongated, flat cells thus they show increased attachment to root the surface. Results: When comparison was carried out between the attachment of fluorosed and nonfluorosed fibroblasts on NFD groups treated with scaling and EDTA, significant results were obtained with increased attachment seen on the group incubated with nonfluorosed fibroblasts (P = 0.029). While on comparison between the attachment of fluorosed and nonfluorosed fibroblasts on NFH group, NFD group treated with SRP and NFD group, no significant results were obtained (P > 0.05). On comparison between the attachment of fluorosed and nonfluorosed fibroblasts on FD group treated with SRP, highly significant results were obtained with increased attachment seen in the group incubated with nonfluorosed fibroblasts (P = 0.001). While the comparison of attachment of fluorosed and nonfluorosed fibroblasts on FH group, FD group treated with SRP + EDTA and FD group revealed no significant results (P > 0.05). Interpretation and Conclusion: SRP proves yet to be a standard requirement for fibroblast attachment to occur both in fluorosed and nonfluorosed teeth. Although, there is no significant difference in attachment between SRP and SRP + EDTA among fluorosed teeth, EDTA does not seem to be a promising agent for root biomodification in fluorosed teeth in given concentration and time of treatment.
Keywords: Cementum, ethylenediaminetetraacetic acid, fibroblast, fluorosis, root planing
|How to cite this article:|
Vandana KL, Girotra N, Jayashree NV, Bhat K. Comparative assessment of fluorosed and nonfluorosed fibroblast attachment on fluorosed and nonfluorosed teeth after scaling and root planning and ethylenediaminetetraacetic acid root biomodification. J Int Clin Dent Res Organ 2014;6:29-35
|How to cite this URL:|
Vandana KL, Girotra N, Jayashree NV, Bhat K. Comparative assessment of fluorosed and nonfluorosed fibroblast attachment on fluorosed and nonfluorosed teeth after scaling and root planning and ethylenediaminetetraacetic acid root biomodification. J Int Clin Dent Res Organ [serial online] 2014 [cited 2019 Oct 22];6:29-35. Available from: http://www.jicdro.org/text.asp?2014/6/1/29/139090
| Introduction|| |
Fluorosis is one of the major public health problems affecting the rural Indian population who are dependent on ground water. In India, several states are affected including Davangere district in Karnataka state. 
It is now an established fact that fluoride ingestion over a period 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 the alveolar crest,  root resorption,  hypermineralization,  while the soft tissue changes include gingival recession,  initial mineralization and globular mineralized deposits in the periodontal ligament area,  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 dental caries is well-documented. On the contrary, 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; , reduced periodontal scores. ,,,, Based on these reports, the fibroblasts of the fluorosed tooth root which is altered in morphology may influence its attachment to the root surface. The morphology of normal fibroblasts is flat and elongated, which enhance their attachment on root surface. Based on fluorosis induced enamel and dentin changes there is a possibility that cementum may exhibit certain mineralization, structural and chemical changes that have not been studied so far. Further, significant difference exists in their fluorosed versus nonfluorosed root response to various nonsurgical therapeutic modalities. It was shown that fluorosed and nonfluorosed teeth respond differently to root biomodification agents  and erbium-doped yttrium aluminium garnet (Er:YAG) laser (140 mJ/pulse and 10 Hz) application.  There are two components that could be altered in a subject's periodontium residing in endemic fluoride geographic belt that is, fibroblasts and root surface which are the determinants of early events of periodontal wound healing such as fibroblast attachment. Considering the normal fibroblast and root surface from nonfluorosed area, the present in vitro study attempted to study the attachment of fibroblasts obtained from fluorosed root. The association of fluorosis and periodontal tissues is thought provoking. Very little has been addressed about this issue in the literature. Considering the above studies on fluoride effects on connective tissue and endemic fluorosis in and around Davangere district, India, there is a need to evaluate the fibroblast attachment on the root surface which is the first step in the formation of collagen of fibers during connective tissue healing. Hence, the objective of the current study was to study the attachment of fluorosed and nonfluorosed fibroblasts on the fluorosed and nonfluorosed root fragments.
