1. Introduction
As a degenerative disease of the cartilage, osteoarthritis’ (OA) main pathological manifestations are the decomposition and fibrotic developments of cartilage extracellular matrix (ECM) resulting in structure and function loss of the articular cartilage [
1]. 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].
The chondrogenesis of the MSCs usually needs the inductive agents, such as the transforming growth factor β3 (TGFβ3), which is a principal factor in cartilage development. However, the over-dose usage of TGFβ3 would cause side-effects, including osteophyte, inflammation and fibroplasia [
4]. Recently, kartogenin (KGN) as a small molecule drug has been proved to facilitate human mesenchymal stem cells (HMSCs) chondrogenesis in a dose-dependent manner (median effective concentration (EC50): 100 nM) [
5], and it provides us with the safer choice of the chondro-inducers that act on MSCs to promote chondrogenesis with less side-effects [
6]. Among the choices of the seed cells, the adipose derived mesenchymal stem cells (ADSCs) is considered a good MSCs candidate for cartilage regeneration, because its harvesting process is less invasive and a great number of cells can be acquired by expanding in vitro culture simply [
7]. The research on the efficient drug delivery strategy would have a great significance in achieving the optimum ADSCs chondrogenesis induced by KGN in cartilage repair.
In recent years, the graphene-based materials show a potential biomedical applications with a good biocompatibility due to its unique structure and geometry characters [
8]. Graphene oxide (GO), as the single-atom-thick layer of graphene sheet with carboxylate, hydroxyl and epoxide functional groups, can adsorb the drugs and biomolecules through π–π interactions [
9] and also function as filler to modify the hydrogel for better biological functions [
10,
11]. Reduced graphene oxide (rGO) is acquired through reducing GO via high-temperature thermal treatment or chemical reducing agents treatment which provided it with more functional groups [
12]. Both GO and rGO have been reported to be the nanocarrier for delivering the drug molecules to induce stem cell differentiation [
13,
14,
15]. Due to the poor solubility and unstable dispersion of GO, the different modification methods were applied to induce the covalent or noncovalent functional groups for the improvement of solubility and stability [
16,
17,
18]. The reduction methods of synthesizing biocompatible rGO from GO were developed with more choices of biological reducing reagents to modify the nanocarrier for broader and safer biological application [
19,
20]. Gelatin as the hydrolyzed collagen is a reducing polypeptide, in which the amino acids on its molecule chain can be oxidized to nitrite [
21]. Our previous synthesized gelatin reduced graphene oxide (rGO@Ge) has been verified as a biocompatible nanocarrier for promoting osteogenesis of the bone marrow mesenchymal stem cells (BMSCs) synergistically with the osteo-inductive drug [
22]. The promised nanocarrier function of rGO@Ge prospects its potential application in delivering KGN for promoting ADSCs chondrogenesis.
With regard to the drug delivery strategy, researchers discovered that the distributions of the nanocarrier in the stem cell could influence its drug delivery function and exert a specific effect on cellular life activities, which resulted in the regulation of stem cell differentiation and proliferation [
23]. Autophagy as the cellular homeostasis mechanism could remove the dysfunctional organelles and macromolecules, and recent observations illustrate that the pathogenesis of the aging-related diseases, including OA, usually involves the change of compromised or defective autophagy [
24]. It has also been reported that the chondrogenesis of MSCs could be promoted via the modulation of autophagy [
25]. This indicates that the augmentation of the cellular homeostasis mechanism (autophagy) may be a new strategy for the modification of the MSCs chondrogenesis in OA treatment. So, in our research, we would firstly clarify the effects of rGO@Ge on the KGN delivery into ADSCs for chondrogenesis and further investigate the entry pathways and degradation of rGO@Ge in ADSCs. Furthermore, the change of autophagy in ADSCs was also evaluated to define the relationship between the chondrogenesis and autophagy modulated by rGO@Ge. Through the serials experiments, we would sketch the possible mechanism of rGO@Ge on the drug delivery and synergistic pro-chondrogenic differentiation effect. To our knowledge, our study is the first to evaluate the correlation between the rGO@Ge-induced autophagy and its promotive effect on MSCs chondrogenesis in vitro. The detailed research about effects of rGO@Ge on promoting ADSCs chondrogenesis with KGN would propose its promising application in the cartilage regeneration repair in vivo.
