Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison...

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Bone marrow mesenchymal stem cells stimulatedby bFGF up-regulated protein expression incomparison with periodontal fibroblasts in vitro

Renato Colenci a, Luciana Reichert da Silva Assunca o b,Suely Regina Mogami Bomfim c, Marjorie de Assis Golim d,Elenice Deffune e, Sandra Helena Penha Oliveira f,*aDDS, School of Dentistry, UNESP – Univ. Estadual Paulista, Sao Paulo, BrazilbDepartment of Stomatology, School of Dentistry, Federal University of Parana, UFPR, Parana, BrazilcDepartment of Clinics, Surgery and Animal Reproduction, School of Veterinary Medicine, UNESP – Univ. Estadual

Paulista, Sao Paulo, Brazild Laboratory of Flow Cytometry, Botucatu Blood Center, School of Medicine, UNESP – Univ. Estadual Paulista,

Sao Paulo, Brazile Laboratory of Cellular Engineering, Botucatu Blood Center, School of Medicine, UNESP – Univ. Estadual Paulista,

Sao Paulo, BrazilfDepartment of Basic Sciences, School of Dentistry, UNESP – Univ. Estadual Paulista, Sao Paulo, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 2 6 8 – 2 7 6

a r t i c l e i n f o

Article history:

Accepted 30 November 2013

Keywords:

Bone marrow

Mesenchymal stem cells

Periodontal ligament

Fibroblasts

bFGF

a b s t r a c t

Objective: The aim of this study was to evaluate, in vitro, the role of bFGF in the proliferation

and expression of collagen type I and fibronectin of dog bone marrow mesenchymal stem

cells (dBMMSCs) in comparison with the expression of the same proteins in dog periodontal

fibroblasts (dPLFs).

Design: dBMMSCs from the iliac crest were cultivated in Dulbecco’s Modified Eagle’s Medium

(DMEM). Flow cytometry analysis (FCA) was used to characterize dBMMSC. Cells were

stimulated with bFGF (1, 5 and 10 ng/mL) after 24 and 48 h. Real time RT-PCR was performed

to verify collagen type I and fibronectin expressions. MTT assay was used to confirm cellular

proliferation. Statistical analyses were performed (ANOVA and Kruskal–Wallis tests; p < 0.05).

Results: FCA showed 55.98% of CD34+ and 32.67% of CD90+ after bone marrow aspiration;

3.33% of CD34+ and 33.0% of CD90+ before P1. After P2, 10.54% of dBMMSCs expressed CD90,

whereas after P3, this number decreased to 1.58%. dPLFs presented 4.04% of CD90+ and 1.05%

of CD34+ after P3. MTT evaluation showed increase in dBMSC proliferation with 5 ng/mL bFGF-

stimulus after 24-h. Both collagen I and fibronectin expression were very similar between the

two cells groups after 24-h stimulation with 1 ng/mL bFGF concentration. Fibronectin and

collagen I expressions were higher after 24-h stimulation with 5 ng/mL bFGF.

Conclusion: dBMMSCs (1 ng/mL-bFGF stimulus after 24 h) are very similar to dPLFs as regards

morphological and immunostaining characteristics, and collagen and/or fibronectin

production. The dBMMSCs presented the highest protein expression rates with 5 ng/mL-

bFGF stimulus after 24-h.

# 2013 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Basic Sciences, UNESP – Rua Jose Bonifacio, 1193, CEP 16015-050 Aracatuba, Sao Paulo, Brazil.Tel.: +55 18 3636 2814; fax: +55 18 3636 3332.

E-mail address: [email protected] (S.H. Penha Oliveira).

