odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate

Archives of Oral Biology 60 (2015) 1545–1553

Contents lists available at ScienceDirect

Archives of Oral Biology journal homepage: www.elsevier.com/locate/aob

Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate Duaa Abuarqouba,* , Abdalla Awidia , Nizar Abuharfeilb,1 a b

Cell therapy center, Jordan University, Amman, Jordan Department of applied Biology, Jordan University of Science and Technology, Irbid, Jordan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 November 2013 Received in revised form 23 June 2015 Accepted 9 July 2015

Introduction: Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. Methodology: DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). Results: The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Conclusion: Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. ã 2015 Elsevier Ltd. All rights reserved.

Keywords: Dental stem cells DPSC SCAP Platelet lysate

1. Introduction Since year 2000, after the discovery of adult stem cells from the dental pulp (DPSCs) (Gronthos, Mankani, Brahim, Robey, & Shi, 2000), several types of dental stem cells have been isolated successively from mature and immature teeth including; stem cells derived from the apical papilla (SCAP) (Sonoyama et al., 2008), stem cells derived from exfoliated deciduous teeth (SHED) (Miura et al., 2003), stem cells from human periodontal ligament (PDL) (Seo et al., 2004), and mesenchymal stem cells from tooth germs (Morsczeck et al., 2009; dAquino et al., 2011). It is considered that these stem cells are undifferentiated

* Corresponding author at: Dua’a Abuarqoub, Cell Therapy Center, University of Jordan, Amman-Jordan. Tel.: +962 799846771; fax: +962 65355000x23961. E-mail address: [email protected] (D. Abuarqoub). 1 Department of biotechnology, Jordan University of Science and Technology, Irbid, Jordan. http://dx.doi.org/10.1016/j.archoralbio.2015.07.007 0003-9969/ ã 2015 Elsevier Ltd. All rights reserved.

mesenchymal cells present in dental tissues and characterized by their; colony forming capacity, unlimited self-renewal and their multipotent differentiation potential (Gronthos et al., 2000) to give rise to distinct cell lineages such as osteoblasts, adipocytes, odontoblast, smooth muscle cells and neurons (Gronthos et al., 2002; Batouli et al., 2003; Iohara et al., 2004; Arthur, Rychkov, Shi, Koblar, & Gronthos, 2008Kadar et al., 2009; Loomba et al., 2012). Ref : (Nakashima, 2005). Some studies highlighted the ability of DPSCs to express chondrogenic markers (Balic, Aguila, Caimano, Francone, & Mina, 2010Karaoz et al., 2010) and some proteins involved in melanogenesis at some stages of in vitro differentiation (Paino et al., 2010). In addition, DPSCs are able to form capillary-like structures when cultured with VEGF (Marchionni et al., 2009). Moreover, DPSCs could change into cardiomyocytes which were used for the repair of myocardial infarction (Gandia et al., 2008). Dental stem cells have been used for tissue- engineering studies to evaluate their potential in clinical applications. Sharpe and

1546

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

different freezing–thawing cycles, to lyse platelets bodies and to collect the released growth factors. The platelet bodies were removed by centrifugation to minimize the effect of aggregation on cell culture. Isolated PL was pooled to minimize donor specific effects.

Young (2005) were pioneers in the use of stem cells in dental tissue engineering. DPSCs were loaded onto scaffolds of collagen sponges, hydroxyapatite (HA), chitosan, biocoral, or PLGA and implanted into animal model to study their ability for bone regeneration (Zhang et al., 2006; Graziano et al., 2008; Yang et al., 2009; Yang et al., 2012). All the mentioned groups reported that DPSCs loaded on scaffolds were able to form bone in vivo, except Zhang et al. (2006) who reported that DPSCs loaded onto spongeous collagen, in mice, were not able to form hard tissues. It is very important to mention that all the studies were done on different animal models. There have been few clinical trials that gave clear evidence of the possibility for bone regeneration in humans by using DPSCs ref (dAquino et al., 2009; Giuliani et al., 2013). SCAP are suitable for cell-based regeneration and preferentially for root formation (Sonoyama et al., 2008). The cultures of SCAP, isolated from impacted third molars, were able to differentiate into odontoblast-like cells with an active mineralization and migratory potential, leading to an organized three-dimensional dentin-like structures in vitro and the ability to produce dentin in vivo (Sonoyama et al., 2008; Laino et al., 2011). Recently, platelet lysate (PL) has attracted major attention for its possible clinical use, due to the detection of anti-fetal bovine serum (FBS) antibodies in most patients infused with MSCs cultures in FBS. This interest has led to the development of therapeutic protocols based on non- transfusional use of hemo-components such as; allogenic and autologous human plasma or serum, cord blood serum or human platelet lysate (HPL). HPL has been used as a substitute for FBS in humans because of its abilities; to stimulate MSC proliferation, maintained their differentiation potential and immunophenotypic characteristics. PL has also been used as an alternative for animal serum in human adipose mesenchymal stem cells (hASCs) culture (Doucet et al., 2005; Kocaoemer, Kern, Kluter, & Bieback, 2007; Schallmoser et al., 2007; Bieback et al., 2009; Capelli et al., 2011; Crespo-Diaz et al., 2011; Govindasamy et al., 2011). Moreover, PL has been reported to increase the proliferation rate of osteoblasts, fibroblasts and periodontal ligament cells (Okuda et al., 2003; Soffer, Ouhayoun, & Anagnostou, 2003). However, the optimum concentration and the effect of PL on SCAP and DPSCs stem cells have not been evaluated yet. The main objective of this study was to isolate two types of dental stem cells DPSCs and SCAP from impacted third molars and to compare their expansion and differentiation potentials in the presence of PL, as an alternative source of FBS.

