The E ﬀ ects of Bone Morphogenetic Protein-4 on Cellular Viability, Osteogenic Potential, and Global Gene Expression on Gingiva-Derived Stem Cell Spheroids

: Bone morphogenetic protein-4 (BMP-4) is engaged in the migration ability of mesenchymal stem cells and the transition of mesenchymal stem cells into osteogenic and adipocytic lines. The aim of this study was to evaluate the e ﬀ ects of BMP-4 on the cellular viability, osteogenic di ﬀ erentiation, and genome-wide mRNA levels using three-dimensional cell spheroids composed of stem cells. Stem cell spheroids were formed using concave microwells in the presence of BMP-4 with ﬁnal concentrations of 0, 2, 6, and 10 ng / mL. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. Osteogenic di ﬀ erentiation was assessed by measuring the level of alkaline phosphatase activity. Global gene expression was assessed using next-generation mRNA sequencing and performing gene ontology and pathway analyses. Spheroids were well-maintained with the addition of BMP-4 up to Day 7. No signiﬁcant di ﬀ erences were observed in cell viability between each group. There were signiﬁcantly higher alkaline phosphatase values in the 2 ng / mL BMP-4 groups when compared with the control ( p < 0.05). A total of 25,737 mRNAs were di ﬀ erentially expressed. Expression of β -catenin (CTNNB1) was increased with higher dosages of BMP-4. The expression of runt-related transcription factor 2 (RUNX2) was increased up to 6 ng / mL. The phosphoinositide-3-kinase–protein kinase B / Akt signaling pathway was associated with the target genes. This study demonstrates that the application of BMP-4 enhanced alkaline phosphatase activity and the expression of CTNNB1 and RUNX2 without a ﬀ ecting cellular viability.


Introduction
Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into the mesenchymal lineage and easily cultured in vitro [1]. A previous study demonstrated that stem cell spheroids of various sizes could be generated from gingival cells using microwells and that the shape and viability of the spheroids could be maintained [2]. Furthermore, cell spheroids made from gingival cells and osteoblast cells were able to maintain shape, viability, and osteogenic differentiation ability [3]. Stem cell therapy has been of great interest in recent years [4]. A two-dimensional culture has long been applied for the evaluation of viability and functionality of stem cells [3]. In more recent years, three-dimensional cultures have been used by applying various methods including the hanging drop two-dimensional culture has long been applied for the evaluation of viability and functionality of stem cells [3]. In more recent years, three-dimensional cultures have been used by applying various methods including the hanging drop method, bioreactor, capsules, and microwells [5]. Three-dimensional cultures have been reported to mimic the in vivo situation more closely [6]. A three-dimensional culture can be categorized by scaffold-based or scaffold-free application [7]. Three-dimensional spheroids can be made of a variety of cells including stem cells with the scaffold-free technique [8]. Spheroids can be used to obtain an overall enhancement in therapeutic potential by improving survival, stemness, angiogenic properties, and anti-inflammatory effects [9].
Bone morphogenetic proteins (BMPs) are powerful growth factors in the transforming growth factor beta superfamily [10]. More than twenty members with various functions have already been identified in humans, with roles in processes such as skeletal formation, hematopoiesis, and neurogenesis [11]. These BMPs are soluble local-acting signaling proteins that may behave in an endocrine, paracrine, or autocrine manner [12]. BMP-4 may be involved in various functions, including enhancing the migration ability of mesenchymal stem cells and the transition from mesenchymal stem cells into the osteogenic and adipocytic lines [13,14]. BMP-4 may act as an important regulator for proper reproductive tissue development [15]. Moreover, BMP-4 is reported to be involved in postnatal tooth cytodifferentiation [16]. BMP-4 has been suggested as a coating material for titanium implants [17]. To the best of the authors' knowledge, there are no previous studies evaluating the effects of BMP-4 on the cell spheroids composed of gingiva-derived stem cells using microwells. In light of the promising findings in previous studies on BMP-4, the aim of the present study was to evaluate the effects of BMP-4 on cellular viability, osteogenic differentiation, and genome-wide mRNA levels using stem cell spheroids.

Formation of Cell Spheroids with Gingiva-Derived Stem Cells
Cell spheroids were made of gingiva-derived mesenchymal stem cells using the concave microwells made of silicone elastomer having 600 μm diameters (H389600, StemFIT 3D; MicroFIT, Seongnam, Korea). The number of cells loaded in each well was 1 × 10 6 . We obtained approval from the Institutional Review Board at Seoul St Mary's Hospital, Seoul, Korea (KC20SISE0695), and informed consent was obtained from the participant. Cell spheroids made were treated with BMP-4 (ProSpec, Ness-Ziona, Israel) at 0, 2, 6, and 10 ng/mL concentrations. The morphological changes in cell spheroids were observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany). The changes in the spheroids' diameter were evaluated on Days 1, 3, 5, and 7. The diameter of the spheroids was determined as described in a previous study [18]. Figure 1 diagrams the overall design of the study.