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http://www.elsevier.com/locate/aob

0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.archoralbio.2013.11.017


1. Introduction

Scientific breakthroughs have opened new horizons for the re-

establishment of tissues damaged by disease and trauma. The

concept of forming new tissue to replace damaged structures

or lost organs is based on the practice of biotechnological

variability, which is well known as Tissue Engineering.1

Tissue regeneration potential has been significantly in-

creased due to the advancements in stem cell research.1 Bone

marrow mesenchymal stem cells (BMSCs) are capable of

generating bone, cartilage, muscle, tendon and fat. The

strategies for treatment with these cells are based on the

assumption that if they are in an appropriate environment

with specific molecular signals, these cells can reconstitute

the original tissue.2

In culture conditions, BMSCs have a fibroblast-like cell

morphology and are capable of adhering to plastic surfaces.3

The use of surface markers has been an accepted method for the

identification of mesenchymal lineages.4 BMSCs in humans and

mice express CD13, CD29, CD44, CD73, CD90 and CD105, but do

not express CD11, CD31, CD34, CD45 and CD235. CD34 is

typically used as a hematopoietic cell lineage marker.5–7

Basic fibroblast growth factor (bFGF) is a potent mitogen

that stimulates the growth and differentiation of a broad

spectrum of mesodermal and neuro-ectodermal cell types.

The bFGF belongs to a large family of small peptides (17–

34 kDa) that mediate cell proliferation, differentiation and

migration.8 Previous studies have reported that the addition of

bFGF to primary cultures of BMSC maintains their osteogenic,

adipogenic and chondrogenic potentials.9 The bFGF has also

the property of regulating periodontal ligament fibroblasts.10

Fibroblasts represent a dynamic population of cells that

exhibit a functional heterogeneity, which also results in

immunophenotypic differences. Koumas et al.11 when com-

paring fibroblasts from human myometrium and orbit,

observed that there are two subpopulations of fibroblasts

according to the expression of CD90, in which CD90+ have a

myofibroblast behaviour, while the CD90� have a lipofibro-

blast characteristic, but so far, there have been no reports

about periodontal ligament fibroblasts.

Fibroblasts are the predominant cells in the periodontal

ligament and are needed to maintain the normal width of the

periodontal ligament by preventing the encroachment of bone

and cementum into the periodontal ligament space.12 Fibro-

blasts derived from the periodontal ligament are responsible

for the metabolism of extracellular-matrix components such

as fibronectin and type I-collagen.13 These proteins have been

implicated in the attachment, migration and differentiation of

cells during periodontal wound healing.14

The observation that bone marrow includes cells that have

the capacity to generate fibroblasts and other mesenchymal

type-cells was first evidenced by the pioneering work of

Friedenstein et al.15 In culture conditions, these cells present

colonies consisting of plastic-adherent and elongated cells

with a fibroblast-like morphology.16 Moreau et al.,17 in a two-

dimension tissue culture model, observed that human bone

marrow stromal cells stimulated with a combination of growth

factors, including bFGF, had the potential to be differentiated

into fibroblasts. Canine bone marrow mesenchymal stem cells

can also be induced to differentiate into fibroblasts and

myofibroblasts under culture conditions.18

The aim of this study was to evaluate, in vitro, the role of

fibroblast growth factor (bFGF) in the proliferation and

expression of collagen type I and fibronectin of dog bone

marrow mesenchymal stem cells (dBMMSCs) in comparison

with the expression of the same proteins in cells obtained

from dog periodontal fibroblast cells (dPLFs).

2. Methods

2.1. Ethical approval

The experimental protocol was approved by the Institutional

Animal Care and Use Committee (School of Dentistry of

Aracatuba, UNESP – University Estadual Paulista, Sao Paulo,

Brazil), Protocol Number 57/06.

2.2. Isolation and culture of dBMMSCs

A total of 2.5 mL bone marrow aspirate was collected from the

anterior iliac crest of five dogs (adult hybrid dogs 12–18 months

old, weighing 10.5–15.2 kg) into a syringe flushed with heparin.

