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Received: 20 July 2018 | Accepted: 30 August 2018

DOI: 10.1002/jcp.27472

ORIGINAL RESEARCH ARTICLE

Dental pulp stem cells senescence and regenerative potential


relationship

Iolanda Iezzi1 | Giorgia Cerqueni1 | Caterina Licini1,2 | Guendalina Lucarini1 |


Monica Mattioli Belmonte1

1
Department of Clinical and Molecular
Sciences‐DISCLIMO, Università Politecnica Abstract
delle Marche, Ancona, Italy Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and
2
Department of Applied Science and
regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs)
Technology (DISAT), Polytechnic of Turin,
Turin, Italy represent a good candidate for oral recovering therapies. Here, we investigated changes in
morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal
Correspondence
Monica Mattioli‐Belmonte, Department of phenotypes of human DPSCs harvested from differently aged donors. Aging is a
Clinical and Molecular Sciences, Marche
physiologic phenomenon occurring with time that hamper body’s capability to maintain
Polytechinc University, Via Tronto 10/a,
60126 Ancona, Italy. homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical,
Email: m.mattioli@univpm.it
quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), and western blot
analyses were performed to gain insight for successful regenerative strategies in elderly.
We observed a decline in DPSCs proliferation and differentiation potential with age.
Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli,
showing different age‐related mineralization capabilities. Similarly, neurogenic differentia-
tion decreased with age. In conclusion, our observations represent a valid tool for the
development of tailored regenerative strategies in an aging society.

KEYWORDS
aging, DMP1, DPSCs, nestin, p16ink4a, SA‐β‐Gal

1 | INTRODUCTION good candidate for regenerative therapies (Gronthos et al., 2002). Dental
pulp stem cells (DPSCs), first isolated by Gronthos, Mankani, Brahim,
Cell and tissue biology studies have extended the focus on oral Robey, and Shi (2000) from DP of third molars, showed a great self‐
medical research to propose in vitro or in vivo approaches for a novel expansion and multilineage differentiation ability (Diomede et al., 2017;
generation of biomimetic tissues structurally and functionally like the La Noce et al., 2014).
native tissue. Aging is a physiologic phenomenon occurring with time (Morgan,
Dental pulp (DP) is a highly specialized ectomesenchymal tissue Kunkel, & Alessio, 2001); it is affected by multiple factors at different
originating from migrating neural crest cells during development (Lindvall, rates, such as lifestyle, environment, and genetics. As people get
Kokaia, & Martinez‐Serrano, 2004). It is a vascularized tissue in the older, the capability to respond to external or internal stresses (i.e., to
central pulp cavity of each tooth, “pulp chamber” (Trubiani, Tripodi, Delle maintain homeostasis) decreases, as well as the body’s functional
Fratte, Caputi, & Di Primio, 2003), composed of soft connective tissue, reserve (Besdine, 2013). Hence, aging leads to a new set of problems
mesenchymal cells, neural fibers, blood vessels, and lymphatics. The also in the regenerative medicine field. Like the other tissues, DP
principal roles of DP are producing dentin and maintaining the biological undergoes to age‐related modifications such as volume reduction,
and physiological viability of teeth (Liu, Gronthos, & Shi, 2006). Due to its decreasing of vascularization, innervation, and cell availability (Balic,
easy accessibility and high cell proliferation potential, DP cells can be a 2018). These processes begin around the middle age (40‐years old),

J Cell Physiol. 2018;1–12. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 1


2 | IEZZI ET AL.