Cell cultures were established using the enzymatic dissociation method (Gronthos et al., 2000). Briefly, teeth were disinfected by 0.5% chlorohexidine (Sage, USA) and cut around the cementum– enamel junction to reveal the pulp chamber using the hand piece. For each third molar, first the apical papilla tissue was removed, and then the tooth was drilled to remove the pulp tissue from the coronal part of the tooth, so that from each single donor tooth, both DPSCs & SCAP cultures could be established. Each tissue was then digested in a solution of 3 mg/ml collagenase type I (GIBCO, Germany) and 4 mg/ml dispase (GI BCO, Germany) for 1 h at 37
C. Single-cell suspensions were obtained by passing the cells through a 70 mm strainer (BD Biosciences, Germany). DPSCs and SCAP were seeded at a density of 104/cm2 using alpha-Modification of Eagle’s Medium [(a-MEM, Lonza, USA), supplemented with 100 mM Lascorbic acid phosphate (Sigma–Aldrich, USA), 100 mg/ml streptomycin (Invitrogen, USA), 2 mM L-glutamine (Invitrogen, USA), 0.25 mg/ml Amphotericin B (Invitrogen, USA) and 100 units/ml penicillin (Invitrogen, USA)]. For cell growth analysis, cells were seeded at 2  105cells/well in 6-well plates. Each well contains a specific concentration of Platelet lysate (PL) as following 1%, 5% and 10%. 10% FBS (Invitrogen, USA) was considered as a positive control and serum free medium was used as a negative control. Then the cells were incubated at 37C in 5% CO2. The cell number was counted every 24 h by using a hemocytometer. Three replicates for each time point of 24, 48, and 72 h, have been performed respectively.

2. Materials and methods

2.4. Flow cytometry

2.1. Platelet lysate preparation

To identify the surface markers of stem cells, DPSCs and SCAP, cells were trypsinized and harvested at passage 3, then washed twice with PBS. 2  106 cells from each type were incubated with different fluorescinated antibodies against isotype controls, FITCCD24, PE-CD34, PerCP Cy5.5-CD105, FITC-CD90 and PE-CD44 and APC-CD73 for half an hour at room temp. Cell fixative was added to the cells, were centrifuged at 1000 rpm for 5 min and re-suspended in PBS. Antibodies were used in concentrations suggested by the manufactures (BD, USA). The expression profile was analyzed by Fluorescein activated cell sorter {(FACS) Canto (BD, USA)}.

For the preparation of platelet lysate (PL), human peripheral blood was taken from donors. All donors were healthy without any clinically evident disease, had not been taking any medications, nonsmokers and non-alcoholic. The collection of samples was performed according to guidelines of the Institutional Review Board. Signed informed consent was obtained from all donors before inclusion in the study. The blood was centrifuged at 1000 rpm for 13 min at 4
C. The supernatant, made of platelets rich plasma (PRP), was taken and frozen at 80
C until use. PRP, taken in a different tube, centrifuged at 1000 rpm for 10 min, frozen at 80
C, and then rapidly thawed at 37
C. Freezing–thawing process was repeated for two times. The resulting platelet lysate (PL) was centrifuged at 5000 rpm for 10 min at 4
C to remove platelet bodies. The PL was filtered through 0.22 m filters to remove platelet membranes. NOTE Among different studies, the optimum concentration of PL for proliferation and differentiation was variable. This variation could be due to the different preparation methods of PL. In this study, hPL was prepared by subjecting peripheral blood into

