Open Access

Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells

  • Authors:
    • Hyunjin Lee
    • Sae Kyung Min
    • Youngmin Song
    • Yun‑Hee Park
    • Jun‑Beom Park
  • View Affiliations

  • Published online on: August 16, 2019     https://doi.org/10.3892/etm.2019.7904
  • Pages: 2867-2876
  • Copyright: © Lee et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study was performed to evaluate the effects of short‑term application of bone morphogenetic protein‑7 (BMP‑7) on human gingiva‑derived mesenchymal stem cells with next‑generation sequencing. Human gingiva‑derived stem cells were treated with a final concentration of 100 ng/ml BMP‑7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT‑qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP‑7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP‑7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP‑7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT‑qPCR and western blot analysis. The short‑term application of BMP‑7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP‑7 using mRNA sequencing.

Introduction

Bone morphogenetic proteins (BMP) are reported to be associated with proliferation and differentiation of numerous types of cells (1,2). In a previous in vitro study, bone morphogenetic protein-7 (BMP-7) was shown to promote odontogenic differentiation of stem cells derived from dental pulp (3). BMP-7 at concentrations of 50 and 100 ng/ml was shown to produce dental pulp stem cells that tended toward odontogenic differentiation through the Smad5 signaling pathway and did not significantly interrupt cell proliferation in vitro (3). A previous report showed that BMP-7 made the fibroblasts derived from a human dermis differentiate into osteoblasts and promoted osteogenesis (4). Similarly, BMP-7 enhanced the human-induced pluripotent stem cells' differentiation potential (5).

Mesenchymal stromal cells from intraoral region drew great attention in tissue regeneration because they can be achieved with a minimally invasive maneuver (6). For several years, gingiva-derived stem cells have been used for various purposes (79). Mesenchymal gingiva-derived stem cells repaired calvarial and mandibular defects, and gingival tissue can serve as a source for stem cell therapy (10). Gingiva-derived stem cells can aggregate into tight three-dimensional spheroids, which can produce their own matrix (8). Gingiva-derived stem cells have demonstrated to have immunoregulatory effects by promoting Treg cell polarization, inducing T-cell apoptosis and suppressing the in vitro polarization of Th17 cell (9). Gingiva-derived stem cells encapsulated in an alginate/hyaluronic acid three-dimensional scaffold have been applied (7). Gingiva-derived stem cells have been used for bone and nerve regeneration (11). This study was aimed at examining the time-dependent impact of BMP-7 on changes of gene expression in the stem cells. mRNA sequencing and data analysis were performed along with gene ontology and pathway analysis. Quantitative analysis of mRNA using real-time polymerase chain reactions of ColI, Sp7 and IBSP and western blot analysis of collagen I, osterix and bone sialoprotein as well as β-actin were performed. The purpose of this study was to demonstrate the effects of BMP-7 on the gingiva-derived stem cells.

Materials and methods

Stem cells collected from human gingivae

Human gingivae were collected from healthy participants following the method used in a previous study (12). Approval of this study was obtained from the Institutional Review Board at Seoul St Mary's Hospital (KC18SESI0199). The participants signed the written consent, and all of the procedures completed in this study followed the relevant regulations and guidelines.

Concisely, de-epithelialization of the obtained tissues was performed, and the tissues were dissected into 1–2-mm2 fragments. The tissues were dissolved using collagenase type IV (Sigma-Aldrich Co., St. Louis, MO, USA) at 2 mg/ml and dispase (Sigma-Aldrich Co.) at 1 mg/ml. The resultant products was filtered with a 70-µm cell strainer (Falcon, BD Biosciences, Franklin Lakes, NJ, USA), and sterile phosphate-buffered saline (PBS, Welgene, Daegu, Korea) was applied to remove the non-adherent cells after incubation for 24 h.

Treatment of the stem cells with BMP-7

The gingivae-derived stem cells were then treated with BMP-7 (CYT-333; ProSpec Co., Nagoya, Japan) at a final concentration of 100 ng/ml. The control group was loaded with the same concentration of the dissolving media of acetic acid. The cells were obtained at 3 or 24 h.