as noted by Bernick and Nedelman since 1975. Moreover, in molars 2.3 | Immunocytochemistry (ICC)
from over 60‐years old subjects an increase in the number of
For ICC staining, 15 × 104 cells per well hDPSCs were cultured on two‐
calcification loci was observed (Bernick & Nedelman, 1975).
well Lab‐Tek® Chamber Slide, fixed in 4% PFA in 0.1 M sodium
At present, there are few and conflicting knowledge about the
phosphate buffer pH 7.4 for 20 min at room temperature (RT) and then
role of aging on DPSC regenerative potential (R. Feng & Lengner,
permeabilized in 0.1% Triton X‐100 in PBS for 10 min. Following
2013), consequently, it becomes crucial to further investigate on this
overnight incubation at 4°C with mouse anti‐human p16ink4A monoclonal
process. Based on these considerations, in this study, we examined in
antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), hDPSCs were
vitro senescence features of human DPSCs (hDPSCs) and their
immunostained using the streptavidin–biotin peroxidase technique
possible role in the impairment of multipotency and differentiation
(Envision Peroxidase Kit; DakoCytomation, Darmstat, Germany). After
potential. Harvested cells were divided in three group (young, middle
the incubation with 0.05% 3,3′‐diaminobenzidine (Sigma‐Aldrich) in
age, and old). Cytofluorimetric, immunohistochemical, quantitative
0.05 M Tris buffer, pH 7.6, with 0.01% hydrogen peroxide, cells were
reverse‐transcription polymerase chain reaction (qRT‐PCR), and
counterstained with hematoxylin, dehydrated in ethanol, and cover-
western blot analyses were performed to ascertain changes in
slipped with Eukitt mounting medium (Electron Microscopy Sciences,
hDPSC osteogenic, odontogenic, and neurogenic capability.
Hatfield, PA). For negative controls, the primary antibody was replaced
with nonimmune serum. The immunocytochemical expression of p16ink4A
2 | MATERIALS AND METHODS was evaluated under Nikon Eclipse E600 light microscope by two
independent observers. Images were captured and processed as above.
2.1 | Sample collection and cell culture
In collaboration with Odontostomatological and Special Surgery Unit, 2.4 | Population doubling time (PDT)
Ospedali Riuniti of Ancona, DP was obtained from third molars of 12
individuals gender matching (mean age, 43 years; range, 20–64 hDPSCs were seeded at an initial density of 5 × 104 cells/well in six‐
years), following approved guidelines set by the Local Ethics well tissue culture plates (VWR®, VWR International Srl, Milan, Italy).
Committee. All patients were aware of the voluntariness of their Cells were detached by trypsinization every 4 days, counted and
participation and provided an informed consent in the form of verbal reseeded at the same density. To examine the time required by cells
authorization. Samples were divided into three age groups: group A of each population to double, PDT was calculated according to the
(21 years; 20–23 years), group B (43 years; 42–45 years) and group C formula:
(64 years; 62–66 years).
PDT = T log 2/(log Nf − log N0) ,
Cells were isolated from DP as described in the previous study
(Gronthos et al., 2000). Briefly, tissue was gently removed and
where T represents the duration of cell culture (in days) and Nf and
immersed in the digestive solution (3.0 mg/ml type I collagenase and
N0 the final and the initial concentration, respectively (Roth, 2006).
4.0 mg/ml dispase) for 1 hr at 37°C, then filtered by 70‐μm cell
strainers to obtain hDPSC suspension. Cells were plated in T25 flasks
and cultured in a complete culture medium DMEM/F12 with 10% 2.5 | Telomere length determination
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and
5% CO2. Telomere length was assessed by Relative Human Telomere
Length Quantification qPCR Assay Kit (ScienCell™, Carlsbad, CA;
92011) according to manufacturer’s instruction. The average
2.2 | Senescence‐associated‐β galactosidase assay telomere lengths of samples designed were directly compared by
(SA‐β‐Gal) telomere primer set that recognized and amplified telomere

The SA‐β‐Gal activity was determined using a SIGMA kit (Sigma‐ sequences.

Aldrich, Milan, Italy) according to manufacturer’s instructions. In


brief, hDPSCs were cultured overnight on two‐well Lab‐Tek®
2.6 | Flow cytometric analysis
Chamber Slides (Sigma‐Aldrich) at a density of 2 × 104 cells/cm2
and fixed with 4% paraformaldehyde (PFA). After overnight incuba- For immunophenotyping 2.5 × 104 cells underwent flow cytometric
tion with SA‐β‐Gal, cells were washed with phosphate‐buffered saline analysis according to the International Society for Cellular Therapy
(PBS), counterstained with hematoxylin and then observed under (ISCT) for identification of human mesenchymal stem cells (hMSCs;
Nikon Eclipse 600 light microscope. Images were captured with a Dominici et al., 2006). hDPSCs were stained for 25 min at RT with
Nikon DSVi1 digital camera and processed with NIS Elements BR anti‐CD90, CD105, CD34, CD73, and HLA‐DR (all from Immuno
3.22 imaging software (all from Nikon Instruments, Florence, Italy). Tools, Friesoythe, Germany) for 30 min at 4°C. For isotype controls,
The senescent cells, blue stained, were counted in at least five the primary antibodies were used instead of FITC‐ or PE‐coupled
randomly selected fields per sample and quantified as a percentage nonspecific mouse immunoglobulin G. Cell fluorescence was
of the total counted cells. Mean ± SD was considered. evaluated in a FACSCalibur flow cytometry system (BD Italia, Milan,
IEZZI ET AL. | 3

