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Effect of inducible bone morphogenetic protein 2 expression on the osteogenic differentiation of dental pulp stem cells in vitro
Bone 132 (2020) 115214
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Full Length Article
Effect of inducible bone morphogenetic protein 2 expression on the osteogenic differentiation of dental pulp stem cells in vitro
T
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Ferenc Tótha, , József M. Gállb, József Tőzsérc, Csaba Hegedűsa a
Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, Debrecen, Hungary Department of Applied Mathematics and Probability Theory, Faculty of Informatics, University of Debrecen, Debrecen, Hungary c Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone morphogenetic protein 2 Gene therapy Osteogenic differentiation Noggin Tet-on
Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily, it is known to be a factor involved in skeletal development and capable of inducing in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) isolated from extracted third molar teeth are an ideal resource for bone tissue engineering and regeneration applications, due to their convenient isolation, safe cryopreservation, and easy maintenance in cell cultures. The aims of this study were to deliver BMP-2 under control of the tetracycline-inducible (tet-on) promoter into dental pulp stem cells and to examine whether these BMP-2 expressing cell lines are capable of promoting osteogenic differentiation in vitro. BMP-2 gene was cloned into the lentiviral transfer plasmid pTet-IRES-EGFP and used to establish the DPSC-BMP-2 cell line. DPSC, DPSCGFP (mock) and DPSC-BMP-2 cell lines were cultured in growth medium or osteogenic medium in the presence or absence of 100 ng/ml doxycycline. To assess differentiation, alkaline phosphatase activity, calcium accumulation and gene transcription levels of different genes involved in osteogenic differentiation (BMP-2, Runx2, alkaline phosphatase, and noggin) were measured. Doxycycline-induced BMP-2 expression induced the differentiation of DPSCs into the preosteoblastic stage but could not favor the further maturation into osteoblasts and osteocytes. We found that while Runx2 gene transcription was continuously upregulated in doxycycline-treated DPSC-BMP-2 cells, the alkaline phosphatase activity and the accumulation of minerals were reduced. As a result of the increased BMP-2 expression, the transcription level of the BMP antagonist noggin was also upregulated, and probably caused the observed effects regarding alkaline phosphatase (ALP) activity and mineral deposition. Our study shows that this system is effective in controlling transgene expression in DPSC cell line. Exploration of all known factors affecting osteogenic differentiation and their interactions is of major importance for the field of regenerative medicine. As the metabolic reaction to the upregulated transgene transcription appears to be cell line-specific, a wrongly selected target gene and/or regulation system could have adverse effects on differentiation.
1. Introduction Regulation of transgene expression in gene therapy is quite often necessary [1,2] in regards to controlling timing, duration, and level of expression. Moreover, the appearance of cell-based therapies in tissue engineering and regenerative medicine, relying on regulated expression is crucial for successful applications [3–6]. This controllable expression is a very important part of stem cell-based bone tissue engineering, in order to avoid possible side effects of uncontrollable gene expression of osteogenic differentiation factors [7–9] for in vivo bone formation and
regeneration. The tetracycline-induced (tet-on) systems, first described by Gossen and Bujardt [10,11] are widely used and well-characterized for regulating transgene expression. This system can be induced by the binding of a tetracycline-dependent transactivator provided by adding the inducer and stopped by withdrawing it. Tetracycline-regulated gene expression is already used for the expression of genes involved in bone formation [9,12–14]. Human dental pulp stem cells (DPSCs) isolated from third molar teeth [15,16] are adult stem cells showing mesenchymal stem cell properties [15]. DPSCs can be induced to differentiate into multiple
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Corresponding author at: Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, H-4002 Debrecen, P.O. Box 400, Hungary. E-mail addresses: [email protected] (F. Tóth), [email protected] (J.M. Gáll), [email protected] (J. Tőzsér), [email protected] (C. Hegedűs). https://doi.org/10.1016/j.bone.2019.115214 Received 25 September 2019; Received in revised form 17 December 2019; Accepted 23 December 2019 Available online 26 December 2019 8756-3282/ © 2019 Published by Elsevier Inc.
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transformed into One Shot Stbl3 E. coli strain (Invitrogen, Carlsbad, CA, USA) and new transformants with BMP-2 gene in the pTet-IRES-EGFP plasmid were verified by DNA sequencing (data not shown). The resultant recombination vector was referred to as pTet-IRES-EGFP-BMP2.
connective-tissue cell lineages, including the osteogenic/odontogenic phenotype [17–19]. Furthermore, DPSCs are highly proliferative and can be safely cryopreserved [20]. Considering their characteristics along with their shorter differentiation path [21]; compared to embryonic stem cells or differentiated cell types; they are a very promising source for regenerative medicine and tissue engineering applications [22,23]. Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily. Many of the family members are involved in skeletal development, regeneration, and repair [24] BMP-2 has been known to be a factor capable of inducing in vitro osteogenic/ odontogenic differentiation [5,25–28] and in vivo bone formation [29–31]. Along with another family member; the bone morphogenetic protein 7, its use is also approved by the American Food and Drug Administration for human clinical application. These recombinant proteins are recommended for the treatment of tibial nonunions, bone fractures, and spine fusion [32–34]. However, as the generally applied doses are much higher than the physiological concentrations, the possibility of an inflammatory response is also a possible threat [35,36], reflecting the need for properly orchestrated delivery [34,36] including gene therapy applications [37]. The effect of the BMP-2 on the osteogenic/odontogenic differentiation of dental pulp cells from different sources showed promising results [28,38–40] in vitro and in vivo as well. However, the effect of stable BMP-2 gene transfer and regulated transgene expression on DPSCs is not yet investigated. In the present study, we have established a DPSC cell line capable of tet-inducible BMP-2 transgene expression, where this expression is activated by the presence of doxycycline. We have used two lentiviral vectors for stable gene transfer, one vector encodes the transgene BMP2 and green fluorescent protein (GFP) separated by internal ribosome entry site, while the other vector encodes the reverse transactivator protein that controls transcription of the transgene. Thereafter; using this cell line, we have investigated the effect of induced BMP-2 transgene expression on osteogenic differentiation.
2.3. Isolation and cell culture DPSCs were isolated from third molar teeth that had been extracted at the Faculty of Dentistry, University of Debrecen, with the modification of a previously described protocol [19]. Briefly, the tooth was cut around by sterile dental fissure burs to expose the pulp chamber, and the pulp tissue was removed from the crown and root. The pulp tissue was then digested in a solution of collagenase type I (3 mg/ml, Sigma Aldrich, St Louis, MO, USA) and dispase (4 mg/ml, Gibco, Waltham, MA, USA) for 1 h at 37 °C. Cell suspension was seeded into 6-well plates (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM F12 (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% Glutamax (Gibco) and 1% Antibiotic-Antimycotic (Gibco), and incubated at 37 °C in 5% CO2. The cells were cultured in the same medium. This medium is referred to in the text as control medium (CM). After the third passage, cells were seeded into 6-well plates and used for transduction. After transduction and antibiotic selection, the mixture of GFPpositive and GFP-negative cells were seeded into 24 well plates and cultured in different media. The culture medium was refreshed 3 times a week. Osteoinductive medium (OM) was prepared by supplementing CM with 10 mM β-glycerolphosphate (Sigma Aldrich, St Louis, MO, USA), 50 μg/ml ascorbic acid (Sigma Aldrich), 0.1 μM dexamethasone (Sigma Aldrich) and 50 nM vitamin D3 (Sigma Aldrich). Media for inducing transgene expression were supplemented by 100 ng/ml doxycycline (Sigma) (indicated as CM+ and OM+ respectively).
