The role of cell surface markers and enamel matrix derivatives on human periodontal ligament mesenchymal progenitor responses in vitro
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)
- et al.
Investigation of multipotent postnatal stem cells from human periodontal ligament
Lancet
(2004) - et al.
A perivascular origin for mesenchymal stem cells in multiple human organs
Cell Stem Cell
(2008) - et al.
Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts
Exp Hematol
(2008) - et al.
Isolation and characterization of CD146+ multipotent mesenchymal stromal cells
Exp Hematol
(2008) - et al.
Perturbed amelogenin secondary structure leads to uncontrolled aggregation in amelogenesis imperfecta mutant proteins
J Biol Chem
(2010) - et al.
Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP)
J Biol Chem
(1995) - et al.
Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement
Cytotherapy
(2006) - et al.
Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment
Cell
(2007) - et al.
Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells
Biochem Biophys Res Commun
(2010) - et al.
Amelogenin binds to both heparan sulfate and bone morphogenetic protein 2 and pharmacologically suppresses the effect of noggin
Bone
(2008)
Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells
Biochem Biophys Res Commun
Stem cells in the periodontal ligament
Oral Dis
Putative stem cells in regenerating human periodontium
J Periodontal Res
Variation in collagen expression by cloned periodontal ligament cells
J Periodontal Res
Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine
J Dent Res
Location of putative stem cells in human periodontal ligament
J Periodontal Res
Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells
Stem Cells Dev
Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp
J Bone Miner Res
Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow
Stem Cells
Novel markers of mesenchymal stem cells defined by genome-wide gene expression analysis of stromal cells from different sources
Exp Cell Res
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
High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue
J Biomed Biotechnol
A method to isolate, purify, and characterize human periodontal ligament stem cells
Methods Mol Biol
Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use
J Clin Periodontol
The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase
Stem Cells Dev
Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro
Cell Tissue Res
Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels
J Clin Periodontol
Novel regulators of bone formation: molecular clones and activities
Science
Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis
J Cell Mol Med
Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow
Cell Tissue Res
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
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