Italy) and data were analysed using FCS Express 6 Plus software (De For cytoplasmic DMP1 detection, cell cultures underwent digestion
Novo Software, Glendale, CA). In addition, physical parameters such with type I collagenase.
as cell size (forward scatter [FSC]) and granularity (side scatter [SSC]) Neurogenic differentiation was generated by seeding hDPSCs at a
were analysed. density of 2.5 × 103 cells/cm2 into six‐well plates (VWR®) in Neurobasal
Medium (Gibco®, Life Technologies, Carlsbad, CA) supplemented with 1%
penicillin/streptomycin, 1% B27 (Gibco®, Life Technologies), 40 ng/ml
2.7 | In vitro cell differentiation
fibroblast growth factor 2, and 20 ng/ml epidermal growth factor (both
For adipogenic, osteogenic, and chondrogenic differentiation, com- from Immuno Tools). A medium was changed every 3–4 days.
mercial kits from Life Technologies Corporation (Carlsbad, CA) were Differentiation was assessed by the detection of nestin and β‐tubulin III
used according to the manufacturer’s instruction. For adipogenic after 2 weeks.
differentiation 104 cells/cm2 were seeded in two‐well Lab‐Tek® Control cultures were represented by cells maintained in an
Chamber Slide and treated with the appropriate medium (STEM- appropriate medium without differentiating factors for the same time.
PRO® Adipogenesis Kit, Gibco life technologies, Grand Island, NY) for
14 days, changing the medium twice a week. Differentiation was
2.8 | qRT‐PCR
assessed by Oil red staining. Briefly, cells fixed in 4% PFA were
exposed to Oil red O solution (0.5% in 100% isopropyl alcohol) for qRT‐PCR reactions were carried out in triplicate. Threshold cycle
30 min at RT, cleared with isopropanol 60%, washed in dH2O, and values were quantified, and expression of each gene was normalized
counterstained with hematoxylin. Cells were immediately observed relative to that of references genes.
under a light microscope to avoid the stain decay and counted. Real‐time assays were performed by the Mastercycler RealPlex2
For osteogenesis, cells were seeded at a density of 1.5 × 10 3 (Eppendorf GmbH) using SsoFast™ EvaGreen Supermix 1×, in a final
cells/cm 2 in two‐well Lab‐Tek ® II Chamber Slide and treated with volume of 10 ml. All PCRs contained 1 μl of complementary DNA.
STEMPRO ® osteogenesis medium for 3 weeks, changing the Each PCR assay was performed in the white plate and comprised at
medium twice a week. Alkaline phosphatase (ALP) staining was 95°C for 30 s for enzyme activation and 40 cycles of denaturation at
performed after 1 week by incubating cells with a solution of 5‐ 95°C for 5 s, annealing, and extension at 60°C for 20 s. Every primer
bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) and nitroblue tetra- was used at 10 μM final concentration. Primer sequences were
zolium (NBT) alkaline‐phosphate substrate (Sigma‐Aldrich) in designed by Primer 3 web (Primer3Web version 4.1.0, http://
100 mM Tris‐HCl (pH 9.5), 100 mM NaCl, 10 mM MgCl2 buffer for primer3.ut.ee/) (and their specificity was tested by BLAST to avoid
1 hr at RT, and rinsed in dH2 O. After 3 weeks mineralization was any appreciable homology to pseudogenes or other unexpected
assessed by Alizarin Red S (ARS) staining (Sigma‐Aldrich). Briefly, targets). In each assay, the messenger RNA (mRNA) of both reference
cells were fixed with 4% PFA in PBS for 10 min, incubated with genes and each gene of interest were measured simultaneously
ARS for 30 min at RT and finally washed in dH2 O. The reaction under identical conditions. Primers showed the same amplification
was observed under Nikon Eclipse 600 light microscope. To efficiency. The specificity of the PCR reactions was confirmed by
quantitatively determine calcium mineral deposits, ARS was melt curve analysis: for each amplicon, the detected melting
extracted with 10% cetylpyridinium chloride in 10 mM sodium temperature was the expected one.
phosphate for 60 min at RT. Concentration was quantified Each assay was performed in triplicate and threshold Cycle
spectrophotometrically at 540 nm (Secoman, Anthelie light, (C t) values for reference genes were used to normalize cellular
version 3.8; Contardi, Cesano Maderno, Italy). mRNA data. In this instance, normalization involved the ratio of
For chondrogenesis, hDPSCs were cultured in pellet culture mRNA concentrations for specific genes of interest (as mentioned
system. For the preparation of each pellet, aliquots of 106 cells in above) to that corresponding to C t medium values for glycer-
1 ml of STEMPRO ®
Chondrogenesis Kit (STEMPRO ®
Chondrogen- aldehyde‐3‐phosphate dehydrogenase (GAPDH) and β‐glucuroni-
esis Kit. Gibco life technologies, Grand Island, NY) were spun down at dase (GUSB; Ragni et al., 2013). The amount of each mRNA was
1,200 rpm for 5 min. Pellets were cultured for 14 days, changing the calculated using the comparative C t method with ΔC t = C t (mRNA)
medium twice a week. Pellets were then fixed in 4% PFA, paraffin − C t (GAPDH/GUSB), and data were expressed as gene relative
embedded, and sectioned. Sections were exposed to a solution of expression (2−ΔCt ). Moreover, to highlight the effect of aging on
Alcian Blue (pH 1; Bio‐Optica, Milano, Italy) for 30 min at RT and cell behavior, ΔΔC t method for the evaluation of fold‐change
observed under Nikon Eclipse 600 light microscope. (2−ΔΔCt ) was used comparing values obtained in cells of groups B
Odontogenic differentiation was obtained by seeding 2.5 × 103 and C with those of group A (Livak & Schmittgen, 2001). The
cells/cm into six‐well plates (VWR ) and cultured for 3 weeks in α‐
2 ®
qPCR efficiency in all our experiments was more than 90%. The
minimum essential medium (α‐MEM) supplemented with 10% FBS, difference between the actual and theoretical (100%) efficiencies
1% penicillin/streptomycin, 10 mM/L β‐glycerophosphate, 10 nM/L would result in an underestimation of the mRNA concentration of
dexamethasone and 50 µg/ml ascorbic acid (all from Sigma‐Aldrich). all the analysed samples.
Differentiation was assessed by the detection of dentin matrix acidic Oligonucleotide sequences for target genes are reported in the
phosphoprotein 1 (DMP1) and dentin sialophosphoprotein (DSPP). Supporting Information Table S1.
4 | IEZZI ET AL.