2. Materials and methods 2.4. Lentivirus preparation and transduction 2.1. Ethics statement Viral particles were produced by transient transfection of 293FT cells, maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), using polyethyleneimine (Sigma). Cells were grown to approximately 70% confluence and a total of 16 μg plasmid was used for the transfection of cells in T75 flasks: 8 μg pTet-IRES-EGFP, pTet-IRES-EGFP-BMP-2 or pLenti CMV rtTA3 Blast (w756-1) (Addgene plasmid 26429). pLenti CMV rtTA3 Blast (w756-1) was a gift from Eric Campeau (transfer vector plasmid), 6 μg psPAX2, 2 μg pMD2.G and 13 μg salmon sperm DNA (Sigma Aldrich), in DMEM containing 1% FBS. The medium was replaced after 6 h and the conditioned media containing virus particles were collected after 3 days, clarified by centrifugation, and was filtered through 0.45 μm polyvinylidene fluoride (PVDF) filter (Millipore, Billerica, MA, USA), then stored in −70 °C. For transduction, 500 μl pLenti CMV rtTA3 Blast (w756-1) and 500 μl pTet-IRES-EGFP-BMP-2 or pTet-IRES-EGFP (mock) virus and 1 ml fresh medium mixed with 8 μg/ml polybrene (Sigma Aldrich) in 6well plates containing DPSC cells at 50% confluency. After overnight incubation, the medium was changed. Medium containing 5 μg/ml blasticidin (Thermo Fisher Scientific) was added to the cells at passage one. After antibiotic selection, GFP expression was induced by the addition of 100 ng/ml doxycycline, and the cells were examined under fluorescence microscopy (Zeiss Axiovert 100, Carl Zeiss Microscopy, Jena, Germany) and by flow cytometry (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) to confirm the efficiency of transduction. DPSC cells transduced by pTet-IRES-EGFP-BMP-2 or pTetIRES-EGFP containing virus were referred to as DPSC-BMP-2 and mock respectively.
Human dental pulp tissue collection and subsequent gene transfer performed in this study were approved by the Ethical Committee of the Hungarian Medical Research Council (approval number: ETT TUKEB 49849-3/2016/EKU). For the use of tissue samples, written information consent was obtained from all human subjects who participated in the investigation. 2.2. Construction of pTet-IRES-EGFP-BMP-2 plasmid BMP-2 complementary DNA was synthesized from 1 μg of total RNA of human 293T cell line using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. BMP-2 coding region of the cDNA was amplified by polymerase chain reaction by using Q5 high fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA) and the following set of primers: 5′-CTAAAGGATCCATGGTGGCCGGGACCCG CTG-3′ (forward) and 5′-GATTTGGATCCCTAGCGACACCCACAACCCT CCAC-3′ (reverse). The forward and reverse primers harbored a recognition site for BamHI (underlined). PCR was performed under the following conditions: 30 s at 93 °C (pre-denaturation), 35 cycles of 10 s at 93 °C (denaturation) and 1 min 50 s at 72 °C (extension), followed by 2 min at 72 °C. After purification, the PCR products were cleaned by Monarch PCR & DNA Cleanup Kit (New England Biolabs) according to the manufacturer's instruction. The cleaned product was digested with BamHI and ligated into the pTet-IRES-EGFP plasmid [41] at the corresponding restriction site. The plasmid pTet-IRES-EGFP was a gift from Maria Lung (Addgene plasmid # 64238). The ligation mixture was then 2
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Fig. 1. Tet-on system in hDPSCs. (A) Differential GFP expression by treatment with a range of doxycycline concentrations (3.125, 6.25, 12.5, 25, 50, 100 ng/ml) in the DPSC-BMP-2 cell line. (B) The percentages of GFP-positive cells after treatment of doxycycline followed by Harmony 4.8 software or (C) FACS analysis (n = 3). Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurement (P ˂ 0.05). (D) Representative western blot showing BMP-2 expressions after treatment with different concentrations of doxycycline.
2.5. Fluorescent microscopy and fluorescence-activated cell sorting (FACS) analyses
removed from the wells, and total RNA was isolated using Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) with on-column DNAse digestion. The concentration of the RNA was determined based on their A260 by a Nanodrop equipment. 1 μg total RNA was used per sample for cDNA synthesis, using random primers (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Genes of interest were detected using Applied Biosystems Taqman Assays: ALP (Hs01029144_m1), BMP-2 (Hs00154192_m1), Noggin (Hs00271352_s1), Runx2 (Hs00231692_m1), and 5× HOT FIREPol Probe qPCR Mix Plus (no ROX) (Solis BioDyne, Tartu, Estonia). Results were normalized to the reference housekeeping gene GAPDH (glycerolaldehyde-3-phosphate dehydrogenase, Hs02758991_g1) in each sample.
4 × 104 cells/well were seeded into a 24 well plate and cultured for 7 days in media containing 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml doxycycline. After 7 days nuclei of the cells were stained with 1:1000 DRAQ5 (Biolegend, San Diego, CA, USA) the cells were examined under fluorescence microscopy by an Opera Phenix™ High Content Screening System (Perkin Elmer, Waltham, MA, USA) and analysed using the Harmony 4.8 software (Perkin Elmer). For flow cytometry analyses (BD FACSCalibur, BD Biosciences) the cells were scraped and washed by PBS, then counted to determine the number of GFP-positive cells. Data were evaluated by FlowJo flow cytometry analysis software. 2.6. BMP-2 immunoblot
2.8. Detection of alkaline phosphatase (ALP) activity
2 × 105 cells were seeded to petri dishes and cultured for 6 days in media containing 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml doxycycline. After 6 days, protein transport from the endoplasmic reticulum to the Golgi apparatus was inhibited by the addition of 10 μg/ml Brefeldin A (Tocris Bioscience, Tocris, United Kingdom). After an additional day the cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% triton-x 100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl and 1% protease inhibitor cocktail, pH 8.0). The protein concentration was determined by Pierce BCA Protein Assay (Thermo Scientific), then 20 μg lysates from each sample was resolved by 10% TGX Stain-Free FastCast acrylamide gel (Bio-rad Laboratories, Hercules, CA, USA) and analysed by western blot using Human BMP-2 Antibody (R&D Systems, Minneapolis, MN, USA) and Anti-GAPDH Antibody (Lifespan Biosciences, Seattle, WA, USA) respectively.
Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. At day 7 and 21 media were removed from the wells, and the cells were lysed by ALP lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM Triton-X 100, 1% PMSF and 1% protease inhibitor cocktail, pH 7.4). Cell lysates were centrifuged for 10 min at 16,000g at 4C° to remove cellular debris. 30 μl of lysate supernatant was added to each well of 96-well plate and then 100 μl of pNitrophenyl Phosphate Liquid Substrate System (Sigma Aldrich, St. Louis, MO, USA) was added to lysates. The absorbance was measured in room temperature at 405 nm by HIDEX Sense microplate reader (HIDEX, Turku, Finland), immediately and after 15 min and ALP activity (absorbance change) was determined by the subtraction of the absorbance measured immediately from the absorbance measured after 15 min of the addition of the substrate. The total protein concentration of lysate supernatants was determined by BCA Protein Assay Kit (Thermo Scientific) and ALP activity was normalized to total protein concentration.
2.7. Quantitative real-time PCR Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. At day 7 and 21 medium were 3
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Fig. 2. Representative gene expression profile of BMP-2 gene in case of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) cell lines cultured for 7 days (A) and 21 days (B) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
2.9. Mineralization assay
3. Results
Mineralization of DPSCs was observed by alizarin red S (Sigma Aldrich) staining. Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. After 21 days of culture with different media (CM, OM, CM+, and OM+), the cells were washed with PBS, fixed with 4% paraformaldehyde, stained with 2% alizarin red S solution (pH 4.2) at room temperature in the dark for 45 mins. The stained cultures were washed with distilled water, then airdried. Stained cultures were examined under the microscope (Olympus SZ61, Olympus Corporation, Shinjuku, Tokyo, Japan), and images were acquired. For quantification of the staining, alizarin red S were extracted with 10% cetylpyridinium chloride (Sigma Aldrich) solution and absorbance was measured by a Hidex Sense Microplate reader at 570 nm.
