Elsevier

Biomaterials

Volume 32, Issue 30, October 2011, Pages 7375-7388
Biomaterials

The role of cell surface markers and enamel matrix derivatives on human periodontal ligament mesenchymal progenitor responses in vitro

https://doi.org/10.1016/j.biomaterials.2011.06.043Get rights and content

Abstract

Periodontitis is a chronic-, infectious-disease of the human periodontium that is characterized by the loss of supporting tissues surrounding the tooth such as the periodontal ligament (PDL), cementum and alveolar bone. Regeneration of the periodontium is dependent on the participation of mesenchymal stem/stromal cells (MSC) resident in the PDL. Enamel matrix derivative (EMD), an extract from immature porcine enamel rich in amelogenin protein but that also contain bone morphogenetic protein (BMP), is used to treat periodontal defects. The effects of EMD on MSC cells of the PDL are not well characterized. In this in vitro study, we identify PDL progenitor cells from multiple individuals and demonstrate that EMD stimulates them. We show that the effect of EMD on cell proliferation and migration is mediated through the amelogenin it contains, while the differentiation of these progenitor cells to cell types of mineralized tissue is mainly due to BMP signaling.

Introduction

In humans, periodontitis is a pervasive, chronic infectious disease of the soft and hard-tissues supporting the teeth. This disease is characterized by the loss of both the hard and soft tissues (e.g. the periodontal tissues) that anchors the tooth in the jaws. Conventional periodontal therapy involves debridement of the root surface to induce local healing by repair pathways. However, regeneration of bone and cementum with a periodontal ligament (PDL) remains a challenge. Recently, the PDL has been shown to be of critical importance in the regenerative process [1] by providing mesenchymal stem/stromal cells (MSC) located within the PDL [2] that contribute to regeneration of the destroyed tissues [3].

Cells of the PDL represent a heterogeneous population [4] and it is not known which subsets of PDL cells are specifically involved in wound healing. However, it is well established that the PDL contains a cell population with MSC properties, such as self-renewal, clonal expansion and multiple lineage differentiation. Furthermore, these MSC-like cells of PDL origins display similarities with bone marrow derived MSC (BMSC), dental pulp stem cells and dental follicle stem cells [5]. The absence of a single specific MSC marker makes analysis of PDL progenitors more difficult, requiring instead the use of a combination of cell surface markers for their identification. Among these, CD146/MUC-18 is one the most employed. CD146 is located on endothelium, smooth muscle, Schwann cells, in some neoplasms and is considered as one of the key markers of perivascular-, multipotent-progenitor cells (e.g. pericytes) in human connective tissues [6] including the PDL [3], [7], [8]. While the precise function of CD146 is not known, it has been linked to various cellular processes including cell adhesion, cytoskeletal reorganization, cell shape, migration, and proliferation through its capacity for transmembrane signaling [9]. Several studies demonstrated that CD146 positive (+) cell populations from numerous connective tissue sites exhibit MSC potential [10], [11], [12], [13], [14], [15]. Because the CD146(+) population is not homogenous, attention has been paid to further refine CD146(+) cell subsets. When associated with the stem cell marker STRO-1, CD146 has been shown to identify PDL cell populations with MSC-like properties [2], [8] involved in regenerating periodontal tissues [3]. Others cell surface markers have been proposed to locate precursors cells within the human PDL, including CD106 (VCAM-1) [16] and the tissue non-specific alkaline phosphatase (TNAP) [17], recently shown to be identical to the MSC antigen 1 (MSCA-1), known to be expressed in human BMSC [18]. To date, the behavior of specific subsets of PDL cells have not been fully characterized and the role of these populations during periodontal healing warrants further elucidation. The differentiation capacity of PDL subsets during the regeneration process also remains unclear.

Multiple treatment modalities have been deployed in the treatment of periodontal defects including bone grafts or bone-substitutes, the use of barrier membrane and biological mediators. One of the goals of periodontal regenerative therapy, and especially the use of bioactive factors, is to trigger specific populations of PDL progenitor cells that would result in optimal periodontal regeneration. One biological mediator called Enamel Matrix Derivatives (EMD) is composed of immature porcine enamel matrix, rich in amelogenin protein, but that also contain bone morphogenetic proteins (BMP) BMP2 and BMP7 [19]. EMD has been used to treat infrabony defects and based upon observations from various animal models, EMD has been suggested to enhances PDL cell proliferation, migration and osteo-cementogenic differentiation [20]. Although EMD and the BMPs it contains has been shown to target MSC [21], [22], [23], the mechanisms of their action on PDL MSC progenitors is controversial.

In this study, cells were recovered from the PDL of 6 donors and their CD146, CD106 and MSCA-1 cell surface expression analyzed. To decipher the effects of EMD on the in vitro behavior of PDL progenitor cells, we used pure recombinant sources of amelogenin and BMP2/7.