Evaluation of Cellular Viability
Qualitative analysis of the cell spheroids for cellular viability was done using the Live/Dead assay kit (Molecular Probes, Eugene, OR, USA). These spheroids were washed twice with the growth media before calcein acetoxymethyl (Molecular Probes) and ethidium homodimer-1 (Molecular Probes) were added for an incubation period of 30 min at room temperature. Then, the spheroids were evaluated using a fluorescence microscope (Axiovert 200; Zeiss, Oberkochen, Germany) on Days 3 and 7.
Quantitative analysis of cell viability was performed using the cell counting kit-8 (CCK-8; Dojindo, Tokyo, Japan) on Days 1, 3, 5, and 7. WST-8 solution was added to the stem cell spheroids, which were then cultured for 45 min at 37 • C. Absorbance was measured using a microplate reader at 450 nm (BioTek, Winooski, VT, USA).

Evaluation of Osteogenic Differentiation Using Alkaline Phosphatase Activity Assays
Cell spheroids were grown in osteogenic medium and were collected on Days 1, 3, 5, and 7. A commercially available alkaline phosphatase assay kit (K412-500, BioVision, Inc., Milpitas, CA, USA) was used for the evaluation of osteogenic differentiation. In short, the resultant supernatant was mixed and incubated with p-nitrophenyl phosphate substrate (BioVision, Inc.) for 40 min at room temperature. Absorbance was measured using a microplate reader at 405 nm (BioTek, Winooski, VT, USA).

Sequencing of mRNA, Gene Ontology, and Pathway Analysis
Construction of a library of RNAs was performed using the SENSE mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria). Briefly, 2 µg of total RNA was processed and incubated with oligo-dT magnetic beads, after which other RNAs except mRNA were eliminated with a washing solution. Random hybridization of starter/stopper heterodimers was applied to the poly(A)RNA still bound to the magnetic beads in order to produce libraries. These heterodimers consisted of Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction was applied to extend the starter to the next hybridized heterodimer. Then, the newly synthesized cDNA insert was bound with the stopper. The release of the library from the beads was done by second-strand synthesis. The library was amplified afterward and bar codes were introduced. High-throughput sequencing was done using HiSeq 2500 (Illumina, San Diego, CA, USA) as paired-end 100 bp sequencing.

Statistical Analysis
All statistical analysis was performed using SPSS 12 for Windows (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance with Tukey's post-hoc test was used to evaluate the differences between each group after performing a test of normality. A p-value less than 0.05 was set as the threshold for statistical significance.

Formation of Cell Spheroids with Human Gingiva-Derived Stem Cells
Spheroids were well-established in each microwell on Day 1 ( Figure 2). Furthermore, no noticeable changes in the shape of the cell spheroids were observed with the addition of BMP-4 at concentrations Coatings 2020, 10, 1055 4 of 15 of 2, 6, or 10 ng/mL. There were no noticeable changes at the longer culturing times. The spheroid diameters are shown in Figure 3. There was a general decrease in the diameter of the spheroids with longer incubation time.

Formation of Cell Spheroids with Human Gingiva-Derived Stem Cells
Spheroids were well-established in each microwell on Day 1 ( Figure 2). Furthermore, no noticeable changes in the shape of the cell spheroids were observed with the addition of BMP-4 at concentrations of 2, 6, or 10 ng/mL. There were no noticeable changes at the longer culturing times. The spheroid diameters are shown in Figure 3. There was a general decrease in the diameter of the spheroids with longer incubation time.   Figure 4A shows qualitative results for the viability of cell spheroids at Day 3 using a Live/Dead assay kit ( Figure 4A). In all cases, the cells in the spheroids produced intense green fluorescence. Red fluorescence was partly noted around the boundary of the spheroids. No significant differences were noted at Day 7 when compared with results of Day 3 ( Figure 4B). Figure  4C shows the quantitative results for cellular viability on Days 1, 3, 5, and 7. No significant   Figure 4A shows qualitative results for the viability of cell spheroids at Day 3 using a Live/Dead assay kit ( Figure 4A). In all cases, the cells in the spheroids produced intense green fluorescence. Red fluorescence was partly noted around the boundary of the spheroids. No significant differences were noted at Day 7 when compared with results of Day 3 ( Figure 4B). Figure 4C shows the quantitative results for cellular viability on Days 1, 3, 5, and 7. No significant differences were observed among the groups on Day 1 (p > 0.05). In general, there were no significant differences among the groups with longer incubation times.  Figure 4A shows qualitative results for the viability of cell spheroids at Day 3 using a Live/Dead assay kit ( Figure 4A). In all cases, the cells in the spheroids produced intense green fluorescence. Red fluorescence was partly noted around the boundary of the spheroids. No significant differences were noted at Day 7 when compared with results of Day 3 ( Figure 4B). Figure  4C shows the quantitative results for cellular viability on Days 1, 3, 5, and 7. No significant differences were observed among the groups on Day 1 (p > 0.05). In general, there were no significant differences among the groups with longer incubation times.