3.1. Doxycycline-sensitive transgene expression pTet-IRES-EGFP-BMP-2 and pLenti CMV rtTA3 Blast (w756-1) plasmid were used to develop an inducible GFP and BMP-2 expression in the DPSC cell line. At first, we used GFP expression to verify successful gene transfer and inducible transgene expression. The tet-on system showed negligible doxycycline dose-dependent transgene expression indicated only by the GFP expression in the cells (Fig. 1A). The maximum percentage of GFP expressing cells observed was 29.3% if determined by the Harmony 4.8 software (Fig. 1B) and 72.8% if determined by FACS (Fig. 1C) when the cells were treated with 100 ng/ml doxycycline. Higher concentrations of doxycycline did not cause a further increase in the number of GFP-positive cells (data not shown). The BMP-2 expression does not show dose-dependency to doxycycline concentration (Fig. 1D), although the addition of doxycycline increased the amount of the expressed protein even at the lowest concentration (3.125 ng/ml).
2.10. Statistics The independent sample t-test and the Welch t-test were used to compare the means of different pairs of populations (e.g. two media or cell lines), depending on the equality of variances. The latter condition was tested by the F test for variances. Error bars on the figures represent the estimation of the standard deviation calculated from three parallel measurements.
3.2. The doxycycline-induced BMP-2 gene expression has upregulated the expression of BMP antagonist noggin, and runt-related expression factor 2, while downregulated the transcription of the alkaline phosphatase gene Gene expression of proteins involved in osteogenic differentiation was examined after 7 and 21 days of culture. After 7 days there are no significant differences between the BMP-2 expression of cell lines 4
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Fig. 3. Representative gene expression profile of ALP, Noggin, and Runx2 genes in case of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSCBMP-2) cell lines cultured for 7 days (above) and 21 days (below) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
and DPSC-BMP-2 cell lines, while in CM+ only the DPSC-BMP-2 cell line showed a significant downregulation compared to the control cell line. We have observed a significant upregulation of the ALP gene expression in control cells incubated in CM+ compared to those were incubated in CM. The same treatment of medium (addition of doxycycline) downregulated the ALP activity of the DPSC-BMP-2 cell line cultured in CM+, while upregulated in OM+ compared to the results measured in CM and OM, respectively (Fig. 3).
cultured in CM or OM (Fig. 2A), although we observed a dramatic upregulation in case of the DPSC-BMP-2 cell line compared to the control cell line and to the expression measured in CM or OM, respectively. We have also observed a slight, but significant downregulation in the mock cell line in CM+ and OM+ medium compared to the control. Similar trends were noticed for the expression of noggin (Fig. 3) in the DPSC-BMP-2 cell line, but not in the mock. The expression of runt-related transcription factor (Runx2) was upregulated in the DPSC-BMP-2 cell line cultured in OM+ media (Fig. 3) compared to the control cell line, and in the mock cell line in CM+ compared to the ones grown in CM. On the contrary, in the DPSC-BMP-2 cell line, ALP gene expression (Fig. 3) was significantly downregulated when incubated in CM+ or OM+ media compared to the control cell line and to those, which were incubated in CM or OM respectively. After 21 days of treatment, expression of BMP-2 (Fig. 2B) in the DPSC-BMP-2 cell line cultured in CM+ and OM+ was still persuasively upregulated compared to the control cell line and to the expression measured in CM or OM, respectively. Also, in the DPSC-BMP-2 cell line, a less prominent, but still significant upregulation were observed in CM and OM as well compared to the control cell line. Moreover, a slight downregulation was noticed in the mock cell line compared to that of the control, when cultured in OM or OM+. The DPSC-BMP-2 cells showed upregulated noggin expression in all media compared to the control cell line, but in case of the OM+ medium it was not a significant change (Fig. 3). Also, significant upregulation was observed with the mock when incubated in CM, compared to the control. We have also noticed, that the DPSC-BMP-2 cell line cultured in OM or OM+ media still showed upregulated Runx2 expression compared to the control, but in OM+ medium this upregulation was not significant (Fig. 3). In CM, the gene expression of ALP was upregulated in both the mock
3.3. Doxycycline-induced BMP-2 expression has a negative effect on the activity of alkaline phosphatase Alkaline phosphatase (ALP) activity of cells was measured after 7 and 21 days of incubation. After 7 days, a significant increase was observed in the ALP activity of the control cells grown in CM+ and OM+ compared to CM and OM, respectively. In CM+ and OM+ significant reduction was noticed in the ALP activity of the DPSC-BMP-2 cell line compared to the control cell line and to CM and OM respectively (Fig. 4A). Contrarily, ALP activity of this DPSC-BMP-2 cell line was significantly increased both in CM and OM compared to the control. The ALP activity of mock cells cultured in OM was also increased compared to that of the control cell line. It should be noted that while the ALP activity is at least slightly increased in almost all cell lines and types of media after 21 days of culture (Fig. 4B) compared to the results measured after 7 days, the activity of the DPSC-BMP-2 cell line was still significantly reduced in CM+ medium compared to the activity measured in CM medium and to the control cell line in CM+. Surprisingly, the ALP activity in OM+ was restored in the DPSC-BMP-2 cell line both when compared to the control cell line or to the DPSC-BMP-2 cells grown in OM, respectively.
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line capable of induced (tet-on) expression of BMP-2, using lentivirusmediated stable gene transfer. We have demonstrated a negligible dosedependent control of GFP transgene expression with doxycycline concentration but found no dose-dependency in the case of BMP-2 transgene expression. The effect of inducible BMP-2 expression on the cells was examined in culture media with the presence (CM+, OM+) or absence (CM, OM) of the inductive agent doxycycline. Investigation of the effect of doxycycline induction on osteogenic differentiation showed that while doxycycline had no impact on ALP activity of the DPSC and mock cell lines, as ALP activity only increased if cultured in OM, which was in line with our expectations. It was significantly reduced in the DPSC-BMP-2 cell line after 7 days in both CM+ and OM+. Furthermore, this effect lasted until 21 days in CM+ but not in OM+. As the CM+ and OM+ media consists of the same ingredients (except for β-glycerophosphate, ascorbic acid, dexamethasone, and vitamin D3 in OM+) the reduced ALP activity observed in OM+ after 7 days of culture were probably corrected by the effect of the additional materials in the basic OM. Examination of calcium deposits revealed that the addition of doxycycline resulted only in a slight reduction of calcium accumulation, notably only in the case of the DPSC-BMP-2 cell line cultured in CM+. In addition, we have observed that cell lines transduced by lentiviral particles; both in OM and OM+, accumulated less calcium compared to the control DPSC cell line. This reduction in the calcium deposition probably caused by the lentiviral transduction and use of polybrene [43], which reportedly can affect proliferation or differentiation potential of the MSCs. The study of osteogenic marker genes showed that BMP-2 expression of DPSC-BMP-2 was dramatically increased in CM+ and OM+, compared to the control cell line, it came along with the increased expression of BMP antagonist noggin. As noggin is able to bind and sequester BMPs [44] and has an effect on differentiation and fate of osteoblasts [45]; including the development of a fracture non-union [46], it may, therefore, have caused the reduced ALP activity and mineralization observed in this cell line in CM+ and OM+. When studying the expression of Runx2, we have not found significant changes in any cell lines or medium compared to the other ones after 7 days, with only one exception. In the case of the DPSC-BMP-2 cell line, the expression of Runx2 was upregulated in OM+ compared to other media. This upregulation in OM+ was detectable after 21 days of culture as well. Increased level of Runx2 induces the differentiation of mesenchymal pluripotent cells into preosteoblasts and immature osteoblasts, while inhibiting osteoblast maturation [47], therefore it can be hypothesized that continuous Runx2 upregulation may contribute to the effect of noggin, and result in reduced ALP activity and mineralization observed in the DPSC-BMP-2 cell line when BMP-2 expression is induced. Studies regarding doxycycline addition revealed reduced endogenous levels of BMP-2 in osteoprogenitor cells [48], probably through inhibition of TGF-β signaling [49], we, however, have not observed this effect in the DPSC or mock cell line. BMP-2 transgene expression controlled by a doxycycline-repressible promoter in C3H10T1/2 mesenchymal stem cells had a positive effect on osteogenesis and chondrogenesis [9,50]. Moreover, another study revealed that doxycycline counteracts BMP-2 induced osteogenic mediators [51], suggesting that proper choice of inducing agent and/or system for controlling expression is also crucial for a successful application.
