RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidate andAddress (in block letters) / DR PRAVIN KUMAR
DEPARTMENT OF PEDODONTICS & PREVENTIVE DENTISTRY,
GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE,
BANGALORE-560002.
2. / Name of the Institution / GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE,
BANGALORE-560002.
3. / Course of study and subject / MASTER OF DENTAL SURGERY
PEDODONTICS AND PREVENTIVE DENTISTRY
4. / Date of Admission to course / 31.05.2012
5. / Title of the research topic :
COMPARATIVE EVALUATION OF RINGER’S LACTATE WITH DEXTROSE NORMAL SALINE (DNS), RICETRAL, EGG WHITE AND INFANT MILK FORMULA ON PERIODONTAL LIGAMENT CELL VIABILITY.
-AN IN VITRO STUDY
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6.7.
8. / BRIEF RESUME OF THE INTENDED RESEARCH WORK
6.1. Need for the study
Tooth avulsion or exarticulation is the loss of a tooth, following trauma.The treatment of choice for an avulsed tooth is immediate replantation. If doubt exists that the tooth can be replanted immediately, the tooth should be stored in a storage medium.1
Success of replantation was initially thought to be mainly associated with the speed
with which the tooth is replanted i.e. shorter the extra alveolar time, better the prognosis but it is changed with time and researchers have demonstrated that storage medium is one of the most important factor than the extra oral time as short period of dry storage shows comparatively poor prognosis than the prolonged storage in a suitable medium. Then the question arises which is the most suitable and best medium and more importantly its cost and ease of availability.2
Various materials have been tested for their potential to serve as a storage medium for avulsed teeth, such as water, saline, milk, and culture medias, with differing degrees of success. However, none of the currently used media are ideal and the search for a suitable storage medium, that can overcome the limitations of these materials continues.1
Lactated ringer’s solution was one of the first laboratory solutions of salts in water that showed to prolong the life of tissues removed from the body.8 Fibroblasts are usually the predominant cells obtained from the cultured PDL tissue. The viability of the PDL fibroblasts is critical for successful healing of replanted avulsed teeth5. Ringer's lactate did not induced significant fibroblast injury.6
Lactate was as effective as glucose in preserving retinal vascular endothelial cell (RVEC) viability in vitro and Hartmann's lactated Ringer's solution was superior to balanced salt solution in maintaining RVEC viability.7
The aim of the present study is to evaluate the effectiveness of ringer’s lactate as a storage media for avulsed tooth in maintaining the PDL cell viability along with Dextrose normal saline, Ricetral, egg white and infant milk formula.6.2 Review of literature
1. Rajendran P, Varghese NO, Varughese JM, Murugaian E.
Conducted a study to determine the efficacy of an oral rehydration solution ‘Ricetral’, in retaining the vitality of periodontal ligament cells.
This study concluded that the ability of Ricetral to retain PDL cell vitality was similar to HBSS and both these solutions were better than milk.1
2. Tiwari M, Wadhwani KK, Agrawal AK.
Conducted a study to compare the capability of different easily available storage media viz., normal saline, milk, dextrose, dextrose normal saline, saliva, tap water, Dulbacco’s Modified Eagle’s Medium and combinations of them as storage/transport media for avulsed tooth, using human PDL cells under in vitro condition over a time period of 30 minutes to 96h.
In conclusion, a Satisfactory result with milk was explained on the basis of its physiological osmolality and markedly fewer bacteria than saliva. DNS+milk showed the best results and also the results obtained by milk and DNS separately were very appreciable.2
3. Khademi AA, Saeed SA.
Evaluated the efficacy of egg white in maintaining the viability of human periodontal ligament (PDL) cells on avulsed teeth
The study concluded that Egg white was a suitable storage medium for an avulsed tooth. It was as good as using HBSS and may be preferable because it is more likely to be available at the site of a traumatic event than HBSS.3
4. Pillegi R, Dumsha TC, Nor JE.
Compared the number of viable PDL cells in different storage media using a collagenase assay.
This study concluded that
1. Milk was comparable to saline and HBSS for storage of avulsed teeth when dry time did not exceed 30 min.
2. The collagenase and dispase assay appeared to be a viable method for evaluating PDL cell viability.4
5. Buffa EA, LubbeAM, Verstraete FJ, Swaim SF.
Conducted an in vitro study to determine the effects of phosphate-buffered saline (PBS), sterile tap water, normal saline, and Ringer's lactate on wound healing
Results showed that Ringer's lactate did not induce any significant fibroblast injury and they stated that further clinical investigation is indicated to establish whether Ringer's lactate is the wound lavage solution of choice compared with normal saline.6
6.3 Research question
Is Ringer’s lactate a possible substitute as a storage media for avulsed tooth
6.4 Objectives of the study
1. To determine the number of viable periodontal ligament cells in avulsed teeth with an extra-oral period of 30 minutes, followed by a 45 minutes immersion in one of the five experimental storage media using collagenase dispase assay.
2. To compare and evaluate the efficacy of new storage media, Ringer’s lactate with that of Dextrose normal saline, Ricetral, egg white and infant milk formula in maintaining the viability of PDL cells which are easily available at places with higher risks of accidents i.e. in school / house.
Materials and Methods
7.1 Source of data
Human teeth with closed apices that are extracted for orthodontic purpose will be obtained from the department of Oral and maxillofacial surgery, Government dental college and research institute, Bangalore.