2.2. In vitro isolation of dental stem cells Normal impacted third molar teeth were collected from three donors aged 18, 19 and 24 years at the stage of root development (two thirds of the root completed). All donors were healthy with no clinically evident disease, had not been taking any medications, non-smokers and non-alcohol. Signed informed consent was obtained from all donors before inclusion in the study. 2.3. Cell culture

3. In vitro mineralization and Alizarin red staining 3.1. Osteo/odontogenic differentiation

a-MEM media were supplemented with 10 mM b-glycerophosphate (Sigma–Aldrich), 50 mg/ml L-ascorbic acid 2-phosphate (Sigma–Aldrich, A), 1 mM dexamethasone (Sigma–Aldrich, USA). These chemicals have the ability to induce the osteo/odontogenic induction for differentiation (differentiation of mesenchymal stem

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

1547

Fig. 1. The morphological characteristics of isolated dental stem cells microscopy (40). (A) DPSCs. (B) SCAP, at day 4 of primary culture. (C) DPSCs reached confluence culture at day 14 (D) SCAP reached confluence culture at day 8.

cells (MSCs) into osteoblasts). For osteo/odontogenic differentiation, DPSCs and SCAP cells were plated at 2  105 cells/well in 6well plates and maintained in the growth media until reach nearly 70% confluence. Then, the cells were maintained in osteogenic media. Each well in the plate contains specific concentration as following: 1%, 5%, and 10% PL. 10% FBS was considered as positive control and normal cell culture media was used as a negative control. Medium was changed every 3 days. To visualize calcium deposits for the detection of mineralization, induced cells were stained with Alizarin red {(pH 4.2) Sigma– Aldrich, USA} for 20 min at room temp and ARS was aspirated. Finally, the cells were washed twice (2) with distilled water and were observed under the microscope (Soffer et al., 2003). The amounts of ALP, osteopontin (OPN) and osteocalcin (OCN), secreted by DPSCs and SCAP, were measured at different days (1, 9 and 15) for each concentration, using Alkaline Phosphatase Assay Kit (Biovision, USA) and Quantikine ELISA (R&D, USA).

4. Real time/polymerase chain reaction (RT)-PCR analysis 4.1. RNA extraction Total RNA was extracted from cells at days 1, 9, 15 and 21 after induction of osteogenic differentiation. Cells were detached by trypsinization {washing with 1X PBS (Invitrogen, USA) and treatment with 1X Trypsin EDTA (0.25%. Invitrogen, USA}. Detached cells were washed with the growth media and centrifuged at 1000 rpm for 5 min. The pellet was re-suspended in 200 ml of the media followed by addition of 600 ml of Trizol (Qiagen, USA) and mixed by using a vortex for 5 min. 200 ml of chloroform were added and mixed by vortex. The mixture was incubated for 2 min at room temperature and centrifuged at 13,000 rpm for 15 min. The aqueous layer was transferred into 600 ml isopropanol and centrifuged for 10 min at 13,000 rpm. After that, 700 ml of 70% ethanol were added to the pellet and centrifuged at 9000 rpm for 5 min. The supernatant was discarded and pellet was kept for air dry. Finally, RNA samples were

Table 1 Oligonucleotide primers for quantitative RT-PCR. Gene

Primer sequence

Bone sialoprotein (BSP)

50 -ATGGAGAGGACGC CACGCCT-30 50 -GGTGCCCTTGCCCTGCCTTC-30

Osteocalcin (OCN)

50 -GACTGTGACGAGTTGGCTGA-30 50 - AAGAGGAAAGAAGGGTGCCT-30

Dentinesialophosphoprotein (DSPP)

50 - GGGACACAGGAAAAGCAGAA-30 50 - TGCTCCATTCCCACTAGGAC-30

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

50 - GAAGGTGAAGGTCGGAGT- 30 50 - GAAGATGGTGATGGGATTTC-30

RT-PCR, real-time polymerase chain reaction (Laino et al., 2011).

1548

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

Fig. 2. Flow cytometric analysis of (A) DPSCs (B) SCAP using surface markers

Fig. 3. The effect of various concentrations of PL on the proliferation of isolated dental stem cells. (A) DPSCs (p < 0.04). (B) SCAP (p < 0.04). Proliferation is measured as cell count doublings over time in hours, using the seeding cell density as 1X

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

rehydrated in Nuclease Free Water (NFW, Promega). Following that, RNA was reverse transcribed by using Reverse transcriptase kit (Fermantas, Canada) to produce cDNA. 4.2. RT/PCR amplification Diluted cDNA (5 ml) was amplified using GoTaq1qPCR Master mix (Promega, USA), mixed with 25 pmol of forward and reverse primers (Table 1). The conditions for the amplification are; 95
C for 2 min as initial denaturation, 95
C for 15 s, 60
C for 60 s, and 72
C for 30 s, for 40 cycles.

