Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the

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Biological and Medical Applications of Materials and Interfaces

Fibrous topography-potentiated canonical Wnt signaling directs the odontoblastic differentiation of dental pulp-derived stem cells Saeed Ur Rahman, Joung-Hwan Oh, Young-Dan Cho, Shin Hye Chung, Gene Lee, Jeong-Hwa Baek, Hyun-Mo Ryoo, and Kyung Mi Woo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19782 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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ACS Applied Materials & Interfaces

Fibrous topography-potentiated canonical Wnt signaling directs the odontoblastic differentiation of dental pulp-derived stem cells Saeed Ur Rahman†, Joung-Hwan Oh†, Young-Dan Cho†, Shin Hye Chung§, Gene Lee †, Jeong-Hwa Baek†‡, Hyun-Mo Ryoo†‡, Kyung Mi Woo†‡

*

† Department of Molecular Genetics, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Republic of Korea § Department of Dental Biomaterials Science, School of Dentistry, Seoul National University, Republic of Korea ‡ Department of Pharmacology & Dental Therapeutics, School of Dentistry, Seoul National University, Republic of Korea

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ABSTRACT Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms which underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, of which levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of β-catenin protein and enhanced its trans-location to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The β-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in-vivo. Taken together, these results demonstrate the nano-fibrous engineered matrix strongly supports odontoblastic


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differentiation of DP-MSCs by enhancing Wnt/β-catenin signaling.

Keywords: Nano-fibrous engineered matrix, dental pulp mesenchymal stem cells, odontoblast, Wnt3a, canonical Wnt/ β-catenin signaling, DSPP



■ INTRODUCTION The extracellular matrix (ECM) formed by its proper cells provides the mechanical, physical, and biochemical cues for cells to control their behaviors. Scaffolds as an artificial ECM would perform vital roles in facilitating cellular behavior and function in tissue regeneration. It has been reported that the performance of scaffolds is largely affected by their characteristics including chemical composition, mechanical stiffness or elasticity, and topography. Considerable efforts have been directed toward developing scaffolds which represent the fibrous architecture of the ECM, since a synthetic nanofibrous matrix might perform a structural role of collagen fibrils and provide a beneficial microenvironment for cells.1-4 Accumulating evidence indicates that engineered nanofibrous matrices can modulate cell responses leading to tissue regenerations for nerves, blood vessels, skin, cartilage, bone, and dentin.5-10 However, the mechanism by which the engineered nanofibrous matrix expedites the tissue regeneration remains unclear. Adult mesenchymal stem cells (MSCs) have been considerably investigated for applications in tissue engineering. MSCs can be separated from bone marrow (BM), adipose tissue, dental pulp tissue (DP), and other tissues.11-13 MSCs are multipotent, which can be differentiated into a specific lineage of cells according to their microenvironments, and the differentiation capacity tends to be distinctive according to the originating tissues. DP-MSCs residing in dental pulps have similar characteristics to BM-MSCs.14 However, differentially expressed genes between DP-MSCs and BM-MSCs have been reported.15 The DP-MSCs are more prone to differentiate into odontoblasts than osteoblasts in experimental culture models compared with BM-MSCs.16,17 Though, studies on dentin regeneration have shown that the regenerative procedures more likely form bone-like mineralized tissues rather than dentin.18,19 In previous studies, we have investigated on nanofibrous scaffolding for bone and dentin