2.9 | Western blot (WB) analysis 3 | RESULTS


The expression of DMP1, nestin, and β‐tubulin III in hDPSC after
3.1 | Senescent features in hDPSCs
odontogenic and neurogenic differentiation was quantified by WB.
hDPSC total proteins were extracted at the fourth passage of To ascertain the presence of age‐related changes in cells derived
subculture in basal cultures (i.e., cells grown in DMEM/F12) and after from the three groups, we analysed the expression of SA‐β‐Gal and
cell differentiation toward odontoblast and neuron phenotypes. p16ink4a, SSC, and FSC cytofluorimetric parameters, as well as
Moreover, to ascertain the possible natural hDPSC differentiation a changes in telomere length. The positive rate of SA‐β‐Gal and
further control was represented by cells cultured in appropriate p16ink4a was remarkably higher in hDPSCs of group C in comparison
media without differentiating cues. Radioimmunoprecipitation assay with cells harvested from younger subjects (Figure 1a,b). As far as
buffer (RIPA Lysis Buffer System; Santa Cruz Biotechnology) was p16ink4a is concerned we detected a significantly (p < 0.05) higher
used for protein extraction. Protein concentration was determined number of positive cells in groups B (81 ± 4%) and C (97 ± 5%) in
using Bradford reagent (Sigma‐Aldrich). Electroblotting was per- comparison to group A (56 ± 3%). Flow cytometry analysis con-
formed using Wet Blotting System (XCell II Blot Module; Invitro- firmed SA‐β‐Gal observations, evidencing an increase of SSC values
gen™). Membranes were incubated overnight with primary with aging. On the contrary, no changes in cell size were observed
antibodies anti‐DMP1 (1:200, Santa Cruz Biotechnology), anti‐nestin (Figure 1c). PDT showed no differences until the passage 13 of
and β‐tubulin III (1:500; Santa Cruz Biotechnology) followed by subculture. After then, cells from groups B and C started to slow
incubation with a secondary antibody conjugated to horseradish and at P17 hDPSCs of group C stopped their proliferation
peroxidase (1:1,000; Santa Cruz Biotechnology). The signals were (Figure 1d).
captured using an Alliance Mini system (UVITEC, Cambridge, UK); qRT‐PCR analysis displayed changes in the expression of
the DMP1, nestin, and β‐tubulin III bands were quantified with stemness genes in relation to age (Table 1). Comparing gene
UVITEC software and their intensity was normalized by comparison expression of hDPSCs from groups B and C with those of group A,
to the GAPDH housekeeping, used as a loading control. The intensity a slight upregulation of sex‐determining region Y (SRY)‐Box 2 (Sox2)
of each band was then compared with the negative controls and any mRNA expression and a downregulation of both octamer‐binding
change was expressed as ratio. transcription factor 4 and Nanog were showed. On the contrary, the
expression of Kruppel‐like factor 4 (Klf4) appeared downregulated in
cells from group B and almost unchanged in hDPSCs from group C

2.10 | Immunofluorescence (IF) (Figure 1e).


At last, the length average of telomere of group A was 1.1‐ and
hDPSCs were seeded at the density of 2.5 × 104 cells per well on two‐ 1.2‐fold longer than that of groups B and C, respectively,
well Lab‐Tek® Chamber Slides and treated with neurogenic strengthening the features of age‐related changes in our hDPSCs
differentiating media for 2 weeks. For the detection of nestin and (Figure 1f).
β‐tubulin III localization IF was performed. After differentiation cells
were fixed in 4% PFA and then permeabilised in 0.1% Triton X‐100 in
PBS. For immunofluorescence morphological analysis, samples were 3.2 | hDPSCs characterization and mesenchymal
blocked with a 2% bovine serum albumin (BSA) solution in PBS for tissues differentiation
30 min. Cells were then incubated overnight at 4°C with mouse anti‐
human–nestin or anti‐human–β tubulin III (1:500; Santa Cruz hDPSCs isolated from the three groups showed no differences in

Biotechnology). After overnight incubation, cells were stained with their ability to adhere to plastic, in morphology (Figure 2a) and in

a goat anti‐mouse FITC‐conjugated secondary antibody (Bethyl their phenotypic profile according to ISCT criteria: Our cells were