1549

using phase-contrast microscope (Olympus, USA). DPSCs cultures were quite heterogeneous, containing cells ranging from narrow spindle-shaped cells to large polygonal ones, and appeared as tightly-packed cuboidal cells in subconfluent cultures (Fig. 1A). SCAP were smaller in size, fibroblast-like or satellite in shape (Fig. 1B). In DPSCs cultures, few numbers of colonies could be detected only after 6–8 days, whereas in SCAP cultures, colonies of high density were already observed by day 2–3. SCAP cell cultures reached confluency after one week, at which time they were trypsinized (passage 1) and processed for further experiment (Fig. 1D). On the other hand, most DPSCs cultures needed 14 days to produce a subconfluent monolayer (Fig. 1C).

4.3. Statistical analysis 5.2. Surface marker analysis Data were analyzed using graphPad Prism (Version 6) and microsoft windows (Excel, 2007). All assays were performed in three independent experiments (n = 3), with duplicates of each and results were expressed as means  standard deviations (SD). A paired t-test and one way ANOVA analysis were used to determine the differences in the proliferation and differentiation rates of DPSCs and SCAP, and to analyze the differences in their gene expression patterns in the presence of platelet lysate (PL) (significance assumed for P < 0.05).

Flow cytometric analysis revealed that these DPSCs were negative for CD34 (7%), CD24 (1%), CD19 (0.1%) and CD14 (0.1%). The DPSCs cultures were positive for CD146 (58%), CD105 (57%), CD44 (93%), CD73 (94%) and CD90 (57%). On the other hand, SCAP were negative for CD34 (6%), CD19 (0.6%) and CD14 (0.6%). SCAP cultures were positive for CD24 (26%) which is a specific marker for these cells, CD146 (76.1%), CD105 (76.1%), CD44 (68.25%), CD73 (62.97%) and CD 90 (68.233%) (Fig. 2).

5. Results

5.3. The effect of PL on the proliferation rates of SCAP and DPSCs

5.1. Morphological and growth characteristics of DPSCs and SCAP

There was no difference in the level of cell proliferation rates after treatment with different concentrations of PL for 24 h. However, after 48 and 72 h, significant differences, among the groups treated with different concentrations of PL, were observed (Fig. 3). Fig. 3 shows the proliferation rates of DPSCs and SCAP in

Significant differences were observed in the morphological and growth characteristics of DPSCs and SCAP cultures established from the dental pulp and adjacent apical papilla of the same tooth

Fig. 4. DPSCs microscopy after 3 weeks of osteogenic induction stained with alizarin Red Stain (40). (A) Negative control. (B) 1%PL. (C) 5% PL. (D) 10% FBS.

1550

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

Fig. 5. SCAP microscopy after 3 weeks of osteogenic induction stained with alizarin Red Stain (40). (A) Negative control. (B) 1%PL. (C) 5% PL. (D) 10% FBS.

comparison with treatments of different PL concentrations. Treatment with 5% PL for 72 h resulted in the highest proliferation rates in DPSCs and SCAP (7X, 7.5X doubling time, respectively). However, treatment with higher concentrations (10% of PL) caused

detachment of the cultures leading to cells death. On the other hand, treatment with lower concentration (1% PL) showed nonsignificant proliferation rate of DPSCs and SCAP cultures. No

Fig. 6. Osteeogenic markers expression (A) ALP (p < 0.47) (B) OCN (p < 0.5) (C) BSP (p < 0.09) (D) DSPP (p < 0.4) (E) OPN (p < 0.05) (F) OCN (p < 0.34) in DPSCs at different days of differentiation, nourished with different concentrations of PL or FBS.

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

1551

Fig. 7. Osteeogenic markers expression (A) ALP (p < 0.49). (B) OCN (p < 0.56) (C) BSP (p < 0.08) (D) DSPP (p < 0.05) (E) OPN (p < 0.038) (F) OCN (p < 0.47) in SCAP at different days of differentiation, nourished with different concentrations of PL or FBS.

expansion was observed in DPSCs and SCAP cultures after treatment with serum free medium (Negative control).

significantly observed for SCAP (P < 0.05) only for the expression of DSPP in 5% PL at day (15) (Figs. 6 and 7).