| Materials and methods|| |
In this study freshly extracted fluorosed premolar teeth were obtained from the Department of Oral Surgery, College of Dental Sciences, Davangere, Karnataka, India. Eight fluorosed and nonfluorosed periodontally diseased teeth and three fluorosed healthy (FH) and non-FH (NFH) teeth were collected. Inclusion criteria comprised of freshly extracted premolar teeth due to orthodontic reasons, for dental fluorosis, the fluorotic enamel stains were confirmed by the clinical examination and history of subjects from natural high water fluoride areas in and around Davangere (water fluoride concentration 1.5-3.0 ppm; Jackson's fluorosis index Type B white areas of,>2 mm in diameter; Type C colored (brown) areas <2 mm in diameter, irrespective of there being white areas). The fluorosed subjects with significantly higher urinary fluoride levels 1.17 ± 0.14 mg/L than the nonfluorosed subjects 0.48 ± 0.19 mg/L. confirmed the fluorosis status. The periodontally healthy and diseased nonfluorosed teeth from nonendemic areas indicated for extraction were included.
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) and periodontally healthy teeth without dental fluorosis, periodontally diseased fluorosed and nonfluorosed teeth from nonendemic areas indicated for extraction. The exclusion criteria included teeth extracted due to root caries, traumatic extraction, proximal caries extending to the cementum, teeth with fillings extending beyond cementoenamel junction, teeth with intrinsic stains caused by other reasons such as porphyria, erythroblastosis fetalis, tetracycline therapy etc.
Distribution of specimens is shown in [Table 1]. A total of 56 root specimens were included in each of fluorosed fibroblast group (Group A) and nonfluorosed fibroblast group (Group B).
|Table 1: Distribution of root specimens in fluorosed and nonfluorosed fibroblast groups|
Click here to view
Immediately after extraction, the teeth were rinsed with sterile normal saline solution (0.9%) and autoclaved.  Four fragments were obtained from each tooth.
Prior to sectioning, the experimental specimens were scaled and root planed using number 11 and number 12 gracey curette to remove any organic deposits and/or debris. Each root fragment was root planed using a total of 20-30 strokes.  The experimental root fragments were burnished with 24% ethylenediaminetetraacetic acid (EDTA) (neutral pH) gel for 2 min as a root biomodification procedure. ,
Both fluorosed and nonfluorosed periodontal fibroblasts were cultured by the standard technique. Human periodontal ligament cell culture was obtained from FH and NFH premolar teeth surgically extracted due to orthodontic treatment in an atraumatic manner. After extraction, the tooth was rinsed with and placed in minimum essential medium with antibiotic supplement. Fibroblast-like cells that were grown from tissue biopsies were identified, incubated, and fed every 3 days until confluence. ,, The cells for the third passage were sub-cultured by a standard method. The sub-cultures were observed under inverted microscope to confirm viability of fibroblasts (LABOMED TCM 400 # 7126000 Inverted Research Microscope, Biosciences).  Using 96-well culture plates, fluorosed periodontal fibroblasts were seeded at 10,000 cells/well in growth media containing Dulbecco's minimum eagle's medium supplemented with 10% fetal bovine serum and antibiotics. 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, cells on segments were rinsed with phosphate buffered saline and fixed with 4% gluteraldehyde. 
The cell attachment on to the surface of the treated root fragments of all the groups at 72 h period was observed in a stereomicroscope , (MAGNUS Model MSZ-BI Stereo zoom microscope) at 20 mm focal length under ×100 magnification and cell counting was performed.
For the cell counting, all root fragments were observed under stereomicroscope at a same work distance 20 mm and same magnification ×100. Photomicrographs were recorded with five megapixel digital camera. On every digitized image, the software overlaid a grid of evenly spaced horizontal and vertical lines (Adobe Photoshop, version CS3, Adobe Systems). This method allowed for counting on the surface of each root fragment. Each root fragment was counted for 10 min.