Samples were diluted 1:1 with 0.9% saline solution, transferred

into 15 mL Ficoll-PaqueTM Plus (Amersham Biosciences,

Uppsala, Sweden) and centrifuged at 400 � g/20 8C for

30 min. The middle layer was transferred to a new tube and

diluted again with 0.9% saline solution. The suspension was

then centrifuged at 450 � g/20 8C for 5 min. Cells were washed

three times, suspended in 10 mL DMEM culture medium

(Invitrogen Corporation, Grand Isle, USA) and manually

counted in a Neubauer chamber using trypan blue. Cells were

seeded in 25 cm2 culture flasks (TPP, Trasadigen, Switzerland)

in 5 mL culture medium at a density of 106 cells/cm2. The

adherent cells were kept under standard culture conditions

(5% CO2 at 378 C, high humidity). For maintenance, the cells

were cultured in DMEM (Invitrogen, Carlsbad, CA) containing

10% foetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 1%

penicillin–streptomycin (Sigma, St. Louis, MO), 2% non-

essential amino acids and 1% of essential amino acids (Sigma,

St. Louis, MO). Culture media were changed three times a

week. Non-adherent cells were removed by medium aspira-

tion after 72 h. Cells were harvested and passage was

performed at near confluence (90%). After time intervals P1

(11 days), P2 (14 days), and P3 (21 days) the cells were used to

perform assays, which were performed in triplicate.

2.3. Isolation and culture of dPLFs

The mandibular lateral incisors of 5 dogs were extracted under

sterile conditions, with the use of an elevator and dental

forceps. The teeth were immediately immersed in an ice-cold

10% DMEM culture medium (Invitrogen, Carlsbad, CA) contain-

ing penicillin–streptomycin (Sigma, St. Louis, MO). The middle

third of the tooth root was grated with a scalpel blade #11 and

the material obtained was transferred to a Petri dish containing

1 mL DMEM culture medium, supplemented with 10% of FBS.

Once a day 1 mL of the medium was added until a volume of

5 mL was completed. The culture media were changed three

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 2 6 8 – 2 7 6 269


times a week at near confluence (90%) and after P3 (21 days); the

dPLFs were submitted to the assays performed in triplicate.

2.4. Haematoxylin and Eosin staining

After P3 (21 days), dBMMSCs and dPLFs cells were stained with

haematoxylin and eosin in order to make a detailed compari-

son of the morphology of the two cell cultures.

2.5. Flow cytometry

The dBMMSCs obtained after P1, P2 and P3 and dPLFs after P3

were used to analyze the patterns of cell-surface molecules.

Expression of CD90/Thy-1 and CD34 in dBMMSCs and dPLFs

was investigated by three-colour flow cytometry. A total of

5 � 105 cells were rinsed and suspended in 100 mL PBS (pH 7.4)

and incubated for 1 h with a rat monoclonal anti-dog CD90/

Thy-1 (1:100) (AbD Serotec, Kidlington, Oxford, UK) and

identified by the secondary rabbit F(ab0)2 anti-rat IgG-R-

Phycoerythrin(RPE) labelled antibody (1:100) (AbD Serotec,

Kidlington, Oxford, UK) and with a conjugated mouse

monoclonal anti-dog CD34-fluorescein isothiocyanate

(FITC)-labelled antibody (1:100) (AbD Serotec, Kidlington,

Oxford, UK). Cells were analyzed in a FACSCalibur flow

cytometer (BD Biosciences, San Jose, CA, USA).

2.6. MTT proliferation assay

Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide staining (MTT) (Sigma, St.

Louis, MO, USA). After P3, dBMMSCs and dPLFs were seeded in

a 96-well plate with a 104 cell/well density. The stimuli were

performed with bFGF at 1, 5 and 10 ng/mL for 24 and 48 h.

Afterwards the cells were incubated in MTT solution (MTT at

0.5 mg/mL in cell culture medium) at 37 8C in 5% CO2 for 4 h.

After incubation, 100 mL of DMSO were added to each well to

dissolve the formazan crystals and the absorbance was

measured at 570 nm with a reference wavelength of 690 nm.

