DR. azin malekfar post graduate student department of conservative dentistry and endodontics



Download 60.62 Kb.
Date conversion26.11.2016
Size60.62 Kb.
DISSERTATION SYNOPSIS

DR. AZIN MALEKFAR

POST GRADUATE STUDENT

DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES AND HOSPITAL, RGC CAMPUS, BANGALORE- 32

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

4th BLOCK, JAYANAGAR, BANGALORE, KARNATKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

NAME OF THE CANDIDATE AND ADDRESS

DR. AZIN MALEKFAR

POST GRADUATE STUDENT,

DEPARTMENT OF CONSERVATIVE DENTISTRY & ENDODONTICS

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES & HOSPITAL, CHOLANAGAR,

R.T. NAGAR POST
BANGALORE - 560 032, KARNATAKA, INDIA


2.

NAME OF THE INSTITUTION

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES & HOSPITAL

3.

COURSE OF THE STUDY AND SUBJECT

MASTER OF DENTAL SURGERY IN CONSERVATIVE DENTISTRY & ENDODONTICS

4.

DATE OF ADMISSION TO COURSE

18.06.2013

5.

TITLE OF THE TOPIC

ISOLATION AND COMPARISION OF DENTAL PULP STEM CELLS FROM INFECTED PULP TISSUES OBTAINED FROM ENDODONTIC TREATMENT AND EXTRACTED TEETH: AN IN VITRO STUDY

6.

BRIEF RESUME OF THE INTENDED STUDY

6.1 NEED FOR THE STUDY

Because of advantages of DPSCs like high proliferation potential, multi-lineage differentiation capacity and immunomodulatory effect(1), concept of dental pulp stem cells banking has recently gained importance because the cells isolated from the human teeth can be stored and utilized for future therapeutic procedures (2).

In order to develop dental pulp stem cell banking, the primary step for using this source of stem cells is to identify and select the best protocol for isolating hDPSCs from various accessible sources of pulp tissue.

Since it has been proved that inflammatory process does not affect the DPSCs properties(3), collecting of these cells from infected dental pulp tissues obtained from endodontic treatment would be of therapeutic value.



Successful isolation of DPSCs from this said source would be considered as a novel approach that can address simplicity and short times of preparation of the cells for dental pulp stem cells banking and their subsequent applications in regenerative medicine.


6.2 REVIEW OF THE LITERATURE


  • In 2000, Dr. Gronthos and colleagues conducted a study for identification of stem cells in human permanent teeth. For the first time DPSCs were isolated as a clonogenic and rapidly proliferative population of cells from enzymatic treatment of pulp of human normal impacted third molars (4).



  • In another study, isolated DPSCs were capable of forming ectopic dentin and associated pulp tissue in vivo. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. Results of this study demonstrated that DPSCs possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation (5).




  • In another set of experiments scientists attempted to develop a new method to cryopreserve DPSCs inside a whole tooth, thus avoiding the need to purify the cells before cryopreservation and reducing the initial costs and workload of tooth banking. Therefore they cryopreserved whole teeth after digging micro-channels into the tooth with laser piercing to allow the cryopreservative to reach the dental pulp and preserve the cells at -80°C. Their data demonstrate that DPSCs isolated from laser pierced cryopreserved teeth show mesenchymal stem cells morphology, immunophenotype, viability and proliferation rate similar to those of cells isolated from fresh, non-cryopreserved teeth (2).



  • Recently it has been reported that DPSCs can be isolated from inflamed dental pulps. Results of this study demonstrated that the morphology, proliferation rate and differentiation potential of these DPSCs were similar to those which were isolated from normal pulp thus indicating that the inflammatory process did not affect the stem cell properties (3).




  • In another study on DPSCs characterization with respect to morphology, growth kinetics, cell surface marker expression and differentiation capacity, scientists’ findings suggest that DPSCs can be expanded with the highest efficiency in KO-DMEM medium supplemented with 10% FBS as best growth medium (6).


6.3 OBJECTIVES OF THE STUDY

  • To isolate DPSCs from vital infected pulp tissues obtained from endodontic treatment and extracted teeth.



  • To evaluate and compare growth kinetics and mesenchymal characterization of derived dental pulp stem cells from these two different approaches.



  • To determine a novel way of DPSCs banking by comparison of the suitability of isolated dental pulp stem cells from these two different approaches.




7.

MATERIALS AND METHODS

7.1 SOURCE OF THE DATA

An in vitro study will be conducted on 10 infected vital pulp tissues obtained from fresh extracted posterior teeth which will be collected from Oral and Maxillofacial Surgery Department and another 10 infected vital pulp tissues obtained by endodontic treatment referred to Conservative Dentistry and Endodontics Department, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Cholanagar, R.T. Nagar Post Bangalore - 560 032, Karnataka, India.

The study will be conducted at Manipal Institute of Regenerative Medicine, Bangalore.

