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Macedonian Journal of Medical Sciences. 2011 Mar 15;
4(1):37-43.
doi:10.3889/MJMS.1857-5773.2011.0147
Basic Science
Identification and Cementoblastic / Osteoblastic
Differentiation of Postnatal Stem Cells from Human Periodontal Ligament
Maha Abd El Fattah1,2, Gang Ding1, Fulan Wei1,
Chunmei Zhang1, Eman Aboul Ezz2, Songlin Wang1,3
1Salivary Gland Disease Center and the Molecular Laboratory for
Gene Therapy and Tooth Regeneration, Capital Medical University School of
Stomatology. Tian Tan Xi Li No.4, Beijing 100050, China; 2Oro-dental
genetics department, National Research Center, Cairo, Egypt; 3Department
of Biochemistry and Molecular Biology, Capital Medical University School of
Basic Medical Sciences, You An Men Wai No.10, Beijing 100069, China
Background.
Periodontal diseases that lead to the destruction of periodontal tissues,
including periodontal ligament (PDL), cementum, and bone, are a major cause
of tooth loss in adults and are a substantial public health burden
worldwide. PDL is a specialized connective tissue that connects cementum and
alveolar bone to maintain and support teeth in situ and preserve tissue
homoeostasis. In this study we aimed to isolate, identify periodontal
ligament stem cells and their osteoblastic/ cementoblastic differentiation.
Methods. Periodontal ligament tissue was obtained from human impacted
third molars (n=5) from different individuals from the oral surgery
department, Department of Oral and Maxillofacial Surgery, Capital Medical
University School of Stomatology (Beijing, China) following which a colony
forming unit – fibroblast assay, identification of periodontal ligament stem
cells (PDLSCs; STRO-1 + & CD146+) by using immunocytofluorescence and
isolation of PDLSCs (STRO-1+) by using flow cytometry and cementoblastic/osteoblastic
exvivo induction were performed.
Results. Mesenchymal stem cells were identified in the periodontal
ligament derived by their capacity to form adherent clonogenic cell
clusters. Ex-vivo expanded periodontal ligament stem cells were found to
express the mesenchymal stem cell markers STRO-1 and CD146. Flow cytometric
study showed that a total of 24.53% of periodontal ligament cell population
stained positive for the STRO-1 antibody and of that population 1.14% were
strongly positive.
Conclusions. The finding of this study indicated that some PDL cells
possess crucial stem cells properties, such as self renewal and express the
mesenchymal stem cell markers (STRO-1 and CD 146) on their cell surface and
small round alizarin red-positive nodules formed in the PDLSC cultures after
4 weeks of induction, indicating calcium accumulation in vitro. Thus, PDL
cells can be used for periodontal regenerative procedures.
..................
Citation: El Fattah MA, Ding G, Wei F,
Zhang C, Ezz EA, Wang S. Identification and Cementoblastic / Osteoblastic
Differentiation of Postnatal Stem Cells from Human Periodontal Ligament.
Maced J Med Sci. 2011 Mar 15; 4(1):37-43.
doi.10.3889/MJMS.1957-5773.2011.0147.
Key words: Stem Cells; Periodontal Ligament; Differentiation;
Cementoblast; Osteoblast.
Correspondence: Prof. Songlin Wang. Salivary Gland Disease Center and
the Molecular Laboratory for Gene Therapy and Tooth Regeneration, Capital
Medical University School of Stomatology. Tian Tan Xi Li No.4, Beijing
100050, PR China. Tel. /Fax +86 10 6706 2012, E-Mail: slwang@ccmu.edu.cn
Received: 05-Jun-2010; Revised: 30-Aug-2010; Accepted: 28-Sep-2010; Online
first: 28-Jan-2011
Copyright: © 2011 El Fattah MA. This is an open-access article
distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Competing Interests: The author have declared that no competing
interests exist.
Periodontal ligament is a soft connective tissue embedded between the
cementum (a thin layer of mineralized tissue covering the roots of the
teeth) and the inner wall of the alveolar bone socket, and helps constrain
teeth within the jaw. Periodontal ligament not only has an important role in
supporting teeth, but also contributes to tooth nutrition, and repair of
damaged tissue [1]. Stem cells are unspecialized cells with two main
defining properties: the ability of self renewal which is the ability of
stem cells to divide and produce more stem cells that are defined by their
clonogenic potential, in other words, a single adult stem cell should be
able to generate a line of genetically identical cells, which then give rise
to all the appropriate, differentiated cell types of the tissue in which it
resides; another is ability of differentiation into other cells [2].