2. Materials and Methods
2.1. Rat ADSCs Isolation and Culture
Adipose derived mesenchymal stem cells (ADSCs) were isolated from 4-week-old male Sprague-Dawley (SD) rats. The inguinal adipose tissue was collected from the SD rats under sterilized condition and rinsed by phosphate buffer saline (PBS) three times. The tissue block was cut into pieces with microsurgical scissors and digested with collagenase type I (1 mg/mL, Sigma-Aldrich, USA) for 60 min at 37 °C, then the digestion product was vortexed for further dispersion. The mixture solution was centrifuged with 1500 rpm for 10 min, the supernatant was discarded and the remaining pellet was resuspended with the Dulbecco’s modified Eagle medium (DMEM) (supplemented with 10% Foetal Bovine Serum (FBS) and 1% penicillin/streptomycin) for the cell expansion culture in a humidified atmosphere of 5% CO2 in air at 37 °C. When the cell colonies became large and confluent, ADSCs were digested by trypsin for passage with medium changes every 3 days. Subsequently, ADSCs acquired from passage 2 to 3 were used in our research. All animal experiments were approved by the Animal Ethics and Welfare Committee of Beijing Stomatological Hospital & School of Stomatology, Capital Medical University (KQYY-202008-003).
2.2. The Effects of KGN on Rat ADSCs
Before the investigation on the rGO@Ge, the chondrogenic differentiation ability of ADSCs was first studied using the DMEM medium supplemented with 100 nM dexamethasone (Sigma-Aldrich), 1% insulin-transferrin-selenium (Sigma-Aldrich), 50 μM ascorbate-2-phosphate, 1 mM sodium pyruvate, 50 mg/mL Bovine Serum Albumin (BSA), 5 μg/mL linoleic acid (Sigma-Aldrich), 1% penicillin/streptomycin and with 10 ng/mL TGFβ3 (R&D Systems) or different concentrations of KGN (Sigma-Aldrich). The medium containing TGFβ3 was used to verify the chondrogenic differentiation ability of rat ADSCs, and the concentration gradient of KGN in the DMEM medium was 100 nM, 500 nM, 1 μM and 10 μM. For evaluation on ADSCs proliferation under different concentrations of KGN, the cells were seeded in a 96-well plate with a density of 4 × 10
3 per well and tested by Cell Counting Kit-8 (CCK8) assays according to manufacturer’s protocol at 1, 4 and 7 days. After incubation with the detection reagent at 37 °C for 1 h, the acquired optical density (OD) value at 450 nm was used to reflect the proliferation ability of ADSCs. The chondrogenic differentiation of ADSCs was further tested by PCR, immunofluorescent (IF) staining, alcian blue (Ab) and toluidine blue (Tb) stainings. ADSCs were seeded in a 24-well plate with a density of 1 × 10
5 per well and the medium containing different KGN concentrations was used to culture cell for evaluation on ADSCs chondrogenesis. The culture medium containing KGN was changed after the first cell seeding 24 h and refreshed every other day in the subsequent experiments. After 14 days culture, the chondrogenic differentiation of ADSCs was terminated and the cell was fixed using 4% paraformaldehyde (PFA) for the subsequent IF (Sox-9 and Col II), Ab and Tb stainings. Meanwhile, the total RNA was extracted for the PCR analysis of the chondrogenic makers (Sox-9, Col II, Aggrecan and Col X), as in the previously described process [
22]. The procedures of the staining and PCR analysis were also briefly described in subsequent sections.