Determination of Cellular Viability
(A)

Evaluation of Alkaline Phosphatase Activity Assay with the Addition of BMP-4
The results of the alkaline phosphatase activity assay at Days 1, 3, 5, and 7 are presented in Figure 5. In general, there were increases in the alkaline phosphatase activity with longer incubation time up to Day 7. Notably, the group treated with 2 ng/mL BMP-4 at Day 3 had a significantly higher activity compared with that of the control group at Day 3 (p < 0.05).

Evaluation of Alkaline Phosphatase Activity Assay with the Addition of BMP-4
The results of the alkaline phosphatase activity assay at Days 1, 3, 5, and 7 are presented in Figure 5. In general, there were increases in the alkaline phosphatase activity with longer incubation time up to Day 7. Notably, the group treated with 2 ng/mL BMP-4 at Day 3 had a significantly higher activity compared with that of the control group at Day 3 (p < 0.05).

Gene Ontology
A total of 25,737 mRNAs were differentially expressed. Scatter plots of the differentially expressed mRNAs are shown in Figure 6. A Venn diagram of the gene ontology analysis of differentially expressed mRNAs is shown in Figure 7. When compared with the 0 ng/mL control group, 1270 mRNAs were upregulated and 1070 mRNAs were downregulated in the 2 ng/mL group. In the 6 ng/mL group, 1536 mRNAs were upregulated and 1889 mRNAs were downregulated compared to controls. In the 10 ng/mL group, 1525 mRNAs were upregulated and 1533 mRNAs were downregulated compared to controls. A clustering analysis of differentially expressed mRNAs related to osteoblast differentiation is shown in Figure 8. The changes in expression of RUNX2 and CTNNB1 are shown in Figure 9. While the expression of CTNNB1 was increased dose-dependently, the expression of RUNX2 was highest at 6 ng/mL and the expression decreased at the higher dose of 10 ng/mL ( Figure 9A,B). The phosphoinositide-3-kinase-protein kinase B/Akt (PI3K/AKT) signaling pathway was involved in the target genes chosen for stem cell differentiation ( Figure 10).

Gene Ontology
A total of 25,737 mRNAs were differentially expressed. Scatter plots of the differentially expressed mRNAs are shown in Figure 6. A Venn diagram of the gene ontology analysis of differentially expressed mRNAs is shown in Figure 7. When compared with the 0 ng/mL control group, 1270 mRNAs were upregulated and 1070 mRNAs were downregulated in the 2 ng/mL group. In the 6 ng/mL group, 1536 mRNAs were upregulated and 1889 mRNAs were downregulated compared to controls. In the 10 ng/mL group, 1525 mRNAs were upregulated and 1533 mRNAs were downregulated compared to controls. A clustering analysis of differentially expressed mRNAs related to osteoblast differentiation is shown in Figure 8. The changes in expression of RUNX2 and CTNNB1 are shown in Figure 9. While the expression of CTNNB1 was increased dose-dependently, the expression of RUNX2 was highest at 6 ng/mL and the expression decreased at the higher dose of 10 ng/mL ( Figure 9A,B). The phosphoinositide-3-kinase-protein kinase B/Akt (PI3K/AKT) signaling pathway was involved in the target genes chosen for stem cell differentiation ( Figure 10).

Discussion
In this study, we examined the effects of BMP-4 on stem cell spheroids under predetermined concentrations of 2, 6, and 10 ng/mL and found that the application of BMP-4 increased alkaline phosphatase activity and the expression of RUNX2 and CTNNB1 without affecting cellular viability.
BMP-4 is reported to act as a regulator for osteogenic differentiation and has been shown to induce endochondral and intra-membranous bone formation [12,24]. In a previous report, BMP-4 carried by liposomes seemed to improve the healing process in alveolar bone [25]. Similarly, the expression of BMP-4 appeared to be associated with normal bone homeostasis and the remodeling of grafted and nongrafted maxillary sites [26]. Additionally, BMP-4 induced osteogenic