7.2 METHOD OF COLLECTION OF DATA

The study will be conducted in 2 groups;

In group I infected vital pulp tissues will be obtained from fresh extracted posterior teeth from 10 patients undergoing molar and premolar extraction due to caries with irreversible pulpitis.

In group II vital infected pulp tissue will be collected from 10 patients undergoing endodontic treatment due to caries with irreversible pulpitis.

A random sampling technique for collection of data will be used. The study will be started after approval of the Ethical Committee of Health Science Faculty of the Sri Rajiv Gandhi Dental College and Hospital and obtaining the informed consent from the patient.

Group I: (DPSCs from extracted teeth)

Inclusion criteria:


  1. Age between 15-25 years old.

  2. Vital infected pulp tissues obtained from freshly extracted carious teeth with irreversible pulpitis.

Exclusion criteria:

1. Teeth with periodontal disease.

2. Teeth with periapical changes.

3. Teeth with open apex.

4. Healthy impacted teeth.

Group II:(DPSCs from pulp tissues obtained from endodontic treatment)

Inclusion criteria:


  1. Age between 15-25 years old.

  2. Vital infected pulp tissues obtained from endodontic treatment on carious teeth with irreversible pulpitis.

Exclusion criteria:

  1. Teeth with periapical changes.

  2. Teeth with open apex.

MATERIALS

  • Culture media contents:

    • Dulbecco’s modified eagle’s media- knock out (DMEM-KO) (Invitrogen)

    • Fetal bovine serum 10% (Hyclone)

    • Ascorbic acid 100µM (Invitrogen)

    • L-glutamax - 5mM (Invitrogen)

    • 100U/ml penicillin-streptomycin (Invitrogen)

  • Transport media:

    • Dulbecco’s modified eagle’s media (Invitrogen)

  • Collagenase blend (Sigma-Aldrich)

  • Liquid nitrogen

  • Positive markers – CD90, CD73 and CD166 (BD Pharmingen)

  • Negative markers - CD 34, CD 45 and HLA-DR (BD Pharmingen)

  • Isotype – PE IgG1 BD (Pharmingen)

ARMAMENTARIUM

  • Barbed broaches

  • Rubber dam kit

  • Endo access bur(No #2&3 )

  • Aluminum foil

  • Surgical scalpel blade # 21

  • Osteotome

  • Bio safety hood – Bio laminar flow chamber

  • Centrifuge machine (Eppendorf, Germany)

  • Pipette and pipetting aids (Eppendorf)

  • Incubator (Thermo Electron corporation, USA)

  • BD falcon- 35mm2 tissue culture flask (BD Bioscience)

  • Petri dish 50mm x 12mm (BD Bioscience)

  • BD falcon- 15 ml conical base tube (BD Bioscience)

  • Flowcytometry tube (BD Bioscience)

  • FACS calibur flow cytometer (BD Biosciences, USA)

  • Inverted microscope (Nikon)

METHOD

Isolation of DPSCs:

DPSCs will be isolated from human dental pulp tissues obtained from both root canal treatment and extracted teeth (n=20). Dental pulp tissues will be washed 2-3 times with DPBS solution under the laminar flow chamber inside the culture room. Then 2mg/ml collagenase blend will be added and the tissue will be minced to smaller pieces to increase the surface area for better enzyme action. Then minced tissues will be kept in the incubator (5% CO2, 37°C) for 1 hr. After incubation, the action of enzyme will be neutralized by addition of the culture medium. The samples will be then centrifuged at 400g for 5min, cell pellet will be collected and plated in 24-well plates and kept in the incubator. Cultures will be fed with fresh media every 48 hr.

DPSCs will be cultured in KO-DMEM supplemented with 10% FBS, 5mM L-glutamine, and 50U/ml penicillin-streptomycin.

Growth kinetic study:

The growth kinetic study will be carried out by calculating the population doubling (PD), cumulative population doubling (CPD) and population doubling time (TD). DPSCs will be dissociated with 0.25% trypsin and counted by Trypan blue exclusion method on a Neubauer Hemocytometer at the end of each passage once cells are 90% confluent and then they will be re-plated for the subsequent passage.

The population doubling will be determined by the formula: PD =LogNh-LogNi/Log2; where Ni = number of cells at seeding time and Nh = number of cells at harvesting time.

CPD = PD of each passage + PD of previous passage

To calculate population doubling time (TD), cells will be plated in each well of the 24-well plate and cells in 3 wells will be counted at a time after 24, 48, 72 hrs and so on till the 192nd hour i.e. 8th day in culture; an average number of cells in every three wells will be considered for analysis. Then the values will be plotted as a graph as cell number versus time (days). The time at which the cell number is double the initially seeded cell-number determines the population doubling time of the cells.

Mesenchymal characterization:

Flowcytometry method: flowcytometry based DPSCs will be performed to characterize these cells for a candidate set of MSC-specific surface markers. Cells will be harvested and counted; then 10µL of tagged antibody will be added into appropriate number of cells. Samples will be stained for 1h at 40C and then will be washed with FACS buffer for 5 min. To fix the cells, 4% PFA will be added to the pellet and re-suspended. The following markers will be used for immunostaining: CD166-PE, CD73-PE, CD90-PE, CD34-FITC, CD45-PE, HLA-DR-PE and data analyses will be optimized against control cells incubated with specific isotypes IgG1-PE, on FACS Calibur (BD). Cells will be identified by light scatter for 10,000 gated events and analyzed using BD Cell Quest Pro software (BD).



