Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures

Abstract

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

Stemness Potency of Human Gingival Cells—Application in Anticancer Therapies and Clinical Trials

“…Osteogenic differentiation was carried out in a number of studies by conducting GMSCs culture in osteoinductive medium with addition of dexamethasone, ascorbic acid, and β-glycerophosphate (among others) [22,25,28,29,32,[35][36][37]. The results of differentiation were confirmed with Alizarin Red S staining to detect calcified deposits in culture, alkaline phosphatase (ALP) activity assay, and gene expression analysis of bone specific markers-Runx2 (Runt-related transcription factor 2), collagen I, ALP, osteocalcin (OCN), and bone sialoprotein (BSP) [22,25,28,29,32,[35][36][37]. Moreover, GMSCs exhibited higher osteogenic potential than BM-MSCs, as indicated by Sun et al [36].…”

Morphological Comparison of Stem Cells Using Two- Dimensional Culture and Spheroid Culture

“…Bone marrow is an attractive source of stem cells, but gaining stem cells from bone marrow may produce greater pain and morbidity [10]. Stem cells can also be achieved intraorally, and gingiva may serve as a more feasible source for stem cells because obtaining gingival-derived stem cells can be done under local anesthesia with less pain and morbidity [11]. Figure 1 shows morphology of the stem cells cultured in an alpha-minimal essential medium (α-MEM, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA), 200 mM of lglutamine (Sigma-Aldrich Co.), and 10 mM of ascorbic acid 2-phosphate (Sigma-Aldrich Co.) on Day 12.…”

“…In a previous report, two-and three-dimensional systems were used for the determination of secreted human vascular endothelial growth factor using a commercially available kit (Quantikine ® ELISA, R&D Systems, Inc., Minneapolis, MN, USA) [6]. The osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids were evaluated by comparing two-and three-dimensional cultures, and the results indicated that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture [11]. The co-culture of various cells including stem cells and primary cells can be done at various ratios [5].…”

3D printed in vitro tumor tissue model of colorectal cancer

“…However, as the mechanisms of tumor progression and the tumor microenvironment have become elucidated, the limitations of the models have become increasingly apparent. Traditional two-dimensional (2D) cultures lack the diversity of internal spatial information, cell types, and the TME 15 . Although animal models can simulate physiological conditions and reflect interactions between various systems, clinical trials have had a low rate of success, requiring a long culture cycle, and are associated with high costs 16 .…”