Brief Resume of the Intended Work Need for the Study

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Brief Resume of the Intended Work
Need for the Study:

The root surface walls of periodontal pockets harbour bacteria and endotoxins, which hinders the reattachment of gingival and periodontal cells1.Though scaling and root planing are followed conventionally as a part of periodontal therapy, it is not completely effective in removing the diseased cementum.Therefore, chemical root conditioning is advocated to achieve a biologically suitable root surface for epithelial and connective tissue attachment2.

Dentinal collagen exposed by demineralization enhances binding of the extracellular matrix glycoprotein to dentin.3 This increases the attachment and growth of fibroblasts on dentin surfaces.4

` Tetracyclines are one of the effective agents used to enhance periodontal regeneration.5 Root dentin treatment by tetracycline removes the smear layer and exposes the dentin with open tubules.6 It promotes the binding of fibronectin to dentin.The adsorbed fibronectin stimulates fibroblast attachment and growth.Tetracyclines reduce excessive collagenase activity.Also, tetracycline is adsorbed to and subsequently released from dentin, maintaining its antimicrobial activity.7

Tetracycline hydrochloride concentration ranges from 0.5% to 200% and application periods between 0.5 to 10 minutes.8,9 Higher concentrations of tetracycline hydrochloride are required to achieve complete demineralization .8,10 The efficacy of application times and optimal concentration of tetracycline hydrochloride to achieve root surface characteristics to support periodontal reconstructive therapy have yet to be established.11

The purpose of this study is to evaluate and compare surface changes of human root dentin at different Tetracycline hydrochloride concentrations of 0,10,25,50,75,100,125 and 150mg/ml solutions, and also different application times i.e, 1,3 and 5 minutes, using Scanning Electron Microscope.The surface characteristics are compared and the width of the dentinal tubule orifices will be measured.

Objectives of the study:
The objectives of this study are to evaluate and compare the surface morphology of human root dentin following-

  1. Application of various concentrations of Tetracycline hydrochloride

  2. Different application times

The concentrations (weight per volume) of tetracycline hydrochloride test solutions are 0,10,25,50,75,100,125 and 150mg/ml

Application times for each group are 1,3and 5 minutes.
Observations are made under Scanning Electron Microscope for the following-S 1)Removal of smear layer on the root surface

2)Morphological changes on the root surface

3)Opening of dentinal tubules.

The width of dentinal tubules will be analysed and compared for each concentration and for each application time.

Materials and Methods
1. Source of data:

Extracted teeth collected from patients visiting Department of Oral and Maxillofacial Surgery,

Krishnadevaraya College of Dental Sciences and hospital.

2. Method of collection of data:

Sample size:

24 freshly extracted periodontally affected single rooted teeth with poor prognosis.

Inclusion criteria

Periodontally affected single rooted teeth with poor prognosis indicated for extraction.

Exclusion criteria

1)Teeth with root caries.

2)Teeth with root surface demonstrating abrasive lesions.

3)Teeth with root surface showing demineralized areas.

4)Teeth with root surface showing hypercementosis.

5)Teeth with fractured root surface

6)Patients who give history of scaling and root planning in the previous 6


7)Teeth with root restorations.

8)Patients who give history of using desensitizers and mouth washes.

3.Sampling technique:
48 specimen samples will be prepared from the proximal surface of 24

periodontally affected single rooted teeth indicated for extraction.

Prior to specimen preparation,root surface apical to cementoenamel junction

is subjected to thorough scaling and root planning using Gracey curettes


Sectioning procedure:
The crown portion of the extracted teeth will be sectioned following the cementoenamel junction as the guideline using high speed diamond bur.Following the separation of the crown from the roots,a longitudinal sectioning will be done at the line marked on the facial and lingual or palatal surface of the roots extending 6mm apically from the CEJ and two proximal halves will be separated with cross section at the termination of 6mm vertical cut.The root blocks will measure individually in 3 dimensions as follows




The specimens will be prepared thoroughly,washed and will be stored in distilled water until further procedures.
Criteria for grouping

A total of 48 dentin specimens are divided into

  • 8 groups for conditioning at tetracycline hydrochloride concentration(weight

per volume) of 0,10,25,50,75,100,125 and 150mg/ml and

- 3 application periods of 1,3, and 5 minutes(i.e, 2 samples per group).