2.3. Synthesis of rGO@Ge and Evaluation on Its Nanocarrier Function
The gelatin reduced graphene oxide (rGO@Ge) was synthesized as our previous study [
22]. Here is a brief description of the method: graphene oxide (GO) was chemically reduced with excessive gelatin through stirring for 24 h at 95 °C, and the resulting reaction solution was centrifuged at 20,000 rpm with hot water washing three times for removing excess gelatin. The synthesized rGO@Ge was dissolved in water and stored at 4 °C until use. The drug loading–releasing property of rGO@Ge was tested with kartogenin (KGN). The 5 μL KGN solution (1 mM) was mixed with different amounts of 100 mg/mL rGO@Ge solution (the amounts were 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μL) in a total 300 μL volume of PBS solution. The mixture system was incubated at room temperature on a shaking table for 2 h and centrifuged with 14,000 rpm for 10 min so as to monitor the free KGN concentration in the supernatant. Through calculation, the loading capacity of rGO@Ge on KGN can be clarified and the nanocarrier with saturable absorption of KGN (rGO@Ge/KGN) was collected for further analysis of the releasing kinetic curve. The 30 mg rGO@Ge/KGN was used to test the releasing of KGN with different pH (7, 5 and 2) in 1mL PBS or acetate buffer solution at 37 °C, and the KGN concentration in the supernatant after centrifugation was tested after shaking for different times (1, 3, 5, 7, 9, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168 and 264 h). The KGN has a characteristic absorption peak at 280 nm in the UV-vis spectrum, and the OD value is linearly related KGN concentration, which provides us with a reliable method for quantitative research on the loading–releasing kinetics.
2.4. The Biocompatibility and Entry Pathways of rGO@Ge in ADSCs
The biological effects of rGO@Ge on ADSCs were tested by live/dead staining, CCK8 assay and flow cytometry analysis so as to evaluate its cytotoxicity and influences on proliferation and apoptosis of ADSCs, respectively. The concentration series of rGO@Ge were 0, 10, 10
2, 10
3 and 10
4 μg/mL in the DMEM medium (supplemented with 10% FBS and 1% penicillin/streptomycin). Calcein-AM/PI staining kit (Yeasen, Shanghai, China) and Annexin V-FITC/PI staining kit (Thermo Fisher Scientific, USA) were used for material cytotoxicity and cell apoptosis analysis. The proliferation of ADSCs in the rGO@Ge medium was tested at 1, 4 and 7 days by CCK8 as described above. The rGO@Ge was labeled with Rhodamine 6G (R6G) as previously described [
22], and ADSCs were incubated with medium containing R6G-labeled rGO@Ge for observation of the nanocarrier distribution in the cell. Through pretreating ADSCs with different endocytosis inhibitors, the amount change of rGO@Ge that entered into cell was studied by flow cytometry quantitative analysis to clarify the main entry pathway of rGO@Ge in ADSCs. The inhibitors included wortmannin (30 μM), amiloride (1 mM) and genistein (40 μM) that inhibited phagocytosis, micropinocytosis and caveolae-dependent endocytosis, respectively [
26]. After the co-culture of ADSCs and rGO@Ge for 24 h, the collected samples were prepared as the ultrathin sections (70 nm) using ultramicrotome (Leica EM UC7), and the transmission electron microscope (TEM) (H-7650; Hitachi, Tokyo, Japan) was used to observe the location and degradation of rGO@Ge in ADSCs.
2.5. ADSCs Chondrogenesis Induced by KGN-Loaded rGO@Ge
After clarification on the biocompatibility and nanocarrier function of rGO@Ge, the studies on the application of KGN-loaded rGO@Ge in promoting ADSCs chondrogenesis were further carried out to evaluate the functions of the drug-nanocarrier complex composites. The concentration of KGN in the composites was set to 1 μM, and the different concentrations of rGO@Ge were mixed with KGN, which composed the different medium with a series of KGN/rGO@Ge concentration ratios (1 μM KGN corresponding to 0, 10, 102, 103 and 104 μg/mL rGO@Ge). For the convenience of description to the different groups, we abbreviated these KGN/rGO@Ge ratios to K/G values, so the above described series medium were named as K/G0 (1 μM KGN: 0 μg/mL rGO@Ge), K/G1 (1 μM KGN: 10 μg/mL rGO@Ge), K/G2 (1 μM KGN: 102 μg/mL rGO@Ge), K/G3 (1 μM KGN: 103 μg/mL rGO@Ge) and K/G4 (1 μM KGN: 104 μg/mL rGO@Ge) in short. The ADSCs were seeded in a 24-well plate with a density of 1 × 105 per well and the different medium with K/G values was used to culture ADSCs for evaluation on the stem cell chondrogenesis. After 14 days of cell culture, the IF, Ab and Tb stainings were carried out to analyze the chondrogenic differentiation of ADSCs. Meanwhile, PCR was also conducted to evaluate the ADSCs chondrogenesis.