Evaluation of the secretion of human vascular endothelial growth factor for paracrine effect

Secretion of human vascular endothelial growth factor was determined at 3 and 24 h using a kit (Quantikine® ELISA, cat. no. DVE00; R&D Systems, Inc., Minneapolis, MN, USA). Preparation of the samples and reagents were performed following the manufacturer's recommendations. The resulting products were diluted ten times. The differences of absorbance levels at 450 and 570 nm were used for the evaluation of paracrine effects.

RNA isolation

Isolation of total RNA was done using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Quality of RNA was evaluated by bioanalyzer (Agilent 2100; Agilent Technologies, Amstelveen, The Netherlands) using the nanochip (RNA 6000, Agilent Technologies), and quantification of RNA was carried out using a spectrophotometer (ND-2000, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Library preparation and sequencing

Library of control and test RNAs were built using SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) following the manufacturer's instructions. Concisely, each 2 µg total RNA was prepared and reacted with magnetic beads coated with oligo-dT. Washing solution was used to remove other RNAs except mRNA. Initiation of library production was performed by the random hybridization of starter/stopper heterodimers to the poly(A) RNA still attached to the magnetic beads. Illumina-compatible linker sequences were part of these starter/stopper heterodimers. The starter was extended to the next hybridized heterodimer by a single-tube reverse transcription and ligation reaction. The stopper ligated the newly-synthesized cDNA insert. The library was released by second strand synthesis from the beads and the library was amplified with introduction of barcodes. Paired-end 100 sequencing performed high-throughput sequencing using HiSeq 2500 (Illumina, Inc., San Diego, CA, USA).

Data analysis

TopHat software tool was used to map mRNA-Seq reads tool in order to gain the alignment file (13). Counts from unique and multiple alignments were used to determine differentially expressed gene from coverage in Bedtools (14). Quantile-quantile normalization method was applied to process the read count data were processed from EdgeR within Rusing Bioconductor (15). Cufflinks were used for assembling transcripts, estimating their abundances and revealing differential expression of genes or isoforms with the alignment files. The expression level of the gene regions was determined by applying method of fragments per kilobase of exon per million fragments. Gene classification was based on searches performed by DAVID (http://david.abcc.ncifcrf.gov/) (16). Pathway analysis was performed on differentially expressed genes basted on the Kyoto encyclopedia of genes and genome pathway databases (17).

Quantification by real-time polymerase chain reaction

Total RNA was isolated from cultured stem cells using Trizol (Invitrogen Corp.) and was reverse transcribed. Primer sequences were as follows: Collagen I Forward 5′:CCAGAAGAACTGGTACATCAGCAA-3′, Reverse 5′:CGCCATACTCGAACTGGAATC-3′, Sp7 Forward 5′:TTGCCAATCACTCTCTTTACC-3′, Reverse 5′: GAGATACCCAAGCCCAGAAT-3′, IBSP Forward 5′:AGGACTGCCAGAGGAAGCAA-3′, Reverse 5′: CACAGGCCATTCCCAAAATG-3′ and β-actin Forward 5′: TGGCACCCAGCACAATGAA-3′ and Reverse 5′: CTAAGTCATAGTCCGCCTAGAAGCA-3′. The housekeeping gene was β-actin in this study, which was used for normalization. SYBR Green Real-Time PCR Master Mixes (Applied Biosystems, Carlsbad, CA, USA) was used to detect mRNA expression through a real-time polymerase chain reaction following the manufacturer's manual.

Western blot analysis

Samples were rinsed with ice-cold PBS twice and treated with a lysis buffer at 3 and 24 h for 30 min. The lysates were centrifuged at 13,500 rpm for 15 min at 4°C . Separation of the samples were performed by gel electrophoresis (Mini-PROTEAN® TGX™ Precast Gels; Bio-Rad, Hercules, CA, USA), transblotted to the membranes (Immun-Blot®; Bio-Rad) and immunoblotted with the corresponding antibodies and the detection kits. Primary antibodies against collagen I (ab6308; Abcam, MA, USA), RUNX2 (ab76956; Abcam), OCN (ab13418; Abcam), Sp7 transcription factor (ab22552; Abcam), bone sialoprotein (ab52128, Abcam), β-actin (SC-516102; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and secondary antibodies were purchased from Santa Cruz Biotechnology. The protein expressions of Collagen I, Sp7, bone sialoprotein and β-actin was quantitatively analyzed using with image processing program (ImageJ, National Institutes of Health, Bethesda, MD, USA).