Periodontal diseases are infectious diseases that are characterized by
destruction of the periodontium including periodontal ligament, cementum,
alveolar bone, and gingiva. Periodontal diseases are the main cause of tooth
loss and are a substantial public health burden worldwide [3]. Regeneration
of the attachment apparatus that is destroyed as a result of periodontitis
has long been the goal of periodontal therapy. Periodontal regeneration
requires new connective tissue attachment to the root surface, a process
that involves the regeneration of periodontal fibers and the insertion of
these fibers into newly formed cementum [4].
Numerous clinical techniques, including bone grafts, root surface
conditioning, barrier membranes and various growth factors, have been
utilized over the years in attempt to achieve periodontal regeneration.
Unfortunately, current therapeutic measures are unable to achieve
predictable regeneration because they results in the reduction of probing
depth and stabilization of attachment loss, but histologically, the
epithelial tissue proliferates at a faster rate than the underlying
mesenchymal tissues (periodontal ligament, alveolar bone and root cementum),
thus underscoring the importance of restoring or providing the cells and
microenvironment capable of initiating and promoting new periodontal tissue
formation [4].
The periodontal ligament has been shown to be of critical importance in the
regenerative process. It has been demonstrated that only the periodontal
ligament but not gingival connective tissue or bone contain cells capable of
establishing new attachment fibers between cementum and bone [5]. STRO-1 and
CD 146 are two early mesenchymal stem cell markers also present on dental
pulp stem cells and bone marrow mesenchymal stem cells.
In this study, we assessed the colony forming unit- fibroblastic, identified
the PDLSCs using immunocyto-fluorescence, flow cytometry, and
cementoblastic/osteoblastic differentiation of the PDLSCs.
Sampling and Cell Culture
Healthy human impacted third molars (free of inflammation) were selected
from five different individuals aged from 19-29 years old from the
Department of Oral and Maxillofacial Surgery, Capital Medical University
School of Stomatology (Beijing, China), after the patients gave their
informed consent. The samples were collected immediately after extraction
and were placed in 50 ml sterile polypropylene tube containing
alpha-modified Eagle’s medium (alpha-MEM) supplemented with antibiotics (100
U/ml penicillin and 100 mg/ml streptomycin). Periodontal ligament tissue was
removed from the surface of the root by sterile lancet, rinsed five times by
phosphate-buffered saline (PBS) supplemented with penicillin and
streptomycin.
Periodontal ligament pieces derived from the different individuals were
mixed together and were minced into tiny pieces, then digested in a solution
of 3 mg/ml collagenase type I (Worthington Biochem, Freehold, NJ, USA) and 4
mg/ml Dispase (Roche, Mannheim, Germany) for 1 hour at 37°C in a water bath.
Single cell suspensions were seeded into a T-25 Flask (Costar, Cambridge,
MA, USA) with alpha-MEM (GIBCO BRL, Grand Island, NY, USA), supplemented
with 15% fetal calf serum (Equitech-Bio Inc., Kerrville, TX, USA), 400
mmol/ml L-glutamine, 20 000 U/ml penicillin and 20 000 ng/ml streptomycin
and then incubated in a humidified atmosphere of 5% CO2 at 37°C.
The flasks were periodically checked every 24 hours by the phase contrast
inverted microscopy and the culture medium was changed 3-times per week, the
cells were sub-cultured when reached confluency about 80 % by using 0.25%
trypsin and 0.02% EDTA.
Colony forming unit-fibroblast (CFU-F) assay
The clonogenic potential of the isolated cells was tested. For this assay
100,000 cells (low density) were plated on a T-25 flask as shown in and
incubated for 10 days. Subsequently, the cultures were fixed with 4%
paraformaldehyde and then stained with 0.1% toluidine blue. Aggregates of
greater than or equal to 50 cells were scored as colonies.
Flowcytometry
When the cells reached confluency (third passage), they were detached with
0.25% trypsin and 0.02% EDTA, the samples were centrifuged at 1000 rpm for 5
min then approximately 2 x 100,000 cells were pelleted in 5 ml polypropylene
tubes and were treated with PBS containing 10% goat serum for 30 min at 4°C
to block the non-specific sites and incubated with the primary antibody
(Mouse IgM anti-human STRO-1 or Mouse IgG anti-human CD146) for 30 min at
4°C, then washed with PBS and resuspended again in phosphate buffered saline
with fluorescin-conjugated secondary antibody (goat anti-mouse IgM or IgG)
for 30 min at 4°C in dark.
After washing with PBS the cells were fixed with 4% paraformaldehyde (to
postpone flowcytometry analysis for a few days). The percentage of cell
surface marker STRO-1 positive cells were analyzed and sorted by Flow
cytometry by collecting 10,000 events, and were analyzed by FACScalibur flow
cytometry (Becton Dickinson).
