Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor-associated Genes in PDLSCs Fazliny Abd Rahman, M.Biotech*, Johari Mohd Ali, PhD†, Mariam Abdullah, MClintDent*, Noor Hayaty Abu Kasim, PhD*, Sabri Musa, MSc‡ *Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, WP Malaysia. †Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, WP Malaysia. Email: [email protected]; Fax: +603-79674957; Tel: +603-79674533. ‡Department of Paediatric Dentistry & Orthodontics, Faculty of Dentistry. Email: [email protected]; Tel: +603-79674802; Fax: +603-79674530. Background: This study was carried out to investigate the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential and expression of growth factor-associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from periodontal ligaments tissue were isolated from human premolars (n=3). The MSCs identity was confirmed by immunophenotyping and tri-lineage differentiation assays. Cell proliferation activity was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCR array was used to profile the expression of 84 growth factor-associated genes. Pathway analysis was used to identify the biological functions and canonical pathways activated by aspirin treatment. The osteogenic potential was evaluated through mineralization assay using Alizarin Red S. Results: Aspirin at 1000 µM enhances osteogenic potential of PDLSCs. Using a fold-change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and 7 downregulated genes. FGF9, VEGFA, IL2, BMP10, VEGFC and FGF2 were markedly upregulated (FC range: 6 to 15), whilst PTN, FGF5, BDNF and DKK1 were markedly downregulated (FC ~32). Of the 84 growth factors associated genes screened, 35 showed high Ct values (Ct >35). Conclusions: Aspirin modulates the expression of growth factors associated genes and enhances osteogenic potential in PDLSCs. Aspirin upregulated the expression of genes that could activate biological functions and canonical pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration and differentiation. These findings suggest that aspirin enhances PDLSCs function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.
KEYWORDS: guided periodontal tissue regeneration, acetylsalicylic acid, periodontal ligament, gene expression profiling, growth factors, periodontitis.
Mesenchymal stem cell (MSCs) have been widely investigated for its potential use in regenerative medicine.1 Since then, the knowledge from the study of bone marrow mesenchymal stem cells (BMMSCs) have provided the ‘gold standard’ approach, to evaluate the use of stem cells for regenerative medicine.1 However, the harvesting of BMMSCs is an invasive procedure and thus, alternatives sources of stem cells have to be explored. Periodontal ligament stem cells (PDLSCs) were first isolated by Seo et al. in 2004,2 and this provided an alternative stem cells source that are easier to be accessed. The PDLSCs showed self-renewal ability and multipotency characteristics that are useful for periodontal therapeutics and regenerative medicine application.2-4 1
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
Growth factors (GFs) are known biological mediators with crucial roles in tissue proliferation and repair. Fibroblast growth factor-2 (FGF2), platelet derived growth factor-BB (PDGF-BB), bone morphogenetic proteins (BMP2/6/12) and brain derived neurotrotrophic factor (BDNF) are among candidate GFs for periodontal regeneration.3 The most studied growth factors in periodontal regeneration are FGF2, IGF, PDGF, TGFβ and the BMPs.5 Aspirin (ASA) has been widely used for decades as a non-steroidal anti-inflammatory drug (NSAID), which is capable of modulating conditions such as cardiovascular disease, cancer and periodontal disease.6, 7 ASA anti-inflammatory action is achieved by the disruption of cyclic prostanoids biosynthesis, through the inhibition of the enzyme cyclooxygenase-2 (COX-2) activity.8 The impact of ASA on stem cells and its potential use have been reported in a number of studies.9-11 Liu and colleagues reported that ASA treatment on stem cells from exfoliated deciduous teeth (SHED) at low dosages (~55 and 277 µM) resulted to the activation of telomerase reverse transcriptase (TERT) activity, which improved SHED osteogenesis and immunomodulatory properties.9 The authors suggested a low dose ASA therapy is a feasible and efficient pharmacologic approach to activating TERT activity in stem cells, which may improve stem cell functions, avoid replicative senescence and achieve the therapeutic effects of stem cells. A recent study reported that administration of ASA and human umbilical cord matrixderived mesenchymal stem cells (hUCMs) reduced the effect of post-ischemic brain injury in the rat induced by artificial stroke.10. Liu and colleagues reported that BMMSC-based calvarial bone defect repair in C57BL/6 mice was enhanced by site-specific aspirin treatment, and this was due to the suppression of TNFα and IFNγ concentrations (and action) at the implantation site.11 The observations from previous studies7 indicated that ASA showed positive impact in reducing human alveolar bone loss and improving periodontal status, which led to the suggestion that ASA provided a favourable conditions for periodontal tissues health.7 However, it was also argued that due to the complexity of periodontitis pathogenesis, it was unlikely that the inhibition of inflammation by NSAIDs was the only factor which contributed to the observed clinical benefit.7 It could be hypothesized that ASA may have positively affect growth factors gene expression in dental stem cells, leading to improvement in periodontal status. Thus, investigating the effect of ASA on stem cells properties, such as those sourced from PDLSCs is of relevance. To the best of our knowledge, there is not much data or studies that have been done to investigate the effect of ASA on the viability and expression of growth factor genes in PDLSCs. The outcome of this study could shed additional insights on the impact of ASA on PDLSCs properties and how it could be of benefit for periodontal regeneration.