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regeneration. The nanofibrous topography promotes differentiation into osteoblasts or odontoblasts, according to their environments, and biomineralization both in vitro and in vivo.10,20,21 Of note, the in vivo experiments, in which the scaffolds alone have been implanted without cells and bioactive molecules, have shown the formation of bone or dentin in certain regions apart from the adjacent pre-existing bone or dentin tissues also. These features suggest that nanofibrous scaffolds may have a differentiation-facilitating effect on MSCs. In support of our observations, it has been reported that an engineered three dimensional (3D) ECM scaffold induced the differentiation of DP-MSCs without any exogenous growth factors.22,23 It is known well that growth factors can exert robust and accurate effects in cellular microenvironments and stimulate the differentiation of MSCs into cells of specific lineages and support their terminal differentiation. Based on the literature and our observations, it was hypothesized in this study that the nanofibrous engineered matrix mediates the expression of signaling molecules (growth factors) specifically and leads to the differentiation of the MSCs into cells of specific linages. The cell signaling pathways associated with growth factors such as bone morphogenetic proteins (BMPs) and Wnt ligands regulate the processes of tooth development in a precisely regulated manner.24, The BMP signals are required in early tooth development from the lamina to the bud stage.26-29 The developing teeth express BMPs in the epithelium and mesenchyme.30 BMP2-mutant mice exhibit defects in the tooth formation. Odontoblasts in BMP-2 knockout mice appear of immature morphology and fail to produce dentin properly.28 Tooth development is arrested at early stages when Noggin, a BMP signaling inhibitor, is over-expressed.26 Treatment of MSCs with BMP4 or BMP7 leads to odontoblast differentiation and a robustly increased expression of DSPP, a major marker molecule of odontoblast differentiation.31,32 Wnt signaling is considered essential in tooth development.



The developmental dental organ broadly expresses Wnt genes.33 The β-catenin has appeared in the nuclei and the reporter activities under the Wnt/β-catenin control have been increased, indicating that activations of the Wnt/β-catenin signaling have been involved in tooth developments.25,33 Functional significances of the canonical Wnt/β-catenin signaling have been extensively investigated. The β-catenin has been intensely expressed and requisite for developing root dentin. The β-catenin-null mice showing a disturbance in its signaling arrest the root development, and the heterozygote mutant mice exhibit aberrant tooth phenotypes.33,34 Deleting LEF1 or forced expressing DKK1 halts the morphogenesis of dental organ.35,36 Overexpression of LEF1 accelerates the differentiation of dental pulp cells into odontoblasts.37 Constitutive active β-catenin has yielded multiple teeth in a transplantation study.38 Despite these developmental reports supporting the role of Wnt signaling in dentinogenesis, there are conflicting reports. A study has shown Wnt-1 induces β-catenin that inhibits alkaline phosphatase (ALP) activity39 thereby suggesting a negative modulation of odontoblast differentiation by canonical Wnt signaling. Moreover, it has also been reported that Wnt10a null mice do not exhibit the significantly altered dentin formation and odontoblast differentiation.40 This study was performed under the hypothesis that an engineered nanofibrous matrix mediates the expression of signaling molecules specifically leading to the differentiation of DP-MSCs into the odontoblast phenotypes and focused on the expression of the odontoblast phenotype marker DSPP. Considering a conventional tissue culture dish (TCD) is made of polystyrene (PS) basically, a PS fiber (PSF) matrix was prepared by electrospinning as previously described,41 and the responses of the DP-MSCs to the PSF matrix were compared with those to the TCD. First, the expressions of various molecules in the DP-MSCs cultured on the PSF matrix were verified including the Wnt and BMP ligands, their physiological


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inhibitors, and odontoblast/osteoblast differentiation markers. The responsiveness of the cells on the PSF matrix to exogenous recombinant BMP2 or Wnt3a proteins was monitored. The effects of the PSF-enhanced Wnt3a production and the subsequent Wnt/β-catenin signaling on DSPP expressions were investigated. Furthermore, using the PSF-enhanced odontoblast differentiation model, it was investigated whether the DSPP transcription was directly controlled by Wnt/β-catenin signaling. Lastly, the association of the PSF-induced Wnt3a with the DSPP expression was confirmed in vivo by transplantation into the subcutaneous tissue of nude mice. ■ EXPERIMENTAL SECTION Preparation of PSF engineered matrix. The nano-fibrous engineered PSF matrix was prepared by electrospinning as previously described.41 Briefly, PS (Sigma, St Louis, USA) was homogeneously dissolved in N,N-Dimethylformamide (DMF, Sigma) (12% w/v) at room temperature. For the electrospinning, the solution was put in a syringe with a 0.2 mm inner diameter metallic needle and run at 0.5 mL/h. A high voltage of 30kV was applied (NanoNC, Seoul, Korea). The fibers were collected on a rotating metal drum covered with aluminum foil. An electrospun PSF sheet was put over a petri dish and fixed. Before using in culture, the PSF matrix was placed in the sterile 70% ethanol and then distilled water. To make PS-solid used in a transplantation experiment, PSF was placed into incubator at 60℃ until the smooth solid films (PS-solid) were formed. The PS-solid was air-dried. Surface topography analysis. The surface topography of the PSF matrix was analyzed as previously described.41 Morphology with or without cells was examined under a scanning electron microscope (SEM; S-4700, Hitachi, Tokyo, Japan). Surface roughness and topographical measurements were determined under a confocal microscope (Carl Zeiss LSM5 Pascal, Oberkochen, Germany). Tensile strength was measured with a universal testing