Alkaline phosphatase activity

In the test groups, the stem cells were treated with BMP-7 for 3 or 24 h. Then media were changed to osteogenic media composed of α-MEM (Gibco, Grand Island, NY, USA), containing 200 mM of L-glutamine (Sigma-Aldrich Co.), 10 mM of ascorbic acid 2-phosphate (Sigma-Aldrich Co.), 100 µg/ml of streptomycin (Sigma-Aldrich Co.), 15% fetal bovine serum (Gibco), 100 U/ml of penicillin, 2 mg/ml of glycerophosphate disodium salt hydrate, and 38 µg/ml of dexamethasone. A commercially available kit (K412-500; BioVision, Inc., Milpitas, CA, USA) were used for the analysis of alkaline phosphatase activity on Day 7.

Statistical analysis

The data were presented as mean ± standard deviations of the results. A test of normality was conducted with Shapiro-Wilk, and differences among the groups were analyzed by one-way analysis of variance with Tukey's post hoc test using a statistical package (SPSS 12 for Windows; SPSS Inc., Chicago, IL, USA).P<0.05 was considered to indicate a statistically significant difference.

Results

Evaluation of gene ontology and pathway analysis

Differentially expressed mRNAs in this study is a total of 25,737. The scatter plots displaying differentially expressed mRNAs, are illustrated in Fig. 1. Gene ontology analysis of mRNA expression is categorized by its importance and the selection criteria were fold change ≥1.3 and log2 normalized read counts ≥4 (Fig. 2). Fig. 3 shows gene ontology analysis of mRNA expression by upregulation and downregulation (fold change of 1.3 or greater, log2 normalized read counts of 4 or greater were selected).

The results of differentially expressed mRNA related to osteoblast differentiation, are displayed in Table I. To analyze the effects of incubation time on the cultured cells, the comparisons were performed between the control at 24 and at 3 h. The investigation demonstrated that upregulation was seen in 9 mRNAs and downregulation was noted in 11 mRNAs. Comparisons between BMP-7 at 24 h and BMP-7 at 3 h showed that 9 mRNAs were upregulated and 20 were downregulated. Comparisons between BMP-7 at 3 h and the control at 3 h exhibited upregulation of 14 mRNAs and downregulation of 10 mRNAs. When the results of BMP-7 at 24 h were compared with the control at 24 h, upregulation of 14 mRNAs and downregulation of 21 mRNAs were seen.

Table I.

Differentially expressed mRNA related to osteoblast differentiation (fold change of 1.3 or greater, log2 normalized read counts ≥4 were selected).

Table I.

Differentially expressed mRNA related to osteoblast differentiation (fold change of 1.3 or greater, log2 normalized read counts ≥4 were selected).

Gene symbolControl-24 h/Control-3 hGene symbolBMP7-24 h/BMP7-3 hGene symbolBMP7-3 h/Control-3 hGene symbolBMP7-24 h/Control-24 h
AMELX2.167BMP42.073LRRC173.748WNT113.428
SOX81.932ALPL2.069WNT112.907ALPL2.946
HDAC41.743WNT111.900SOX82.304BMP42.805
CHRD1.523SHOX21.800BMP32.150SHOX22.684
NF11.478RUNX21.734HDAC41.808RUNX21.829
DNAJC131.415TMSB4Y1.592AMELX1.741TWIST11.723
BMP21.407WNT31.476PTH1R1.568NOG1.709
GLI21.365CREB3L11.418DNAJC131.447SMO1.518
SMAD31.301SNAI21.309BMP41.445PTHLH1.514
PENK0.761SMAD30.752NF11.440IFT801.513
SNAI20.691HSPE10.731VCAN1.434ITGA111.390
SEMA7A0.655IGFBP50.718SHOX21.400WNT31.389
HSPE10.654FASN0.706BMP21.390SNAI21.386
SNAI10.638RSL1D10.674IFT801.321PENK1.347
EPHA20.634IGFBP30.661SNAI20.732PHB0.749
PTHLH0.592FHL20.639TMSB4Y0.718LIMD10.749
JUNB0.585FZD10.636JUNB0.693RSL1D10.724
IGFBP30.585SYNCRIP0.628EPHA20.662GLI20.714
NOG0.371ALYREF0.626FBL0.659SMAD30.682
ALPL0.312MYBBP1A0.596MEF2D0.643FBL0.680
GTPBP40.579NOG0.581SNAI10.666
AMELX0.577SEMA7A0.516SYNCRIP0.661
SEMA7A0.519ALPL0.444FASN0.656
BMP30.511SNAI10.439FHL20.638
DDX210.507 MYBBP1A0.637
MSX20.440 CHRD0.629
CBFB0.427 FZD10.623
LRRC170.268 ALYREF0.606
SOX80.057 GTPBP40.562
DDX210.526
CBFB0.519
MSX20.512
AMELX0.464
SEMA7A0.409
SOX80.068
Secretion of human vascular endothelial growth factor