Fig. 4. ALP activity of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) hDPSCs cultured for 7 days (A) and 21 days (B) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
3.4. Doxycycline-induced BMP-2 expression has no effect on mineralization, while lentiviral transduction reduced the calcium accumulation of mock and DPSC-BMP-2 cell lines Mineralization was measured in CM, CM+, OM, and OM+ after 21 days of incubation. Data presented in Fig. 5 suggest that there is no difference between the mineralization of the mock or DPSC-BMP-2 cell lines in CM medium, while calcium accumulation in the DPSC-BMP-2 cell line in CM+ was slightly reduced, compared to that of the control. Both mock and DPSC-BMP-2 cell lines cultured in OM or OM+, showed significantly reduced calcium accumulation compared to the control cell line.
4. Discussion BMP-2 protein is known to induce osteogenic/odontogenic differentiation of mesenchymal stem cells, however, its effect on the differentiation of DPSCs is quite controversial [39,40,42]. Although exogenous modulation of gene expression represents a useful tool for studying the regulation of osteogenic differentiation and is extremely applicable for cell-based therapy, studies so far have used recombinant BMP-2 to enhance osteogenic differentiation. The effect of temporary or stable gene expression in DPSCs has not yet been investigated. In the present study, we have established a human dental pulp cell
5. Conclusion In conclusion, the induction of BMP-2 expression in DPSC-BMP-2 cell line increased the expression of the osteogenic mediator Runx2 and the BMP antagonist noggin, which resulted in reduced ALP activity and calcium deposition in the cell line. The cell line also showed less potency to differentiate into osteoblasts compared to the mock and DPSC cell line. As the use of DPSCs as resources is still a very attractive 6
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Fig. 5. Mineralization of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) hDPSCs cultured for 21 days in different media. Cells were stained with alizarin red (A) and quantified with the use of cetylpyridinium chloride (B). Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
could have a significant impact on the successful clinical application of DPSCs for accelerating bone healing.
alternative for tissue engineering and regenerative medicine, further exploration of the effect of known factors and their interactions affecting osteogenic differentiation is essential. These findings; along with a carefully chosen vector system for controlled gene expression,
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CRediT authorship contribution statement
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Ferenc Tóth: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization, Project administration. József M. Gáll: Formal analysis, Writing - original draft, Visualization. József Tőzsér: Writing - review & editing, Funding acquisition. Csaba Hegedűs: Conceptualization, Methodology, Writing - review & editing, Funding acquisition, Supervision. Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgments We thank the Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, GINOP-2.3.2-15-2016-00020 TUMORDNS grant and Endre Kókai for The Opera Phenix equipment and Harmony 4.8 software access, and Zsuzsanna Polgár for help with imaging and image analysis. The work is supported by the GINOP-2.3.2-15-2016-00011 and GINOP-2.3.2-15-2016-00022 projects. The projects are co-financed by the European Union and the European Regional Development Fund. This work was also financed in part by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Biotechnology thematic program of the University of Debrecen. References [1] C. Toniatti, H. Bujard, R. Cortese, G. Ciliberto, Gene therapy progress and prospects: transcription regulatory systems, Gene Ther. 11 (2004) 649–657, https://doi.org/ 10.1038/sj.gt.3302251. [2] S. Goverdhana, M. Puntel, W. Xiong, J.M. Zirger, C. Barcia, J.F. Curtin, E.B. Soffer, S. Mondkar, G.D. King, J. Hu, S.A. Sciascia, M. Candolfi, D.S. Greengold, P.R. Lowenstein, M.G. Castro, Regulatable gene expression systems for gene therapy applications: progress and future challenges, Mol. Ther. 12 (2005) 189–211, https://doi.org/10.1016/j.ymthe.2005.03.022. [3] B.S. Bruder, P. Scott, Fox, Tissue Engineering of Bone: Cell Based Strategies. : Clinical Orthopaedics and Related Research (367 Suppl), Clin. Orthop. Relat. Res. (1999) S68–S83 (by PubMed), , Accessed date: 22 July 2017. [4] M.T.P. Albuquerque, M.C. Valera, M. Nakashima, J.E. Nör, M.C. Bottino, Tissueengineering-based strategies for regenerative endodontics, J. Dent. Res. 93 (2014) 1222–1231, https://doi.org/10.1177/0022034514549809. [5] J. Ma, S.K. Both, F. Yang, F.-Z. Cui, J. Pan, G.J. Meijer, J. a Jansen, J.J.J.P. van den Beucken, Concise review: cell-based strategies in bone tissue engineering and regenerative medicine, Stem Cells Transl. Med. 3 (2014) 98–107, https://doi.org/10. 5966/sctm.2013-0126. [6] J.N. Fisher, G.M. Peretti, C. Scotti, Stem Cells for Bone Regeneration: From Cellbased Therapies to Decellularised Engineered Extracellular Matrices, vol. 2016, (2016), https://doi.org/10.1155/2016/9352598. [7] H. Ide, T. Yoshida, N. Matsumoto, K. Aoki, Y. Osada, T. Sugimura, M. Terada, Growth regulation of human prostate cancer cells by bone morphogenetic protein2, Cancer Res. (1997) 5022–5027 http://cancerres.aacrjournals.org/content/ canres/57/22/5022.full.pdf (accessed July 22, 2017). [8] F. Pouliot, A. Blais, C. Labrie, Overexpression of a dominant negative type II bone morphogenetic protein receptor inhibits the growth of human breast cancer cells 1, Cancer Res. 63 (2003) 277–281 https://pdfs.semanticscholar.org/ed27/ 812eab596d85c81cd2451a1a29aba768d8d2.pdf (accessed July 22, 2017). [9] I.K. Moutsatsos, G. Turgeman, S. Zhou, B.G. Kurkalli, G. Pelled, L. Tzur, P. Kelley, N. Stumm, S. Mi, R. Müller, Y. Zilberman, D. Gazit, Exogenously regulated stem cell-mediated gene therapy for bone regeneration, Mol. Ther. 3 (2001) 449–461, https://doi.org/10.1006/mthe.2001.0291. [10] M. Gossen, H. Bujardt, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters, Cell Biol. J. Gehring. 89 (1992) 5547–5551 http://www.pnas.org/content/89/12/5547.full.pdf , Accessed date: 22 July 2017. [11] M. Gossen, A.L. Bonin, S. Freundlieb, H. Bujard, Inducible gene expression systems for higher eukaryotic cells, Curr. Opin. Biotechnol. 5 (1994) 516–520, https://doi. org/10.1016/0958-1669(94)90067-1. [12] Y. Gafni, G. Pelled, Y. Zilberman, G. Turgeman, F. Apparailly, H. Yotvat, E. Galun, Z. Gazit, C. Jorgensen, D. Gazit, Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system, Mol. Ther. 9 (2004) 587–595, https://doi.org/10.1016/j.ymthe.2003.12.009. [13] C.A. Gersbach, J.M. Le Doux, R.E. Guldberg, A.J. García, Inducible regulation of Runx2-stimulated osteogenesis, Gene Ther. 13 (2006) 873, https://doi.org/10. 1038/sj.gt.3302725.
