Isolation and Characterization of Alkaline Phosphatase Positive Periodontal Ligament Fibroblasts

摘要

In order to establish the basis of periodontal regeneration, calcification mechanisms of periodontal ligament fibroblasts PDLF should be investigated. It is known that PDLF produce osteoblasts-related extracellular matrix proteins and show higher alkaline phosphatase ALP activity compared with gingival fibroblasts GF. It is also known that PDLF populations are heterogeneous and consist of several cell subsets that differ in their function. The aim of the present study was to isolate the osteogenic population of PDLF. Human PDLF were isolated by scraping the root surface of teeth extracted for orthodontic reasons. Cells were allowed to outgrow from the tissue fragments and subcultured. The cells were then divided into two populations, ALP-positive and ALP-negative, using magnet and magnetic beads conjugated with monoclonal antibody against tissue nonspecific ALP. Expression of receptors for growth factors and osteoblasts-related molecules was compared between these two populations using flowcytometry and RT-PCR. The expression of FGC receptor and TGF-beta receptor was significantly higher in ALP-positive PDLF than in ALP-negative PDLF. ALP-positive PDLF also expressed higher mRNA levels of osteocalcin and bone sialoprotein compared with ALP-negative PDLF. These results indicate that osteoblasts-like PDLF subsets can be isolated from the heterogenous PDLF populations by our novel method. Isolation and ex vivo expansion of such cells may be useful to clarify the mechanism of periodontal tissue regeneration.