2.6. Analysis on the Intracellular ROS and Autophagy Changes in ADSCs Treated with Different K/Gs Media
The intracellular Reactive Oxygen Species (ROS) in ADSCs was measured using the ROS Assay Kit (Yeasen, Shanghai, China). The ADSCs were cultured with different K/Gs media for 24 h and the medium containing 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) fluorescent probe was used to replace the different K/Gs media for detecting the ROS levels in different groups according to the manufacturer’s protocol. After 30 min of incubation with the fluorescent probe, the ADSCs were washed with PBS three times for the subsequent fluorescent microscope imaging and flow cytometry quantitative analysis. To explore the change of autophagy in ADSCs that was induced by the KGN/rGO@Ge composites, the K/G0, 1, 2, 3 and 4 series media were also used to treat the ADSCs for 24 h and the incubated cells were fixed with 4% PFA for the LC3B IF staining to evaluate the autophagy activity. In the meantime, parallel experiments were also carried out to verify the changes of autophagy-related protein expression in ADSCs for autophagy analysis. To determine the role of the Erk1/2 signal pathway in the pro-autophagy effect of rGO@Ge and the role of autophagy in the pro-chondrogenic effect of rGO@Ge, the Erk1/2 signal pathway inhibitor PD98059 (CST, USA) and autophagy inhibitor 3-methyladenine (3-MA; Selleck, USA) were added into the K/G3 medium for the evaluation on the changes of autophagy and chondrogenesis in ADSCs. During the chondrogenesis of ADSCs that were treated with K/G3 medium containing PD98059 or 3-MA, the culture medium was changed every 3 days with the addition of the Erk1/2 signal pathway and autophagy inhibitors.
2.7. Immunofluorescence (IF), Alcian Blue (Ab) and Toluidine Blue (Tb) Stainings
As for the immunofluorescence staining, the fixed cell samples were firstly washed with PBS three times and then incubated with 0.5% Triton X-100 with 5 min for membrane rupture. The cells were blocked with 10% BSA for 1 h and incubated overnight with different primary antibodies at 4 °C according to the corresponding experiments, including anti-LC3B antibody (Abcam, USA), anti-Sox-9 antibody (Abcam, USA) and anti-Col II antibody (Abcam, USA). Then, the samples were washed with PBST (PBS with 0.05% Tween-20) three times and incubated with the secondary antibodies. Subsequently, the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) and the cytoskeleton was stained with FITC-labeled phalloidin. The images of the IF staining were acquired by the confocal microscope and fluorescent microscope. The chondrogenic differentiation of ADSC samples were also stained with alcian blue and toluidine blue staining solutions (Sangon Biotech, Shanghai, China) for 30 min, washed with PBS and observed under the light microscope. The propanol dissociated solution was used to dissolve the alcian blue dye, and the OD value at 600 nm was detected for quantitative analysis of the glycosaminoglycan (GAG) content in the Ab stained samples.
2.8. Real-Time Quantitative PCR Analysis
Firstly, the total RNA was extracted from the ADSCs chondrogenesis samples with TRIzol reagent (Life Technologies, USA) according to the manufacturer’s protocol. Subsequently, the RNA was reverse transcribed into the complementary DNA (cDNA) using the reverse transcription kit (Takara, Japan). At last, the real-time quantitative PCR was conducted using SYBR Green Master Mix (Roche, USA) by ROCHE480 real-time PCR system to detect the changes of chondrogenic makers, including Sox-9, Col II, Aggrecan and Col X. The sequences of primers for each gene were listed in
Table 1. The tested genes were normalized using β-actin as the internal reference. The expression level of each gene was analyzed using the comparative Ct (2
−∆∆Ct).