Results were expressed as mean � SEM of six replicates, and

the assays were performed in triplicate.

2.7. dBMMSCs stimuli

After P3 dBMMSCs were seeded in a 24-well plate and

stimulated with bFGF at 1, 5 and 10 ng/mL for 24 and 48 h.

After P3 a group of dBMMSCs and a group of dPLFs, both

without stimulus, were used as control.

2.8. Real-time quantitative RT-PCR

Total RNA from dBMMSCs and dPLFs was isolated with TRIzol,

in accordance with the manufacturer’s instructions (Invitrogen

Life Technologies, Carlsbad, CA). The RNA concentration was

measured by spectrophotometry. First strand cDNAs were

synthesized using 1 mg of total RNA and Superscript II RNase H�

reverse transcriptase (Invitrogen Life Technologies). Pro-colla-

gen I and fibronectin mRNA levels were measured by means of

the SYBR Green system and amplified in the Stepone Real-Time

PCR system (Applied Biosystems). The housekeeping gene

b-actin was used as internal positive control. The primers were

as follows: b-actin (forward, 50-CTC CAT CAT GAA GTG TGA CGT

T-30; reverse, 50-ATC TCC TTC TGC ATC CTG TCA G-30), Pro-

collagen I (forward, 50-GTG TGT ACA GAA CGG CCT CA-30;

reverse, 50-TCG CAA ATC ACG TCA TCG-30) and fibronectin

(forward, 50-AGG TTG TTA CCG TGG GCA-30; reverse, 50-GCA

TAA TGG GAA ACC GTG TAG-30). Primers were drawn using the

Primer Express software (Applied Biosystems). Reactions were

carried out using a volume of 20 mL, and each sample was run in

duplicate. The PCR thermal cycling conditions used in the

experiments were those recommended by manufacturer. The

pro-collagen I and fibronectin mRNA expression levels in each

sample were normalized to the b-actin mRNA level. The results

were analyzed using the comparative threshold cycle (CT)

method. Results were presented in fold increase of the pro-

collagen I and fibronectin mRNA expression in cells treated with

bFGF compared with untreated cells. Results were expressed as

mean � SEM of six replicates, and the assays were performed in

triplicate.

2.9. Statistical analysis

Two-way ANOVA with Bonferroni correction was used for

MTT analysis, and the Kruskal–Wallis test with Dunn’ post test

were applied for RT-PCR analysis. p values lower than 0.05

were considered statistically significant.

3. Results

3.1. dBMSC and dPLFs cultures

The majority of the cells in the dBMSC culture exhibited a

round morphology with some isolated colonies showing a

fibroblastic morphology on day 3 (Fig. 1A). On day 8, the

predominance of spindle shaped cells was evident (Fig. 1B). A

similar morphology could be noted in dBMMSCs and dPLFs on

day 13 (Fig. 1C and D). The similarity of morphology between

dBMMSCs and dPLFs was also confirmed by H.E. staining after

P3 (Fig. 1E and F).

3.2. Flow cytometry analysis

Flow cytometry analysis of BMA showed that 55.98% cells were

CD34+ (Fig. 2A), indicating that most of the cells obtained

belong to the hematopoietic lineage. For CD90+ expression,

32.67% of the sample was positive for the mesenchymal

marker (Fig. 2B).

After P1 (11 days), no more hematopoietic cells were

labelled (Fig. 2C) and 33% percent of cells were positive for the

mesenchymal lineage marker (Fig. 2D), indicating that in 11

days of culture after the puncture, the percentage of the

mesenchymal lineage was maintained.

After P2, flow cytometry analysis revealed 10.54% of CD90+

(Fig. 2E) and the same percentage for CD34 as in P1 (Fig. 2D).

After P3, no significant labelling for the hematopoietic marker

was observed (Fig. 2F), confirming that the remaining cell

population did not belong to this lineage. Fig. 2G shows that

there was negligible marking (3.94%) with anti-CD90.

Fig. 2H shows the results of flow cytometry analysis

performed of DPLFS after P3, for anti-CD34. No significant



labelling (2.88%) confirmed the negativity of these cells for the

characteristic staining of the hematopoietic lineage. Fig. 2I

shows that after the addition of the primary antibody (anti-

CD90), followed by addition of the secondary antibody, there

was also negligible marking (4.04%).

3.3. MTT

Fig. 3 shows that there was an increase in dBMSC cell

proliferation according to time only for the control group,

whereas for all other groups, the cell density fluctuated with

the increase in time and did not maintain a linear ascent or

descent. Although there was no significant relationship

between the concentration of 5 ng/mL bFGF and the other

groups, this concentration presented the highest proliferation

rate after 24 h of stimulus.

3.4. RT-PCR

Fig. 4A and B shows the results of gene expression. In panel A,

the dBMMSCs expressed more fibronectin at the concentration

of 5 ng/mL at 24 h after stimulation. Similar results were

observed in the fibronectin gene expression between

dBMMSCs (control) and dPLFs in panel A. On the other hand,

after 48 h, the concentration of 1 ng/mL presented higher

expression of fibronectin, which decreased with the increase

in concentration of bFGF.

In panel B, there was also a similarity in collagen I gene

expression between dBMMSCs and dPLFs. The collagen gene

expression of dBMMSCs (bFGF 1 ng/mL stimulation) and

dPLFs decreased after 48 h. The dBMMSCs presented higher

collagen I gene expression at concentration of 5 ng/mL

after 24 h. After 48 h, there was a decrease in the relative

Fig. 1 – The dBMSC culture. Panel A: The arrows indicate the first adherent fibroblast-like cells (optical magnification 400T) in

the 3rd of culture. Panel B: 8th day of dBMSC culture, showing the prevalence of the spindle-shaped cells (optical magnification

200T). Panel C and D: dBMMSCs and dPLFs, respectively, on the 13rd day of culture (optical magnification 100T). Panel E and F:

dBMMSCs and dPLFs, respectively, after P3 days in culture with H.E. staining (optical magnification 400T).



expression of collagen I gene values for all concentrations of

bFGF stimulus.

4. Discussion

Recently, several studies have indicated that PDL fibroblastic

cells have biological characteristics in common with bone

marrow mesenchymal cells, suggesting that the mineralized

tissue-forming cell and fibroblastic lineages may originate

from a common early progenitor cell.19–21

The adherence of the cells and their morphology corrobo-

rate the studies that confirm their adhesion to the culture flask

surface, and morphology similar to that of fibroblasts, as being

characteristics of mesenchymal cells.22,23 Lee et al.24 and

Romanov et al.25 performed the culture of MSCs in a low

Fig. 2 – dBMC flow cytometry analysis (FCA). Panel A/B: FCA after bone marrow aspiration: 55.98% CD34+/32.67% CD90+. Panel

C/D: FCA before dBMMSCs 1st passage: 3.33% CD34%/33% CD90+. Panel E/F: FCA before dBMMSCs 2nd passage: 1.72%

CD34%/10.54% CD90+. Panel G/H: FCA after dBMMSCs 3rd passage: 4.83% CD34+/3.94 CD90+. Panel I/J: FCA after dPLC 3rd

passage: 2.88% CD34+/4.04% CD90+.



glucose medium and obtained colonies of adherent cells with

fibroblastoid morphology only after the first 5 days of culture,

while our results showed the appearance of cells with this

morphology on day 3 after plating (Fig. 1A). This difference can

be explained by the glucose concentration, since in our study

we used a culture medium with a normal concentration.