Laboratories Inc., Montgomery, Alabama; dilution, 1:100). Nuclei positive for CD73, CD90, and CD105, and negative for CD45, HLA‐

were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and DR, and CD14 (Figure 2b). The analysis of their differentiation
®
samples were mounted using Vectashield mounting medium (Vector capability exhibited a reduction of the adipogenic and osteogenic

Laboratories Inc, Burlingame, CA). For negative controls, the primary potential in cells from group C in comparison with both groups A and

antibody was replaced with nonimmune serum. Cells were observed B, as evidenced by Oil red, ALP and ARS stainings. On the contrary,

with a Nikon E600 Fluorescence microscope. no changes in chondrogenic potential were observed (Figure 2c).
Age‐related modifications of genes involved in osteogenic differ-
entiation were detected, with a decrease in runt‐related transcription
factor 2 (Runx2), β‐catenin, and bone γ‐carboxyglutamic acid‐
2.11 | Statistical analysis
containing protein (BGLAP) expression (Table 1). It was interesting
Statistical analysis was performed by Prisma 4 Software (GraphPad to observe that, after 3 weeks in odontogenic media, the expression
software, La Jolla, CA). Mean and SD of three different experiments of mRNA for Bmp2, markedly upregulated before differentiation,
was reported. Data were analysed by analysis of variance and appeared downregulated in groups B and C in comparison with group
Bonferroni’s T test. Statistical significance was tested at p < 0.05. A. On the contrary, no significant changes were detected for the
IEZZI ET AL. | 5

F I G U R E 1 (a) Detection of SA‐β‐Gal in


hDPSCs isolated from three different age
groups. (b) Immunocytochemical analysis of
p16ink4a in the differently aged hDPSCs.
(c) Histogram of cytofluorimetric detection
of changes in cell internal complexity (SSC)
and size (FSC). (d) Histogram of the
evaluation of PDT during subculturing of
hDPSCs isolated from three different age
groups. (e) Histogram depicts changes in
stemness gene mRNA expression in the
differently aged hDPSCs. Data are
expressed as fold‐regulation (2−ΔΔCt ; see
Section 2), the dotted black line indicates
the mRNA expression of group A; *p < 0.05
versus group A, #p < 0.05 versus group B.
(f) Table showing the qPCR quantification
of telomere length in the three different
age groups. FSC: forward scatter; hDPSCs:
human dental pulp stem cells; mRNA:
messenger RNA; PDT: population doubling
time; qPCR: quantitative polymerase chain
reaction; SA‐β‐Gal: senescence‐associated‐β
galactosidase assay; SSC: side scatter
[Color figure can be viewed at
wileyonlinelibrary.com]

other osteogenic genes except for BGLAP (Figure 2d and Supporting ARS showed a marked increase in cells of groups B and C in
Information Table S2). comparison with hDPSCs of group A (Figure 3a). This was
As odontogenic differentiation is concerned, even if no sustained by the changes of odontogenic gene expression and
significant morphological age‐related changes were observed, DMP1 protein. After differentiation, a decrease in the expression
6 | IEZZI ET AL.

of DMP1 and DSPP mRNAs was observed in hDPSCs of groups B III (Figure 4c). Nestin and β‐tubulin III IF detection evidenced
and C in comparison to cells of group A (Figure 3b and Appendix age‐related differences in their intensity and localization
Table 2). WB analysis showed a faint expression of DMP1 (Figure 4a).
fragments in undifferentiated cells, except for the N‐terminal
one in cells from group B. The expression of cytoplasmic DMP1
increased in cells undergoing odontogenic differentiation as well 4 | D I S C U SS I O N
as in hDPSCs cultured without an odontogenic media (Figure 3c).
In hDPSCs treated with odontogenic medium, the majority of Although great progress has been made in dentistry, the
DMP1 was in the form of 105 kDa, while in cells cultured, without treatment of pulpitis and periodontitis continues to have post-
odontogenic cues, it was in the size of 37 kDa fragments. Protein operative complications. Nowadays regenerative approaches, as
aggregates (120 kDa), accumulated on top of the sodium dodecyl well as biomaterials commonly used in dentistry, have been
sulfate–polyacrylamide gel electrophoresis gel, were also pre- considered for patients without a specific age target. The world’s
sent, especially in cells of group C cultured for 3 weeks without population is aging, and it has been appraised that by 2050 the
odontogenic induction. Mineralization nodules were detected number of people 65 years of age and older will reach 1.5 billion
only in cells treated with differentiating media, indicating that (Richard & John, 2011). Hence, the concept of customized
DMP1 favored mineralization (Figures 3a,d). Moreover, ARS therapies must meet this contingency. Like others, DP tissue
quantization evidenced age‐related variations in the mineraliza- undergoes age‐depended changes and the decline of its regen-
tion capability of hDPSCs after osteogenic or odontogenic stimuli erative capacity might be related to stem cell senescence (Chu,
(Figure 3e). 2013). Up to now, few and conflicting data are available on
possible age‐related changes in the regenerative potential of
DPSCs (Chu, 2013; Yi et al., 2017). These cells possess a great
3.3 | Neurogenic differentiation
self‐expansion and differentiation capabilities toward osteogenic
Following 2 weeks of induction with neurogenic medium, hDPSCs (Teti et al., 2015), odontogenic (Gronthos et al., 2002) and
decreased in proliferation and acquired a stellate morphology neurogenic (Gervois et al., 2015) phenotypes, making them an
with no differences among the groups (Figure 4a). qRT‐PCR interesting cell source for regenerating pulp and periodontal
analysis indicated that nestin and β‐tubulin III mRNA expression tissues, and in the treatment of neurodegenerative diseases.
profile decreased with aging (Figure 4b). WB protein expression Based on these considerations, we investigated changes in
patterns of early (nestin) and intermediate (β‐tubulin III) neuronal morphology, proliferation, and in vitro differentiation potential in
markers showed an increase of nestin 90 kDa in hDPSCs with age, hDPSCs isolated from differently aged donors. Cells were split into
whilst no changes were detected for nestin 120 kDa and β‐tubulin juvenile (group A), middle age (group B), and aged (group C) clusters