2.9. Western Blotting Analysis
The cell samples were lysed by Radio-Immunoprecipitation Assay (RIPA) Buffer (Beyotime, China) containing phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) for protein extraction. The total cell proteins were denatured with SDS-PAGE loading buffer, separated by SDS-PAGE gel and transferred to Polyvinylidene Fluoride (PVDF) membrane (Millipore, USA). Then the membranes were blocked with 10% BSA and incubated with primary antibodies including anti-ULK1 (Abcam, USA), anti-Beclin-1 (Abcam, USA), anti-LC3 (Abcam, USA), p-Erk1/2 (CST, USA), Erk1/2 (CST, USA) and anti-β-actin (CST, USA) overnight at 4 °C. Subsequently, the blots were washed with TBST (Tris Buffered Saline with 0.05% Tween 20) three times and incubated with the Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:10000, CST) for 45 min. The chemiluminescence reagent (Thermo, USA) was used to expose and identify the protein bands using ChemiDoc Touch System (Bio-Rad, USA), and quantitative analysis of the protein expression level was calculated relative to the β-actin control using the ImageJ software (NIH, USA).
2.10. Statistical Analysis
All the experiments were repeated three times and the quantitative data were expressed as mean ± SD. The statistical analysis of multiple comparisons was performed using a one-way ANOVA and Student–Newman–Keul tests with the SPSS 13.0 software (SPSS Inc., USA). p values < 0.05 were considered statistically significant.
4. Discussion
In our study, we utilized the rat ADSCs as the mesenchymal stem cell to evaluate the effects of KGN and its rGO@Ge complex on MSCs chondrogenic differentiation. The results concerning the ADSCs chondrogenesis induced by KGN illustrate that the efficient drug concentration was 1 μM, and the proper concentration of the drug could promote the ADSCs proliferation and chondrogenic differentiation. These observations were consistent with the recent studies about the effect of KGN on other MSCs chondrogenic differentiation, such as umbilical cord mesenchymal stem cells [
27] and bone marrow mesenchymal stem cells [
28]. The decreased proliferation ratio and chondrogenic gene expressions in the 10 μM KGN group were detected in our study, which potentially resulted from the over-differentiation of ADSCs into hypertrophic chondrocytes with less formation of the cartilage matrix and expression of chondrogenic genes under the induction of the exorbitant KGN. It has also been reported that KGN at low concentrations was beneficial to cell proliferation and the over-high KGN concentrations would promote cell differentiation with decreased promotion on proliferation [
29]. The successful chondrogenesis of rat ADSCs also provided more experimental evidence for its promised seed cell function in cartilage regeneration repair. Meanwhile, the detection of the Sox-9 and Col II chondrogenic markers in the KGN induced ADSCs in our study certified its chondrogenesis potential as a stem cell, so ADSCs could be used as the seed cell for the subsequent experiments about the rGO@Ge biological functions. The PCR analysis also supported the ADSCs chondrogenesis potential with a promised application in cartilage regeneration repair.
The rGO@Ge as the reduced graphene oxide (rGO) had the essential properties of rGO, in which more functional groups on the rGO nanosheet surface could extend its application in biomedical fields including neural [
30], cardiac [
31] and bone [
32] tissue engineering. Compared with the synthetic substrate GO, the gelatin on the surface of rGO@Ge guaranteed it the better stability in different media as described in our previous study [
22]. Meanwhile, the biomacromolecule gelatin attached on rGO@Ge also prospects its promised biocompatibility and potential biodegradation compared with the un-modified GO. The further analysis on its function as nanocarrier for KGN delivery in our study showed its widespread locations in the cytoplasm and nucleus of ADSCs, which guaranteed its efficient delivery of the KGN molecule into the MSCs to exert its promoting chondrogenic differentiation effect. The live/dead staining illustrated the good biocompatibility of rGO@Ge even at a high dose of 10 mg/mL, whereas its promotion effect on ADSCs proliferation had attained the optimum efficiency at 1 mg/mL with a decreased cell viability at 10 mg/mL. The analysis on the ADSCs apoptosis influenced by different rGO@Ge concentrations showed the great increased apoptotic ratio of ADSCs at 10 mg/mL, which explained the decrease of cell proliferation ratio due to the high dose of rGO@Ge. The analysis about the entry pathways of rGO@Ge showed that ADSCs take in the nanocarrier mainly via phagocytosis and macropinocytosis mechanisms. The wortmannin-inhibited ADSCs