In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.
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.
Huang
George T-J
GT
Division of Associated Clinical Specialties, Section of Endodontics, UCLA School of Dentistry, Los Angeles, CA, USA. GHuang@umaryland.edu
Sonoyama
Wataru
W
Chen
James
J
Park
Sang Hyuk
SH
D001483:Base Sequence; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D003782:Dental Pulp / Q000166:cytology* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009804:Odontoblasts / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins; D013499:Surface Properties; D023822:Tissue Engineering; D000099311:Dentin Sialophosphoprotein
D024042:Collagen Type I; D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein