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Tissue and Cell xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Tissue and Cell


journal homepage: www.elsevier.com/locate/tice

Effects of decellularized matrices derived from periodontal ligament


stem cells and SHED on the adhesion, proliferation and osteogenic
differentiation of human dental pulp stem cells in vitro
Boon Chin Heng, Shaoyue Zhu, Jianguang Xu, Changyong Yuan, Ting Gong,
Chengfei Zhang
Comprehensive Dental Care, Endodontics, Faculty of Dentistry, The University of Hong Kong, Pokfulam, Hong Kong

a r t i c l e i n f o a b s t r a c t

Article history: A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited pro-
Received 26 November 2015 liferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal
Received in revised form in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This
21 December 2015
study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated
Accepted 21 December 2015
deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC
Available online xxx
culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded
DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of
Keywords:
Dental pulp
more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-
Extracellular matrix DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR
Focal adhesion data showed signicantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control.
Mitosis Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus
Osteogenesis TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase
assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-
DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing
conditions.
2015 Elsevier Ltd. All rights reserved.

1. Introduction Nevertheless, a major bottleneck to the therapeutic applications


of DPSC is the limited proliferative capacity of these cells and their
In recent years, numerous studies have demonstrated that den- tendency to undergo senescence after a limited number of passages
tal pulp stem cells (DPSC) have promising therapeutic applications within ex vivo culture (Wu et al., 2015). Only a relatively small quan-
not only in regenerative dentistry (Aurrekoetxea et al., 2015; Cao tity of autologous DPSC can be isolated from each individual patient,
et al., 2015), but can also be utilized for tissue engineering, repair and it is often necessary to carry out extensive ex vivo expansion
and regeneration of a variety of non-dental and non-oral tissues of these cells to attain sufcient numbers of cells required for suc-
including bone (Kwon et al., 2015), cartilage (Rizk and Rabie, cessful transplantation or tissue engineering. Additionally, ex vivo
2013), blood vessels (Li et al., 2014), cardiac muscle (Gandia et al., culture may also be necessary for induction of DPSC into desired
2008), brain (Yamagata et al., 2013) and spinal cord (Yamamoto lineages for therapeutic applications.
et al., 2014). Amongst the major advantages of DPSC are that The major technical challenge faced in the ex vivo culture of
these cells are readily available and easily isolated from biologi- DPSC is that the 2D in vitro culture milieu is often sub-optimal
cal waste routinely produced during dental treatment, and that the and differs considerably from the physiological 3D microenviron-
transplantation of autologous DPSC within the same patient can cir- ment of the stem cell niche in vivo, which may in turn compromise
cumvent the immunological barrier in cell-based therapy (Aimetti the proliferative and differentiation capacity of these adult stem
et al., 2014; Tatullo et al., 2015). cells. One potential strategy to overcome this deciency may be to
utilize an appropriate extracellular matrix (ECM) substratum for
the ex vivo culture of DPSC. Physiologically in vivo, cells are natu-
Corresponding author. rally surrounded by extracellular matrix within the stem cell niche,
E-mail address: zhangcf@hku.hk (C. Zhang). which is composed of a diverse array of high molecular weight

http://dx.doi.org/10.1016/j.tice.2015.12.004
0040-8166/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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2 B.C. Heng et al. / Tissue and Cell xxx (2015) xxxxxx

structural proteins (i.e. collagen, laminin and bronection), proteo- l-ascorbic acid for at least 1 week prior to DECM derivation. The
glycans and glycosaminoglycans, as well as growth factors (Walters conuent cell sheets were rinsed with PBS and treated with PBS
and Gentleman, 2015). Indeed, it is well-known that ECM-cellular containing 20 mM NH4 OH and 0.5% Triton X-100 for 5 min at 37 C.
interactions play key roles in orchestrating development and inu- DECM remaining in the wells were then rinsed again with PBS and
encing the lineage fate of adult stem cells, in addition to regulating subjected to DNAse treatment for 1 h at 37 C, prior to being rinsed
tissue homeostasis and repair (Walters and Gentleman, 2015). in deionized water and subjected to air-drying. The DECM thus
Nevertheless, most commercially-available extracellular matrix obtained from the conuent cell sheets within 6-well plates were
coatings for in vitro culture such as Matrigel are often derived stored at 4 C prior to being utilized in subsequent experiments.
from animal sources or cancer cell lines, i.e. Matrigel being derived
from Engelbeth-Holm swarm mouse sarcoma (Orkin et al., 1977). 2.4. Immunohistochemistry for detection of collagen and
This in turns poses a major technical barrier and regulatory hur- bronectin expression on the decellularized matrices
dle for clinical therapy, because the culture of human cells on
such commercially-available products would invariably lead to the SHED and PDLSC were seeded at a density of 20,000 cells/cm2 on
adhesion of either xenogenic or tumorigenic proteins on the cell glass cover slips placed within 6-well culture plates and cultured
membrane, which could in turn provoke an immunological reac- to conuence in the presence of 50 g/ml of l-ascorbic acid for at
tion upon transplantation within the patient, even if these cells least 1 week, prior to derivation of DECM, as previously described.
were autologous in origin. Hence, it is imperative to look for an The cover slips with DECM were initially blocked in PBS supple-
alternative source of human-derived ECM substrata for the ex vivo mented with 10% (v/v) FBS for 2 h. This was followed by washing
culture of DPSC. three times in PBS and incubation at room temperature for 2 h
One promising source of such material could be other dental and with primary antibodies specic for collagen (Cat. No. SC-8784-R,
oral stem cells that are also readily available and easily isolated Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or bronectin
from biological waste routinely produced by dental treatment. (Cat No. FBN11, ThermoFisher Scientic Inc., Waltham, MA, USA),
This study will therefore attempt to extract decellularized matrix which were diluted in 1% (w/v) BSA/PBS at appropriate dilution fac-
(DECM) from stem cells derived from human exfoliated deciduous tors, according to the manufacturers instructions. Subsequently,
teeth (SHED) and periodontal ligament stem cells (PDLSC), and uti- the cover slips with DECM were then washed in 1% (w/v) BSA/PBS
lize these as substrata for the ex vivo culture of DPSC under both and incubated in the dark for 2 h at room temperature with either
non-inducing and osteogenic-inducing conditions. The adhesion, goat anti-rabbit secondary antibody that was conjugated to TRITC
proliferation and osteogenic differentiation of ex vivo cultured DPSC (Cat No. ab6718, Abcam Inc., Cambridge, UK) or goat anti-mouse
on these DECM would be assessed and compared by various tech- secondary antibody that was conjugated to Alexa uor 488 (Cat
niques including immunocytochemistry, WST-8 assay, qRT-PCR, No. ab150117, Abcam Inc., Cambridge, UK). The secondary anti-
alizarin red S staining and alkaline phosphatase assay. bodies were diluted in 1% (w/v) BSA/PBS at appropriate dilution
factors, according to the manufacturers instructions. Following
2. Materials and methods another wash with 1% (w/v) BSA/PBS, the cover-slips with DECM
were mounted on glass slides with FluorSave mounting reagent
2.1. Cells, reagents, culture media, supplements and labware (Merck Millipore Inc., Darmstadt, Germany), prior to imaging under
an Olympus IX81 confocal uorescent microscope (Olympus Inc.,
Human DPSC (Cat No. DP003F) and SHED (Cat No. DP004F) were Tokyo, Japan) with the appropriate excitation/emission wavelength
obtained from AllCells LLC. (Alameda, CA, USA). Human PDLSC were for Alexa uor 488 and TRITC.
obtained as a gift from the School of Stomatology of Fujian Medical
School (Fujian, China) and were isolated as previously described 2.5. Quantication of total protein, collagen and
(Seo et al., 2004). Unless otherwise stated, all chemical reagents glycosaminoglycan contents of the decellularized matrices
were obtained from SigmaAldrich Inc. (St. Louis, MO, USA), all cul-
ture media and associated culture supplements were obtained from The total protein contents of PDLSC-DECM and SHED-DECM
Life Technologies Inc. (Carlsbad, CA, USA), while all labware were were determined by utilizing a bicinchoninic acid (BCA) protein
obtained from Becton-Dickinson Inc. (Franklin Lakes, NJ, USA). assay kit (Cat No. 23225, ThermoFisher Scientic Inc., Waltham,
MA, USA), following the manufacturers protocol. Briey, the DECM
2.2. Cell culture within each well of a 6-well plate was incubated with 0.5 ml of
assay reagent for 2 h at 37 C, under mild agitation. Absorbance
The culture milieu utilized for expansion culture of DPSC, SHED readings were then measured at 570 nm, and used to calcu-
and PDLSC was identical, and comprised of -minimum essen- late the corresponding protein concentrations from a standard
tial medium (-MEM) supplemented with 10% (v/v) fetal bovine curve.
serum (FBS) and 1% (v/v) penicillinstreptomycin antibiotic solu- The collagen contents of PDLSC-DECM and SHED-DECM were
tion. Fresh culture media was replenished every 34 days. For determined by colorimetric analysis utilizing a hydroxyproline
routine sub-culture, 0.5% (w/v) Trypsin-EDTA was used to disso- assay kit (Cat No. MAK008, SigmaAldrich Inc., St. Louis, MO, USA),
ciate conuent monolayers. A humidied 5% CO2 incubator set at following the manufacturers protocol. Briey, the DECM were
37 C was utilized for all cell cultures. All subsequent experimental scraped from the surface of the 6-well plates in deionized H2 O
protocols involving human DPSC, PDLSC and SHED were approved and hydrolyzed in 12 M HCl at 120 C for 3 h. Subsequently, 100 L
by the Institutional Review Board (IRB) of the University of Hong of supernatant was then mixed with 200 L of assay reagent in
Kong. duplicates, and incubated at 60 C for 24 h, followed by transfer
of 200 L of the sample reaction mixture into a 96-well plate
2.3. Derivation of decellularized matrices for absorbance readings at 560 nm. Subsequently, the hydroxy-
proline concentrations were determined from the corresponding
DECM were derived from conuent cell sheets of SHED and absorbance readings via a standard curve. The collagen contents
PDLSC (passage 510) according to previously published protocol of PDLSC-DECM and SHED-DECM were then computed based
(Decaris et al., 2012). Briey, SHED and PDLSC were cultured to on an assumption of a collagen-to-hydroxyproline ratio of 10:1
conuency within 6-well dishes, in the presence of 50 g/ml of (w/w).

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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The GAG contents of PDLSC-DECM and SHED-DECM were quan- 2.8. WST-8 assay of proliferation of dental pulp stem cells on the
tied by utilizing the Blyscan Sulfated Glycosaminoglycan Assay Kit decellularized matrices
(Cat No. B1500, Biocolor Ltd., Carrickfergus, County Antrim, UK), fol-
lowing the manufacturers protocol. Briey, the DECM within each Sub-senescent DPSC (passage 1015, >50 days of continu-
well of a 6-well plate was digested with 0.5 ml of papain extraction ous in vitro culture) were seeded at a density of 2000 cells/cm2
reagent (0.1 mg/mL papain) at 65 C for 3 h. Subsequently, 100 L on 6-well plates containing either PDLSC-DECM, SHED-DECM or
of supernatant was then mixed with 1 ml of the assay reagent bare TCPS (control), and subsequently cultured for a period of 9
to form a colored precipitate that was collected by centrifuga- days at 37 C within a humidied 5% CO2 incubator. The culture
tion at 10,000 g for 10 min, followed by resuspension in 0.5 ml medium was composed of -MEM with 10% (v/v) FBS and 1% (v/v)
of dissolution buffer, to enable absorbance readings at 660 nm. The penicillinstreptomycin antibiotic solution. After 1, 3, 5, 7, and 9
GAG concentrations were then determined from the corresponding days of culture, the WST-8 assay (CCK-8 kit, Dojindo Molecular Lab-
absorbance readings via a standard curve. oratories Inc., Kumamoto, Japan) was used to quantify the density
of viable cells within each well. The cells were incubated for 2 h
at 37 C with culture media supplemented with 10% (v/v) WST-
2.6. Adhesion of dental pulp stem cells on the decellularized 8 reagent (500 L/well of 6-well plate), followed by absorbance
matrices readings at 450 nm with a SpectraMAX 340 microplate reader
(Molecular Devices, Sunnyvale, CA, USA). A standard curve was used
Sub-senescent DPSC (passage 1015, >50 days of continu- to determine densities of adherent viable cells. The proliferation
ous in vitro culture) were seeded at a density of 80,000 cells indices at 1, 3, 5, 7, and 9 days of culture were calculated as a ratio
per well (9.62 cm2 ) of a 6-well plate containing either PDLSC- of cell density on that particular day to that of the TCPS control on
DECM, SHED-DECM or bare TCPS (control). The culture media day 1. After 9 days of culture, samples were collected for qRT-PCR
utilized was -MEM supplemented with 10% (v/v) FBS and 1% (v/v) analysis of stemness marker expression.
penicillinstreptomycin antibiotic solution. The cells were allowed
to adhere for either 2 or 24 h within a humidied 5% CO2 incu- 2.9. Osteogenic induction of dental pulp stem cells
bator set at 37 C. The non-adherent cells were then rinsed away
with PBS, and the density of adherent cells was quantied with To induce osteogenic differentiation of sub-senescent DPSC,
the WST-8 assay (CCK-8 kit, Dojindo Molecular Laboratories Inc., the cells (passage 1015, >50 days of continuous in vitro culture)
Kumamoto, Japan), according to the manufacturers instructions. were seeded at a density of 300,000 cells per well of a 6-well
Briey, this involved incubation of adherent cells with 10% (v/v) plate containing either PDLSC-DECM, SHED-DECM or bare TCPS
WST-8 reagent supplemented in culture media (500 L per well (control) in non-inducing growth medium. The following day, a
of 6-well plate) for 2 h at 37 C, followed by absorbance readings 7080% conuent monolayer of DPSC was obtained and the culture
at 450 nm with a microplate reader. The density of adherent DPSC milieu was changed to osteogenic induction medium comprising
was determined from standard curves based on known cell densi- -MEM supplemented with 10% (v/v) FBS, 108 M dexametha-
ties. The percentage of adherent cells was then calculated according sone, 10 mM -glycerophosphate, 50 g/ml ascorbic acid and 1%
to the formula Nadherent /Nseeding 100%, where Nadherent is the den- (v/v) penicillinstreptomycin antibiotic solution. The duration of
sity of adherent cells after 2 or 24 h, and Nseeding is the initial cell osteogenic induction culture was up to 21 days. Fresh osteogenic
seeding density i.e. 80,000 cells per well of a 6-well plate. induction medium was replenished every 34 days. After 7, 14 and
21 days of osteogenic induction culture, samples were collected for
either qRT-PCR analysis of osteogenic marker expression, assay of
2.7. Immunocytochemistry for detection of vinculin expression by alkaline phosphatase activity or alizarin red S staining for detection
newly-adherent dental pulp stem cells on the decellularized of mineralized calcium deposition.
matrices
2.10. Assay of alkaline phosphatase activity
Sub-senescent DPSC (passage 1015, >50 days of continuous
in vitro culture) were seeded at a density of 80,000 cells per well Alkaline phosphatase (ALP) activity in whole cell lysates was
(9.62 cm2 ) of a 6-well plate containing either PDLSC-DECM, SHED- quantied by the SensoLyte pNPP Alkaline Phosphatase Assay Kit
DECM or bare TCPS (control), and the cells were allowed to adhere (Cat No. AS-72146, AnaSpec Inc., Fremont, CA, USA), following the
for 4 h within a humidied 5% CO2 incubator set at 37 C. The manufacturers protocol. Briey, on day 14 of osteogenic induc-
cells were xed in 4% (w/v) paraformaldehyde overnight at 4 C, tion, the cells cultured on either PDLSC-DECM, SHED-DECM or bare
and blocked in PBS supplemented with 10% (v/v) FBS for 2 h. The TCPS (control) were lysed in 0.2% (v/v) Triton X-100 in deionized
xed cells were then incubated for 2 h at room temperature with a water. Subsequently, 50 l cell lysate and 50 l pNPP substrate
rabbit monoclonal primary antibody specic for vinculin (Cat. No. solution were added into each well of a 96-well plate and incu-
42H89L44, ThermoFisher Scientic Inc., Waltham, MA, USA) that bated for 30 min at room temperature. Absorbance readings were
was diluted in 1% (w/v) BSA/PBS (1:200 dilution). The cells were taken at 405 nm, and the concentration of alkaline phosphatase
then washed in 1% (w/v) BSA/PBS and incubated in the dark with a in each sample was calculated according to a standard curve.
goat anti-rabbit secondary antibody that was conjugated to Alexa The alkaline phosphatase activity was normalized to total cellu-
Fluor 488 (Cat No. ab150077, Abcam Inc., Cambridge, UK) at room lar protein concentration determined by the Bicinchoninic acid
temperature for 2 h. The secondary antibody was diluted in 1% (w/v) (BCA) assay (Cat No. 23225, ThermoFisher Scientic Inc., Waltham,
BSA/PBS at a dilution factor of 1:200. Following another wash with MA, USA).
1% (w/v) BSA/PBS, the cell nuclei were stained with DAPI, prior to
imaging under a Nikon Eclipse TiTM uorescent microscope (Nikon 2.11. Alizarin red S staining to assess mineralized calcium
Inc., Tokyo, Japan) with the appropriate excitation/emission wave- deposition
length for DAPI and Alexa Fluor 488 . The uorescent images of
each sample obtained from these two urophores were superim- After 21 days of osteogenic induction, calcium mineralization
posed utilizing the ImageJ software (National Institute of Health, was assessed by alizarin red S staining. Briey, after xation with
Bethseda, MD, USA). 10% (w/v) formaldehyde for 15 min at room temperature, the xed

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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Table 1
Primer sequences utilized for qRT-PCR analysis.

Gene Primer sequences

Nestin F 5 -GAAACAGCCATAGAGGGCAAA-3
R 5 -TGGTTTTCCAGAGTCTTCAGTGA-3
OCT4 (Octamer transcription factor F 5 -CAAAGCAGAAACCCTCGTGC-3
4) R 5 -TCTCACTCGGTTCTCGATACTG-3
Nanog F 5 -TGATTTGTGGGCCTGAAGAAAA-3
R 5 -GAGGCATCTCAGCAGAAGACA-3
Stemness markers
CD90 F 5 -CGCTCTCCTGCTAACAGTCTT-3
R 5 -CAGGCTGAACTCGTACTGGA-3
CD73 F 5 -ATTGCAAAGTGGTTCAAAGTCA-3
R 5 -ACACTTGGCCAGTAAAATAGGG-3
CD44 F 5 -AGAAGGTGTGGGCAGAAGAA-3
R 5 -AAATGCACCATTTCCTGAGA-3

OSX (Osterix) F 5 -TGCTTGAGGAGGAAGTTCAC-3


R 5 -AGGTCACTGCCCACAGAGTA-3
RUNX2 (Runt related transcription F 5 -TACGACTGGACGCTGGTGC-3
factor 2) R 5 -TTCATGGGTCGCTTGACGT-3
BMP2 (Bone morphogenetic F 5 -TTCCACCATGAAGAATCTTTGGA-3
protein 2) R 5 -CCTGAAGCTCTGCTGAGGTGAT-3
COL1 (Collagen Type I) F 5 -GAGGGCCAAGACGAAGACATC-3
Osteogenic markers
R 5 -CAGATCACGTCATCGCACAAC-3
BSP (Bone Sialoprotein) F 5 -GCGAAGCAGAAGTGGATGAAA-3
R 5 -TGCCTCTGTGCTGTTGGTACTG-3
OCN (Osteocalcin) F 5 -AGCAAAGGTGCAGCCTTTGT-3
R 5 -GCGCCTGGGTCTCTTCACT-3
OMD (Osteomodulin) F 5 -TCCAAGAAATTTGGAACACC-3
R 5 -TGACCATTAGTGCTTCGTTG-3

DMP-1 (Dentin Matrix Protein 1) F 5 -CTCCGAGTTGGACGATGAGG-3


R 5 -TCATGCCTGCACTGTTCATTC-3
Odontogenic markers
DSPP (Dentin F 5 -GCCATTCCAGTTCCTCAACTT-3
Sialophosphoprotein) R 5 -CATGCACCAGGACACCACTT-3

GAPDH (Glyceraldehyde F 5 -GGCATGGACTGTGGTCATGAG-3


Housekeeping gene
3-phosphate dehydrogenase) R 5 -TGCACCACCAACTGCTTAGC-3

Fig. 1. Immunocytochemistry for detection of (A) collagen type I, and (B) bronectin on PDLSC-DECM; and for detection of (C) collagen type I, and (D) bronectin on
SHED-DECM.

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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cells were washed in distilled water, and stained with 1% (w/v)


alizarin red S (pH 4.14.2) for 20 min at room temperature. The
images of the stained mineralized nodules were then captured
with a scanner, after rinsing with deionized water. To quantify the
intensity of alizarin red S staining, the stained cells were incubated
overnight with 10% (w/v) sodium dodecyl sulfate (SDS) at 37 C.
The lysates were then collected for absorbance readings at 405 nm.
To normalize with respect to cell density, the DNA content of the
supernatant was determined by the Quant-iT PICO Green assay
(Cat No. P7589, ThermoFisher Inc., Waltham, MA, USA).

2.12. Real-time quantitative polymerase chain reaction


(qRT-PCR)

Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen,
Valencia, CA, USA). 1.0 g of total RNA was reverse-transcribed
for each sample utilizing the SuperScript VILO Master Mix (Life
Technologies, Grand Island, NY, USA). Quantitative real-time PCR
(qRT-PCR) was performed using the SYBR Select Master Mix
(Applied Biosystems, Grand Island, NY, USA) with the StepOne Real-
Time PCR System (Applied Biosystems, Grand Island, NY, USA).
The primer sequences utilized for qRT-PCR analysis are shown
in Table 1. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)
was utilized as the internal housekeeping gene. The amplica-
tion parameters utilized for qRT-PCR were as follows: 2 min at
50 C, 20 s at 95 C, and 40 cycles of 3 s at 95 C, followed by 30 s
at 60 C. The relative cycle threshold (Ct) was determined with
the 2Ct method and normalized against endogenous GAPDH
gene expression. The fold change in the expression of each gene
in DPSC cultured on either PDLSC-DECM or SHED-DECM was com-
pared against DPSC cultured on the TCPS control. There were three
experimental replicates for the qRT-PCR analyses.

2.13. Statistical analysis

All experimental samples were collected in triplicates and all


data were expressed as the mean standard deviation. Differences
between experimental data sets were assessed by the Students t-
test using the SPSS 19.0 Statistics Software (SPSS Inc, Chicago, IL,
USA). Values of P < 0.05 were considered statistically signicant.

3. Results

3.1. Immunocytochemistry for detection of collagen and Fig. 2. Comparison of (A) total protein, (B) collagen, and (C) glycosaminoglycan
bronectin within decellularized matrices (GAG) contents of DECM derived from SHED and PDLSC. *: P < 0.05.

The immunocytochemistry results for detection of collagen type seen in Fig. 2C, PDLSC-DECM also exhibited a signicantly higher
I and bronectin within PDLSC-DECM and SHED-DECM are shown GAG content compared to SHED-DECM (0.37 0.08 g/cm2 versus
in Fig. 1. There appear to be morphological differences in the orga- 0.25 0.06 g/cm2 respectively, P < 0.05).
nization and distribution of collagen type I and bronectin in the
two different decellularized matrices, with PDLSC-DECM appearing 3.3. Adhesion of dental pulp stem cells on decellularized matrices
more sponge-like (Fig. 2A and B), and SHED-DECM exhibiting a
more brous appearance (Fig. 2C and D). The WST-8 assay was utilized to assess and compare the adhe-
sion of DPSC on PDLSC-DECM, SHED-DECM and TCPS (Fig. 3) at 2 h
3.2. Quantication of the total protein, collagen and and 24 h after cell seeding. At the 2 h timepoint, it was observed that
glycosaminoglycan contents of decellularized matrices there was a signicantly higher percentages of adherent DPSC on
PDLSC-DECM and SHED-DECM versus the TCPS control (72.9 1.4%
The total protein, collagen and GAG contents of PDLSC- and 59.1 2.8% versus 6.6 1.5% respectively, P < 0.05). By contrast
DECM versus SHED-DECM were assessed and compared (Fig. 2). at the 24 h timepoint, the differences in the percentages of adher-
The BCA protein assay (Fig. 2A) showed that SHED-DECM ent DECM on PDLSC-DECM and SHED-DECM versus the TCPS control
had a signicantly higher total protein content compared were not signicantly different (83.9 5.8% and 79.8 1.8% versus
to PDLSC-DECM (8.80 0.61 g/cm2 versus 7.17 0.22 g/cm2 69.4 9.3% respectively, P > 0.05).
respectively, P < 0.05). By contrast, the hydroxyproline assay Immunocytochemistry for detection of vinculin, a key focal
(Fig. 2B) showed that PDLSC-DECM had a signicantly higher adhesion protein (Fig. 4A, C and E), demonstrated that DPSC seeded
collagen content compared to SHED-DECM (2.68 0.10 g/cm2 on PDLSC-DECM and SHED-DECM displayed much higher expres-
versus 1.38 0.18 g/cm2 respectively, P < 0.05). Additionally, as sion of vinculin and hence focal adhesions, as compared to TCPS,

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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Fig. 3. Percentages of adherent DPSC on TCPS, SHED-DECM and PDLSC-DECM at 2 h


and 24 h after seeding. *: P < 0.05 w.r.t. TCPS control, : P < 0.05 between SHED-DECM Fig. 5. Proliferation of DPSC on DECM derived from SHED and PDLSC, as compared
and PDLSC-DECM. to TCPS. *: P < 0.05 w.r.t. TCPS control, : P < 0.05 between PDLSC-DECM and SHED-
DECM.

Fig. 4. Immunocytochemistry for detection of vinculin expression by DPSC seeded on (A) PDLSC-DECM, (C) SHED-DECM, and (E) TCPS, at 4 h after seeding. The green
uorescence represents vinculin, while the blue uorescence represent nuclear DNA staining with DAPI. B, D and F are the corresponding phase-contrast light microscopy
images of A, C and E respectively. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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B.C. Heng et al. / Tissue and Cell xxx (2015) xxxxxx 7

Fig. 6. qRT-PCR analysis of stemness markers: (A) Nestin, (B) OCT4, (C) Nanog, (D) CD90, (E) CD73, and (F) CD44 expression by DPSC cultured on TCPS, SHED-DECM and
PDLSC-DECM. *: P < 0.05 w.r.t. TCPS control.

at the 4 h timepoint. The phase contrast light microscopy images and qRT-PCR analysis of the expression of key osteogenic markers
(Fig. 4B, D and F) revealed that DPSC seeded on PDLSC-DECM and (Fig. 8).
SHED-DECM displayed more extensive spreading as compared to The alizarin red S staining results after 21 days of osteogenic
DPSC seeded on TCPS. induction showed that DPSC cultured on PDLSC-DECM and SHED-
DECM exhibited higher levels of mineralized calcium deposition
3.4. Proliferation of dental pulp stem cells on decellularized compared to the TCPS control, as manifested by more intense
matrices staining (Fig. 7A). As seen in Fig. 7B, spectrophotometric quan-
tication of the staining intensity at an absorbance wavelength of
The proliferation of DPSC seeded on PDLSC-DECM, SHED-DECM
405 nm revealed that the alizarin red S staining intensity (normal-
and TCPS was assessed by the WST-8 assay (Fig. 5). It was observed
ized with respect to cell number) for DPSC cultured on PDLSC-DECM
that the proliferation of DPSC on both types of DECM yielded con-
and SHED-DECM was signicantly higher than the TCPS control
sistently higher cell densities compared to the TCPS control, over
(5.51 0.47 and 2.00 0.19 folds respectively versus the TCPS con-
the entire 9 days duration of culture. At 5, 7 and 9 days of cul-
trol, P < 0.05). Additionally, it was also observed that the alizarin
ture, there were signicantly higher cell densities of DPSC grown
red S staining intensity was signicantly higher for DPSC cultured
on SHED-DECM versus PDLSC-DECM.
on PDLSC-DECM versus SHED-DECM (P < 0.05).
Subsequently after 9 days of culture, DPSC grown on the dif-
At 7, 14 and 21 days of culture, DPSC cultured on PDLSC-DECM
ferent substrata were collected for qRT-PCR analysis of stemness
consistently displayed signicantly higher alkaline phosphatase
gene marker expression (Fig. 6). The results showed that only nestin
activity compared to the TCPS control (P < 0.05). By contrast, DPSC
was differentially expressed, with DPSC grown on PDLSC-DECM
cultured on SHED-DECM consistently displayed signicantly lower
and SHED-DECM displaying higher expression of nestin compared
alkaline phosphatase activity compared to the TCPS control at all
to the TCPS control (2.36 0.58 and 2.27 0.38 folds respectively
three timepoints (P < 0.05).
versus the TCPS control, P < 0.05). By contrast, there were no sig-
The qRT-PCR analysis data revealed that DPSC cultured
nicant differences in the expression levels of the other analyzed
on PDLSC-DECM exhibited more differential expression of key
stemness gene markers (OCT4, Nanog, CD90, CD73 and CD44) by
osteogenic marker expression versus the TCPS control, as com-
DPSC cultured on the different substrata (P > 0.05).
pared to DPSC cultured on SHED-DECM (Fig. 8). At 7 days of
3.5. Osteogenic induction of dental pulp stem cells on osteogenic induction, DPSC cultured on PDLSC-DECM displayed
decellularized matrices signicantly higher expression of OSX and BMP2 (P < 0.05), but sig-
nicantly lower expression of COL1 (P < 0.05) compared to the TCPS
The osteogenic induction of DPSC cultured on PDLSC-DECM, control. By contrast, DPSC cultured on SHED-DECM displayed sig-
SHED-DECM and TCPS was assessed and compared by alizarin red nicantly lower expression of only COL1 at 7 days of osteogenic
S staining (Fig. 7A and B), alkaline phosphatase assay (Fig. 7C), induction. At 14 days of osteogenic induction, DPSC cultured on

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
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signicantly higher expression of only BSP (P < 0.05). There were no


detectable expression levels (after 60 amplication cycles) of DSPP
in DPSCs cultured on either PDLSC-DECM, SHED-DECM or TCPS;
after 7, 14 and 21 days of osteogenic induction (data not shown).

