**1. 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 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.