machine (Lloyd LF Plus, Lloyd Instruments, Fareham, UK), while the tensile force was applied at a speed of 5 mm/min until rupture. Four specimens in each group were tested, and the elastic moduli were calculated. The contact angle was measured by the sessile drop method with a video camera-equipped image analyzer (Phoenix 300, Surface electro optics, Seoul, Korea). Cell culture. Human DP-MSCs were isolated from dental pulp tissues from patients (IRB authorization number S-D20070004). Both the DP-MSCs and MDPC-23 pre-odontoblasts were maintained in a basal medium [BM; DMEM (Hyclone, Logan, USA) with 10% (v/v) fetal bovine serum (FBS, Hyclone) and 100 U/mL of penicillin/streptomycin (Hyclone)] at 37 °C in a humidified incubator with 5% CO2. For the odontoblastic differentiation, a differentiation medium [DM; the BM with 50 µg/mL ascorbic acid (Sigma) and 10 mM βglycerophosphate (Sigma)] was used. Before starting the actual experiments, the primary DPMSCs were expanded, and the aliquots were stored at passage 3. For the actual each experiment, the cells at the passage of 5 or 6 were used and the seeding density was 5×103 cells/cm2. Adhesion assay. DNA contents in the cells adhered for 15 h were measured using Quant-iT Picogreen reagent (Invitrogen, Grand Island, USA) according to manufacturer’s instruction. The fluorescence was read using Fluostar Optima (BMG Labtech Gmbh, Ortenberg, Germany). Quantitative real-time PCR (qPCR). The qPCRs were performed from total RNAs which were extracted using RNAiso Plus reagents (Takara, Tokyo, Japan). Synthesis of cDNA using PrimeScript™ RT reagent kit (Takara) was followed by qPCRs. Amplifications were performed and analyzed in a Real-time PCR system (Applied Biosystems, USA). GAPDH


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was used for the normalization. The primers sequences are listed in Supporting Information (Table S1, S2). ALP activity. The cells cultured for 4 days were homogenized in distilled water. The activity was estimated with p-nitrophenyl phosphate (p-NPP, Sigma) as a substrate in 0.1 M glycine-NaOH (pH 10.3). Western blot analysis. Cells were lysed using a buffer [12.5 mM Tris-HCl (pH6.8, Biosesang, Suwon, Korea), 20% glycerol (Sigma), 4% SDS (Sigma), 10% mercaptoethanol (Biosesang), and a complete protease inhibitor cocktail solution (Sigma)]. Proteins in the lysate were fractionated in 10-12% SDS-PAGE and transferred onto PVDF membrane. Blocking with 5% non-fat skim milk in TBST buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) was followed by the incubations with specific primary antibodies and then HRP-conjugated secondary antibodies. The membrane was treated with an ECL reagent (iNtron Biotechnology, Sungnam, Korea) and read using a Bio-Image Analyzer (Bio-Rad, Hercules, USA). β-actin was used as a loading control. TOP flash luciferase reporter assay. Cells cultured for 2 days were transfected with βcatenin/TCF luciferase reporter (TOP flash) or mutant binding site (FOP flash) constructs using a Genefectine reagent (Genetrone Biotech, Qwangmyeong, Korea). The Renilla luciferase reporter (pRL-TK) was used as an internal control. The firefly luciferase activity after 2 days of transfection was measured in the cell lysates using Dual-Glo Luciferase Lysis Buffer (Promega, Madison, USA). The Renilla luciferase activity was also measured and used in normalization. Immunofluorescence assay. The cultured cells were subjected to fixation with 4% paraformaldehyde, permeabilization with PBS containing 0.1% Triton X-100 (PBS-T), and