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Full Length Article
Effect of inducible bone morphogenetic protein 2 expression on the osteogenic differentiation of dental pulp stem cells in vitro
T
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Ferenc Tótha, , József M. Gállb, József Tőzsérc, Csaba Hegedűsa a
Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, Debrecen, Hungary Department of Applied Mathematics and Probability Theory, Faculty of Informatics, University of Debrecen, Debrecen, Hungary c Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone morphogenetic protein 2 Gene therapy Osteogenic differentiation Noggin Tet-on
Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily, it is known to be a factor involved in skeletal development and capable of inducing in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) isolated from extracted third molar teeth are an ideal resource for bone tissue engineering and regeneration applications, due to their convenient isolation, safe cryopreservation, and easy maintenance in cell cultures. The aims of this study were to deliver BMP-2 under control of the tetracycline-inducible (tet-on) promoter into dental pulp stem cells and to examine whether these BMP-2 expressing cell lines are capable of promoting osteogenic differentiation in vitro. BMP-2 gene was cloned into the lentiviral transfer plasmid pTet-IRES-EGFP and used to establish the DPSC-BMP-2 cell line. DPSC, DPSCGFP (mock) and DPSC-BMP-2 cell lines were cultured in growth medium or osteogenic medium in the presence or absence of 100 ng/ml doxycycline. To assess differentiation, alkaline phosphatase activity, calcium accumulation and gene transcription levels of different genes involved in osteogenic differentiation (BMP-2, Runx2, alkaline phosphatase, and noggin) were measured. Doxycycline-induced BMP-2 expression induced the differentiation of DPSCs into the preosteoblastic stage but could not favor the further maturation into osteoblasts and osteocytes. We found that while Runx2 gene transcription was continuously upregulated in doxycycline-treated DPSC-BMP-2 cells, the alkaline phosphatase activity and the accumulation of minerals were reduced. As a result of the increased BMP-2 expression, the transcription level of the BMP antagonist noggin was also upregulated, and probably caused the observed effects regarding alkaline phosphatase (ALP) activity and mineral deposition. Our study shows that this system is effective in controlling transgene expression in DPSC cell line. Exploration of all known factors affecting osteogenic differentiation and their interactions is of major importance for the field of regenerative medicine. As the metabolic reaction to the upregulated transgene transcription appears to be cell line-specific, a wrongly selected target gene and/or regulation system could have adverse effects on differentiation.
1. Introduction Regulation of transgene expression in gene therapy is quite often necessary [1,2] in regards to controlling timing, duration, and level of expression. Moreover, the appearance of cell-based therapies in tissue engineering and regenerative medicine, relying on regulated expression is crucial for successful applications [3–6]. This controllable expression is a very important part of stem cell-based bone tissue engineering, in order to avoid possible side effects of uncontrollable gene expression of osteogenic differentiation factors [7–9] for in vivo bone formation and
regeneration. The tetracycline-induced (tet-on) systems, first described by Gossen and Bujardt [10,11] are widely used and well-characterized for regulating transgene expression. This system can be induced by the binding of a tetracycline-dependent transactivator provided by adding the inducer and stopped by withdrawing it. Tetracycline-regulated gene expression is already used for the expression of genes involved in bone formation [9,12–14]. Human dental pulp stem cells (DPSCs) isolated from third molar teeth [15,16] are adult stem cells showing mesenchymal stem cell properties [15]. DPSCs can be induced to differentiate into multiple
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Corresponding author at: Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, H-4002 Debrecen, P.O. Box 400, Hungary. E-mail addresses: [email protected] (F. Tóth), [email protected] (J.M. Gáll), [email protected] (J. Tőzsér), [email protected] (C. Hegedűs). https://doi.org/10.1016/j.bone.2019.115214 Received 25 September 2019; Received in revised form 17 December 2019; Accepted 23 December 2019 Available online 26 December 2019 8756-3282/ © 2019 Published by Elsevier Inc.
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transformed into One Shot Stbl3 E. coli strain (Invitrogen, Carlsbad, CA, USA) and new transformants with BMP-2 gene in the pTet-IRES-EGFP plasmid were verified by DNA sequencing (data not shown). The resultant recombination vector was referred to as pTet-IRES-EGFP-BMP2.
connective-tissue cell lineages, including the osteogenic/odontogenic phenotype [17–19]. Furthermore, DPSCs are highly proliferative and can be safely cryopreserved [20]. Considering their characteristics along with their shorter differentiation path [21]; compared to embryonic stem cells or differentiated cell types; they are a very promising source for regenerative medicine and tissue engineering applications [22,23]. Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily. Many of the family members are involved in skeletal development, regeneration, and repair [24] BMP-2 has been known to be a factor capable of inducing in vitro osteogenic/ odontogenic differentiation [5,25–28] and in vivo bone formation [29–31]. Along with another family member; the bone morphogenetic protein 7, its use is also approved by the American Food and Drug Administration for human clinical application. These recombinant proteins are recommended for the treatment of tibial nonunions, bone fractures, and spine fusion [32–34]. However, as the generally applied doses are much higher than the physiological concentrations, the possibility of an inflammatory response is also a possible threat [35,36], reflecting the need for properly orchestrated delivery [34,36] including gene therapy applications [37]. The effect of the BMP-2 on the osteogenic/odontogenic differentiation of dental pulp cells from different sources showed promising results [28,38–40] in vitro and in vivo as well. However, the effect of stable BMP-2 gene transfer and regulated transgene expression on DPSCs is not yet investigated. In the present study, we have established a DPSC cell line capable of tet-inducible BMP-2 transgene expression, where this expression is activated by the presence of doxycycline. We have used two lentiviral vectors for stable gene transfer, one vector encodes the transgene BMP2 and green fluorescent protein (GFP) separated by internal ribosome entry site, while the other vector encodes the reverse transactivator protein that controls transcription of the transgene. Thereafter; using this cell line, we have investigated the effect of induced BMP-2 transgene expression on osteogenic differentiation.
2.3. Isolation and cell culture DPSCs were isolated from third molar teeth that had been extracted at the Faculty of Dentistry, University of Debrecen, with the modification of a previously described protocol [19]. Briefly, the tooth was cut around by sterile dental fissure burs to expose the pulp chamber, and the pulp tissue was removed from the crown and root. The pulp tissue was then digested in a solution of collagenase type I (3 mg/ml, Sigma Aldrich, St Louis, MO, USA) and dispase (4 mg/ml, Gibco, Waltham, MA, USA) for 1 h at 37 °C. Cell suspension was seeded into 6-well plates (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM F12 (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% Glutamax (Gibco) and 1% Antibiotic-Antimycotic (Gibco), and incubated at 37 °C in 5% CO2. The cells were cultured in the same medium. This medium is referred to in the text as control medium (CM). After the third passage, cells were seeded into 6-well plates and used for transduction. After transduction and antibiotic selection, the mixture of GFPpositive and GFP-negative cells were seeded into 24 well plates and cultured in different media. The culture medium was refreshed 3 times a week. Osteoinductive medium (OM) was prepared by supplementing CM with 10 mM β-glycerolphosphate (Sigma Aldrich, St Louis, MO, USA), 50 μg/ml ascorbic acid (Sigma Aldrich), 0.1 μM dexamethasone (Sigma Aldrich) and 50 nM vitamin D3 (Sigma Aldrich). Media for inducing transgene expression were supplemented by 100 ng/ml doxycycline (Sigma) (indicated as CM+ and OM+ respectively).