2.0 MATERIALS AND METHODS 2.1 Isolation and Culturing of Human PDLSCs From Periodontal Ligament Tissue This study was approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [Medical Ethics Clearance Number: DF CO1107/0066 (L)] and with patient’s informed consent. The PDLSCs were isolated from normal human premolars. The donors were aged between 18 to 35 years old (n=3) and the teeth were being extracted for orthodontics treatment purposes. The PDLSCs were isolated by standard protocol with some modifications.2 Periodontal ligament tissues were separated from the root surface of permanent teeth using a sterilized scalpel. The PDL tissues were minced into small fragments prior to its digestion in a solution of 2
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
1 mg/mL collagenase type I§ for 30 minutes at 37°C. After neutralization with a 10% fetal bovine serum* (FBS), the cells were centrifuged and seeded in T75 culture flasks† using a culture medium containing KO-DMEM, 10% FBS, 0.5% and 10000 µg/mL of penicillin/streptomycin‡, 1% 1x glutamax§ and incubated at 37oC in the presence of 5% CO2. The medium was replaced every three days until the cells reach 70-80% confluency. The PDLSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation as previously described.2, 4 2.2 Flow Cytometry The PDLSCs were immunophenotyped using a flow cytometerǁ using MSC human phenotyping kit¶ according to the manufacturer’s protocol. The following antibodies were used to mark the cell surface: epitopes-CD90-phycoerythrin (PE), CD73-APC, CD105-PE and CD14-PerCP, CD20-PerCP, CD34-PerCP, and CD45-PerCP. The results were analyzed using the flow cytometer software#. 2.3 MTT Cell Viability Assay The PDLSCs viability was assessed using 3-(4,5-dimethylthiazohl-2-yl)-2,5-diphenyltetrazolium bromide** (MTT) reagent. ASA†† was dissolved in ethanol to make a stock solution of 1M. The experimental group was treated with ASA at 10 μM to 10 mM. The PDLSCs were seeded at 3x104 cells in a 96-well plate and incubated overnight at 37oC in 5% CO2 atmosphere. The experimental group was treated at the required ASA concentrations for 24, 48 and 72 hour. The MTT solution was added thereafter, and the cells were further incubated at 37◦C for 4 hour. The culture media was then removed and dimethyl sulfoxide (DMSO) was added into each well. The absorbance was then measured at 575 nm. The cell viability was determined by calculating the ratio of optical density (OD) values between the ASA-treated and vehicle-control cells. 2.4 Growth Factors Gene Expression Profiling The gene expression profiling was done using the human growth factors PCR array‡‡. The array profiles 84 growth factor associated genes that correspond to the following: angiogenic growth factors, regulators of apoptosis, cell differentiation and developmental regulators. The PDLSCs were cultured for 24 hour in the absence (experimental control) or presence of 1000 µM ASA, using culture media as described in section 2.1. Total RNA was then isolated using RNA easy Mini kit§§ and the RNA was then reverse-transcribed into cDNAǁǁ , according to manufacturer’s instructions. The cDNA amplification was performed using the SYBR green mastermix¶¶ PCR array kit, using real-time PCR thermalcycler##. The expression values of target genes were normalized using GAPDH gene. For data analysis, the real time PCR software*** was used to calculate the fold change of gene expression between the non-treated and the ASA treated PDLSCs based on the ΔΔCT method. A cutoff cycle threshold (CT) value of 35 was assigned for estimation of fold change. Upregulation was defined as having a fold change of > 2.0. 2.5 Array Validation The PCR array results were validated by quantitative real-time PCR (qRT-PCR). The following genes were selected for the validation assay, with the corresponding reagents set†††: FGF2Hs00266645_m1; FGF9-Hs00181829_m1; BMP2-Hs00154192_m1, FGF7-Hs00940253_m1, VEGFA-Hs00900055_m1; VEGFC-Mm00437310_m1, IL2-Hs00907779_m1; IL4Hs00174122_m1; IL10-Hs00961622_m; BMP10-Hs00205566_m1, BMP3-Hs00609638_m1; 3
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