Preparation of Tetracycline hydrochloride solutions:
Fresh tetracycline hydrochloride solutions will be prepared at room temperature by weighing pure tetracycline hydrochloride powder and dissolving it in 20 to 35 ml of distilled water, using a magnetic stirrer to mix the solution to a final volume of 50ml.The concentrations(weight per volume) of tetracycline hydrochloride test solutions are 0,10,25,50,75,100,125,and 150mg/ml.The pH of each solution will be tested with a hand-held, battery operated pH meter and recorded.

The specimens in each group will be conditioned with one of the 8 concentrations and the dentin surface is burnished for 1,3 or 5 minutes with a solution-soaked cotton pellet changed every 30 seconds;the area will then be flushed for 30 seconds with distilled water.

Specimen preparation for SEM:
Immediately after applying Tetracycline hydrochloride and rinsing,the dentin specimens are fixed in 2.5% Glutaraldehyde in phosphate buffer (pH 7.3) for 24 hours at 40C, washed three times in same phosphate buffer, post-fixed in phosphate-buffered 1.5% osmium tetroxide for 2 hours,and washed three more times in phosphate buffer.The specimens will then be dehydrated in a graded series of aqueous ethanol solutions(50%,70%,85%,95%,and 100% ethanol) for 10 minutes each.After two more 10- minute washings in absolute ethanol,the samples will then be dried overnight in a desiccator jar.
The specimens thus prepared will be subjected to Scanning Electron Microscopic analysis to evaluate-

  • Smear layer removal

  • Morphological changes on the root surface

  • Opening of dentinal tubules;the width of dentinal tubules for each concentration of Tetracycline hydrochloride solution and for each application time.

4. Statistical analysis
The means and standard deviation of diameter of the tubule orifices for each solution is determined for 1,3, and 5 minute application periods.One-Way analysis of variance is used to examine effect of application times and tetracycline hydrochloride concentrations on the width of dentinal tubule orifices.All groups are tested by multiple range test (Tukey test).Paired t-test is performed for each combination of application time and concentration.

List of References

  1. Andriaens PA,De Boever JA,Loesche WJ-Bacterial invasion in root cementum

and radicular dentin of periodontally diseased teeth in humans A reservoir of

periodontopathic bacteria. J Periodontol 1988 Apr;59 4):222-230

  1. Coldrion NB,Yukna RA,Weir J,Caudill RF-A quantitative study of cemenum

removal with hand curettes. J Periodontol 1990 May;61(5):293-299.
3) Polson AM,Proye MP-Fibrin linkage:A precursor for new attachment.

J Periodontol 1983;54:141-147.

4) Polson AM,Hanes PJ-Cell and fiber attachment to demineralized dentin:A comparison of normal and periodontitis affected root surfaces.

J Clin Periodontol 1987;14:357-365

5)Bjorvatu K-Antibiotic compounds and enamel demineralization.An invitro study. Acta Odontol Scand 1982;40:341-352

6)Wikesjo UME,Baker PJ,Christersson LA,et al-A biochemical approach to periodontal regeneration:Tetracycline treatment conditions dentin surfaces.

J Periodon Res 1986;21:322-329
7) Delazari FMC,Gerlach RF,Joly JC,Lima AFM-Scanning Electron Microscopy Study of the effect of Tetracycline HCl on smear layer removal and fibrin network formation .Braz Dent J (1999)10(2):81-87
8) Hanes PJ,O’Brien NJ,Garnick JJ-A morphological comparison of radicular

dentin following root planning and treatment with citric acid and

tetracycline HCl. J Clin Periodontol 1991;18:660-668
9)Madison JG,Hohett SD-The effects of different tetracyclines on the dentin

root surface of instrumented,periodontally involved human teeth:A comparative

scanning electron microscopic study. J Periodontol 1997;68:739-745
10)Sterret JD,Simmons J,Whitford G,Russell CM-Tetracycline demineralization

of dentin:the effects of concentration and application time.

J Clin Periodontol 1997; 24:457-463
11)Nadir Babay SEM study on the effect of two different demineralization methods

with saturated tetracycline hydrochloride on diseased root surfaces.

The Journal of Contemporary Dental Practice ,Volume 2, No.2 ,Spring ISSUE ,2001
12)A.Gulden Isik, Berna Tarim, Abeer A.Hafez, Funda Saglam Yalcin,

Utku Onan, and Charles F. Cox-A comparative scanning electron microscopic study

on the characteristics of demineralized dentin root surface usingdifferent tetracycline HCl

sconcentrations and application times-J Periodontol 2000;71:219-225.
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