Increased extracellular matrix deposition during chondrogenic differentiation of dental pulp stem cells from individuals with neurofibromatosis type 1: an in vitro 2D and 3D study

Abstract

Background: Neurofibromatosis 1 (NF1) presents a wide range of clinical manifestations, including bone alterations. Studies that seek to understand cellular and molecular mechanisms underlying NF1 orthopedic problems are of great importance to better understand the pathogenesis and the development of new therapies. Dental pulp stem cells (DPSCs) are being used as an in vitro model for several diseases and appear as a suitable model for NF1. The aim of this study was to evaluate in vitro chondrogenic differentiation of DPSCs from individuals with NF1 using two-dimensional (2D) and three-dimensional (3D) cultures.

Results: To fulfill the criteria of the International Society for Cellular Therapy, DPSCs were characterized by surface antigen expression and by their multipotentiality, being induced to differentiate towards adipogenic, osteogenic, and chondrogenic lineages in 2D cultures. Both DPSCs from individuals with NF1 (NF1 DPSCs) and control cultures were positive for CD90, CD105, CD146 and negative for CD13, CD14, CD45 and CD271, and successfully differentiated after the protocols. Chondrogenic differentiation was evaluated in 2D and in 3D (pellet) cultures, which were further evaluated by optical microscopy and transmission electron microscopy (TEM). 2D cultures showed greater extracellular matrix deposition in NF1 DPSCs comparing with controls during chondrogenic differentiation. In semithin sections, control pellets hadhomogenous-sized intra and extracelullar matrix vesicles, whereas NF1 cultures had matrix vesicles of different sizes. TEM analysis showed higher amount of collagen fibers in NF1 cultures compared with control cultures.

Conclusion: NF1 DPSCs presented increased extracellular matrix deposition during chondrogenic differentiation, which could be related to skeletal changes in individuals with NF1.

Keywords: Cell culture; Cell differentiation; Chondrogenesis; Neurofibromatosis 1.

Fig. 1

Flow cytometry of DPSCs from NF1 and control cultures at passage 3 in culture. a: Dot plot showing CD45/SSC complexity and fluorescence. The regions of CD45+ and CD45- cells are indicated. b-f: Histograms showing the expression of CD105, CD90, CD34, CD146 and CD271, respectively, in CD45- cells. Gray line: negative control; Black line: NF1 cultures and dotted line: control cultures

Fig. 2

DPSCs were induced to differentiate towards adipogenic, osteogenic, and chondrogenic lineages in a 2D culture. Phase-contrast microscopy. a-b: Oil Red O staining shows lipids deposits in red after 21 days of adipogenic differentiation. c-d: von Kossa staining identifies calcium deposits at the end of osteogenic protocol. e-f: Alcian blue staining shows the presence of GAGs. A, C, E: CT12. B, D, F: NF87

Fig. 3

Percentage of GAGs stained area after chondrogenic differentiation in 2D cultures from NF1 and control cultures

Fig. 4

Control and NF1 pellets stained with hematoxylin and eosin after chondrogenic differentiation. e-f: Pellets with fusiform cells at the periphery. g-h: Pellets’ central area consisting of large, polygonal cells, with clear cytoplasm, reminding the morphology of chondroblasts. a, e, g: CT10. b: CT11. c: NF37. d, f, h: NF87

Fig. 5

Control and NF1 pellets stained with Alcian blue after chondrogenic differentiation. GAGs are stained in blue. e, j: Cartilage-like fragment is seen. a, e, g: CT10. b: CT11. c: NF37. d, f, h: NF87

Fig. 6

Ultrastructural analysis of control (a, c, e) and NF1 pellets (b, d, f) after chondrogenic differentiation. a-d: Semithin sections stained with toluidine blue. c-d: Intra and extracellular vesicles (green and red arrows, respectively). c: Control culture, vesicles are small and of uniform size. d: NF1 culture, vesicles present heterogeneity of sizes. e-f: V: matrix vesicles; N: nucleus; white arrows: collagen fibers