Abstract
Introduction
Revascularization of necrotic pulp has been successful in the resolution of periradicular
inflammation; yet, several case studies suggest the need for cell-based therapies
using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration.
Because the availability of MSCs may be limited, especially in an aged population,
the current study reports an alternative approach in generating MSCs from epidermal
keratinocytes through a process called epithelial-mesenchymal transition (EMT).
Methods
We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient
transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation
capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation
capacities.
Results
Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin),
whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting
the EMT phenotype acquired extended replicative potential and an increased telomere
length compared with the control cells. Similar to the established MSCs, the NHEKs
with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent
progressive proliferation and differentiation. Upon differentiation, these EMT cells
expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily
formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic
differentiation.
Conclusions
The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells
may be used for tissues engineering purposes, including that of dental pulp.
Key Words
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Article info
Publication history
Published online: September 06, 2014
Identification
Copyright
© 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.