4. Discussion

The natural physiological microenvironment of the adult stem


cell niche is poorly recapitulated by conventional in vitro culture
protocols utilizing bare polystyrene culture surfaces, resulting in
a less than conducive environment for supporting the growth and
maintenance of the stemness phenotype of DPSC ex vivo. Optimi-
zing the in vitro culture milieu for DPSC may extend the limited
proliferative capacity of these cells ex vivo, as well as mitigate their
tendency to undergo senescence within in vitro culture. This could
in turn facilitate the therapeutic applications of DPSC in tissue engi-
neering and regenerative dentistry/medicine.
Indeed, a number of previous studies have investigated the use
of ECM-based substrata and scaffolds as a strategy for optimizing
the in vitro culture milieu for either the ex vivo proliferation or dif-
ferentiation of DPSC (Smith et al., 2015; Park et al., 2013; Wang
et al., 2015; Zhang et al., 2012; Ravindran et al., 2014). The study of
Smith et al. (2015) extracted ECM from bovine incisor pulp, and uti-
lized this as a substratum for the primary culture of rat dental pulp
cells, and observed that the stem cell phenotype was better main-
tained through culture on the pulp ECM. Subsequently, the same
study demonstrated enhanced osteogenic differentiation of the rat
dental pulp cells cultured on the bovine pulp ECM (Smith et al.,
2015). Enhanced odontogenic differentiation of DPSC on a porcine
fetal enamel matrix derivative was reported by the study of Park
et al. (2013). In the study of Wang et al. (2015), DPSC was cultured
in the presence of decellularized porcine urinary bladder matrix,
and enhancement in proliferation and differentiation potential, as
well as inhibition of apoptosis was observed; but there was no
effect of the ECM substrata on cell morphology and stemness
marker expression. Zhang et al. (2012) showed that DPSC cultured
on demineralized dentin matrix exhibited enhanced odontogenic
differentiation, as evidenced by upregulated dentin sialophos-
phoprotein (DSPP) expression and increased alkaline phosphatase
activity. Similar enhancement in the odontogenic differentiation of
DPSC was reported by the study of Ravindran et al. (2014), which
utilized DECM derived from DPSC itself. Nevertheless, it must be
noted that most of these aforementioned studies utilized extra-
cellular matrix components derived from animal sources, which
in turn poses a major immunological and safety barrier to clinical
therapeutic applications.
A promising source of human-derived ECM for ex vivo culture
of DPSC are other dental and oral stem cells that are also readily
isolated from biological waste routinely produced by dental treat-
ment. DECM derived from DPSC itself will not be investigated in
this study, as it has already been examined in a previous study
Fig. 7. Osteogenic induction of DPSC cultured on TCPS, SHED-DECM and PDLSC- (Ravindran et al., 2014). Moreover, this study sought to investi-
DECM. (A) Alizarin red S staining after 21 days of osteogenic induction, (B) gate whether DECM derived from other histological sources on the
corresponding quantication of alizarin red S staining intensity at an absorbance same extracted tooth from which DPSC is isolated, can be utilized
wavelength of 405 nm, and (C) alkaline phosphatase assay at 7, 14 and 21 days of
osteogenic induction. *: P < 0.05 w.r.t. TCPS control, : P < 0.05 between PDLSC-DECM
to enhance the ex vivo culture of DPSC. Hence, PDLSC will be exam-
versus SHED-DECM. ined in this study as a potential source of ECM substrata, because
these are isolated from a different histological source (i.e. mid-
third portion of the root surface) on extracted tooth (Zhu and Liang,
PDLSC-DECM displayed signicantly higher expression of RUNX2, 2015), as opposed to DPSC, which are harvested from the tooth pulp
BMP2, COL1, BSP and DMP1 (P < 0.05) compared to the TCPS con- chamber (Potdar and Jethmalani, 2015). Additionally, this study
trol; whereas DPSC cultured on SHED-DECM displayed signicantly will also investigate SHED as a potential source of ECM substrata,
lower expression of only COL1 (P < 0.05). At 21 days of osteogenic because SHED and DPSC originate from the same histological source
induction, DPSC cultured on PDLSC-DECM displayed signicantly (pulp chamber), the only difference being that DPSC are derived
higher expression of BMP2, BSP and OMD (P < 0.05) compared to from permanent tooth, whereas SHED are derived from primary
the TCPS control; whereas DPSC cultured on SHED-DECM displayed tooth (Kashyap, 2015). Previous studies have demonstrated that

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
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Fig. 8. qRT-PCR analysis of key osteogenic markers: (A) OSX, (B) RUNX2, (C) BMP2, (D) COL1, (E) BSP, and (F) DMP1, (G) OCN, and (H) OMD expression by DPSC cultured on
TCPS, SHED-DECM and PDLSC-DECM after 7, 14 and 21 days of osteogenic induction. *: P < 0.05 w.r.t. TCPS control.

Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
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SHED, being at an earlier developmental stage, possess a much et al., 2013). It is known to be a key marker of adult stem cell renewal
higher proliferative potential than DPSC (Abdullah et al., 2014; (Park et al., 2010). Hence, enhanced nestin expression by DPSC cul-
Nakamura et al., 2009). Hence, there is a possibility that ECM tured on SHED-DECM and PDLSC-DECM could imply that DECM
derived from SHED may contain specic factors that could enhance enhance the self-renewal capacity of these cells ex vivo.
the proliferative potential of DPSC within ex vivo culture. After osteogenic induction culture for 21 days, it was observed
Immunocytochemistry for detection of collage type I and that osteogenic differentiation of DPSC was markedly enhanced
bronectin revealed differences in the morphology and topography by PDLSC-DECM, whereas there was only a slight or marginal
of PDLSC-DECM and SHED-DECM, which may in turn be respon- enhancement of DPSC osteogenesis with SHED-DECM. This result
sible for the differential results observed with these two different is consistent with previous studies that demonstrated enhanced
types of decellularized matrices. Numerous studies have previously osteogenesis with DECM derived from a variety of different
demonstrated that matrix topography exert a profound inuence sources (Rao Pattabhi et al., 2014; Hoch et al., 2015; Kim
on cell adhesion (Karuri et al., 2004), proliferation (Liliensiek et al., et al., 2015). Furthermore, co-culture of PDLSC with other cell
2006; Dalby et al., 2002) and osteogenic differentiation (Song et al., types have demonstrated that PDLSC could secrete factors that
2015; Faghihi and Baghaban Eslaminejad, 2014). promote osteogenic induction. Tansriratanawong et al. (2014)
The presence of both PDLSC-DECM and SHED-DECM enhanced reported that expression of osteogenic genes was upregulated
short-term adhesion of DPSC (4 h after seeding), as compared to the in de-differentiated fat cells upon co-culture with PDLSC. Simi-
bare TCPS surface. Immunocytochemistry for detection of vinculin, larly, studies by both Kook et al. (2014) and Inanc et al. (2007)
a key focal adhesion protein, showed that newly-adherent DPSC reported enhanced osteogenic differentiation of embryonic stem
expressed more focal adhesions on PDLSC-DECM and SHED-DECM, cells through co-culture with PDLSC. Hence, it would therefore
as compared to the TCPS control. These results are consistent with be expected that DECM derived from PDLSC would have similar
previous studies that reported enhanced adhesion of mesenchy- enhancing effects on osteogenesis, which was conrmed by the
mal stem cells on DECM derived from various different sources results of this study. Because there were no detectable expres-
(Iop et al., 2009; Xin et al., 2015; Matuska and McFetridge, 2015). sion levels of the late odontogenic differentiation marker DSPP
Characterization of the total protein, collagen and GAG content in all experimental groups, this would imply that in vitro culture
of PDLSC-DECM and SHED-DECM showed that although PDLSC- with DECM could not overcome the impaired odontogenic capac-
DECM had a lower total protein content than SHED-DECM, the ity (Mehrazarin et al., 2011) of the sub-senescent DPSC (passage
collagen and GAG contents were higher. Both collagen and GAG are 1015, >50 days of continuous in vitro culture) utilized in this study.
known to play key roles in the process of cell adhesion (Schubert Nevertheless, it is unknown whether the odontogenic differentia-
and LaCorbiere, 1980; Heino, 2007; Bruns and Gross, 1980; Heino tion capacity of DPSC could be preserved if the cells were cultured
et al., 2009), which could in turn explain why the percentage of on DECM from early passage onwards. This would be the subject of
rapidly adhering DPSC on PDLSC-DECM was signicantly higher our future investigations.
than SHED-DECM at 2 h after seeding.
Assessment of DPSC proliferation on PDLSC-DECM and SHED- 5. Conclusion
DECM with the WST-8 assay demonstrated consistently higher cell
numbers than the TCPS control, over the entire culture duration In conclusion, this study demonstrated that DECM derived from
of 9 days. This is consistent with previous studies which reported other dental and oral stem cells such as PDLSC and SHED, can exert
that DECM from other sources enhanced cellular proliferation (Ng a profound effect on the adhesion, proliferation and osteogenic
et al., 2014; Rao Pattabhi et al., 2014; Antebi et al., 2015). The study differentiation capacity of DPSCs. SHED-DECM was demonstrated
of Ng et al. (2014) reported enhanced ex vivo expansion of adult to be more suitable for ex vivo expansion of DPSC, whereas
mesenchymal stem cells on DECM derived from fetal mesenchymal PDLSC-DECM was demonstrated to be more suitable for osteogenic
stem cells. Similarly, both the study of Rao Pattabhi et al. (2014) and induction of DPSC. These human-derived DECM could serve as a
Antebi et al. (2015) reported that DECM derived from mesenchy- viable alternative to animal-derived ECM substrata for the ex vivo
mal stem cells could promote the proliferation of these cells, in culture of DPSC, thereby facilitating the clinical therapeutic appli-
addition to maintaining their stemness and differentiation capac- cations of these cells in regenerative medicine and dentistry.
ity.
Because SHED is in fact an earlier developmental stage of DPSC
Conict of interest
with much higher proliferative capacity (Abdullah et al., 2014;
Nakamura et al., 2009); there is possibility that DECM derived from
The authors hereby declare no relevant conicts of interests in
these cells could be more effective in enhancing the proliferation of
the publication of this article.
DPSC compared to PDLSC-DECM. Indeed, the results demonstrate
this to be case, with SHED-DECM yielding signicantly higher cell
numbers than PDLSC-DECM after 5, 7 and 9 days of culture. Alter- Acknowledgement
natively, the higher proliferation of cell numbers on SHED-DECM
may also be due to its higher total protein content versus PDLSC- This work was supported by the General Research Fund (GRF)
DECM. The qRT-PCR analysis of stemness marker expression after grants from the Research Grants Council of Hong Kong (grant no.
nine days of culture, demonstrated signicant upregulation of only HKU17126914) to C. Zhang.
nestin expression by DPSC cultured on SHED-DECM and PDLSC-
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Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004
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Please cite this article in press as: Heng, B.C., et al., Effects of decellularized matrices derived from periodontal ligament stem cells
and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell (2015),
http://dx.doi.org/10.1016/j.tice.2015.12.004

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