T A B L E 1 mRNA relative expression in native cells

Group A Group B Group C

Mean SD Mean SD Mean SD Bonferroni’s T test


Sox2 9.66E−06 4.83E−07 2.13E−05 2.13E−05 1.66E−05 1.66E−05 Group C versus groups A and B Group B versus Group A
Oct4 4.69E−04 2.34E−05 3.15E−04 1.58E−05 2.90E−04 1.45E−05 n.s.
Nanog 8.93E−05 4.46E−06 8.83E−05 4.42E−06 1.66E−05 8.30E−07 Group C versus groups A and B
Klf4 3.43E−02 1.71E−03 3.04E−02 1.52E−03 4.21E−02 2.11E−03 Group C versus groups A and B
Bmp2 3.48E−04 1.74E−05 2.98E−03 1.49E−04 1.81E−03 9.04E−05 Group C versus groups A and B
Runx2 6.96E−03 3.48E−04 2.09E−03 1.05E−04 3.08E−03 1.54E−04 Group C versus groups A and B Group B versus group A
β‐Catenin 2.37E−02 1.19E−03 1.51E−02 7.57E−04 2.50E−02 1.25E−03 Group B versus groups A and C
BGLAP 1.41E−04 7.03E−06 1.14E−05 5.70E−07 3.30E−05 1.65E−06 Group C versus groups A and B
DMP1 4.69E−05 2.35E−06 6.79E−05 3.40E−06 5.73E−05 2.87E−06 Group C versus group A and B Group B versus group A
DSPP 2.16E−05 1.08E−06 3.30E−05 1.65E−06 2.59E−05 1.30E−06 Group C versus group A and B
Nestin 1.01E−02 5.04E−04 4.52E−03 2.26E−04 8.75E−03 4.38E−04 Group C versus group A and B Group B versus group A
β‐tubulin 1.−03 6.78E−05 1.71E−03 8.55E−05 1.71E−03 8.57E−05 n.s.

Note. BGLAP: bone γ‐carboxyglutamic acid‐containing protein; Bmp2: bone morphogenetic protein 2; DMP1: dentin matrix acidic phosphoprotein 1;
DSPP: dentin sialophosphoprotein; Klf4: Kruppel like factor 4; n.s: not significant; Oct4: octamer‐binding transcription factor 4; Runx2: runt‐related
transcription factor 2; Sox2: sex‐determining region Y (SRY)‐box 2.
IEZZI ET AL. | 7

F I G U R E 2 (a) Representative
phase‐contrast images of hDPSCs isolated
from the three different age group.
(b) Cytofluorimetric analysis of the
detection of MSC surface markers in
hDPSCs isolated from the three groups
(white plots indicate FITC and PE negative
controls). (c) Differentiation of hDPSCs
isolated from the differently aged groups
toward adipocytes (Oil red staining; scale
bars = 10 µm); osteoblasts (ARS and ALP
stainings; scale bars = 50 µm), and
chondrocytes (Alcian blue staining; scale
bars = 50 µm). (d) Histogram depicts
changes in mRNA expression of genes
involved in osteogenic differentiation in
the differently aged DPSCs before and
after (post) odontogenic differentiation.
Data are expressed as fold‐regulation
(2−ΔΔCt ; see Section 2), the dotted black line
indicates the mRNA expression of group A;
*p < 0.05 versus group A, #p < 0.05 versus
group B. ALP: alkaline phosphatase; ARS:
Alizarin red S; hDPSCs: dental pulp stem
cells; human dental pulp stem cells; mRNA:
messenger RNA; MSC: mesenchymal stem
cells; PDT: population doubling time;
qPCR: quantitative polymerase chain
reaction [Color figure can be viewed at
wileyonlinelibrary.com]

based on the donors’ age (Bernick & Nedelman, 1975). No significant senescence in cultured cells and in tissues from various animals
age‐related morphological differences were detected. On the (Dimri et al., 1995). P16ink4a is a protein involved in cell cycle
ink4a
contrary, we noticed a significant increase of SA‐β‐Gal and p16 progression (X. Feng et al., 2014), and along with SA‐β‐Gal activity, is
nuclear expression in group C compared with both groups A and B. an established biomarker of senescent cells (Dimri et al., 1995).
SA‐β‐Gal is a lysosomal enzyme highly correlated with cellular These changes were in accordance with the cytofluorimetric
8 | IEZZI ET AL.