PLAN FOR DATA ANALYSIS

Results will be presented as mean ± standard error of the mean (SEM). Statistical comparisons will be performed using appropriated methods, Student’s t-test and one or two way ANOVA.



7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED IN PATIENTS OR OTHER HUMANS OR ANIMALS

Yes.


  • Patients will be subjected to intra-oral periapical radiography.

  • Pulp vitality test.

7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?

Yes.



8.

LIST OF REFERENCES




1. Huang GT-J, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009 Sep;88(9):792–806.

2. Gioventù S, Andriolo G, Bonino F, Frasca S, Lazzari L, Montelatici E, et al. A novel method for banking dental pulp stem cells. Transfus Apher Sci Off J World Apher Assoc Off J Eur Soc Haemapheresis. 2012 Oct;47(2):199–206.

3. Pereira LO, Rubini MR, Silva JR, Oliveira DM, Silva ICR, Poças-Fonseca MJ, et al. Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps. Int Endod J. 2012 Dec;45(12):1080–90.

4. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13625–30.

5. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002 Aug;81(8):531–5.

6. Kanafi MM, Pal R, Gupta PK. Phenotypic and functional comparison of optimum culture conditions for upscaling of dental pulp stem cells. Cell Biol Int. 2013 Feb;37(2):126–36.




9.

Signature of the candidate




10.

10.1 Remarks of the guide




10.2 Name and designation of Guide( in block letters)


Dr. KUSUM VALLI. S

PROFESSOR AND HOD

DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODOTICS,

SRI RAJIV GANDHI COLLEGE OF


DENTAL SCIENCES AND HOSPITAL

10.3 Signature




11.

11.1 Co-guide (If any)

DR. RAMESH. R. BHONDE

DEAN AND PROFESSOR

MANIPAL INSTITUTE OF REGERNERATIVE MEDICINE-BANGALORE,

MANIPAL UNIVERSITY



11.2 Signature




12.

12.1 Head of the department


Dr. KUSUM VALLI. S

PROFESSOR AND HEAD OF THE

DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODOTICS,

SRI RAJIV GANDHI COLLEGE OF


DENTAL SCIENCES AND HOSPITAL

12.2 Signature




13.

13.1 Remarks of the principal




13.2 Principal of the institution

Dr. VAIBHAVI JOSHIPURA

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES AND HOSPITAL



13.3 Signature




DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES AND HOSPITAL, RGC CAMPUS, BANGALORE- 32

CONSENT FORM

Patient Name:

Age and Sex:

Contact information:


Dear Patient,
I, Dr. Azin Malekfar, a post graduate student in the Department of Conservative Dentistry and Endodontics, Bangalore, have examined you. The details of the case findings and treatment for the same have been explained to you and found that your tooth is required endodontic treatment as one of the procedures.
I am conducting a study entitled, “Isolation of dental pulp stem cells from infected pulp tissues obtained from endodontic treatment and extracted teeth: an in vitro study”.
Since your dental pulp tissue obtained from your treated tooth would be utilized for this study, I kindly request you to give your consent for the same.
From,
Dr. Azin Malekfar

Postgraduate student

Department of Conservative Dentistry and Endodontics,

Sri Rajiv Gandhi College of Dental Sciences & Hospital, Cholanagar,

R.T. Nagar Post
Bangalore - 560 032, Karnataka, India

The above procedure has been explained to me in the language best understood by me. Hereby, I agree to give consent for the above mentioned procedure.

Date:

Place Signature of the patient


DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES AND HOSPITAL, RGC CAMPUS, BANGALORE- 32

CONSENT FORM

Patient Name:

Age and Sex:

Contact information:


Dear Patient,
I, Dr. Azin Malekfar, a post graduate student in the Department of Conservative Dentistry and Endodontics, Bangalore, have examined you. The details of the case findings and treatment for the same have been explained to you and found that your tooth is required extraction as one of the procedures.
I am conducting a study entitled, “Isolation of dental pulp stem cells from infected pulp tissues obtained from endodontic treatment and extracted teeth: an in vitro study”.
Since your extracted tooth would be utilized for this study, I kindly request you to give your consent for the same.
From,
Dr. Azin Malekfar

Postgraduate student

Department of Conservative Dentistry and Endodontics,

Sri Rajiv Gandhi College of Dental Sciences & Hospital, Cholanagar,

R.T. Nagar Post
Bangalore - 560 032, Karnataka, India

The above procedure has been explained to me in the language best understood by me. Hereby, I agree to give consent for the above mentioned procedure.



Date:

Place Signature of the patient


The database is protected by copyright ©dentisty.org 2016
send message

    Main page