2. Materials and methods 2.4. Lentivirus preparation and transduction 2.1. Ethics statement Viral particles were produced by transient transfection of 293FT cells, maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), using polyethyleneimine (Sigma). Cells were grown to approximately 70% confluence and a total of 16 μg plasmid was used for the transfection of cells in T75 flasks: 8 μg pTet-IRES-EGFP, pTet-IRES-EGFP-BMP-2 or pLenti CMV rtTA3 Blast (w756-1) (Addgene plasmid 26429). pLenti CMV rtTA3 Blast (w756-1) was a gift from Eric Campeau (transfer vector plasmid), 6 μg psPAX2, 2 μg pMD2.G and 13 μg salmon sperm DNA (Sigma Aldrich), in DMEM containing 1% FBS. The medium was replaced after 6 h and the conditioned media containing virus particles were collected after 3 days, clarified by centrifugation, and was filtered through 0.45 μm polyvinylidene fluoride (PVDF) filter (Millipore, Billerica, MA, USA), then stored in −70 °C. For transduction, 500 μl pLenti CMV rtTA3 Blast (w756-1) and 500 μl pTet-IRES-EGFP-BMP-2 or pTet-IRES-EGFP (mock) virus and 1 ml fresh medium mixed with 8 μg/ml polybrene (Sigma Aldrich) in 6well plates containing DPSC cells at 50% confluency. After overnight incubation, the medium was changed. Medium containing 5 μg/ml blasticidin (Thermo Fisher Scientific) was added to the cells at passage one. After antibiotic selection, GFP expression was induced by the addition of 100 ng/ml doxycycline, and the cells were examined under fluorescence microscopy (Zeiss Axiovert 100, Carl Zeiss Microscopy, Jena, Germany) and by flow cytometry (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) to confirm the efficiency of transduction. DPSC cells transduced by pTet-IRES-EGFP-BMP-2 or pTetIRES-EGFP containing virus were referred to as DPSC-BMP-2 and mock respectively.
Human dental pulp tissue collection and subsequent gene transfer performed in this study were approved by the Ethical Committee of the Hungarian Medical Research Council (approval number: ETT TUKEB 49849-3/2016/EKU). For the use of tissue samples, written information consent was obtained from all human subjects who participated in the investigation. 2.2. Construction of pTet-IRES-EGFP-BMP-2 plasmid BMP-2 complementary DNA was synthesized from 1 μg of total RNA of human 293T cell line using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. BMP-2 coding region of the cDNA was amplified by polymerase chain reaction by using Q5 high fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA) and the following set of primers: 5′-CTAAAGGATCCATGGTGGCCGGGACCCG CTG-3′ (forward) and 5′-GATTTGGATCCCTAGCGACACCCACAACCCT CCAC-3′ (reverse). The forward and reverse primers harbored a recognition site for BamHI (underlined). PCR was performed under the following conditions: 30 s at 93 °C (pre-denaturation), 35 cycles of 10 s at 93 °C (denaturation) and 1 min 50 s at 72 °C (extension), followed by 2 min at 72 °C. After purification, the PCR products were cleaned by Monarch PCR & DNA Cleanup Kit (New England Biolabs) according to the manufacturer's instruction. The cleaned product was digested with BamHI and ligated into the pTet-IRES-EGFP plasmid [41] at the corresponding restriction site. The plasmid pTet-IRES-EGFP was a gift from Maria Lung (Addgene plasmid # 64238). The ligation mixture was then 2
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Fig. 1. Tet-on system in hDPSCs. (A) Differential GFP expression by treatment with a range of doxycycline concentrations (3.125, 6.25, 12.5, 25, 50, 100 ng/ml) in the DPSC-BMP-2 cell line. (B) The percentages of GFP-positive cells after treatment of doxycycline followed by Harmony 4.8 software or (C) FACS analysis (n = 3). Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurement (P ˂ 0.05). (D) Representative western blot showing BMP-2 expressions after treatment with different concentrations of doxycycline.
2.5. Fluorescent microscopy and fluorescence-activated cell sorting (FACS) analyses
removed from the wells, and total RNA was isolated using Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) with on-column DNAse digestion. The concentration of the RNA was determined based on their A260 by a Nanodrop equipment. 1 μg total RNA was used per sample for cDNA synthesis, using random primers (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Genes of interest were detected using Applied Biosystems Taqman Assays: ALP (Hs01029144_m1), BMP-2 (Hs00154192_m1), Noggin (Hs00271352_s1), Runx2 (Hs00231692_m1), and 5× HOT FIREPol Probe qPCR Mix Plus (no ROX) (Solis BioDyne, Tartu, Estonia). Results were normalized to the reference housekeeping gene GAPDH (glycerolaldehyde-3-phosphate dehydrogenase, Hs02758991_g1) in each sample.
4 × 104 cells/well were seeded into a 24 well plate and cultured for 7 days in media containing 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml doxycycline. After 7 days nuclei of the cells were stained with 1:1000 DRAQ5 (Biolegend, San Diego, CA, USA) the cells were examined under fluorescence microscopy by an Opera Phenix™ High Content Screening System (Perkin Elmer, Waltham, MA, USA) and analysed using the Harmony 4.8 software (Perkin Elmer). For flow cytometry analyses (BD FACSCalibur, BD Biosciences) the cells were scraped and washed by PBS, then counted to determine the number of GFP-positive cells. Data were evaluated by FlowJo flow cytometry analysis software. 2.6. BMP-2 immunoblot
2.8. Detection of alkaline phosphatase (ALP) activity
2 × 105 cells were seeded to petri dishes and cultured for 6 days in media containing 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml doxycycline. After 6 days, protein transport from the endoplasmic reticulum to the Golgi apparatus was inhibited by the addition of 10 μg/ml Brefeldin A (Tocris Bioscience, Tocris, United Kingdom). After an additional day the cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% triton-x 100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl and 1% protease inhibitor cocktail, pH 8.0). The protein concentration was determined by Pierce BCA Protein Assay (Thermo Scientific), then 20 μg lysates from each sample was resolved by 10% TGX Stain-Free FastCast acrylamide gel (Bio-rad Laboratories, Hercules, CA, USA) and analysed by western blot using Human BMP-2 Antibody (R&D Systems, Minneapolis, MN, USA) and Anti-GAPDH Antibody (Lifespan Biosciences, Seattle, WA, USA) respectively.
Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. At day 7 and 21 media were removed from the wells, and the cells were lysed by ALP lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM Triton-X 100, 1% PMSF and 1% protease inhibitor cocktail, pH 7.4). Cell lysates were centrifuged for 10 min at 16,000g at 4C° to remove cellular debris. 30 μl of lysate supernatant was added to each well of 96-well plate and then 100 μl of pNitrophenyl Phosphate Liquid Substrate System (Sigma Aldrich, St. Louis, MO, USA) was added to lysates. The absorbance was measured in room temperature at 405 nm by HIDEX Sense microplate reader (HIDEX, Turku, Finland), immediately and after 15 min and ALP activity (absorbance change) was determined by the subtraction of the absorbance measured immediately from the absorbance measured after 15 min of the addition of the substrate. The total protein concentration of lysate supernatants was determined by BCA Protein Assay Kit (Thermo Scientific) and ALP activity was normalized to total protein concentration.
2.7. Quantitative real-time PCR Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. At day 7 and 21 medium were 3
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Fig. 2. Representative gene expression profile of BMP-2 gene in case of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) cell lines cultured for 7 days (A) and 21 days (B) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
2.9. Mineralization assay
3. Results
Mineralization of DPSCs was observed by alizarin red S (Sigma Aldrich) staining. Cells were seeded into a 24-well plate at a density of 2 × 104/well. Three parallels were set for each group. After 21 days of culture with different media (CM, OM, CM+, and OM+), the cells were washed with PBS, fixed with 4% paraformaldehyde, stained with 2% alizarin red S solution (pH 4.2) at room temperature in the dark for 45 mins. The stained cultures were washed with distilled water, then airdried. Stained cultures were examined under the microscope (Olympus SZ61, Olympus Corporation, Shinjuku, Tokyo, Japan), and images were acquired. For quantification of the staining, alizarin red S were extracted with 10% cetylpyridinium chloride (Sigma Aldrich) solution and absorbance was measured by a Hidex Sense Microplate reader at 570 nm.
