Influence of rAm and rAb in Differentiation of Odontogenic Cells in vitro

Overview

Objectives/Goals An extract of pig enamel proteins is being used to repair tissues destroyed by periodontal disease without knowing what is the active ingredient. In this project I want to determine if the major enamel proteins (amelogenin (Am) or ameloblastin (Ab)) can function like growth factors to repair tissues. Methods/Materials Cells were grown in Petri dishes containing permissive media until they reach confluence and then placed on differentiation media containing recombinant Am or Ab protein or just the media. Cells were grown for different days, media was changed every other day and changes in morphology were documented before mRNA was isolated. Cell differentiation was determined using Reverse Transcription # Polymerase Chain Reaction (RT-PCR). RT was done by adding a Poly-T, 4 deoxy-nucleotides and reverse transcriptase enzyme to the mRNA and incubated at 42oC for 1 hour. PCR was done by with 1 µl of cDNA, dNTPs, Ex Taq polymerase and specific primers for proteins associated with tooth formation. The PCR products are analyzed using agarose gel Electrophoresis and the genes expressed are visualized using UV light. Results The results indicate that there are no morphological changes in cells incubated with rAM or rAB as compared to the control. The results from the RT-PCR indicate that there is a band for β-actin primer for each of the cells used. Osteonectin primer: there is no differences at 7 days, the expression goes down at 14 days in culture, and at 28 days, rAm down regulates the expression of osteonectin, rAb just a little. BMP4: day 7, no effect, follows a similar profile as osteonectin except at 35 days rAm and rAb down regulated its expression. NFI-X primer: at 14 days, rAb up-regulates expression of NFI-X, later it down regulates. For the DSPP primer bands do not appear. Conclusions/Discussion No morphological changes were found in any of the cells tested in the presence of rAb or rAm. All mRNAs obtained were of good quality to do the studies. rAm down regulates the expression of osteonectin and BMP-4 after 14-28 days in culture. rAb up-regulates the expression of NFI-X at 14 days and then it down-regulates it. This suggest that Am and Ab might act as growth factors and induce cell differentiation besides being involved in the production of an enamel extracellular matrix.

Competition history

• CSEF 2002 Pharmacology/ Toxicology · Entry S1424

Resources

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