Walls, Angus

Travers, Paul

Dutta, Arindam

2022-09-23T13:58:16Z

2022-09-23T13:58:16Z

2022-09-23

INTRODUCTION: Regenerative endodontic procedures have been practiced approximately over the past twenty years. These regenerate pulp-like tissue within the root canals of immature permanent teeth with pulpal necrosis which may also have periapical disease. The procedure involves disinfecting the root canal first, followed by conditioning the root surface dentine before a scaffold (such as a blood clot) is introduced into the canal space, along with mesenchymal stem cells (MSCs). Removal of the smear layer is usually undertaken with ethylenediaminetetraacetic acid (EDTA), which is also able to liberate extracellular matrix proteins embedded within dentine that help support the differentiation of MSCs. Maleic acid (MA) is another chelator which has been introduced to endodontics approximately ten years ago. It’s ability to extract dentine matrix proteins (DMP) and their subsequent influence on MSCs is unknown. AIMS: Characterise the DMP extracted by EDTA (EDMP) or MA (MDMP) from dentine and evaluate their effects on odontoblastic differentiation of dental pulp stem cells (DPSC). METHODS: Dentine powder was prepared from human healthy caries-free third molar teeth and exposed to EDTA or MA and total soluble proteins recovered were measured. The constituent proteins in each sample were also analysed using mass spectrometry (MS) & transforming growth factor 1 (TGF1) concentration was estimated using Enzyme-linked immunosorbent assay (ELISA). The proteins were then added to culture medium and exposed to DPSC which had also been characterised. DPSC grown in osteogenic medium (OM) served as the positive control & MDMP+OM and EDMP+OM as additional experimental groups. The influence of DMPs on odontoblastic differentiation of DPSCs was evaluated by using reverse transcription-quantitative polymerase chain reaction assessing relative gene expression of COL1A1, RUNX2, MSX2, DLX5, NAT10, OCN, OPN, DMP1 and DSPP and Alizarin red assay to quantify mineralisation. RESULTS & CONCLUSIONS: Even though the total protein quantified in MDMP was lower than EDMP, proteomic profiles differed, with more proteins predicted in MDMP. TGF1 as a fraction of the total protein quantum was about 2-3 times higher in MDMP than EDMP. These findings may indicate that while dentine structure may be better preserved with MDMP, biologically relevant proteins may be extracted better by MA than EDTA. The DPSCs were not true stem cells, but were at least unipotent and displayed all canonical stem cell markers. DPSCs exposed to both MDMP and EDMP did not differentiate into odontoblasts and genes associated with mineralisation were generally suppressed. This may have relevance in formulating novel pulp regeneration strategies

https://hdl.handle.net/1842/39381

en

The University of Edinburgh

dentine matrix proteins

dental pulp stem cells

TGFB1

maleic acid

EDTA

RT-qPCR

mass spectrometry

flow cytometry

Dentine matrix proteins solubilised by 17% ethylenediaminetetraacetic acid and 7% maleic acid and their influence on human dental pulp stem cells: an in vitro study

Thesis or Dissertation

Doctoral

DDS Doctor of Dental Surgery