Section snippets

Materials

Sources and concentrations of manufactured antibodies and reagents are summarized in Table 1. Recombinant poly(His) tagged mouse 180 amino acid amelogenin, rp(H)M180 [24] was used at 5 μg/mL. Preparation of the STRO-4 monoclonal antibody anti-heat shock protein 90β, has been recently described [25]. All others reagents were from Sigma (St Louis, MO, USA).

HPDL cells isolation and cell culture

Human PDL cells (hPDL) were isolated from non-impacted premolars extracted for orthodontic reasons obtained from six healthy donors (four

Characterization of hPDL cells

We analyzed a panel of 13-cell surface markers for their expression on donor hPDL cells (Supplementary Figure 1A). The staining pattern for each of the hematopoietic markers (79a, 45, 14 and HLADR), CD34, and ELF97 was negative, whereas the expression levels for non-hematopoietic cell markers (CD90, 73, 105, and STRO-4) were uniformly positive (>95% of positive cells) with the exception of three markers, CD146, CD106, and MSCA-1, whose expression showed marked variability between donors (32% ± 

Discussion

For many years considerable efforts have been made towards developing strategies for the regeneration of tooth-supporting tissues destroyed by periodontal disease. Evidence has shown that the periodontal ligament (PDL) contains a heterogeneous mix of progenitor cells that represents the main source for the healing of those tissues destroyed by periodontitis [2]. The present study highlights the usefulness of cell surface markers, especially CD146, to identify the cell subset within the

Conclusion

The data presented in this paper aids in defining the in vitro behavior of human PDL progenitors and their progeny during the biological events involved in periodontal wound healing. We identified the effect of EMD on hPDL cells sub-populations by using their main components, amelogenin protein and BMP2 and 7 on the PDL progenitors. This knowledge can be used to improve the involvement of endogenous progenitor cells with MSC-like characteristics that are resident in the PDL to contribute to

Acknowledgements

We want to thank B. Alliot-Licht (Faculty of Odontology, Nantes-France) and JC. Farges (Faculty of Odontology, Lyon-France) for reading the manuscript, F. Maupas-Schwalm, M. Bichard-Breaud and JM. Botella (Biochemistry -Rangueil; Toulouse-France) for helping in biochemical experiments, S. Kemoun for helping in the statistical studies, M. Gadelorge and P. Bourin (STROMALab, UMR CNRS/UPS/EFS 5273 et INSERM U1031, Toulouse-France) for discussing about stem cells, S. Allard and D. Sapede for

References (62)

  • K. Fukiage et al.

    Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

    Biochem Biophys Res Commun

    (2008)
  • S. Ivanovski et al.

    Stem cells in the periodontal ligament

    Oral Dis

    (2006)
  • N.H. Lin et al.

    Putative stem cells in regenerating human periodontium

    J Periodontal Res

    (2008)
  • H. Limeback et al.

    Variation in collagen expression by cloned periodontal ligament cells

    J Periodontal Res

    (1983)
  • G.T. Huang et al.

    Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine

    J Dent Res

    (2009)
  • S.C. Chen et al.

    Location of putative stem cells in human periodontal ligament

    J Periodontal Res

    (2006)
  • J. Xu et al.

    Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells

    Stem Cells Dev

    (2009)
  • S. Shi et al.

    Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp

    J Bone Miner Res

    (2003)
  • D. Baksh et al.

    Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow

    Stem Cells

    (2007)
  • N. Kaltz et al.

    Novel markers of mesenchymal stem cells defined by genome-wide gene expression analysis of stromal cells from different sources

    Exp Cell Res

    (2011)
  • K.C. Russell et al.

    In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment

    Stem Cells

    (2010)
  • R.C. Schugar et al.

    High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue

    J Biomed Biotechnol

    (2009)
  • K. Mrozik et al.

    A method to isolate, purify, and characterize human periodontal ligament stem cells

    Methods Mol Biol

    (2010)
  • T. Iwata et al.

    Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use

    J Clin Periodontol

    (2010)
  • M. Sobiesiak et al.

    The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase

    Stem Cells Dev

    (2010)
  • P. Kemoun et al.

    Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro

    Cell Tissue Res

    (2007)
  • D.D. Bosshardt

    Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

    J Clin Periodontol

    (2008)
  • J.M. Wozney et al.

    Novel regulators of bone formation: molecular clones and activities

    Science

    (1988)
  • A. Haze et al.

    Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis

    J Cell Mol Med

    (2009)
  • Y.C. Huang et al.

    Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow

    Cell Tissue Res

    (2010)
  • S. Gronthos et al.

    Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues

    Stem Cells Dev

    (2009)
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