Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

Abstract

Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs) may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1) has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH) has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+) and STRO-1(-) hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+) hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+) hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R) than STRO-1(-) hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+) hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+) hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+) hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.

Figure 1

Characterization of hPDLSCs. (a) The morphology of colony-forming hPDLSCs (magnification: 50x). (b) Differentiation potential of hPDLSCs. Differentiated cells were stained with Alizarin red (left; magnification: 100x) or Oil Red O (right; magnification: 630x). (c) Immunophenotypic profiling was performed to detect CD90, CD105, CD166, CD146, Stro-1, and CD34.

Figure 2

STRO-1(+) hPDLSCs exhibited strong osteogenic capacity. (a) Immunostaining for Stro-1 in isolated STRO-1(+) and STRO-1(−) hPDLSCs (magnification: 50x). The surface marker Stro-1 was stained with FITC and visualized using fluorescence microscopy. Nuclei were stained with DAPI (blue). (b) ALP activity in STRO-1(+) and STRO-1(−) hPDLSCs. (c) Osteogenic differentiated cells were stained with Alizarin red (magnification: 100x). (d) Real-time PCR and western blot analyses of Runx2 expression in STRO-1(+) and STRO-1(−) hPDLSCs. The expression of each target was normalized to that of GAPDH. Data are presented as the means ± SDs of three independent experiments performed in duplicate. ^∗ P < 0.05, ^∗∗ P < 0.01.

Figure 3

Real-time PCR and western blot analyses of PTH1R expression in STRO-1(−) and STRO-1(+) hPDLSCs. The expression of each target was normalized to that of GAPDH. Data are presented as means ± SDs of three independent experiments performed in duplicate. ^∗∗ P < 0.01.

Figure 4

The osteogenic capacity of STRO-1(+) hPDLSCs was enhanced by PTH. (a) Real-time PCR and western blot analyses of PTH1R, RUNX2, and SP7 expression in STRO-1(+) hPDLSCs before and after PTH treatment and induction by osteogenic-induction medium (OM). The expression of each target was normalized to that of GAPDH. (b) ALP activity in STRO-1(+) hPDLSCs. (c) Cells subjected to osteogenic differentiation were stained with Alizarin red (magnification: 100x). Data are presented as the means ± SDs of three independent experiments performed in duplicate. ^∗ P < 0.05, ^∗∗ P < 0.01.