Immunocytofluoresence
Third passage PDLSCs were sub-cultured into 24-well cell culture plates,
when cells reached semi-confluency they were fixed with 4% paraformaldehyde
for 30 min and then blocked with PBS containing 10% goat serum at room
temperature for 30 min and incubated with primary antibody at dilution 1
ml:200
ml of
PBS overnight at 4°C, after washing the samples were incubated with
fluorescein-conjugated secondary antibody (goat anti-mouse IgM) for 60
minutes in dark.
Antibodies used are: A. Primary antibodies: 1- Anti-STRO-1,
mouse monoclonal IgM anti-human STRO-1 (from Millipore) 1ml
primary antibody:200 ml
PBS; 2- Anti-CD 146, mouse monoclonal IgG anti-human CD 146 (from Abcam), 1
ml
primary antibody:200 ml
PBS. B. Fluorescent conjugated secondary antibodies: 1- Goat
anti-mouse IgM; 2- Goat anti-mouse IgG.
For nuclei staining DAPI (4, 6-diamino-2-phenylindol) was applied for 5
minutes in dark, after washing with PBS the samples were examined by
fluorescence microscopy (Olympus) with 40 X magnification power.
Cementoblastic/Osteoblastic differentiation
To investigate the potential of PDLSCs to cementoblastic/osteoblastic
differentiation, established secondary PDLSCs cultures were supplemented
with L-ascorbate-2-phosphate, dexamethasone (Sigma), and inorganic phosphate
to induce mineralisation in vitro. Calcium accumulation was detected by
staining with 2% alizarin red S (pH 4·2).
To isolate PDLSCs, single cell suspensions were generated from human
periodontal ligament from extracted third molars, following
collagenase/dispase digestion.
Isolation of Clonogenic Populations of Periodontal ligament stem
cells
Figure 1: The arrow is denoting a un- stained CFU-F (more
than 50 cells).
Periodontal ligament derived cells have the ability to form adherent
clonogenic cell clusters (colony forming unit-fibroblast, CFU-F), shown by
the formation of about 180 colonies, generated from 100,000 single cells
when cultured at low density and the cells within each colony were
characterized by a typical fibroblast-like morphology as shown in Figure 1 &
2.
Figure 2: The arrows are denoting several colonies stained
by toulidine blue.
Immunocytofluorescence
 
Figure 3: Immunocytochemical staining showed that cultured
PDLSCs were expressed STRO-1 (40 X magnification power).
Ex-vivo expanded PDLSCs were found to express the cell - surface molecules
STRO-1 shown in Figure 3 and CD146 shown in Figure 4.
Figure 4: Immunocytochemical staining showed that cultured
PDLSCs expressed CD 146(40 X magnification power).
Flow cytometric analysis
Figure 5: The percent of PDLSCs positive for STRO-1 was
24.53%.
Surface marker STRO-1 positive cells were analyzed and sorted by flow
cytometry. A total of 24.53% of periodontal ligament cell population stained
positive for the STRO-1 antibody as shown in Figure 5 and of that population
1.13% were strongly positive as shown in Figure 6.
Figure 6: Strongly positive STRO-1 cells represent around
1.13% of the total cell population.
Cementoblastic/Osteoblastic differentiation
Figure 7: Small round alizarin red-positive nodule formed in the PDLSC
cultures after 4weeks of induction, indicating calcium accumulation in vitro
(by 4 X magnification power).
Small round alizarin red-positive nodules formed in the PDLSCs cultures
after 4 weeks of induction, indicating calcium accumulation in vitro Figure
7 & 8.
Figure 8: Showing Orange-Red staining within the cells indicating
mineralized nodule formation (by 40 X magnification power).
Periodontal ligament cells play an important role, not only in the
maintenance of the periodontium but also in promoting periodontal
regeneration. Recent studies have shown that proper manipulation of
periodontal ligament cells is essential for tissue engineering [6, 7].
Seo et al [8] investigated the presence of multipotent stem cells from human
periodontal ligament and they found that ex-vivo expanded PDLSCs mainly
expressed the cell surface molecules STRO-1 and CD 146. They also suggested
that PDLSCs represent a novel population of multipotent stem cells, as shown
by their capacity to develop into cementoblast-like cells, adipocytes in
vitro, cementum/PDL-like tissue in vivo, and by their high expression of
scleraxis (a specific transcription factor associated with tendon cells).
PDLSCs also showed the capacity to form collagen fibers similar to Sharpey’s
fibers, connecting to a cementum-like tissue, suggesting the potential to
regenerate PDL attachment. These data lend further support to the notion
that PDLSCs are a unique population of postnatal stem cells.