The results clearly demonstrated that the vascular endothelial growth factor was secreted at 3 and 24 h irrespective of culture period (Fig. 4). No significant change in secretion of the vascular endothelial growth factor was seen at 3 h with the addition of bone morphogenetic protein (P>0.05). Similarly, no statistically significant changes were noted with the loading of bone morphogenetic protein at 24 h (P>0.05).

Validation of mRNA expression

Quantitative real-time PCR revealed that mRNA levels of collagen I were higher in the 24-h control group than in 3-h control group (P>0.05; Fig. 5A). Application of BMP-7 increased the expression of collagen I at 3 and 24 h. The results showed that application of BMP-7 at 24 h produced a decrease of Sp7 (P>0.05; Fig. 5B). The results showed that application of BMP-7 at 24 h showed a decrease of IBSP, but no significant differences were noted (P>0.05; Fig. 5C).

Western blot analysis

Western blot analysis was done to analyze protein expression of collagen I, Sp7 and bone sialoprotein following the application with BMP-7 at 3 and 24 h compared with the untreated control group at 3 and 24 h (Fig. 6A). Normalization of the protein expressions showed that the control at 24 h showed 88.4±31.8% expression of collagen I, and the group treated with BMP-7 yielded 154.5±8.2% and 75.1±16.0% of expression of collagen I at 3 and 24 h, respectively, compared to control values at 3 h as a baseline of 100% (100.0±14.3%) (P>0.05, Fig. 6B).

The expression of Sp7 in the control group at 24 h did not show significant change compared with the control at 3 h. Normalization of the protein expressions revealed that the control at 24 h showed 86.8±20.7% expression of Sp7, and the group treated with BMP-7 yielded 93.2±40.6% and 75.4±13.9% of expression of Sp7 at 3 and 24 h, respectively, compared to control values at 3 h as a baseline of 100% (100.0±3.6%) (P>0.05, Fig. 6C).

The relative expression of bone sialoprotein is shown in Fig. 6C. Normalization of the protein expressions demonstrated that the control at 24 h showed 146.8±16.4% expression of bone sialoprotein, and the group treated with BMP-7 yielded 82.5±15.1% and 135.1±9.6% of expression of bone sialoprotein at 3 and 24 h, respectively, when the control value at 3 h were considered 100% (100.0±5.0%). TGF-β signaling pathway was associated with the target genes selected for osteoblast differentiation (Fig. 7).

Alkaline phosphatase activity

The data of the alkaline phosphatase activity assays at Day 7 are demonstrated in Fig. 8. The absorbance values at 405 nm at Day 7 for control at 3 h, BMP-7 at 3 h, control at 24 h and BMP-7 at 24 h were 0.060±0.002, 0.061±0.001, 0.062±0.001, and 0.062±0.001, respectively (P>0.05).

Discussion

In this report, we evaluated the effects of BMP-7 on stem cells under predetermined concentrations at 3 and 24 h. mRNA sequencing and validation of the expression was done with qualitative real-time PCR and Western blot analysis. It was seen that the application of BMP-7 produced increased expression of collagen I of human gingiva-derived mesenchymal stem cells.

