**4. 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 [24,28–32]. 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 [38–40]. 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].

#### **5. 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 when stem cells are utilized. BMP-4 can be suggested as a coating material for stem cell cultures. Spheroids impregnated with BMP-4 can be suggested for the bone regeneration field as stem cell therapy.

**Author Contributions:** Conceptualization, J.-Y.T., Y.-H.P., Y.K. and J.-B.P.; methodology J.-Y.T., Y.-H.P. and J.-B.P.; validation, J.-Y.T., Y.-H.P. and J.-B.P.; formal analysis, J.-Y.T., Y.-H.P. and J.-B.P.; writing—original draft preparation, J.-Y.T., Y.-H.P., Y.K. and J.-B.P.; and writing—review and editing, J.-Y.T., Y.-H.P., Y.K. and J.-B.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C4001624). This research was also funded by Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea.

**Conflicts of Interest:** The authors have no competing interests regarding this study.