INHBB-Hs00173582_m1; GAPDH- Hs02758991_g1. All determinations were normalized using GAPDH as the reference gene. 2.6 The Effects of ASA on PDLSCs Osteogenic Potential and Proliferation Rate The PDLSCs cells were expanded in culture media (section 2.1) and maintained in 5% CO2 incubator at 37◦C until they reached 70 to 80% confluency. Then, the PDLSCs (n=3) were exposed to ASA [ 0 (control), 10, 200, 500, 1000 μM] in osteogenic media containing 10% FBS, 1% L-alanyl-L-glutamine, 100 nM dexamethasone, 10 mmol/L β-glycerol phosphate and 0.2 mM of ascorbic acid. The cells were cultured for 21 days and the media was changed every 3 days. The MTT assay was conducted at days 0, 3, 7, 17, 17 and 21. The mineralization assay was done to evaluate PDLCs osteogenic potential and the cells were stained with 2% Alizarin Red S (ARS) at the designated days. The Alizarin Red S positive area (ARSA) was analyzed using software‡‡‡ and the result is presented as percentage of ARSA over the total area of the image. 2.7 Statistical Analysis The results are presented as means ± standard deviations (SDs) of at least 3 replicates. Two-way ANOVA with Bonferroni post-test was performed using software§§§. P values of 35) in the treated and untreated cells, suggesting they were expressed only at very low/background levels: BMP3, BMP8B, CLC, CSF2, CSF3, EREG, FGF11, FGF13, FGF14, FGF17, FGF22, FGF6, GDF10, GDF11, IGF2, IL11, IL1A, IL1B, INHBA, JAG2, LEFTY1, LEFTY2, LTBP4, MDK, NDP, NRG2, NRG3, NRTN, OSGIN1, PSPN, SLCO1A2, SPP1, TDGF1, THPO, TYMP. 3.4 Validation of PCR Array Result by qRT-PCR The qRT-PCR results were generally in agreement with the PCR array data. The upregulation of the following genes were confirmed: FGF2, FGF7, FGF9, VEGFA, VEGFC, BMP2, BMP10, IL2, IL4, IL10, INHBB and CSF3 (Table 2). 3.5 Pathway Analyses: Molecular Relationship, Biological Functions and Canonical Pathway Analyses Pathway enrichment analysis¶¶¶ was carried out to identify the plausible association between the upregulated genes with biological functions and canonical pathways. This may provide insights on the biological effect of ASA on PDLSCs. The top 15 significant biological functions (Figure 4A) identified include the following: cellular development, cellular growth and proliferation, cell cycle, connective tissue development and function, cell signaling and organ development. The top 15 canonical pathways identified include the following: fibroblast growth factor (FGF) signaling, integrin-linked kinase (ILK) signaling, regulation of epithelial-mesenchymal transition (EMT) pathway, actin cytoskeleton signaling, VEGF signaling, human embryonic stem cell pluripotency, BMP signaling pathway, TGF-β signaling, as well as role of NANOG in mammalian embryonic stem cell pluripotency (Figure 4B). The molecular interaction analyses¶¶¶ indicated that the genes upregulated by ASA treatment are linked to cellular proliferation and differentiation; development of epithelial tissue and invasion of endothelial cells which have direct interaction to angiogenesis pathway. 3.6 Evaluation of Osteogenic Potential of PDLSCs The results indicated that ASA modulates the proliferative capacity and osteogenic potential of PDLSCs. The MTT assay indicated that 500 µM and 1000 µM ASA treatments induced higher proliferative capacity compared to untreated cells (Figure 2). For the osteogenic potential assay, statistically significant differences (P
DOI: 10.1902/jop.2016.150610
Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor-associated Genes in PDLSCs Fazliny Abd Rahman, M.Biotech*, Johari Mohd Ali, PhD†, Mariam Abdullah, MClintDent*, Noor Hayaty Abu Kasim, PhD*, Sabri Musa, MSc‡ *Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, WP Malaysia. †Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, WP Malaysia. Email: [email protected]; Fax: +603-79674957; Tel: +603-79674533. ‡Department of Paediatric Dentistry & Orthodontics, Faculty of Dentistry. Email: [email protected]; Tel: +603-79674802; Fax: +603-79674530. Background: This study was carried out to investigate the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential and expression of growth factor-associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from periodontal ligaments tissue were isolated from human premolars (n=3). The MSCs identity was confirmed by immunophenotyping and tri-lineage differentiation assays. Cell proliferation activity was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCR array was used to profile the expression of 84 growth factor-associated genes. Pathway analysis was used to identify the biological functions and canonical pathways activated by aspirin treatment. The osteogenic potential was evaluated through mineralization assay using Alizarin Red S. Results: Aspirin at 1000 µM enhances osteogenic potential of PDLSCs. Using a fold-change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and 7 downregulated genes. FGF9, VEGFA, IL2, BMP10, VEGFC and FGF2 were markedly upregulated (FC range: 6 to 15), whilst PTN, FGF5, BDNF and DKK1 were markedly downregulated (FC ~32). Of the 84 growth factors associated genes screened, 35 showed high Ct values (Ct >35). Conclusions: Aspirin modulates the expression of growth factors associated genes and enhances osteogenic potential in PDLSCs. Aspirin upregulated the expression of genes that could activate biological functions and canonical pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration and differentiation. These findings suggest that aspirin enhances PDLSCs function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.