Application with gingiva-derived stem cell inhibited macrophage foam cell formation and reduction of inflammatory macrophage activation (18). Previous reports showed that long incubation of gingiva-derived stem cells lead to neural precursor cells in vitro (19). Three-dimensional bioprinted constructs made with gingiva-derived stem cells promoted facial nerve regeneration (8).

BMP-7 expressing stem-cell sheets were applied in the previous studies (2022). In a previous report, BMP-7 overexpressing adenovirus vector was used to transfect mesenchymal stem cells (22). In a non-union fracture model, BMP-7 expressing mesenchymal stem cells enhanced bone regeneration (20). Similarly, BMP-7 expressing stem cells produced enhanced bone healing in critical-sized defects when compared with the control stem cell sheets (23). Moreover, application of BMP-7 overexpressing stem cells derived from bone marrow decelerated the development of disc degeneration (21).

It is widely accepted that differentiation of mesenchymal stem cells is associated with gene transcription and courses of molecular signaling (24). Researchers previously used RNA sequencing to analyze transcriptional profiling of osteoblast differentiation (25). Moreover, RNA sequencing and quantitative reverse transcription polymerase chain reactions were applied for characterization of three-dimensional organoids (26). RNA Sequencing has also been applied to compare the differences between mesenchymal stem cells derived from bone marrow from healthy control and diseased participants (27). Researchers previously tested the functionality of mesenchymal stem cells, the precursors of osteoblasts, using RNA sequencing to measure the epigenetic changes with aging (28). This study tested gingiva-derived stem cells' responsiveness to BMP-7 showed differentially expressed mRNA related to osteoblast differentiation in each group.

Researchers previously tested the expression of osteoblast-associated genes and proteins, including COL1A1, Runx2, IBSP, SPARC, SPP1 and BGALP (29). In this study, several gene expressions were tested in mesenchymal stem cells, including Runx2, Sp7 and collagen I (2931). Researchers previously demonstrated that romidepsin promoted osteogenic differentiation of human mesenchymal stem cells and that it showed increased SP7 (Osterix) and alkaline phosphatase expression (32). They also showed that transfection of Runx2/SP7 enhanced the osteogenic differentiation of mesenchymal stem cells (33). This study showed that BMP-7 upregulates genes related to osteoblast differentiation, including collagen I, in gingiva-derived stem cells. Alkaline phosphatase activity is considered an early-stage marker for osteoblast differentiation, the lack of difference may be dues to various reasons including the treatment time of BMP-7 and the stage of tested cells (34,35).

Conclusively, the effects of BMP-7 on stem cells were assessed using mRNA sequencing, and the validation of the expression was done with quantitative real-time polymerase chain reactions and Western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was related to target genes chosen for osteoblast differentiation. This study may provide new insights into the role of BMP-7 using mRNA sequencing.

Acknowledgements

Not applicable.

Funding

Thε present study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, Information and Communication Technology & Future Planning (Grant no. NRF-2017R1A1A1A05001307).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

HL, SM, YS, YP, and JP contributed to study conception and design. HL, SM, YS, YP, and JP performed the experiments; HL, SM, YS, YP, and JP analyzed the data and HL, SM, YS, YP, and JP wrote the manuscript. All authors reviewed the manuscript.

Ethical approval and consent to participate

The protocol of the study was reviewed and the Institutional Review Board of the Catholic University of Korea, College of Medicine approved the design of this study (KC18SESI0199). Written informed consent was gathered from the participants according to the Act on Legal Codes for Biomedical Ethics and Safety and the Declaration of Helsinki.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Lee H, Min SK, Song Y, Park YH and Park JB: Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells. Exp Ther Med 18: 2867-2876, 2019
APA
Lee, H., Min, S.K., Song, Y., Park, Y., & Park, J. (2019). Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells. Experimental and Therapeutic Medicine, 18, 2867-2876. https://doi.org/10.3892/etm.2019.7904
MLA
Lee, H., Min, S. K., Song, Y., Park, Y., Park, J."Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells". Experimental and Therapeutic Medicine 18.4 (2019): 2867-2876.
Chicago
Lee, H., Min, S. K., Song, Y., Park, Y., Park, J."Bone morphogenetic protein‑7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva‑derived stem cells". Experimental and Therapeutic Medicine 18, no. 4 (2019): 2867-2876. https://doi.org/10.3892/etm.2019.7904