# Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study
## 1 Introduction  
Mesenchymal stem cells (MSCs) have self-renewal and multilineage differentiation potency. In humans, they can be harvested from various tissues such as bone marrow, adipose tissue, and umbilical cord blood [1]. In addition, they have been successfully extracted from the periodontal ligaments of permanent teeth [2]. Periodontal ligament stem cells (PDLSCs) are easy to obtain, have a greater proliferation rate than bone marrow stem cells, are capable of differentiating into osteoblasts, cementoblasts, adipocytes, and chondrocytes, and can form a structure similar to periodontal ligaments in vitro [3]. In addition, the relationship between periodontitis and a stem cell has been revealed in recent years [4, 5, 6].  
Cell cultures are essential for the study of MSCs. Conventional cell culture methods are two-dimensional (2D) methods in which cells are cultured on the bottom of a culture-compatible polystyrene plate. Such cultures create an environment for studying biological processes or cell mechanisms under specific experimental conditions and maintain various types of proliferation [7]. However, in nature, cells grow three-dimensionally ratherthan in a 2D plane. Therefore, it is difficult to study cell-to-cell signal transduction and the functions of cells in a conventional 2D culture environment [8]. To overcome these limitations, cell research using three-dimensional (3D) culture technology is being actively pursued. Such cultures have the advantage of emulating the original characteristics of the cell's environment so that the cell can display form, function, and activity as close to natural as possible [9; 10]. In addition, 3D cell culture has attracted attention because it can function as a substitute for animal experimentation [11].  
There are two general methods of 3D cell culture. The first involves creating a scaffold of microstructures the cells can use to move, proliferate, differentiate, and contact each other [11]. However, scaffolds have limitations that can negatively affect cell stability and cell behavior during incubation [12]. In the case of hydrogel among the scaffold techniques, there is also a problem that the hydrogel collapses during the media replacement process [13]. In the scaffold-free method, a pellet or centrifugation tube culture, low-adhesion plate culture, bioreactor, hanging drop culture, or a pellet culture and liquid overlay are used to create 3D microstructures without scaffolding [7; 11; 12; 14]. In these methods, cells assemble an endogenous extracellular matrix to form their own preferred microenvironment [11; 15; 16].  
Cells cultured in a 3D environment behave differently than those in a 2D environment [14]. For example, primary articular chondrocytes and hepatocytes cultured in a 2D environment rapidly lose their normal phenotypes, but in 3D cultures, the normal phenotype is maintained [17]. In addition, 3D-cultured stem cells exhibit better osteoblastic, adipogenic, and neuronal differentiation than 2D-cultured stem cells [15; 18; 19]. Similarly, 3D-cultured human dental pulp cells show higher expression of osteoclast, dentin sialophosphoprotein, and alkaline phosphatase than those cultured under 2D conditions [20]. The expression of these proteins is influenced by cell-cell and cell-matrix interactions, which are difficult to imitate in 2D culture. In addition, the expression of multilinearage differentiation-related genes, such as Nanog and Oct4, and pluripotency transcription factor-related genes, is higher in 3D-cultured cells than those grown in 2D culture [21]. These studies demonstrate how cell culture conditions can influence gene expression [14; 22]. However, because previous studies have usually used microarrays and polymerase chain reaction (PCR), few studies have analyzed genome-wide expression.  
This pilot study compared the morphology, viability, stemness, and functional gene expression of PDLSCs cultured using either 3D or conventional 2D methods.