MATERIALS USED IN THE STUDY:
· Collagenase type III
· Trypsin solution
· Phosphate buffer saline (PBS)
· Fetal bovine serum
· Ringer’s lactate
· Dextrose normal saline
· Ricetral
· Egg white
· Infant milk formula
· Trypan blue
ARMAMENTARIUM:
· 15ml falcon tubes
· Centrifugation machine
· Micropipettes
· Neubauer’s chamber
· Light microscope with haemocytometer
7.2 Methods of collection of data
SAMPLE SIZE:
Sixty teeth that are extracted for orthodontic purposes.
INCLUSION CRITERIA:
1. Human permanent teeth with closed apices will be collected. The age of patients should be within 30 years.
EXCLUSION CRITERIA:
1. Teeth extracted from patients with moderate to severe periodontal disease
2. Teeth with extensive carious lesions
3. Traumatically extracted teeth
4. Teeth that may have been damaged by any reason (e.g. While handling after extraction)
7.3 Method of allocation of data
Sixty non carious premolar teeth extracted for orthodontic therapeutic purposes will be selected for the study. The tooth will be left extra orally for about 30 minutes, during which it will be left on the ground for 10 minutes to simulate an avulsion injury, then the teeth will be held with forceps by the coronal region and the coronal 3mm of PDL will be scraped with a curette to remove the cells that may have been damaged.
The teeth in the experimental group will be stored in one of the following five
groups (10 teeth each) for 45 minutes.
Group I. Teeth specimen stored in Ringer’s lactate
Group II. Teeth specimen stored in DNS
Group III. Teeth specimen stored in Ricetral
Group IV. Teeth specimen stored in Egg white
Group V. Teeth specimen stored Infant milk formula
Positive control: Five freshly extracted teeth will be immediately assayed for cell vitality.
Negative control: Five freshly extracted teeth will be kept extra oral for 8 hours with no follow up storage time and then be assayed for cell vitality.
7.4 Method of study
Each sample will be gently rinsed in phosphate buffer saline (PBS) to remove blood and debris adhering to the roots. Isolation of the cells from the periodontal ligament will be carried out enzymatically, using Collagenase Type III and Trypsin
Each PBS-rinsed sample will be immersed in 1 ml of this enzyme solution for 10 min in a sterile 15-ml Falcon tube. The solution will be agitated using a micropipette for the last 2–3 min of immersion, to facilitate detachment of cells. At the end of 10 min of enzyme treatment, the teeth will be removed from the solution and 1 ml of the solution will be pipetted to a microtube. Ten microliters of fetal bovine serum will be added to it. The tube will then be centrifuged at 90 g for 4 min. The supernatant will be removed and the pellet will be dissolved in 1 ml of PBS. This will be stained using 0.4% (w/v) trypan blue in a 1:1 ratio (100 µl of solution with 100 µl of dye). After 10 minutes,
10 µl of the stained solution will be taken on a Neubauer’s counting chamber and the cells will be viewed under 10 x magnification.1
7.5 Does the study require any investigations or interventions to be conducted on patients or humans or animals? If so, please describe briefly.
No.
7.6 Has the Ethical clearance been obtained from your institution?
Not applicable.
7.8 Statistical analysis
Statistical analysis of the data will be carried out using nonparametric tests, Krusker Wallis H-test, and Mann–Whitney U-test.
Duration of the study
The duration of the study is for a period of one and a half years (approximately).
LIST OF REFERENCES:
1. Rajendran P, Varghese NO, Varughese JM, Murugaian E. Evaluation, using extracted human teeth, of Ricetral as a storage medium for avulsions – an in vitro study Dental Traumatol 2011; 27: 217–220.
2. Tiwari M, Wadhwani KK, Agrawal AK. Evaluation of Periodontal Ligament Cells Viability in Different Storage Media - In Vitro Study. JIDA, Vol. 5, No. 4, April 2011
3. Khademi AA, Saeed SA. New Storage Medium for an Avulsed Tooth. The Journal of Contemporary Dental Practice 2008; 9(6):25-32.
4. Pillegi R, Dumsha TC, Nor JE. Assessment of post traumatic PDL cell viability by a novel collagenase assay. Dent Traumatol 2002; 18:186-9.
5. Saad Al-Nazhan, Afaf Al-Nasser. Viability of Human Periodontal Ligament Fibroblasts in Tissue Culture After Exposure to Different Contact Lens Solutions. The Journal of Contemporary Dental Practice 2006; 7(4):1-9.
6. Buffa EA, LubbeAM, Verstraete FJ, Swaim SF.The effects of wound lavage solutions on canine fibroblasts: an in vitro study.Vet Surg. 1997 Nov-Dec;26(6):460-6.
7. Mourad KM, Edelhauser HF, Capone A Jr, Lynn MJ, Geroski DH. Effect of intraocular irrigating solutions on the viability of cultured retinal vascular endothelial cells. Curr Eye Res. 1997 Mar; 16(3):239-43.
8. The History ofLactated Ringer's|eHow.com Available from http://www.ehow.com/about_5607430_history-lactated-ringer_s.html (accessed 12 Oct 2012)
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9. / Signature of the Candidate10. / Remarks of the Guide
11. / 11.1. Name and Designation of Guide
11.2 Signature / DR. K.S. SURESH
PROFESSOR & H.O.D
DEPARTMENT OF PEDODONTICS AND PREVENTIVE DENTISTRY
GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE,
BANGALORE-560002
11.3. Co-Guide
11.4 Signature / NO
11.7. Head of the Department
11.8. Signature / DR. K.S. SURESH
PROFESSOR & H.O.D
DEPARTMENT OF PEDODONTICS AND PREVENTIVE DENTISTRY
GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE,
BANGALORE-560002
12. / 12.1. Remarks of the Chairman and Principal
12.2. Signature:
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