showed the small vacuoles without R6G-labeled rGO@Ge, and the FACS analysis also showed the significant decrease of R6G fluorescence intensity. The inhibited macropinocytosis by amiloride also elicited a decreased uptake of rGO@Ge by ADSCs, and the flow cytometry illustrated the less quantitative difference with the control compared with the wortmannin-treated group. The genistein inhibited endocytosis pathway had less effect on the rGO@Ge’s entry into ADSCs. The clarification about the mechanism of the rGO@Ge entry pathways provided us with more information about its nanocarrier function for drug molecules delivery. The TEM analysis of the rGO@Ge in ADSCs also showed its widespread locations, and its closer distribution to the nucleus might have favored its synergistic effect on chondrogenesis with efficient KGN delivery. Meanwhile, the potential small fragment form of the rGO@Ge degradation residue was similar to the Nitric Oxide (NO)-dependent biodegradation of GO reported recently [
33]. Meanwhile, the rGO could also induce the formation of NO [
34], which potentially explains the biocompatibility and biodegradation of rGO@Ge and showed clearer tracks of rGO@Ge nanocarrier in ADSCs. The previous studies about the mechanism of the graphene materials in cancer treatment discovered the mitochondria targeting and oxidative stress phenomenon [
35,
36]. In our study, the TEM also showed that the location of rGO@Ge was adjacent to mitochondria and rough endoplasmic reticulum, which implied the possible mechanism of the nanomaterials in causing ROS production. The proper ROS level caused by rGO@Ge in the ADSCs would further influence the cellular behaviors and the chondrogenic differentiation capacity of the MSCs.
The tests about the pro-chondrogenic effect of the KGN/rGO@Ge composites on ADSCs showed the synergistic effect of rGO@Ge with KGN on promoting chondrogenesis. The K/G3 group as the best efficient complex composite was composed of 1 μM KGN concentration and 1 mg/mL rGO@Ge. So, in the K/G3 group, 1 mL complex medium contained 1 nM KGN and 1 mg rGO@Ge, which had the same order of magnitude as the rGO@Ge loading capacity of KGN (2 nM KGN loaded by 1 mg rGO@Ge). The IF staining of chondrogenic differentiation markers including Sox-9 and Col II, the alcian blue and toluidine blue stainings and the PCR analysis on the chondrogenic relevant markers all illustrated the optimum efficiency of the K/G3 group in ADSCs chondrogenesis. The previous studies reported that the graphene-based materials could cause the reactive oxygen species (ROS) production to exert its biological functions on the cellular behaviors [
37,
38], and the ROS in the cytoplasm was shown to be an essential regulator of the autophagy activity in the cartilage homeostasis mechanisms [
24]. Recent studies also showed that the regulation of autophagy could have a chondro-protective effect on osteoarthritis therapy and cartilage repair [
39,
40]. In our research, we also observed the ROS triggered by rGO@Ge, and the autophagy activity could be influenced by the different rGO@Ge concentrations. The increased ROS caused by the dose increasement of nanomaterial elicited a regulating effect on the autophagy, the gradual increased autophagy with rGO@Ge dose increasement in a relatively low concentration range guaranteed a promised pro-chondrogenic effect and the over-high nanomaterial concentration would cause a reverse effect on the ADSCs chondrogenesis due to the excessive ROS damage. The over-high rGO@Ge concentration caused severe ADSCs apoptosis, and the autophagy activity was also inhibited abnormally. All of the changes led to the decreased ADSCs chondrogenesis compared with the differentiation potential induced by a proper nanomaterial concentration range (10~1000 μg/mL). The worst cellular behaviors and chondrogenesis caused by over-high rGO@Ge implied that the proper nanomaterial concentrations needed be selected carefully so as to reserve its good stimulus function and avoid the severe damage for exerting its favorable biological effects on MSCs. Meanwhile, the ROS/ERK and mTOR signaling pathway was also previously reported as the mechanism of the autophagy modification [
41], and we also observed that the Erk1/2 signal pathway was activated in the rGO@Ge-induced augmentation of autophagy, which indicated the possible mechanism of rGO@Ge on autophagy modification.
The above results of the KGN/rGO@Ge composites show us the synergistic effects of the nanocarrier with KGN for promoting ADSCs chondrogenesis through autophagy modification in vitro. So, the proper complex of KGN and rGO@Ge would have a promised application in cartilage repair in vivo and the animal OA model experiments were needed to conduct for the further research.