In vitro characterization of human dental pulp cells: various isolation methods and culturing environments

Abstract

Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.

How has dental pulp stem cells isolation been conducted? A scoping review

“…However, this process can cause cell damage and induce cell death if not done properly. 23 All evaluated articles in our study described the enzyme used for DPSCs dissociation. Only five families of enzymes were mentioned: collagenase, dispase, trypsin, thermolysin and DNAase.…”

“…18 Few studies have compared the enzymatic and explant techniques for DPSCs isolation. 23,29,31 In a well-designed study, Hilkens and collaborators showed that cells isolated by the enzymatic or the explant techniques had the same proliferating rate and colony formation capacity, and could successfully differentiate into adipogenic, chrondrogenic and osteogenic cell types, indicating that both isolation methodologies could be applied to obtain suitable autologous DPSCs. 29 One particular aspect of the explant technique is the size of tissue fragments.…”

The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot Engineering

“…New blood vessels also penetrated the cells/PGA implants in vivo 3 weeks after the implantation. Since the isolation and characterization of DPSCs and SHED (6,16), using these stem cells for dentin/pulp tissue regeneration has drawn great interest (47)(48)(49).…”

“…Collagen has been considered as a convenient pulp cell carrier and could conveniently be injected into canal space to regenerate pulp clinically, yet collagen as the matrix has been found to contract significantly when carrying pulp cells (47,57), which may considerably affect pulp tissue regeneration. Our in vivo experiments using mouse as a study model showed that when pulp cells were cast into collagen gel and placed into a canal space, the contraction interfered in the pulp regeneration.…”

What is the clinical applicability of regenerative therapies in dentistry?

“…The enzymatic digestion process involves the application of the enzymes collagenase I and dispase in direct contact with the tissue, in order to disaggregate the tissue and obtain cell suspension. In this way, the released stem cells can adhere quickly to the bottom of the culture dish with greater ease 14 . With the explant technique, the tissue does not undergo any method of cell dissociation, however, the tissue is fragmented into several pieces and deposited on culture dishes (Figure 2).…”