FCA showed that there was a progressive loss in CD90

expression during the course of the culture passages,

suggesting that dBMMSCs had undergone a differentiation

process and expressed the same immunophenotype profile

verified in dPLFs after the third passage (CD90�/CD34�). The

lack of CD90 in the dBMSC culture passages shows that they

may apparently compose the group with a lipofibroblast

profile, as defined by Koumas et al.11 According to Wiesmann

et al.,26 the lack of CD90 expression after mechanical

stimulation indicates the differentiation of these cells into

the osteoblastic lineage, but our results showed that even

without the mechanical stimulation, dBMMSCs lost the CD90

expression and shared other similarities with the dPLFs, such

as protein expression and morphology. A decrease in

positivity for CD90 has also been associated with a loss of

immunosuppressive activity in human mesenchymal stromal

cells.27

FGF-2 or bFGF has been reported to be a potent mitogen for

MSC, and that incubation with bFGF maintains the multi-

lineage differentiation potential of MSC throughout many

mitotic divisions.9 Moreover, bFGF has the potential of

enriching BMSC primary cultures for early mesenchymal

progenitor cells.24 The bFGF supplementation of BMSC

primary cultures selects a subpopulation of earlier progenitors

with a significantly longer lifespan, which maintain their

differentiation potential for over 50 population doublings

in vitro.24 Although no statistically significant difference

was seen among the different bFGF concentrations in the

proliferation analysis (MTT), our results showed that there

was a slight improvement in the performance of dBMMSCs

stimulated with bFGF at the concentration of 5 ng/mL after

24 h of stimulus. Moreau et al.17 evaluated the effects of bFGF

in addition to other growth factors, and in their study it was

possible to see that bFGF did not increase the proliferation of

dBMMSCs. Conversely, other studies have reported that bFGF

supports the proliferation potential of mesenchymal stem

cells.28–30

The presence of collagen type I and fibronectin in the

matrix produced was an important finding. Fibronectin and

collagen type I are considered key structural components of

the periodontal ligament.31,32 Moreau el al.17 observed that

before bFGF, the combination of transforming growth factor-b

(TGF-b) with epidermal growth factor (EGF) stimulation

increased collagen I production. In our study, only the bFGF

stimulation improved fibronectin (1 and 5 ng/mL) and collagen

I (5 ng/mL) gene expression. Treatment of BMSCs with a low

concentration of FGF-2 (3 ng/mL) was shown to stimulate

proliferation and up-regulated the expression of extracellular

matrix protein such as collagen I, collagen III, and fibronec-

tin.33 Another strategy for ligament tissue engineering is the

use of bFGF-releasing nanofibrous scaffolds. Sahoo et al.

demonstrated that bFGF incorporated into bioactive nanofi-

bers facilitated BMSC proliferation, increased production and

deposition of collagen and induced tendon/ligament-like

fibroblastic differentiation, indicating their potential in ten-

don/ligament tissue engineering applications, since they

provide sustained bFGF levels over a period of 7 days.34

In the presence of bFGF, Solchaga et al. observed that

human mesenchymal stem cells developed a homogenous

cartilaginous matrix, but with no difference in size or

histological appearance with regard to different concentra-

tions of bFGF (1, 5, or 10 ng/mL).35 In this study, in the hMSCs

Fig. 3 – MTT proliferation assay of dBMMSCs. Cells were stimulated with 1, 5 and 10 ng/mL of bFGF and the proliferation was

evaluated 24 and 48 h afterwards. Results were expressed as mean W SEM of six replicates, and the assays were performed

in triplicate.



growth curve, two modalities were observed in the presence of

1, 5 or 10 ng/mL bFGF stimulus: some cultures showed a

threshold effect with better results at the concentration of

5 ng/mL bFGF stimulus; the other demonstrated a linear dose–

response relationship with a higher rate of cell growth at the

concentration of 10 ng/mL. Our data showed a better perfor-

mance of bFGF at the concentration of 5 ng/mL after 24 h of

stimulus, with the upregulation of gene expression for

periodontal ligament-specific extracellular matrix proteins.

Eijk et al.36 observed that bone marrow stromal cells up-

regulated collagen expression on day 12 of culture when

compared with anterior cruciate ligament fibroblasts. Accord-

ing to these authors, BMSCs represent a more suitable cell

source for the development of tissue-engineered ligament.