5.4. In vitro osteo/odontogenic differentiation and alizarin red staining

6. ELISA

To determine the functional effects of PL on the osteogenic differentiation, in vitro osteogenic differentiation analysis with various concentrations of PL was conducted. After 21 days of treatment, cells of DPSCs and SCAP cultures within the confluent monolayers, started to aggregate and form clusters of high density. There were multiple single mineralized nodules/sites throughout the whole adherent monolayer. Mineralization gradually increased, covering almost 70% of the adherent monolayer, 3 weeks after induction of differentiation. However, visible differences were observed in the patterns of mineralization in two types of cells. The density of calcium deposits increased when stained by ARS (Figs. 4 and 5). The best pattern of differentiation was noted with 5% PL while at 10% PL cells were detached before the end of their differentiation.

Our results showed that the amount of ALP {DPSC (P < 0.47) and SCAP (P < 0.49)} and OCN {DPSCs (P < 0.34) and SCAP (P < 0.47)} produced by DPSCs and SCAP treated with different concentrations of PL, was non-significant. However, the concentration of OPN {DPSCs (P < 0.052) and SCAP (P < 0.038)} was significant (ANOVA, Paired t-test Table 2). Table 2 shows the P values of Paired t-test and ANOVA for DPSCs and SCAP, after the treatments with different concentrations of PL for proliferation, differentiation assays and osteodentin markers.

5.5. Gene expression analysis Quantitative RT-PCR analysis of RNA extracted from DPSCs and SCAP showed that osteo/odontogenic differentiation has induced the expression of different osteodentin markers, osteocalcin (OCN), bone sialoprotien (BSP) and dentin sialophosphoprotien (DSPP). Osteogenic differentiation increases the expression of BSP and OCN gradually up to day 21. The highest level of BSP and OCN expression was observed at 5% PL {BSP: DPSC (P < 0.09) and SCAP (P < 0.08); OCN: DPSCs (P < 0.5) and SCAP (P < 0.56)}, in a short period of time (15 days) (Figs. 6 and 7). Odontogenic differentiation was also

7. Discussion Autologous human serum has been shown to be equivalent to FBS in terms of supporting the proliferation and differentiation capacity of hMSCs and could be utilized for clinical therapies (Doucet et al., 2005; Kocaoemer et al., 2007; Schallmoser et al., 2007; Bieback et al., 2009; Capelli et al., 2011; Crespo-Diaz et al., 2011; Stute et al., 2004; Kobayashi et al., 2005). It contains a pool of endogenous growth factors such as; TGF-b, PDGF, FGF and IGF, which enhances the expansion of MSCs (Sotiropoulou, Perez, Salagianni, Baxevanis, & Papamichail, 2006). Few studies have demonstrated the positive effects of human PL (PRP) on the proliferation and mineralized differentiation of other types of MSCs like human dental stem cells, dental pulp stem cells (DPSCs) (Lee et al., 2011) and periodontal ligament stem cells (PDLSCs) (Lee et al., 2011).

1552

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553

Table 2 P values for Paired t-test (A) DPSCs (B)SCAP & (C) One way ANOVA, after the treatment with different concentrations of PL. (A) Cell proliferation

ALP assay

1% PL

1% PL

5%PL

0.01 0.2

0.008 0.047 0.7 0.9

5%PL

Neg. control 0.06 Pos. control 0.8

10% PL

0.005 0.09 0.2 0.6

1% PL

5% PL

10%PL

0.6 0.6

0.08 0.54

0.11 0.4

DSPP

OCN

Neg.control pos. control

BSP 10% PL

1%PL

5%PL

10%PL

1%PL

5%PL

10%PL

0.05 0.05

0.002 0.01

0.05 0.034

0.5 0.4

0.01 0.7

0.004 0.3

(B) Cell proliferation

ALP assay

1%PL

1%PL

5%PL

10% PL

Neg. 0.008 0.002 0.01 control 0.46 0.7 pos. control 0.9

5% PL

1% PL

5% PL

10%PL

0.048 0.13

0.02

0.1

0.05

0.28

0.76

0.66

0.6

0.5

0.5

0.7

DSPP

OCN

Neg. control pos. control

BSP 10% PL

1%PL

5%PL

10%PL

1%PL

5%PL

10%PL

0.06 0.2

0.008 0.7

0.01 0.7

0.004 0.001

0.007 0.01

0.008 0.009

(C)