In this study, in order to investigate the proportion of cells in
periodontal ligament cell populations that possess stem cell properties,
quantitative analysis of STRO-1 by flow cytometry and the identification of
CD 146 and STRO-1 by immunocytofluoresence were carried out for isolation
and identification of stem cells and also ex-vivo
cementoblastic/osteoblastic differentiation was carried out. Supporting the
aim of the present study, several research laboratories have joined efforts
to find the potential (gold standard) antibody for stem cell identification
[9, 10].
In the present study, periodontal ligament derived cells was found to be
able to form adherent clonogenic cell clusters (CFU-F), where 180 colonies
were generated from 100,000 single cells when cultured at low density. The
cells within each colony were characterized by a typical fibroblast-like
morphology analogous to the progeny of human dental pulp stem cells [11].
In accordance, a study by Seo et al [8] showed the ability of periodontal
ligament derived cells to form adherent clonogenic cell clusters of
fibroblast-like cells by the formation of about 170 single colonies,
generated from 100,000 single cells cultured at low density.
On the other hand, the colony-forming cells derived from dental pulp tissue
was about 22–70 colonies generated from 10,000 cells and the colony-forming
cells from the bone marrow was about 2–3 colonies generated from 10,000
cells when plated over similar plating densities [11].
In another study by Gronthos et al [12] approximately 40 single-colony
clusters were generated from 10, 000 pulp cells in culture, while in a study
by Miura et al [13] colony-forming ability from the bone marrow stromal
cells was even less where only 14 colonies was generated from 100,000 cells.
Colonies formed by periodontal ligament and pulp cells occurred at an
apparently higher frequency in comparison to bone marrow mesenchymal stem
cells. However, this is most likely because of the basic differences in the
composition of the two connective tissues. Whereas periodontal ligament and
pulp is comprised largely of fibrous tissue, in bone marrow aspirates,
hematopoietic cells constitute the majority of the cell population (99.9%).
When bone marrow core biopsies are flushed free of hematopoietic cells and
then subjected to collagenase treatment, the incidence of CFU-F dramatically
increases to more than 10-fold compared with marrow aspirates [11].
Consequently, when considering the number of CFU-F from a connective tissue
devoid of hematopoietic cells, the numbers would appear to be fairly
equivalent.
Mesenchymal stem cells are morphologically heterogenous as well as for
features as surface epitopes [14]; accordingly several antibodies as STRO-1,
CD 146, CD 44, CD 166, and CD 105 have been used to routinely characterize
the expanded mesenchymal cell populations [15, 16].
In the present study, identification of PDLSCs in ex-vivo expanded
periodontal ligament cells were carried out by immunocytofluorescence, using
anti-STRO-1 (Mouse IgM anti-human STRO-1) and anti-CD 146 (Mouse IgG
anti-human CD 146), implying that PDLSCs might also be derived from a
population of perivascular cells, as previously reported by Seo et al [8].
Also, quantitative analysis of STRO-1 positive cells was done using flow
cytometry device capable of counting cells of different types in a mixture,
since different types of cells can be distinguished by quantitating
structural features with different ability to scatter laser light and emit
fluorescent light.
In the results of the present study, the percentage of the strongly positive
stained cells was 1.13%, while in a study by Gay et al [17] conducted on
human periodontal ligament cells and stained for STRO-1 using flow
cytometric analysis, they reported that human periodontal ligament tissue
contains about 27% STRO-1 positive cells with 3% strongly positive cells.
The osteogenic potential of PDL cells has been assessed previously with
several cell-culture methods, and the ability of such cultures to form a
mineralized matrix has been noted. Our data show the potential of PDLSCs to
form calcified deposits in vitro, as previously shown with other mesenchymal
stem-cell populations such as dental pulp stem cells [11, 18].
However, because of the heterogeneity of STRO-1/CD146-positive mesenchymal
stem cells, it is possible that PDLSCs used in this experiment may represent
a heterogeneous stem-cell-enriched population that contains some early
progenitor cells. Attempts to identify unique markers for PDLSCs with
molecular and genetic approaches are mandatory.
Importantly, PDL collected from one tooth, especially from younger
individuals may give rise to many stem cells which are able to assist in
regeneration, because of their capacity to proliferate ex vivo.
Finally, this study showed that human PDL contains population of postnatal
stem cells that can be isolated, expanded in vitro and have osteogenic
potential, providing a unique reservoir of stem cells from an accessible
tissue resource with minimally invasive procedures. Therefore, the
periodontal ligament apparatus represents a viable alternative to obtain
potentially high numbers of cells for regenerative procedures without the
necessity of bone marrow aspiration or more invasive procedures that will
result in increased morbidity.
We recommended for further studies to be carried out identifying unique
markers for PDLSCs, and molecular and genetic approaches are mandatory.
This work was supported by grants from the National Basic Research Program
of China (No. 2007CB947304 to S.W.), Beijing Major Scientific Program
(D0906007000091 to S.W.) and National Research Center of Egypt.
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