3.1. Doxycycline-sensitive transgene expression pTet-IRES-EGFP-BMP-2 and pLenti CMV rtTA3 Blast (w756-1) plasmid were used to develop an inducible GFP and BMP-2 expression in the DPSC cell line. At first, we used GFP expression to verify successful gene transfer and inducible transgene expression. The tet-on system showed negligible doxycycline dose-dependent transgene expression indicated only by the GFP expression in the cells (Fig. 1A). The maximum percentage of GFP expressing cells observed was 29.3% if determined by the Harmony 4.8 software (Fig. 1B) and 72.8% if determined by FACS (Fig. 1C) when the cells were treated with 100 ng/ml doxycycline. Higher concentrations of doxycycline did not cause a further increase in the number of GFP-positive cells (data not shown). The BMP-2 expression does not show dose-dependency to doxycycline concentration (Fig. 1D), although the addition of doxycycline increased the amount of the expressed protein even at the lowest concentration (3.125 ng/ml).
2.10. Statistics The independent sample t-test and the Welch t-test were used to compare the means of different pairs of populations (e.g. two media or cell lines), depending on the equality of variances. The latter condition was tested by the F test for variances. Error bars on the figures represent the estimation of the standard deviation calculated from three parallel measurements.
3.2. The doxycycline-induced BMP-2 gene expression has upregulated the expression of BMP antagonist noggin, and runt-related expression factor 2, while downregulated the transcription of the alkaline phosphatase gene Gene expression of proteins involved in osteogenic differentiation was examined after 7 and 21 days of culture. After 7 days there are no significant differences between the BMP-2 expression of cell lines 4
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Fig. 3. Representative gene expression profile of ALP, Noggin, and Runx2 genes in case of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSCBMP-2) cell lines cultured for 7 days (above) and 21 days (below) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
and DPSC-BMP-2 cell lines, while in CM+ only the DPSC-BMP-2 cell line showed a significant downregulation compared to the control cell line. We have observed a significant upregulation of the ALP gene expression in control cells incubated in CM+ compared to those were incubated in CM. The same treatment of medium (addition of doxycycline) downregulated the ALP activity of the DPSC-BMP-2 cell line cultured in CM+, while upregulated in OM+ compared to the results measured in CM and OM, respectively (Fig. 3).
cultured in CM or OM (Fig. 2A), although we observed a dramatic upregulation in case of the DPSC-BMP-2 cell line compared to the control cell line and to the expression measured in CM or OM, respectively. We have also observed a slight, but significant downregulation in the mock cell line in CM+ and OM+ medium compared to the control. Similar trends were noticed for the expression of noggin (Fig. 3) in the DPSC-BMP-2 cell line, but not in the mock. The expression of runt-related transcription factor (Runx2) was upregulated in the DPSC-BMP-2 cell line cultured in OM+ media (Fig. 3) compared to the control cell line, and in the mock cell line in CM+ compared to the ones grown in CM. On the contrary, in the DPSC-BMP-2 cell line, ALP gene expression (Fig. 3) was significantly downregulated when incubated in CM+ or OM+ media compared to the control cell line and to those, which were incubated in CM or OM respectively. After 21 days of treatment, expression of BMP-2 (Fig. 2B) in the DPSC-BMP-2 cell line cultured in CM+ and OM+ was still persuasively upregulated compared to the control cell line and to the expression measured in CM or OM, respectively. Also, in the DPSC-BMP-2 cell line, a less prominent, but still significant upregulation were observed in CM and OM as well compared to the control cell line. Moreover, a slight downregulation was noticed in the mock cell line compared to that of the control, when cultured in OM or OM+. The DPSC-BMP-2 cells showed upregulated noggin expression in all media compared to the control cell line, but in case of the OM+ medium it was not a significant change (Fig. 3). Also, significant upregulation was observed with the mock when incubated in CM, compared to the control. We have also noticed, that the DPSC-BMP-2 cell line cultured in OM or OM+ media still showed upregulated Runx2 expression compared to the control, but in OM+ medium this upregulation was not significant (Fig. 3). In CM, the gene expression of ALP was upregulated in both the mock
3.3. Doxycycline-induced BMP-2 expression has a negative effect on the activity of alkaline phosphatase Alkaline phosphatase (ALP) activity of cells was measured after 7 and 21 days of incubation. After 7 days, a significant increase was observed in the ALP activity of the control cells grown in CM+ and OM+ compared to CM and OM, respectively. In CM+ and OM+ significant reduction was noticed in the ALP activity of the DPSC-BMP-2 cell line compared to the control cell line and to CM and OM respectively (Fig. 4A). Contrarily, ALP activity of this DPSC-BMP-2 cell line was significantly increased both in CM and OM compared to the control. The ALP activity of mock cells cultured in OM was also increased compared to that of the control cell line. It should be noted that while the ALP activity is at least slightly increased in almost all cell lines and types of media after 21 days of culture (Fig. 4B) compared to the results measured after 7 days, the activity of the DPSC-BMP-2 cell line was still significantly reduced in CM+ medium compared to the activity measured in CM medium and to the control cell line in CM+. Surprisingly, the ALP activity in OM+ was restored in the DPSC-BMP-2 cell line both when compared to the control cell line or to the DPSC-BMP-2 cells grown in OM, respectively.
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line capable of induced (tet-on) expression of BMP-2, using lentivirusmediated stable gene transfer. We have demonstrated a negligible dosedependent control of GFP transgene expression with doxycycline concentration but found no dose-dependency in the case of BMP-2 transgene expression. The effect of inducible BMP-2 expression on the cells was examined in culture media with the presence (CM+, OM+) or absence (CM, OM) of the inductive agent doxycycline. Investigation of the effect of doxycycline induction on osteogenic differentiation showed that while doxycycline had no impact on ALP activity of the DPSC and mock cell lines, as ALP activity only increased if cultured in OM, which was in line with our expectations. It was significantly reduced in the DPSC-BMP-2 cell line after 7 days in both CM+ and OM+. Furthermore, this effect lasted until 21 days in CM+ but not in OM+. As the CM+ and OM+ media consists of the same ingredients (except for β-glycerophosphate, ascorbic acid, dexamethasone, and vitamin D3 in OM+) the reduced ALP activity observed in OM+ after 7 days of culture were probably corrected by the effect of the additional materials in the basic OM. Examination of calcium deposits revealed that the addition of doxycycline resulted only in a slight reduction of calcium accumulation, notably only in the case of the DPSC-BMP-2 cell line cultured in CM+. In addition, we have observed that cell lines transduced by lentiviral particles; both in OM and OM+, accumulated less calcium compared to the control DPSC cell line. This reduction in the calcium deposition probably caused by the lentiviral transduction and use of polybrene [43], which reportedly can affect proliferation or differentiation potential of the MSCs. The study of osteogenic marker genes showed that BMP-2 expression of DPSC-BMP-2 was dramatically increased in CM+ and OM+, compared to the control cell line, it came along with the increased expression of BMP antagonist noggin. As noggin is able to bind and sequester BMPs [44] and has an effect on differentiation and fate of osteoblasts [45]; including the development of a fracture non-union [46], it may, therefore, have caused the reduced ALP activity and mineralization observed in this cell line in CM+ and OM+. When studying the expression of Runx2, we have not found significant changes in any cell lines or medium compared to the other ones after 7 days, with only one exception. In the case of the DPSC-BMP-2 cell line, the expression of Runx2 was upregulated in OM+ compared to other media. This upregulation in OM+ was detectable after 21 days of culture as well. Increased level of Runx2 induces the differentiation of mesenchymal pluripotent cells into preosteoblasts and immature osteoblasts, while inhibiting osteoblast maturation [47], therefore it can be hypothesized that continuous Runx2 upregulation may contribute to the effect of noggin, and result in reduced ALP activity and mineralization observed in the DPSC-BMP-2 cell line when BMP-2 expression is induced. Studies regarding doxycycline addition revealed reduced endogenous levels of BMP-2 in osteoprogenitor cells [48], probably through inhibition of TGF-β signaling [49], we, however, have not observed this effect in the DPSC or mock cell line. BMP-2 transgene expression controlled by a doxycycline-repressible promoter in C3H10T1/2 mesenchymal stem cells had a positive effect on osteogenesis and chondrogenesis [9,50]. Moreover, another study revealed that doxycycline counteracts BMP-2 induced osteogenic mediators [51], suggesting that proper choice of inducing agent and/or system for controlling expression is also crucial for a successful application.
