Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts.
BACKGROUND
Periodontal ligament fibroblasts (PDLFs) are the cells essential for periodontal regeneration. PDLFs comprise a heterogeneous cell population and consist of several cell subsets that differ in their function. It is known that PDLFs produce osteoblast-related extracellular matrix proteins and show higher alkaline phosphatase (ALP) activity compared with gingival fibroblasts (GFs), implying that PDLFs have osteogenic characterisitics. The aim of the present study was to isolate the osteogenic population of PDLFs according to their expression of ALP.


METHODS
PDLFs and gingival fibroblasts were separated into two populations, ALP-positive and ALP-negative, with an immunomagnetic method using a monoclonal antibody against human bone type ALP and magnetic beads conjugated with a secondary antibody. Expression of basic fibroblast growth factor (bFGF) receptor and transforming growth factor (TGF)-beta receptor was investigated in these two populations. Osteoblast-related molecules, osteocalcin, and bone sialoprotein; ALP activity; and effect of bFGF on proliferation were also compared.


RESULTS
Effective separation was confirmed in both PDLFs and GFs by flow cytometry. The expression of FGF receptor (FGFR) and TGF-beta receptor was significantly higher in ALP-positive PDLFs than in ALP-negative PDLFs. ALP-positive PDLFs also expressed higher mRNA levels of osteocalcin and bone sialoprotein compared with ALP-negative PDLFs. The mitogenic effect of bFGF on ALP-positive PDLFs was greater than that of ALP-negative PDLFs.


CONCLUSIONS
These results indicate that osteoblastic and/or cementoblastic PDLF subsets could be isolated from the PDLF populations using an immunomagnetic method. Magnetic isolation of PDLFs may be a useful tool to obtain the cells which will potentially induce mineralization on the root surface.
Murakami
Yuya
Y
Section of Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. murakami.peri@tmd.ac.jp
Kojima
Takehisa
T
Nagasawa
Toshiyuki
T
Kobayashi
Hiroaki
H
Ishikawa
Isao
I
D000469:Alkaline Phosphatase / Q000032:analysis*; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000145:classification / Q000201:enzymology*; D005434:Flow Cytometry; D005881:Gingiva / Q000166:cytology; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D015675:Osteocalcin / Q000032:analysis; D010012:Osteogenesis; D010513:Periodontal Ligament / Q000166:cytology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis; D012795:Sialoglycoproteins / Q000032:analysis; D018709:Statistics, Nonparametric
C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D018125:Receptors, Transforming Growth Factor beta; D012795:Sialoglycoproteins; D016222:Fibroblast Growth Factor 2; D015675:Osteocalcin; C496353:FGFR2 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; D000469:Alkaline Phosphatase