higher ALP activity in comparison to FBS, but at a very late stage. 10% PL was able to speed up both, the osteogenic differentiation and ALP activity, in short period of time (15 days instead of 21days). However, 10% PL cannot be considered as an optimum concentration for osteogenic differentiation as it leads to cells death and detachment, due to the small size of the cultured area (unpublished data). Chen et. al., showed that 5% PL resulted in the highest activity of ALP for DPSCs. Furthermore, treatment with 1% or 2% UCB-PRP also enhanced ALP activity in various dental stem cells (DPSCs, PDLSCs and SHED) but treatment with concentrations higher than 2% UCB-PRP resulted in a decline in ALP activity. These observed effects of UCB-PRP on cell differentiation (Lee et al., 2011) are in consistent with our findings. Regarding the genetic expression patterns, 5% PL is considered as the optimum concentration for induction of expression of certain bone markers in DPSCs and SCAP (Figs. 6 and 7). The expression of these markers (osteocalcin OCN, bone sialoprotein BSP, and dentin sialophosphoprotein DSPP) (Morsczeck et al., 2009; Batouli et al., 2003; Yang et al., 2012; Laino et al., 2011; Chen et al., 2012), is an evidence for the osteogenic/dentinogenic differentiation of DPSCs and SCAP. It has been reported recently that certain inhibitors could enhance the expression levels for osteoblast differentiation (OPN and BSP) in DPSC (Paino et al., 2014) selectively. On the basis of our findings we can report that 5% platelet lysate was optimum concentration for the proliferation and osteo/ dentinogenesis of DPSCs and SCAP for a short period of time (15 days). This finding can be used for further implications in bone regeneration, tooth repair, dentin formation and for advanced dental tissue engineering.

Name of test

SCAP

DPSCs

Trypan blue Elisa

Cell Proliferation ALP activity OPN concentration OCN concentration

P < 0.04 P < 0.49 P < 0.038 P < 0.47

P < 0.04 P < 0.47 P < 0.052 P < 0.34

Competing interest

Q-PCR

BSP DSPP OCN

P < 0.08 P < 0.05 P < 0.56

P < 0.09 P < 0.4 P < 0.5

Acknowledgments

In this study, proliferation rates were variable among different concentrations of PL for both DPSCs and SCAP, when compared with FBS. The optimum concentration of PRP varied from 50% (Ferreira et al., 2005), 10% (Lucarelli et al., 2003) to less than 1% (Soffer et al., 2003). Previously, for example, 0.5 -1% (Soffer et al., 2003; Chen, Sun, Wang, Kong, & Chen, 2012) PRP was the optimum concentration for cellular proliferation and mineralization rates. However, Ferreira et al. (2005) found 50% as the optimum concentration for osteoblast proliferation. On the other hand, 10% PRP was sufficient to induce a marked cell proliferation of MSC (Lucarelli et al., 2003; Castegnaro et al., 2011), derived from adipose tissue. Similarly, 2% PRP (extracted from umbilical cord blood) showed the highest proliferation rate of dental stem cells DPSCs, PDLSCs and SHED (Lucarelli et al., 2003). Govindasamy et. al. (2011) showed that PL was able to increase the expansion of DPSCs two-fold as compared to FBS. In addition, 5% PL was the optimum concentration for MSC and DPSCs proliferation and osteogenesis. (Chen et al., 2012; Chevallier et al., 2010). Previous studies showed that the optimum concentration for the formation of calcium deposits was 2% PRP for DPSCs and PDLSCs, while 1% PRP resulted in the highest calcium deposits in SHED (Lee et al., 2011). In this study, all the various concentrations of PL showed a higher ALP and OPN activity as compared to FBS. 5% PL was able to induce the osteogenic differentiation in a short period of time(15 days) with an observable amount of ALP activity (Figs. 6 and 7). However, the lower concentration (1%PL) showed

There are no conflicts of interest.