KEYWORDS: guided periodontal tissue regeneration, acetylsalicylic acid, periodontal ligament, gene expression profiling, growth factors, periodontitis.
Mesenchymal stem cell (MSCs) have been widely investigated for its potential use in regenerative medicine.1 Since then, the knowledge from the study of bone marrow mesenchymal stem cells (BMMSCs) have provided the ‘gold standard’ approach, to evaluate the use of stem cells for regenerative medicine.1 However, the harvesting of BMMSCs is an invasive procedure and thus, alternatives sources of stem cells have to be explored. Periodontal ligament stem cells (PDLSCs) were first isolated by Seo et al. in 2004,2 and this provided an alternative stem cells source that are easier to be accessed. The PDLSCs showed self-renewal ability and multipotency characteristics that are useful for periodontal therapeutics and regenerative medicine application.2-4 1
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
Growth factors (GFs) are known biological mediators with crucial roles in tissue proliferation and repair. Fibroblast growth factor-2 (FGF2), platelet derived growth factor-BB (PDGF-BB), bone morphogenetic proteins (BMP2/6/12) and brain derived neurotrotrophic factor (BDNF) are among candidate GFs for periodontal regeneration.3 The most studied growth factors in periodontal regeneration are FGF2, IGF, PDGF, TGFβ and the BMPs.5 Aspirin (ASA) has been widely used for decades as a non-steroidal anti-inflammatory drug (NSAID), which is capable of modulating conditions such as cardiovascular disease, cancer and periodontal disease.6, 7 ASA anti-inflammatory action is achieved by the disruption of cyclic prostanoids biosynthesis, through the inhibition of the enzyme cyclooxygenase-2 (COX-2) activity.8 The impact of ASA on stem cells and its potential use have been reported in a number of studies.9-11 Liu and colleagues reported that ASA treatment on stem cells from exfoliated deciduous teeth (SHED) at low dosages (~55 and 277 µM) resulted to the activation of telomerase reverse transcriptase (TERT) activity, which improved SHED osteogenesis and immunomodulatory properties.9 The authors suggested a low dose ASA therapy is a feasible and efficient pharmacologic approach to activating TERT activity in stem cells, which may improve stem cell functions, avoid replicative senescence and achieve the therapeutic effects of stem cells. A recent study reported that administration of ASA and human umbilical cord matrixderived mesenchymal stem cells (hUCMs) reduced the effect of post-ischemic brain injury in the rat induced by artificial stroke.10. Liu and colleagues reported that BMMSC-based calvarial bone defect repair in C57BL/6 mice was enhanced by site-specific aspirin treatment, and this was due to the suppression of TNFα and IFNγ concentrations (and action) at the implantation site.11 The observations from previous studies7 indicated that ASA showed positive impact in reducing human alveolar bone loss and improving periodontal status, which led to the suggestion that ASA provided a favourable conditions for periodontal tissues health.7 However, it was also argued that due to the complexity of periodontitis pathogenesis, it was unlikely that the inhibition of inflammation by NSAIDs was the only factor which contributed to the observed clinical benefit.7 It could be hypothesized that ASA may have positively affect growth factors gene expression in dental stem cells, leading to improvement in periodontal status. Thus, investigating the effect of ASA on stem cells properties, such as those sourced from PDLSCs is of relevance. To the best of our knowledge, there is not much data or studies that have been done to investigate the effect of ASA on the viability and expression of growth factor genes in PDLSCs. The outcome of this study could shed additional insights on the impact of ASA on PDLSCs properties and how it could be of benefit for periodontal regeneration.