blocking in PBS-T with 5% BSA. Then, the cells were incubated with anti-Wnt3a, DSP, or βcatenin antibodies, followed by Alexa Fluor 488 conjugated-goat anti-rabbit secondary antibody (Invitrogen, Grand Island, USA). Rhodamine phalloidin (Invitrogen) was added to visualize the actin. A mounting medium with DAPI (Vector labs, Burlingame, USA) was used to identify nuclei. The samples were examined using a confocal microscope (LSM 700, Carl Zeiss). The image analyses were performed using the ImageJ (NIH) software. Knockdown with siRNAs. Scrambled pooled siRNA against Wnt3a or β-catenin (Dharmacon, Lafayette, USA) were used to knockdown their expression according to the manufacturer’s instruction. The cells cultured for 2 days were transfected with specific siRNA and analyzed the expression of RNA transcripts and proteins after additional 2 days. DSPP promoter assay and site-directed mutagenesis. The DSPP promoter deletion constructs were prepared and analyzed as previously described.42 Site-directed mutagenesis to mutate the β-catenin binding sites on the DSPP promoter was performed. The M-624 and M-435 constructs were generated by site-directed mutagenesis using D-624 and D-435 constructs as a template. Mutant oligonucleotides designated for specific sites (mutated bases in lowercase) are listed as L1 and L2 in Supporting Information (Table S3). The constructs or an empty pGL-3 basic plasmid was transfected with pRL-TK vector. The luciferase reporter activity was measured as described above in TOP flash assay. Chromatin immunoprecipitation (ChIP) assay. The ChIP assay was performed as previously described.43 The cultured cells were fixed with 1% formaldehyde, swollen using a swelling buffer with protease inhibitors (Sigma) on ice, and homogenized. The pellets were incubated in a nuclei lysis buffer [1% SDS, 10 mM EDTA, and 50 mM Tris-Cl (pH 8.0), and protease inhibitors] and sonicated to break the DNA into fragments. Samples diluted in a ChIP dilution buffer (1% Triton X-100, 150 mM NaCl, 2 mM EDTA pH 8.0, 20 mM Tris pH ACS Paragon Plus Environment

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8.0, and protease inhibitors) were immunoprecipitated using protein G beads and anti βcatenin antibody. The precipitates were incubated in a 1% SDS in TE at 65℃. After the treatment with Proteinase K, DNA fragments were extracted and purified with a DNA Clean & Concentrator (Takara). The PCRs were performed for all three regions using the primers as shown in Supporting Information (Table S4). Transplantation. All the procedures were approved by the Institute of Laboratory Animal Resources at Seoul National University (authorization number SNU-121210-5). The MDPC23 pre-odontoblasts seeded on PSF and PS-solid were cultured for 12 h prior to transplantation. Nude mice (5-week-old male) sedated with isoflurane (Haha Pharm, Seoul, Korea) were scrubbed with Povidone. Incision was made on the back skin and the cells-PSF or -PS solid were transplanted into the dorsal subcutaneous space. The wound was closed with clips. After 8 days, the transplants were retrieved, fixed in 4% paraformaldehyde, and subjected to the histology. The paraffin section with 4 µm thickness was used for H-E staining or immunostaining analyses. For Immunostaining, sections were pre-treated with 0.3% H2O2 after de-paraffinization. The sections were incubated with 1% BSA in PBS and then with the primary antibodies specific for DSP or Wnt3a in 1% BSA. For negative control IgG1 were used. It was followed by secondary biotinylated anti-rabbit IgG. The sections were then treated with Vectastain ABC reagent, mounted and observed. The image analyses were performed using the ImageJ (NIH) software. Statistical analysis. All the experiments in this study were repeated. Representative data are presented with averages and standard deviations. Statistically significant differences were analyzed using ANOVA (p

Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the

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