We are greatly thankful for all the participants in this study and for Nazneen Aslam for helping in manuscript editing and proofreading. This work was supported by grant # (217/2011) from the Deanship of Research at the Jordan University of Science and Technology. References Arthur, A., Rychkov, G., Shi, S., Koblar, S. A., & Gronthos, S. (2008). Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells, 26, 1787–1795. Balic, A., Aguila, H. L., Caimano, M. J., Francone, V. P., & Mina, M. (2010). Characterization of stem and progenitor cells in the dental pulp of erupted andmurine molars. Bone, 46, 1639–1651. Batouli, S., Miura, M., Brahim, J., Tsutsui, T. W., Fisher, L. W., & Gronthos, S., et al., (2003). Comparison of stem cell-mediated osteogenesis and dentinogenesis. Journal of Dental Research, 82, 976–981. Bieback, K., Hecker, A., Kocaomer, A., Lannert, H., Schallmoser, K., & Strunk, D., et al., (2009). Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 2331–2341. Capelli, C., Gotti, E., Morigi, M., Rota, C., Weng, L., & Dazzi, F., et al., (2011). Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate. Cytotherapy, 13, 786–801. Castegnaro, S., Chieregato, K., Maddalena, M., Albiero, E., Visco, C., & Madeo, D., et al., (2011). Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue. Current Stem Cell Research & Therapy, 6, 105e14. Chen, B., Sun, H. H., Wang, H. G., Kong, H., & Chen, F. M. (2012). The effects of human platelet lysate on dental pulpstem cells derived from impacted human third molars. Biomaterials (20), 5023–5035. Chevallier, N., Anagnostou, F., Zilber, S., Bodivit, G., Maurin, S., & Barrault, A., et al., (2010). Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate. Biomaterials, 31, 270e8.

D. Abuarqoub et al. / Archives of Oral Biology 60 (2015) 1545–1553 Crespo-Diaz, R., Behfar, A., Butler, G. W., Padley, D. J., Sarr, M. G., & Bartunek, J., et al., (2011). Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability. Cell Transplantation, 20, 797e811. d’Aquino, R., De Rosa, A., Lanza, V., Tirino, V., Laino, L., Graziano, A., Desiderio, V., Laino, G., Papaccio, G., 2009. Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes, November, 18:75–83. d’Aquino, R., Tirino, V., Desiderio, V., Studer, M., De Angelis, G. C., & Laino, L., et al., (2011). Human neural crest-derived postnatal cells exhibit remarkable embryonic attributes either in vitro or in vivo. European Cells & Materials, 21, 304–316. Doucet, C., Ernou, I., Zhang, Y., Llense, J. R., Begot, L., & Holy, X., et al., (2005). Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. Journal of Cellular Physiology, 36, 228–236. Ferreira, C. F., Carriel Gomes, M. C., Filho, J. S., Granjeiro, J. M., Oliveira Simões, C. M., & Magini Rde, S. (2005). Platelet-rich plasma influence on human osteoblasts growth. Clinical Oral Implants Research, 16, 456e60. Gandia, C., Armiñan, A., García-Verdugo, J. M., Lledó, E., Ruiz, A., & Miñana, M. D., et al., (2008). Human dental pulp stem cells improve left ventricular function, induce angiogenesis and reduce infarct size in rats with acute myocardial infarction. Stem Cells, 26, 638–645. Giuliani, A., Manescu, A., Langer, M., Rustichelli, F., Desiderio, V., & Paino, F., et al., (2013). Three years after transplants in human mandibles, histological and inline holotomography revealed that stem cells regenerated a compact rather than a spongy bone: biological and clinical implications. Stem Cells Translational Medicine, 2(4), 316–324. Govindasamy, V., Ronald, V. S., Abdullah, A. N., Ganesan Nathan, K. R., Aziz, Z. A., & Abdullah, M., et al., (2011). Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications. Cytotherapy, 13, 1221. Graziano, A., d’Aquino, R., Cusella-De Angelis, M. G., De Francesco, F., Giordano, A., & Laino, G., et al., (2008). Scaffold’s surface geometry significantly affects human stem cell bone tissue engineering. Journal of Cellular Physiology, 214, 166–172. Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., & Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America, 25, 13625–13630. Gronthos, S., Brahim, J., Li, W., Fisher, L. W., Cherman, N., & Boyde, A., et al., (2002). Stem cell properties of human dental pulp stem cells. Journal of Dental Research, 81, 531–535. Iohara, K., Nakashima, M., Ito, M., Ishikawa, M., Nakashima, A., & Akamine, A. (2004). Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenic protein 2. Journal of Dental Research, 83, 590–595. Kadar, K., Kiraly, M., Porcsalmy, B., Molna, B., Racz, G. Z., & Blazsek, J., et al., (2009). Differentiation potential of stem cells from human dental origin—promise for tissue engineering. Journal of Physiology and Pharmacology, 60(47), 167–175. Karaoz, E., Dogan, B. N., Aksoy, A., Gacar, G., Akyuz, S., & Ayhan, S., et al., (2010). Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochemistry and Cell Biology, 133, 95–112. Kobayashi, T., Watanabe, H., Yanagawa, T., Tsutsumi, S., Kayakabe, M., & Shinozaki, T., et al., (2005). Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. Journal of Bone & Joint Surgery, 87(10), 1426–1433. Kocaoemer, A., Kern, S., Kluter, H., & Bieback, K. (2007). Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells, 25, 1270–897 (25):13625-30. Laino, G., Guida, L., Rullo, R., Bakopoulou, A., Leyhausen, G., Volk, J., Tsiftsoglou, A., Garefis, P., & Koidis, P., et al., (2011). Comparative analysis of in vitro osteo/ odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Archives of Oral Biology, 56(7), 709–721.