Results were presented as mean rank for quantitative data. Since the data were in cells, nonparametric tests were used for intra (Kruskal-Wallis test) and inter (Mann-Whitney test) group comparisons. For all the tests, P < 0.05 was considered for statistical significance.
| Results|| |
A total of 112 root fragments from both FH and NFH and diseased teeth were included in this in vitro study to evaluate the effect of scaling and root planning (SRP) and EDTA root biomodification on fluorosed and nonfluorosed root fragments. All the wells in the cell culture plate were observed under inverted microscope and their viability was confirmed before incubation. All the cells showed viability both on fluorosed and nonfluorosed root fragments. The attachment of fluorosed fibroblasts to the root surface was assessed by stereo microscope and counted using grid applied through software.
The fluorosed fibroblast attachment (FL-FA) and non-FL-FA (NFL-FA) to fluorosed group and nonfluorosed group that consisted of untreated healthy (FH and NFH); untreated diseased (fluorosed diseased [FD] and non-FD [NFD]); scaled diseased (FD + S and NFD + S) and scaled + EDTA treated diseased (FD + SE and NFD + SE) root fragments have been evaluated.
All the cells showed viability both on fluorosed and nonfluorosed root fragments. The FL-FA and NFL-FA to FH (P = 0.167), NFH (P = 0.44), FD (P = 0.3) and NFD (P = 0.6) was nonsignificant. Treatment wise, NFL-FA to NFD + SRP and EDTA (P = 0.029) and FD + SRP (P = 0.001) was significant. The FL-FA and NFL-FA to NFD + SRP (P = 0.338) and FD + SRP EDTA (P = 1) was not significant [Graph 1]. [Additional file 1]
The FL-FA and NFL-FA to different NFH, NFD and N + D + S group did not reveal any significant difference.
The scaled + acid treated NFD group showed significantly better NFL-FA than FL-FA. The altered FL-FA and normal NFL-FA to different groups of altered fluorosed root such as FH, FD, FD + S and FD + SE, was compared. Both FL-FA and NFL-FA showed no significant difference in attachemtn except for the FD + S group. In the FD + S group, the normal type NFL-FA better to the roots than fluorosed fibroblast.
There was statistically significant greater NFL-FA to SRP + EDTA treated root fragments than fluorosed fibroblasts.
| Discussion|| |
The two reasons that lead to this study are, the in vitro toxic effects of fluoride on morphology and functions of fibroblasts ,,, and difference in therapeutic response of fluorosed and nonfluorosed roots to nonsurgical periodontal treatment. , Following periodontal therapy, the initial step in periodontal wound healing such as fibroblast attachment between fluorosed and nonfluorosed roots requires to be evaluated.
There is a possibility that fluorosed fibroblasts exposed to fluoride ions constantly may or may not express any of the functional or morphological changes that would interfere with fibroblast attachment to root surfaces. Second, the cementum of fluorosed root that plays a vital role in fibroblast attachment has not been elucidated. Based on two unexplored factors pertaining to fluorosis, fluorosed fibroblasts and fluorosed root were used that served as test group. The ability of FL-FA to healthy, diseased and treated diseased fluorosed roots is compared to the control group which consisted of NFL-FA to nonfluorosed root.
The results of the study are presented as follows there was no significant difference in FL-FA and NFL-FA to NFH roots. The NFL-FA to nonfluorosed root is a physiologic process that could be possibly attributed to the availability of collagen and its products, which might facilitate fibroblast attachment as suggested by few authors. ,,,, The tendency for a FL-FA to nondemineralized 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 neutrophils, macrophages, and fibroblasts. ,,
There was no significant difference in FL-FA and NFL-FA to NFD root fragments. Different root surface qualities are reported to influence fibroblast functions; e.g., endotoxins from periodontal pathogens may penetrate the root surface and have been implicated in inhibiting fibroblast proliferation, synthesis, and attachment. ,,,,,,,, Rough surface have been considered to endorse cell attachment.  The hypermineralisation of diseased root is reported to act as a barrier to fibroblast attachment. , Contrary to the belief that root treatment is a must for initial fibroblast attachment, root instrumentation does not appear to be an essential prerequisite for initial attachment of fibroblast like cells, provided the cells are healthy and present in the immediate environment.  The finding that cells attach to diseased and nondiseased roots does not necessarily mean that connective tissue reattachment will occur on noninstrumented diseased roots. It would appear instead that events subsequent to initial cell attachment are more likely to be those responsible for limiting regeneration.  There is no consistent literature on fibroblast attachment to healthy and untreated diseased roots.