Discussion
In this study, we examined the effects of BMP-4 on stem cell spheroids under predetermined concentrations of 2, 6, and 10 ng/mL and found that the application of BMP-4 increased alkaline phosphatase activity and the expression of RUNX2 and CTNNB1 without affecting cellular viability.
BMP-4 is reported to act as a regulator for osteogenic differentiation and has been shown to induce endochondral and intra-membranous bone formation [12,24]. In a previous report, BMP-4 carried by liposomes seemed to improve the healing process in alveolar bone [25]. Similarly, the expression of BMP-4 appeared to be associated with normal bone homeostasis and the remodeling of grafted and nongrafted maxillary sites [26]. Additionally, BMP-4 induced osteogenic differentiation of mouse skin-derived fibroblasts and dermal papilla cells [24]. Furthermore, a study testing the effects of abnormal BMP-4 expression in the blood of diabetic participants found that low expression of BMP-4 hindered the osteogenic function of bone marrow-derived stem cells [27]. This study also clearly showed that BMP-4 increased osteogenic differentiation of stem cell spheroids composed of gingiva-derived mesenchymal stem cells.
The effects of BMP-4 concentration were tested in previous reports [24,[28][29][30][31][32]. Application of 20 ng/mL BMP-4 to primary osteoblastic cells derived from the calvaria resulted in an enhancement in fibronectin synthesis [28]. Treatment with 70 ng/mL of BMP-4 stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblasts [29]. Similarly, the use of 30 ng/mL BMP-4 was associated with an increase in osteoprotegerin synthesis in osteoblast-like MC3T3-E1 cells [30]. At a concentration of 50 ng/mL, BMP-4 induced osteogenic differentiation of mouse skin-derived fibroblasts and dermal papilla cells [24]. Treatment with 500 ng/mL BMP-4 resulted in in vitro osteogenic differentiation of C2C12 cells derived from mouse muscle [31]. Primary human mesenchymal stem cells were treated with 100, 200, or 500 ng/mL BMP-4 and cells were stained with Alizarin red to detect calcium deposition, and the results showed that the 500 ng/mL dose produced the highest value [32]. Moreover, MG63 and Sao2 osteosarcoma cell lines were treated with 25 ng/mL BMP-4 to evaluate the cell cycle distributions, and the results showed that BMP-4 seemed to increase the percent of cells in the G0/G1 phases and decrease the percent of cells in the synthetic and/or G2/M phases [33]. This study showed that the application of 2 or 6 ng/mL BMP-4 could increase the osteogenic differentiation of stem cell spheroids and the expression of related genes. The variety of effects seen across concentrations may be partly due to the differences in cell types, culture conditions, and culture times [34,35].
In a previous report, modification of the roughened anodized titanium implant was done by wet coating with growth factors [36]. In another study, the coating of the titanium implants was obtained by absorption of growth factors after coating the surface with the collagen [17]. The results showed that coating with collagen, chondroitin sulphate, and BMP-4 showed the highest bone-to-implant contact. Enhanced coating can be obtained by applying various methods including chemical bonding, polymer layer, and covering layer [37].
BMP-4 has been proposed to act on various pathways [38][39][40]. A previous report showed that BMP-4 affected the osteogenic differentiation and mineralization of bone marrow-derived stem cells through Wnt/β-catenin activation [38]. This study suggested the involvement of the PI3K/AKT pathway, and a previous report showed that the mineralization of osteoblasts occurred through the PI3K/AKT pathway [40].
Sequencing was performed to measure genome-wide mRNA expression levels and to investigate the possible mechanisms behind the observed effects of BMP-4. RUNX2 and CTNNB1 (which affect β-catenin expression) are major regulators for osteoblastic lineage [41,42]. RUNX2 is reported to be essential for osteogenic differentiation and is weakly expressed in uncommitted mesenchymal cells but shows up-regulated expression in preosteoblasts [43]. The expression of the osteoblast marker gene RUNX2 was significantly up-regulated in cell spheroids composed of adipose-derived stem cells [44]. β-catenin is reported to be involved in activation of the osteogenic-related signaling pathway [45,46]. β-catenin is also reported to control the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts [47]. Furthermore, β-catenin is involved in mediating the viability of osteoblasts [48]. In this report, expression levels of both CTNNB1 and RUNX2 were up-regulated with the application of BMP-4. The focusing on RUNX2 and CTNNB1 expression with agonists may produce enhanced functionality. BMP-4 can be suggested as a coating material for the stem cell culture for enhancing for osteogenic differentiation [49]. Moreover, spheroids can be made with stem cells mixed with BMP-4 or impregnated with BMP using fibers [50].

Conclusions
This study evaluated the effects of BMP-4 on cellular viability, osteogenic differentiation, and global mRNA expression using stem cell spheroids composed. Together, these results revealed that the application of BMP-4 increased alkaline phosphatase activity and CTNNB1 and RUNX2 expression without affecting cellular viability. Based on this research, the coating with BMP-4 can be applied