1553

Lee, J. Y., Nam, H., Park, Y. J., Lee, S. J., Chung, C. P., & Lee, G., et al., (2011). The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells. In Vitro Cellular & Developmental Biology Animal, 47(2), 157–164. Loomba, K., Bains, R., Bains, V. K., Loomba, A., Nadig, R. R., & Shrivastava, T. V. (2012). Tissue engineering and its application in endodontics: an overview. ENDO (London, England), 6, 105–112. Lucarelli, E., Beccheroni, A., Donati, D., Sangiorgi, L., Cenacchi, A., & Del Vento, A. M., et al., (2003). Platelet-derived growth factors enhance proliferation of human stromal stem cells. Biomaterials, 24, 3095e100. Marchionni, C., Bonsi, L., Alviano, F., Lanzoni, G., Di Tullio, A., & Costa, R., et al., (2009). Angiogenic potential of human dental pulp stromal (stem) cells. International Journal of Immunopathology and Pharmacology, 22(49), 699–706. Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L. W., & Robery, P. G., et al., (2003). SHED: stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Science, 100(10), 5807–5812. Morsczeck, C., Petersen, J., Völlner, F., Driemel, O., Reichert, T., & Beck, H. C. (2009). Proteomic analysis of osteogenic differentiation of dental follicle precursor cells. Electrophoresis, 30(7), 1175–1184. Nakashima, M. (2005). Tissue engineering in endodontics. Australian Endodontic Journal, 31, 111–113. Okuda, K., Kawase, T., Momose, M., Murata, M., Saito, Y., & Suzuki, H., et al., (2003). Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factorbeta and modulates the proliferation of periodontally related cells in vitro. Journal of Periodontology (74), 849–857. Paino, F., La Noce, M., Tirino, V., Naddeo, P., Desiderio, V., Pirozzi, G., De Rosa, A., Laino, L., Altucci, L., & Papaccio, G. (2014). Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement. Stem Cells, 32 (January (1)), 279–289. Paino, F., Ricci, G., De Rosa, A., d’Aquino, R., Laino, L., & Pirozzi, G., et al., (2010). Ectomesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. European Cells & Materials, 20, 295–305. Schallmoser, K., Bartmann, C., Rohde, E., Reinisch, A., Kashofer, K., & Stadelmeyer, E., et al., (2007). Human platelet lysate can replace fetal bovine serum for clinicalscale expansion of functional mesenchymal stromal cells. Transfusion, 47, 1436e46. Seo, B. M., Miura, M., Gronthos, S., Bartold, P. M., Batouli, S., & Brahim, J., et al., (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 364(9429), 149–155. Sharpe, P. T., & Young, C. S. (2005). Test-tube teeth. Scientific American, 293(2), 34–41. Soffer, E., Ouhayoun, J. P., & Anagnostou, F. (2003). Fibrin sealants and platelet preparations in bone and periodontal healing. Oral Surgery, Oral Medicine, 8, 521–528. Sonoyama, W., Liu, Y., Yamaza, T., Tuan, R. S., Wang, S., & Shi, S., et al., (2008). Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. Journal of Endodontics, 34, 166–171. Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N., & Papamichail, M. (2006). Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells, 24(2), 462–471. Stute, N., Holtz, K., Bubenheim, M., Lange, C., Blake, F., & Zander, A. R. (2004). Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Experimental Hematology, 32(12), 1212–1225. Yang, X., van der Kraan, P. M., Bian, Z., Fan, M., Walboomers, X. F., & Jansen, J. A. (2009). Mineralized tissue formation by BMP2- transfected pulp stem cells. Journal of Dental Research, 88, 1020–1025. Yang, X., Han, G., Pang, X., & Fan, M. (2012). Chitosan/collagen scaffold containing bone morphogenetic protein-7 DNA supports dental pulp stem cell differentiation in vitro and in vivo. Journal of Biomedical Materials Research Part A . Zhang, W., Walboomers, X. F., van Kuppevelt, T. H., Daamen, W. F., Bian, Z., & Jansen, J. A. (2006). The performance of human dental pulp stem cells on different three-dimensional scaffold materials. Biomaterials, 27, 5658–5668.