FIGURE 3 Continued.
IEZZI ET AL. | 9

detection of an augmented internal complexity (i.e., SSC values) with cells from group C. This suggests that in vitro our cells possess a
aging. spontaneous odontogenic potential, even being unable to form
We additionally investigated if hDPSCs progressively lose their mineralized nodules as suggested by ARS. After hDPSC odontoblastic
self‐renewal potential during aging, undergoing a dysregulation of differentiation, 37 kDa fragment of DMP1 decreased in the
proliferative activities and declining functional capacity as described cytoplasm in support of ECM mineralization (Maciejewska
for other MSCs (Oh, Lee, & Wagers, 2014). The evaluation of the et al., 2009).
gene expression profile of Sox2, Oct4, Nanog, and Klf4 transcription The decrease of differentiation potential toward mineralized
factors experienced changes in all age groups. These molecules are tissues (i.e., bone and dentin) with age was also sustained by the
critical to preserve stem cell phenotype and, therefore, their changes the expression of Bmp2, β‐catenin, Runx2, and BGLAP
impairment could be responsible for the loss of stemness properties (osteocalcin) after odontogenic differentiation (Ferretti et al., 2014).
and pluripotency commitment of DPSCs (Gronthos et al., 2000; In hDPSCs, β‐catenin is upregulated during in vitro odontogenic
Huang et al., 2014). The different regulation of Klf4 detected during differentiation (Han et al., 2014) and its overexpression is linked to
aging could be at least in part due to its role in the odontoblastic the suppression of mineralization (Scheller, Chang, & Wang, 2008).
differentiation and inhibition of hDPSC proliferation (Lin et al., 2011). On the contrary, Runx2 expression must be downregulated in
A further confirmation of age‐related changes in our hDPSCs was odontoblast to reach full differentiation for a correct dentinogenesis
represented by the shortening of telomere length in both groups B (Chen et al., 2005). In this regard, the increase of mineralized nodules
and C in comparison with cells of group A (Mokry et al., 2010). in hDPSCs of group B treated with odontogenic medium may be
No changes in the expression pattern of the common ISCT connected to the diffusing calcific degeneration of pulp, a pathologic
markers (Dominici et al., 2006; Teti et al., 2015) were detected condition occurring as a response to aging (Piattelli & Trisi, 1993).
independently from the age of donors. On the contrary impaired A vast body of evidence indicates that neural crest‐derived
osteogenic and adipogenic potentials were found in hDPSCs of DPSCs express specific markers of neuronal cell (Arthur, Rychkov,
groups B and C in comparison with group A, noticing a marked Shi, Koblar, & Gronthos, 2008; Chai et al., 2000). Consequently, they
decline in group C, as suggested by previous work (Yi et al., 2017). might provide a promising source of stem cells for therapeutic
Concerning odontoblastic differentiation, qRT‐PCR showed a marked application in neurodegenerative diseases. The comparison of our
reduction of DMP1 and DSSP mRNAs in cells from aged subjects in biological and morphological analyses evidenced not only an age‐
comparison to young ones. The decrease in DMP1 and DSSP related decrease of markers of neurogenic differentiation (X. Feng
expression was less marked in cells cultured without odontogenic et al., 2013), but also an incorrect localization of β‐tubulin III
media in comparison to differentiated hDPSCs. DMP1 is an acidic no (Martens et al., 2012).
collagenous protein naturally present in bone and dentin extra- In conclusion our results confirmed that the differentiation
cellular matrix (ECM) as proteolytically processed fragments (Qin potential of hDPSCs could be differently impaired by aging. These
et al., 2003). DMP1 occurs predominantly as a C‐terminal (57 kDa) observations must be considered for the development of customized
and a N‐terminal (37 kDa) fragments, both promoters of HA strategies based on the possibility to encourage osteogenesis,
formation and growth. Further aggregates occur as DMP1 proteo- pulpogenesis, and dentinogenesis as well as for application in cell‐
glycan fragment (DMP1‐PG; Qin, D’Souza, & Feng, 2007), an inhibitor therapy to treat neurological disorders. DPSCs of young donors may
of mineralization (Gericke et al., 2010). DMP1 is implicated in a provide an ideal source of stem cells that could extend their
complex biological process: It is initially localized in the nucleus, therapeutic application in oral (i.e., alveolar bone, DP, and dentin
where it acts as transcriptional factor, during cell maturation DMP1 regeneration) and neurodegenerative (i.e., Alzheimer and Parkinson)
is in the cytoplasm and, further, Ca2+ is responsible for its export into diseases. Aged DPSCs could be used as in vitro tool for the study of
the ECM, where it regulates the nucleation of HA and the formation biomaterials solving the challenges of an aging population. Moreover,
of calcified tissue (Narayanan et al., 2003). The WB of cytoplasmic considering the immunomodulative properties of MSCs, further in
DMP1 expression, showed an increase in DMP1 full‐length form vivo investigation also assessing this phenomenon in DPSCs during
(105 kDa) and N‐terminal fragment in hDPSCs after three weeks of aging, will be hugely important, making them potential novel
culture without differentiating medium, with a marked amount in immunotherapeutic resources in inflammatory age‐related diseases.