2.0 MATERIALS AND METHODS 2.1 Isolation and Culturing of Human PDLSCs From Periodontal Ligament Tissue This study was approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [Medical Ethics Clearance Number: DF CO1107/0066 (L)] and with patient’s informed consent. The PDLSCs were isolated from normal human premolars. The donors were aged between 18 to 35 years old (n=3) and the teeth were being extracted for orthodontics treatment purposes. The PDLSCs were isolated by standard protocol with some modifications.2 Periodontal ligament tissues were separated from the root surface of permanent teeth using a sterilized scalpel. The PDL tissues were minced into small fragments prior to its digestion in a solution of 2
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
1 mg/mL collagenase type I§ for 30 minutes at 37°C. After neutralization with a 10% fetal bovine serum* (FBS), the cells were centrifuged and seeded in T75 culture flasks† using a culture medium containing KO-DMEM, 10% FBS, 0.5% and 10000 µg/mL of penicillin/streptomycin‡, 1% 1x glutamax§ and incubated at 37oC in the presence of 5% CO2. The medium was replaced every three days until the cells reach 70-80% confluency. The PDLSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation as previously described.2, 4 2.2 Flow Cytometry The PDLSCs were immunophenotyped using a flow cytometerǁ using MSC human phenotyping kit¶ according to the manufacturer’s protocol. The following antibodies were used to mark the cell surface: epitopes-CD90-phycoerythrin (PE), CD73-APC, CD105-PE and CD14-PerCP, CD20-PerCP, CD34-PerCP, and CD45-PerCP. The results were analyzed using the flow cytometer software#. 2.3 MTT Cell Viability Assay The PDLSCs viability was assessed using 3-(4,5-dimethylthiazohl-2-yl)-2,5-diphenyltetrazolium bromide** (MTT) reagent. ASA†† was dissolved in ethanol to make a stock solution of 1M. The experimental group was treated with ASA at 10 μM to 10 mM. The PDLSCs were seeded at 3x104 cells in a 96-well plate and incubated overnight at 37oC in 5% CO2 atmosphere. The experimental group was treated at the required ASA concentrations for 24, 48 and 72 hour. The MTT solution was added thereafter, and the cells were further incubated at 37◦C for 4 hour. The culture media was then removed and dimethyl sulfoxide (DMSO) was added into each well. The absorbance was then measured at 575 nm. The cell viability was determined by calculating the ratio of optical density (OD) values between the ASA-treated and vehicle-control cells. 2.4 Growth Factors Gene Expression Profiling The gene expression profiling was done using the human growth factors PCR array‡‡. The array profiles 84 growth factor associated genes that correspond to the following: angiogenic growth factors, regulators of apoptosis, cell differentiation and developmental regulators. The PDLSCs were cultured for 24 hour in the absence (experimental control) or presence of 1000 µM ASA, using culture media as described in section 2.1. Total RNA was then isolated using RNA easy Mini kit§§ and the RNA was then reverse-transcribed into cDNAǁǁ , according to manufacturer’s instructions. The cDNA amplification was performed using the SYBR green mastermix¶¶ PCR array kit, using real-time PCR thermalcycler##. The expression values of target genes were normalized using GAPDH gene. For data analysis, the real time PCR software*** was used to calculate the fold change of gene expression between the non-treated and the ASA treated PDLSCs based on the ΔΔCT method. A cutoff cycle threshold (CT) value of 35 was assigned for estimation of fold change. Upregulation was defined as having a fold change of > 2.0. 2.5 Array Validation The PCR array results were validated by quantitative real-time PCR (qRT-PCR). The following genes were selected for the validation assay, with the corresponding reagents set†††: FGF2Hs00266645_m1; FGF9-Hs00181829_m1; BMP2-Hs00154192_m1, FGF7-Hs00940253_m1, VEGFA-Hs00900055_m1; VEGFC-Mm00437310_m1, IL2-Hs00907779_m1; IL4Hs00174122_m1; IL10-Hs00961622_m; BMP10-Hs00205566_m1, BMP3-Hs00609638_m1; 3
