Gelatin reduced Graphene Oxide Nanosheets as Kartogenin Nanocarrier Induces Rat ADSCs Chondrogenic Differentiation Combining with Autophagy Modification.

Delong Jiao,Ning Zhang,Wenting Yu,Yuxing Bai,Ke Zhang,Jing Wang

doi:10.3390/ma14051053

Delong Jiao, Ning Zhang + Show 4 more

Open Access

https://doi.org/10.3390/ma14051053

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Abstract

Biocompatible reduced graphene oxide (rGO) could deliver drugs for synergistically stimulating stem cells directed differentiation with influences on specific cellular activities. Here, we prepared a biodegradable gelatin reduced graphene oxide (rGO@Ge) to evaluate its functions in promoting rat adipose derived mesenchymal stem cells (ADSCs) chondrogenic differentiation through delivering kartogenin (KGN) into the stem cell efficiently. The optimum KGN concentration (approximately 1 μM) that promoted the proliferation and chondrogenic differentiation of ADSCs was clarified by a series of experiments, including immunofluorescent (IF) staining (Sox-9, Col II), alcian blue (Ab) staining, toluidine blue (Tb) staining and real-time quantitative PCR analysis of the chondrogenic markers. Meanwhile, the biocompatibility of rGO@Ge was evaluated to clearly define the nonhazardous concentration range, and the drug loading and releasing properties of rGO@Ge were tested with KGN for its further application in inducing ADSCs chondrogenic differentiation. Furthermore, the mechanism of rGO@Ge entering ADSCs was investigated by the different inhibitors that are involved in the endocytosis of the nanocarrier, and the degradation of the rGO@Ge in ADSCs was observed by transmission electron microscopy (TEM). The synergistic promoting effect of rGO@Ge nanocarrier on ADSCs chondrogenesis with KGN was also studied by the IF, Ab, Tb stainings and PCR analysis of the chondrogenic markers. Finally, the intracellular Reactive Oxygen Species (ROS) and autophagy induced by KGN/rGO@Ge complex composites were tested in details for clarification on the correlation between the autophagy and chondrogenesis in ADSCs induced by rGO@Ge. All the results show that rGO@Ge as a biocompatible nanocarrier can deliver KGN into ADSCs for exerting a pro-chondrogenic effect and assist the drug to promote ADSCs chondrogenesis synergistically through modification of the autophagy in vitro, which promised its further application in repairing cartilage defect in vivo.

Highlights

The alcian bule (Ab) and toluidine blue (Tb) stainings (Figure 1A) showed the adipose derived mesenchymal stem cells (ADSCs) chondrogenic differentiation induced by transforming growth factor β3 (TGFβ3), the glycosaminoglycan (GAG) in the cartilage matrix nodule was stained with alcian blue and the secreted chondroitin sulfate was stained with toluidine blue
All the results prove that the cultured ADSCs had differentiated into chondrocytes
After the verification of the ADSCs chondrogenesis, we evaluated the effects of different KGN concentrations on ADSCs proliferation and chondrogenic differentiation

Summary

Introduction

The autologous cartilage grafts repair has a limited repair capacity due to the poor blood supply for chondrocyte proliferation and migration resulting in the unsatisfactory therapeutic effect [2]. With the development of cartilage engineering, the low regenerative capacity of chondrocytes in grafts restricts its application as the seed cell, so the mesenchymal stem cells (MSCs) have been introduced into the cartilage regeneration as a promising cell source with the ability of self-renewal and multiple differentiation potential [3]. * p < 0.05 indicates a significant difference compared to the corresponding ferent rGO@Ge concentration media, Merge+BF: the merge of Live, Dead and Background Field control group at each time point. Flow cytometry analysis of the ADSCs apoptosis with the different rGO@Ge concentration stimulus.

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