There was no significant difference in FL-FA and NFL-FA to scale and root planed NFD root fragments. This could be possibly attributed to the configuration and composition of scaled root surfaces which 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. 
There was statistically significant greater NFL-FA to SRP + EDTA treated root fragments than fluorosed fibroblasts. There are two main purposes of acid demineralization; different degrees of collagen exposure and smear layer removal produced due to instrumentation. Few authors have suggested that the inconsistencies in the effect of different root biomodification agents on fibroblast attachment may in part be explained by inadequate demineralization of the periodontitis affected root. ,, 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 second objective of this study was to compare FL-FA and NFL-FA to fluorosed root fragments wherein, two unexplored entities like fluorosed root and fibroblasts that would have been possibly altered, were dealt for the 1 st time in literature. There was no significant difference in FL-FA and NFL-FA to healthy fluorosed root fragments. The need of the hour is to explore the chemical composition of healthy fluorosed cementum in terms of collagen content and its byproducts or chemotactic stimulants for fibroblasts.
No significant difference was found in FL-FA and NFL-FA to FD roots. The fibroblast attachment to diseased root surface is not consistent in literature. , 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.
The NFL-FA to fluorosed scaled roots was higher. The reason for this difference requires to be elucidated. However, it can be possibly explained as follows; the smear layer is composed of very small particles of mineralized collagen matrix and generally 1-5 μ thick which has been recently shown that protein associated with layer enhanced cell migration, attachment, differentiation and proliferation. ,,, Removal of superficial cementum provided a suitable surface for cell attachment.  The configuration and composition of 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.
Two purposes of acid demineralization are collagen exposure and smear layer removal produced due to instrumentation. As fibroblast adhesion and locomotion is facilitated by collagen, ,, and smear layer prevents it. The delicate balance of these two factors is vital to fibroblast attachment and both the factors determine the number of fibroblast attachment. In case of higher concentration acid demineralization, there are chances of over demineralization and possible degradation of collagen (instead of collagen exposure), which results in a surface that would prevent fibroblast attachment. The fluorosed and NFL-FA to FD root fragments treated with SRP + EDTA was observed without any significant difference 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 on inadequate removal of smear layer by EDTA, an in vitro study  and an in vivo animal study  by Blomlöf et al. reported effective removal of smear layer using EDTA. Few studies reported significantly greater number of fibroblast attachment to specimens treated with root biomodification. ,, Unfortunately the overdemineralization of fluorosed root leading to loss of collagen may be responsible for poor fluorosed and NFL-FA. The possible hypomineralization of fluorosed cementum would have resulted in overdemineralization after 24% EDTA application. No similar comparative studies exist in the literature. However, this study finding can be indirectly supported by the report of Vandana et al. who observed the incomplete removal of smear layer using EDTA treated fluorosed root, which are the evidences of hypomineralization.  Another study conducted by Dhingra, Vandana et al. showed that FH group (73.33%) showed more amount of melting of surface than NFH group (66.67%) on the application of Er:YAG laser (140 mJ/pulse and 10 Hz) at ×2500 magnification. Any conclusive interpretation from these results is difficult to decipher at present.  Hence during clinical situations, the degree of mechanical debridement and chemical biomodification has to be done cautiously. The concentration of the biomechanical agent requires to be determined to treat fluorosed roots.
Overall conclusion of this study include that both types of fibroblasts showed similar attachment to different root fragments except for the NFL-FA to scaled fluorosed root.
As a matter of fact, present study is a first attempt to elucidate the fibroblast attachment with respect to fluorosed roots. The Medline search reveals no comparative literature similar to the present study. Two major factors in periodontal wound healing are structural and chemical quality of root surface and fibroblasts. Further research should be directed to study and compare the morphological and functional aspects of human periodontal ligament fibroblasts obtained from the dental fluorosis subjects residing in endemic fluorosed area with that of human periodontal fibroblasts from nonendemic areas. Second structural (mineralisation changes) and chemical composition of fluorosed cementum requires to be studied. Further, usage of histologic or scanning electron microscopy analysis can be considered for better analysis of attachment.
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