F I G U R E 3 (a) Representative images displaying the different morphology and the formation of mineralized nodules in hDPSCs after 3‐weeks
culture in odontogenic media. (b) Histogram depicts changes in the expression of DMP1 and DSPP mRNAs in the differently aged hDPSCs
before and after (post) odontogenic differentiation and in DPSCs maintained for 3 weeks in ctrl. Data are expressed as fold‐regulation (2−ΔΔCt ;
see Section 2), the dotted black line indicates the mRNA expression of group A. (c) Western blot analysis of DMP1 protein expression;
histogram depicts changes in differently aged hDPSCs (post) odontogenic differentiation and in DPSCs maintained for 3 weeks in ctrl in
comparison to group A (dotted black line). (d) Histogram of Alizarin red quantization in differently aged hDPSCs (post) odontogenic
differentiation and in hDPSCs maintained for 3 weeks in ctrl. (e) Histogram of Alizarin red quantization in hDPSCs after osteogenic or
odontogenic differentiation. *p < 0.05 versus group A, #p < 0.05 versus group B. ctrl: control media; DMP1: dentin matrix acidic phosphoprotein
1; DPSCs: dental pulp stem cells; DSPP: dentin sialophosphoprotein; hDPSCs: human dental pulp stem cells; mRNA: messenger RNA [Color
figure can be viewed at wileyonlinelibrary.com]
10 | IEZZI ET AL.

F I G U R E 4 (a) Representative images


displaying the morphology of hDPSCs after
culturing in neurogenic media and the
immunofluorescence detection of nestin
and β‐tubulin III (scale bars = 50 µm). (b)
Histogram depicts changes in the
expression of nestin and β‐tubulin III
mRNAs in the differently aged hDPSCs
after neurogenic differentiation. Data are
expressed as relative expression (2−ΔCt ; see
Section 2). (c) Western blot analysis of
nestin and β‐tubulin III protein expression;
*p < 0.05 versus group A, #p < 0.05 versus
group B. hDPSCs: human dental pulp stem
cells; mRNA: messenger RNA [Color figure
can be viewed at wileyonlinelibrary.com]
IEZZI ET AL. | 11

A C K N O W L E D GM E N T S Ferretti, C., Vozzi, G., Falconi, M., Orciani, M., Gesi, M., Di Primio, R., &
Mattioli‐Belmonte, M. (2014). Role of IGF1 and IGF1/VEGF on human
The authors thank Dr. Alessandra Nori and Dr. Vittorio Zavaglia of mesenchymal stromal cells in bone healing: Two sources and two
the Odontostomatological and Special Surgery Unit, Ospedali Riuniti fates. Tissue Engineering, 20(17‐18), 2473–2482.
Gericke, A., Qin, C., Sun, Y., Redfern, R., Redfern, D., Fujimoto, Y., …
of Ancona, for their helpful patient selection and sample collection.
Boskey, A. L. (2010). Different forms of DMP1 play distinct roles in
The authors are grateful to Dr. Manuela Dicarlo for her valid mineralization. Journal of Dental Research, 89(4), 355–359.
collaboration in flow cytometry analysis. Gervois, P., Struys, T., Hilkens, P., Bronckaers, A., Ratajczak, J., Politis, C.,
… Martens, W. (2015). Neurogenic maturation of human dental pulp
stem cells following neurosphere generation induces morphological
CO NFLICTS OF INTE RES T and electrophysiological characteristics of functional neurons. Stem
Cells and Development, 24(3), 296–311.
The authors declare that there are no conflicts of interest. Gronthos, S., Brahim, J., Li, W., Fisher, L. W., Cherman, N., Boyde, A., … Shi,
S. (2002). Stem cell properties of human dental pulp stem cells. Journal
OR CID of Dental Research, 81(8), 531–535.
Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., & Shi, S. (2000).
Monica Mattioli Belmonte http://orcid.org/0000-0002-2087-2776 Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.
Proceedings of the National Academy of Sciences United States of America,
97(25), 13625–13630.
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