Fig. 4. ALP activity of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) hDPSCs cultured for 7 days (A) and 21 days (B) in different media. Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line.
3.4. Doxycycline-induced BMP-2 expression has no effect on mineralization, while lentiviral transduction reduced the calcium accumulation of mock and DPSC-BMP-2 cell lines Mineralization was measured in CM, CM+, OM, and OM+ after 21 days of incubation. Data presented in Fig. 5 suggest that there is no difference between the mineralization of the mock or DPSC-BMP-2 cell lines in CM medium, while calcium accumulation in the DPSC-BMP-2 cell line in CM+ was slightly reduced, compared to that of the control. Both mock and DPSC-BMP-2 cell lines cultured in OM or OM+, showed significantly reduced calcium accumulation compared to the control cell line.
4. Discussion BMP-2 protein is known to induce osteogenic/odontogenic differentiation of mesenchymal stem cells, however, its effect on the differentiation of DPSCs is quite controversial [39,40,42]. Although exogenous modulation of gene expression represents a useful tool for studying the regulation of osteogenic differentiation and is extremely applicable for cell-based therapy, studies so far have used recombinant BMP-2 to enhance osteogenic differentiation. The effect of temporary or stable gene expression in DPSCs has not yet been investigated. In the present study, we have established a human dental pulp cell
5. Conclusion In conclusion, the induction of BMP-2 expression in DPSC-BMP-2 cell line increased the expression of the osteogenic mediator Runx2 and the BMP antagonist noggin, which resulted in reduced ALP activity and calcium deposition in the cell line. The cell line also showed less potency to differentiate into osteoblasts compared to the mock and DPSC cell line. As the use of DPSCs as resources is still a very attractive 6
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Fig. 5. Mineralization of control (DPSC), mock transfected (mock) and BMP-2 expressing (DPSC-BMP-2) hDPSCs cultured for 21 days in different media. Cells were stained with alizarin red (A) and quantified with the use of cetylpyridinium chloride (B). Values are expressed as sample means; error bars represent the estimates of standard deviations calculated from three parallel measurements. In the t-tests, * denotes the case P ˂ 0.05 compared to the control group (DPSC) in the same media, whereas # denotes P ˂ 0.05 compared to the untreated (CM or OM) group of the same cell line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
could have a significant impact on the successful clinical application of DPSCs for accelerating bone healing.
alternative for tissue engineering and regenerative medicine, further exploration of the effect of known factors and their interactions affecting osteogenic differentiation is essential. These findings; along with a carefully chosen vector system for controlled gene expression,
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CRediT authorship contribution statement
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Ferenc Tóth: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization, Project administration. József M. Gáll: Formal analysis, Writing - original draft, Visualization. József Tőzsér: Writing - review & editing, Funding acquisition. Csaba Hegedűs: Conceptualization, Methodology, Writing - review & editing, Funding acquisition, Supervision. Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgments We thank the Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, GINOP-2.3.2-15-2016-00020 TUMORDNS grant and Endre Kókai for The Opera Phenix equipment and Harmony 4.8 software access, and Zsuzsanna Polgár for help with imaging and image analysis. The work is supported by the GINOP-2.3.2-15-2016-00011 and GINOP-2.3.2-15-2016-00022 projects. The projects are co-financed by the European Union and the European Regional Development Fund. This work was also financed in part by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Biotechnology thematic program of the University of Debrecen. References [1] C. Toniatti, H. Bujard, R. Cortese, G. Ciliberto, Gene therapy progress and prospects: transcription regulatory systems, Gene Ther. 11 (2004) 649–657, https://doi.org/ 10.1038/sj.gt.3302251. [2] S. Goverdhana, M. Puntel, W. Xiong, J.M. Zirger, C. Barcia, J.F. Curtin, E.B. Soffer, S. Mondkar, G.D. King, J. Hu, S.A. Sciascia, M. Candolfi, D.S. Greengold, P.R. Lowenstein, M.G. Castro, Regulatable gene expression systems for gene therapy applications: progress and future challenges, Mol. Ther. 12 (2005) 189–211, https://doi.org/10.1016/j.ymthe.2005.03.022. [3] B.S. Bruder, P. Scott, Fox, Tissue Engineering of Bone: Cell Based Strategies. : Clinical Orthopaedics and Related Research (367 Suppl), Clin. Orthop. Relat. Res. (1999) S68–S83 (by PubMed), , Accessed date: 22 July 2017. [4] M.T.P. Albuquerque, M.C. Valera, M. Nakashima, J.E. Nör, M.C. Bottino, Tissueengineering-based strategies for regenerative endodontics, J. Dent. Res. 93 (2014) 1222–1231, https://doi.org/10.1177/0022034514549809. [5] J. Ma, S.K. Both, F. Yang, F.-Z. Cui, J. Pan, G.J. Meijer, J. a Jansen, J.J.J.P. van den Beucken, Concise review: cell-based strategies in bone tissue engineering and regenerative medicine, Stem Cells Transl. Med. 3 (2014) 98–107, https://doi.org/10. 5966/sctm.2013-0126. [6] J.N. Fisher, G.M. Peretti, C. Scotti, Stem Cells for Bone Regeneration: From Cellbased Therapies to Decellularised Engineered Extracellular Matrices, vol. 2016, (2016), https://doi.org/10.1155/2016/9352598. [7] H. Ide, T. Yoshida, N. Matsumoto, K. Aoki, Y. Osada, T. Sugimura, M. Terada, Growth regulation of human prostate cancer cells by bone morphogenetic protein2, Cancer Res. (1997) 5022–5027 http://cancerres.aacrjournals.org/content/ canres/57/22/5022.full.pdf (accessed July 22, 2017). [8] F. Pouliot, A. Blais, C. Labrie, Overexpression of a dominant negative type II bone morphogenetic protein receptor inhibits the growth of human breast cancer cells 1, Cancer Res. 63 (2003) 277–281 https://pdfs.semanticscholar.org/ed27/ 812eab596d85c81cd2451a1a29aba768d8d2.pdf (accessed July 22, 2017). [9] I.K. Moutsatsos, G. Turgeman, S. Zhou, B.G. Kurkalli, G. Pelled, L. Tzur, P. Kelley, N. Stumm, S. Mi, R. Müller, Y. Zilberman, D. Gazit, Exogenously regulated stem cell-mediated gene therapy for bone regeneration, Mol. Ther. 3 (2001) 449–461, https://doi.org/10.1006/mthe.2001.0291. [10] M. Gossen, H. Bujardt, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters, Cell Biol. J. Gehring. 89 (1992) 5547–5551 http://www.pnas.org/content/89/12/5547.full.pdf , Accessed date: 22 July 2017. [11] M. Gossen, A.L. Bonin, S. Freundlieb, H. Bujard, Inducible gene expression systems for higher eukaryotic cells, Curr. Opin. Biotechnol. 5 (1994) 516–520, https://doi. org/10.1016/0958-1669(94)90067-1. [12] Y. Gafni, G. Pelled, Y. Zilberman, G. Turgeman, F. Apparailly, H. Yotvat, E. Galun, Z. Gazit, C. Jorgensen, D. Gazit, Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system, Mol. Ther. 9 (2004) 587–595, https://doi.org/10.1016/j.ymthe.2003.12.009. [13] C.A. Gersbach, J.M. Le Doux, R.E. Guldberg, A.J. García, Inducible regulation of Runx2-stimulated osteogenesis, Gene Ther. 13 (2006) 873, https://doi.org/10. 1038/sj.gt.3302725.
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