Stimulation of Chondrocyte and Bone Marrow Mesenchymal Stem Cell Chondrogenic Response by Polypyrrole and Polypyrrole/Gold Nanoparticles

Abstract

Bone marrow mesenchymal stem cells (BMMSCs) possess a strong ability to differentiate into the chondrogenic lineage, which is important for cartilage regeneration. External stimuli, such as electrical stimulation (ES), are frequently studied for chondrogenic differentiation of BMMSCs; however, the application of conductive polymers such as polypyrrole (Ppy), has never been used for stimulating BMMSCs chondrogenesis in vitro before. Thus, the aim of this study was to evaluate the chondrogenic potential of human BMMSCs after stimulation with Ppy nanoparticles (Ppy NPs) and compare them to cartilage-derived chondrocytes. In this study, we tested Ppy NPs without and with 13 nm gold NPs (Ppy/Au) for BMMSCs and chondrocyte proliferation, viability, and chondrogenic differentiation for 21 days, without the use of ES. The results demonstrated significantly higher amounts of cartilage oligomeric matrix protein (COMP) in BMMSCs stimulated with Ppy and Ppy/Au NPs, as compared to the control. The expression of chondrogenic genes (SOX9, ACAN, COL2A1) in BMMSCs and chondrocytes were upregulated by Ppy and Ppy/Au NPs, as compared to controls. Histological staining with safranin-O indicated higher extracellular matrix production in Ppy and Ppy/Au NPs stimulated samples, as compared to controls. In conclusion, Ppy and Ppy/Au NPs stimulate BMMSC chondrogenic differentiation; however, BMMSCs were more responsive to Ppy, while chondrocytes possessed a stronger chondrogenic response to Ppy/Au NPs.

Keywords: bone marrow mesenchymal stem cells; chondrocytes; chondrogenic differentiation; polypyrrole; polypyrrole/gold nanoparticles.

Figure 1

SEM image of synthesized AuNPs. Inset: UV-Vis spectra of AuNPs suspension.

Figure 2

SEM images of (A) Ppy and (B) Ppy/Au NPs.

Figure 3

BMMSCs and chondrocytes cultivated without and with Ppy or Ppy/Au NPs (10 μg/mL) for 7 days. (A): macroscopic view of the cell plate. (B): cells visualized under light microscopy, X40. w/o NP control cells, cultivated under the same conditions, but without NPs.

Figure 4

The proliferation of BMMSCs and chondrocytes cultivated with and without Ppy and Ppy/Au NPs (10 μg/mL) after 1, 3 and 7 days, measured using Alamar blue dye, which fluorescence measured with a spectrophotometer at 560/590 nm. w/o NP control cells, cultivated under the same conditions, but without NPs (n = 3). Data are presented as mean ± SD. * Horizontal bars represent p ≤ 0.05.

Figure 5

BMMSC viability after cultivating with/without Ppy and Ppy/Au NPs (10 μg/mL) for 21 days, after stained with viability dyes calcein-green for live cells and propidium iodide for dead cells (Live/Dead kit). (A): cells visualized under fluorescent microscopy, using blue (488 nm) and green (532 nm) lasers, ×100. (B): quantified percentage of Live/Dead cells. w/o NP control cells, cultivated under the same conditions, but without NPs.

Figure 6

Levels of COMP in BMMSCs and chondrocytes medium after 21 days of chondrogenic differentiation in monolayer (ELISA), with TGF-β3 (10 ng/mL), Ppy (10 μg/mL), Ppy/Au NPs (10 μg/mL), as well as without TGF-β3 (w/o TGF-β3) and without NPs (w/o NP). Data are presented as mean ± SD. * Horizontal bars represent p ≤ 0.05.

Figure 7

Chondrogenic gene (SOX9, ACAN, COL2A1) expression in BMMSCs and chondrocytes, after 21 days of chondrogenic differentiation in monolayer with TGF-β3 (10 ng/mL), Ppy (10 μg/mL), Ppy/Au NPs (10 μg/mL), as well as without TGF-β3 (w/o TGF-β3) and without NPs (w/o NP). Relative mRNA level was normalized to two housekeeping genes (B2M and RPS9) and expressed as 2−ΔCt*1000, the ratio of Ppy and Ppy/Au NP stimulated samples vs. non-stimulated controls. Data are presented as mean ± SD. * represent differences p ≤ 0.05, which was calculated vs. controls without NPs.

Figure 8

Chondrogenic differentiation of BMMSCs and chondrocytes (macroscopic and microscopic views). Cells were incubated in a chondrogenic differentiation medium with/without TGF-β3 (10 ng/mL) and with/without Ppy (10 μg/mL), Ppy/Au NPs (10 μg/mL) for 21 days, after cell pellets were histologically analysed. Histological sections of cell pellets, stained with safranin-O and visualized under light microscopy ×40/×100 magnification. w/o NP control cells without NPs. Cartilage positive control staining for cartilage histological sections with safranin-O.