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Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment

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An Erratum to this article was published on 16 December 2016

Abstract

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1−/CD146+ and STRO-1−/CD146− subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

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Abbreviations

ALP:

Tissue-nonspecific alkaline phosphatase

BSA:

Bovine serum albumin

BSP:

Bone sialophosphoprotein

CAP:

Cementum attachment protein

CEMP1 or CP23:

Cementum protein 1

DAPI:

4’,6-diamidino-2-phenylindole dihydrochloride

FACS:

Fluorescence-activated cell sorting.

hPDLSCs:

Human periodontal ligament stem cells

MSCs:

Mesenchymal stem cells

OCN:

Osteocalcin

PDL:

Periodontal ligament

RUNX2:

Runt-related transcription factor 2

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Acknowledgments

This work was supported in part by a grant from the National Institutes of Health R01 DE019156 (G.T.-J.H.), by an Endodontic Research Grant from the American Association of Endodontists Foundation (G.T.-J.H.) and a Research Fund from the University of Tennessee Health Science Center.

Authors’ contributions

PG and ZY designed and performed the experimental work, acquired, assembled, analyzed the data and drafted/revised the manuscript. QTT analyzed, interpreted the data, performed statistical analysis and wrote/revised part of the manuscript. FURB and XZ performed some of the experimental work, assembled, analyzed the data and drafted/revised part of the manuscript. GTJH conceived, designed, performed experimental works and supervised the overall project, analyzed and interpreted the data and finalized the manuscript. All authors have read and approved the manuscript for publication.

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    Authors and Affiliations

    1. Department of Endodontics, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, 02118, USA

      Philippe Gauthier & George T.-J. Huang

    2. Faculté de médecine dentaire, Département d’endodontie, Université Laval, Québec, QC, G1V 0A6, Canada

      Philippe Gauthier & George T.-J. Huang

    3. Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, TN, 38163, USA

      Zongdong Yu, Fazal-Ur-Rehman Bhatti & Xiaofei Zhu

    4. Department of Preventive Medicine, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, 38105, USA

      Quynh T. Tran

    5. VIP Dental Service and Geriatric Dentistry, School and Hospital of Stomatology, Peking University, Beijing, China

      Xiaofei Zhu

    6. Lab, Cancer Research Building, University of Tennessee Health Science Center, 19 S. Manassas St. Lab Rm 225, Office 222, Memphis, TN, 38163, USA

      George T.-J. Huang

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    1. Philippe Gauthier
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    Correspondence to George T.-J. Huang.

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    Additional information

    Philippe Gauthier and Zongdong Yu contributed equally to this work.

    An erratum to this article is available at http://dx.doi.org/10.1007/s00441-016-2550-3.

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    Supplemental Fig. 1

    Measurement of in vivo mineral tissue formation by PDLSCs. (a) A low -magnification view of a section showing the entire mass of the resected tissue containing aggregated HA/TCP granules encapsulated by the subcutaneous fibrous connective tissue. The image was analyzed by ImageJ to calculate the total area of the mineral tissues that include the HA/TCP granules, newly formed mineral tissues and small amounts of fibrous tissues among the mineral tissues. Manual outlining the total area with ImageJ to allow quantitation of the outlined area was carefully performed as indicated by the encircled black dashed line. Some samples that shown large areas (similar to the size of a granule) of soft tissues (encircled by the red dashed lines) within the tissue mass were excluded. Sample from a control group. Scale bar: 1 mm. (b) A closer view of the mineral tissues showing scattered newly formed mineral tissues on the surface of HA/TCP granules. These mineral tissues were manually encircled (white dashed line) and numbered with ImageJ and the areas calculated. All the small mineral areas calculated were summed together as the area of newly formed mineral tissues. Sample from a VC treated group. Scale bar: 100 μm. HA: HA/TCP (GIF 723 kb)

    High Resolution Image (TIF 45252 kb)

    Supplemental Fig. 2

    RT-PCR showing gene expression of cementum markers (CAP, CEMP1) and osteogenic markers (ALP, RUNX2, BSP, OCN) in PDLSCs cultured in GM as controls (C) and in OM (O) after 0, 3, 7, 10, 15 and 21 days. Data from cells derived from one donor. (GIF 111 kb)

    High Resolution Image (TIF 591 kb)

    Supplemental Fig. 3

    PDLSC cultures with or without osteogenic stimulation. (a, a’, a”) PDLSCs isolated from PDL at p0 with regular GM. Scale bars: a, 400 μm; a’, 200 μm; a”, 100 μm. (b, b’) PDLSCs from two different donors b, b’, under OM stimulation for 3 weeks with mineral deposits (more amount and diffused in b’). Scale bar: 50 μm for both b & b’. (c) PDLSCs under OM stimulation for 2 or 4 weeks (wks) and stained with Alizarin Red S. Scale bar: 50 μm. (GIF 193 kb)

    High Resolution Image (TIF 3718 kb)

    Supplemental Fig. 4

    PDLSC cultures with or without VC stimulation. (a-d) PDLSCs stimulation for 3 weeks in regular GM with (b, d) or without (a, c) 20 μg/mL VC. (a, b are from a donor; c, d are from a different donor and the cells were used for mouse transplantation experiment – data in Fig. 5). Scale bar: 100 μm for all 4 images. (GIF 364 kb)

    High Resolution Image (TIF 5331 kb)

    Supplemental Fig. 5

    Mineral tissue formation in vivo by PDLSCs. Sample sources and experimental conditions are the same as those in Fig. 5. (a-c) Less calcified mineral structures with less organized fiber orientations. Blue arrowheads indicate Sharpey’s fiber-like structures. (d-e) More calcified mineral tissue with lamellar lines (red arrowheads). Ctrl: cells grown in regular medium; VC: cells treated with VC. (a, b, e) From donor 1 VC group; (c, d) from donor 3 Ctrl group; (f) from donor 3 VC group. Scale bar: (a, b) 50 μm for both images. (c-f) 50 μm for all 4 images. (GIF 686 kb)

    High Resolution Image (TIF 12276 kb)

    Supplemental Fig. 6

    Immunohistochemical analysis of tissue regenerated in vivo by PDLSCs. Sample sources and experimental conditions are the same as those in Fig. 6. HA: hydroxyapatite/tri-calcium phosphate; M: mineral tissue; Ct: fibrous connective tissue. Arrows: in CAP group, blue arrows indicate mineral tissues with brown staining, red arrows indicate Sharpey’s fiber-like structures; in CEMP1 group, blue arrows indicate mineral tissues with brown staining, red arrows indicate immunoreactivities in cells of the fibrous connective tissues; in Mito (mitochondria) group, red arrows indicate stronger staining in cells lining against the mineral tissue and weaker staining in cells in the adjacent soft connective tissue; in Ctrl-IgG group, blue arrows indicate mineral tissue. Ctrl-IgG group: Top image is pre-immune mouse IgG isotype control, bottom image is pre-immune rabbit IgG isotype control. Scale bars: in CAP and CEMP1, 300 μm for left images, 50 μm for right images; in Mito, 50 μm for all 4 images; in Ctrl-IgG, 100 μm for both images. (GIF 1389 kb)

    High Resolution Image (TIF 10182 kb)

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    Gauthier, P., Yu, Z., Tran, Q.T. et al. Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment. Cell Tissue Res 368, 79–92 (2017). https://doi.org/10.1007/s00441-016-2513-8

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