Journal of Periodontology; Copyright 2016
DOI: 10.1902/jop.2016.150610
INHBB-Hs00173582_m1; GAPDH- Hs02758991_g1. All determinations were normalized using GAPDH as the reference gene. 2.6 The Effects of ASA on PDLSCs Osteogenic Potential and Proliferation Rate The PDLSCs cells were expanded in culture media (section 2.1) and maintained in 5% CO2 incubator at 37◦C until they reached 70 to 80% confluency. Then, the PDLSCs (n=3) were exposed to ASA [ 0 (control), 10, 200, 500, 1000 μM] in osteogenic media containing 10% FBS, 1% L-alanyl-L-glutamine, 100 nM dexamethasone, 10 mmol/L β-glycerol phosphate and 0.2 mM of ascorbic acid. The cells were cultured for 21 days and the media was changed every 3 days. The MTT assay was conducted at days 0, 3, 7, 17, 17 and 21. The mineralization assay was done to evaluate PDLCs osteogenic potential and the cells were stained with 2% Alizarin Red S (ARS) at the designated days. The Alizarin Red S positive area (ARSA) was analyzed using software‡‡‡ and the result is presented as percentage of ARSA over the total area of the image. 2.7 Statistical Analysis The results are presented as means ± standard deviations (SDs) of at least 3 replicates. Two-way ANOVA with Bonferroni post-test was performed using software§§§. P values of 35) in the treated and untreated cells, suggesting they were expressed only at very low/background levels: BMP3, BMP8B, CLC, CSF2, CSF3, EREG, FGF11, FGF13, FGF14, FGF17, FGF22, FGF6, GDF10, GDF11, IGF2, IL11, IL1A, IL1B, INHBA, JAG2, LEFTY1, LEFTY2, LTBP4, MDK, NDP, NRG2, NRG3, NRTN, OSGIN1, PSPN, SLCO1A2, SPP1, TDGF1, THPO, TYMP. 3.4 Validation of PCR Array Result by qRT-PCR The qRT-PCR results were generally in agreement with the PCR array data. The upregulation of the following genes were confirmed: FGF2, FGF7, FGF9, VEGFA, VEGFC, BMP2, BMP10, IL2, IL4, IL10, INHBB and CSF3 (Table 2). 3.5 Pathway Analyses: Molecular Relationship, Biological Functions and Canonical Pathway Analyses Pathway enrichment analysis¶¶¶ was carried out to identify the plausible association between the upregulated genes with biological functions and canonical pathways. This may provide insights on the biological effect of ASA on PDLSCs. The top 15 significant biological functions (Figure 4A) identified include the following: cellular development, cellular growth and proliferation, cell cycle, connective tissue development and function, cell signaling and organ development. The top 15 canonical pathways identified include the following: fibroblast growth factor (FGF) signaling, integrin-linked kinase (ILK) signaling, regulation of epithelial-mesenchymal transition (EMT) pathway, actin cytoskeleton signaling, VEGF signaling, human embryonic stem cell pluripotency, BMP signaling pathway, TGF-β signaling, as well as role of NANOG in mammalian embryonic stem cell pluripotency (Figure 4B). The molecular interaction analyses¶¶¶ indicated that the genes upregulated by ASA treatment are linked to cellular proliferation and differentiation; development of epithelial tissue and invasion of endothelial cells which have direct interaction to angiogenesis pathway. 3.6 Evaluation of Osteogenic Potential of PDLSCs The results indicated that ASA modulates the proliferative capacity and osteogenic potential of PDLSCs. The MTT assay indicated that 500 µM and 1000 µM ASA treatments induced higher proliferative capacity compared to untreated cells (Figure 2). For the osteogenic potential assay, statistically significant differences (P