Although they did not use any stimulating factor, our study

reinforces hypothesis that bFGF is a suitable adjuvant to

improve ligament production by bone marrow stem cells.

With particular regard to periodontal tissue engineering, an

in vivo study also reported that dBMMSCs were more effective

in promoting alveolar bone regeneration when compared with

periodontal ligament stem cells.37 These findings corroborate

with our results, in which dBMMSCs showed significantly

higher fibronectin and collagen type I gene expression when

stimulated with 5 ng/mL bFGF after 24 h in comparison with

dPLFs. Although dBMMSCs stimulated with 1 ng/mL and dPLFs

were very similar after 24 h, our findings indicate that the

concentration of 5 ng/mL could be appropriate for developing

periodontal connective tissue and can be associated with

scaffolds that promote a continuous release, as suggested by

Sahoo et al.,34 being suitable for in vivo application.

Injuries to periodontal ligament by disease or trauma are

common events.38 The regeneration of lost periodontal

ligament (PDL) and alveolar bone is the purpose of periodontal

tissue engineering.39 Recently, Tcacencu et al.40 using a rat

Fig. 4 – Fibronectin and collagen expression in dBMSC and PLC. Cells were stimulated with 1, 5 and 10 ng/mL of bFGF and the

proliferation was evaluated 24 and 48 h afterwards. Results were expressed as mean W SEM of six replicates, and the

assays were performed in triplicate. Panel A: Relative fibronectin gene expression. *dBMMSCs stimulated with bFGF 5 ng/mL

after 24 h of stimulus expressed more fibronectin gene than dPLFs ( p < 0.05). **dBMMSCs stimulated with bFGF 1 ng/mL

expressed more fibronectin gene than dBMMSCs stimulated with bFGF 10 ng/mL after 48 h of stimulus ( p < 0.05). Panel B:

Relative collagen gene expression. *dBMMSCs stimulated with bFGF 5 ng/mL after 24 h of stimulus expressed more collagen

gene than dBMMSCs with no stimulus ( p < 0.05). **dBMMSCs stimulated with bFGF 5 ng/mL expressed more collagen gene

than dBMMSCs stimulated with bFGF 1 ng/mL after 48 h of stimulus ( p < 0.05).



periodontal wound model, analyzed the effects of local

treatment with human mesenchymal stem cells (hMSCs).

The authors found that transplanted hMSCs can contribute to

alveolar bone preservation after a periodontal surgical trauma.

Bone marrow mononuclear cells have also been used to

enhance the periodontal healing of replanted dog teeth.41

According to our results, dBMMSCs are very similar to

dPLFs throughout the culture passages, as regards morpho-

logical and immunostaining characteristics, and collagen and/

or fibronectin production, indicating that dBMSC have the

potential to differentiate into dPLFs. This process can be

intensified by FGF stimulus with 5 ng/mL and would be useful

in techniques that aim to regenerate the periodontal ligament

in vivo or in situ.

Funding

This work was supported by Sao Paulo State Research

Foundation (FAPESP) (Grant No. 06/59420-5). R. Colenci was

supported by a scholarship from Sao Paulo State Research

Foundation (FAPESP). L.R.S. Assuncao was supported by a

scholarship from Brazilian research funding agency

(CAPES).

Competing interests

The authors report no conflicts of interest related to this study.

Ethical approval

The experimental protocol was approved by the Institutional

Animal Care and Use Committee (School of Dentistry of

Aracxatuba, UNESP – University Estadual Paulista, Sao Paulo,

Brazil), Protocol Number 57/06.

Acknowledgments

This work was supported by Sao Paulo State Research

Foundation (FAPESP) (Grant No. 06/59420-5). R. Colenci was

granted a scholarship from Sao Paulo State Research Founda-

tion (FAPESP). L.R.S. Assuncao was granted a scholarship from

Brazilian research funding agency (CAPES).

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