2017 Volume 65 Issue 8 Pages 762-767
Using tissue engineering technique to repair cartilage damage caused by osteoarthritis is a promising strategy. However, the regenerated tissue usually is fibrous cartilage, which has poor mechanical characteristics compared to hyaline cartilage. Chondrocyte hypertrophy plays an important role in this process. Thus, it is very important to find out a suitable way to maintain the phenotype of chondrocytes and inhibit chondrocyte hypertrophy. Curcumin deriving from turmeric was reported with anti-inflammatory and anti-tumor pharmacological effects. However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering as seed cells. So we investigated the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs through examination of cell viability, glycosaminoglycan synthesis and specific gene expression. We found curcumin had no effect on expression of chondrogenic markers including Sox9 and Col2a1 while hypertrophic markers including Runx2 and Col10a1 were down-regulated. Further exploration showed that curcumin inhibited chondrocyte hypertrophy through Indian hedgehog homolog (IHH) and Notch signalings. Our results indicated curcumin was a potential agent in modulating cartilage homeostasis and maintaining chondrocyte phenotype.
Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and disability.1) Seventy percent of the population older than 65 years is suffering from varying degrees of OA.2) The pathogenesis of cartilage injury caused by OA is the disruption of the homeostasis between catabolism and anabolism of extracellular matrix.3) However, limited by the intrinsic regeneration capacity of cartilage tissue, repairing damaged articular cartilage currently is a significant challenge.4,5) With the advances in materials science, cell biology, biomechanics, using tissue engineering technology to construct cartilage provides a novel method for repairing damaged cartilage.6) Mesenchymal stem cells (MSCs) have potential of differentiation into chondrocytes and have got the priority in the seed cells choosing list because of their traits in rich resources, easy to get and low immunogenicity.7,8) However, the regenerated tissue-engineered cartilage usually is fibrous cartilage rather than hyaline cartilage.9) The chondrocytes in fibrous cartilage proceed in hypertrophic differentiation and secret Col10a1 and matrix metalloproteinase 13 (Mmp13).10) Therefore, suppressing chondrocyte hypertrophy is the key issue to build a stable hyaline cartilage.
Curcumin is effective substance of turmeric and possesses various pharmacological effects, including antioxidant, anti-bacterial, anti-tumor and anti-inflammatory.11–13) A recent study has reported that curcumin may be a potential therapeutic candidate for Helicobacter pylori associated diseases.14) It has been demonstrated that curcumin could effectively suppress amyloid β (Aβ)-induced cytotoxicity and apoptosis by inhibition of ROS-mediated oxidative damage and regulation of extracellular signal-regulated kinase (ERK) pathway.15) However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Therefore, the objective of this study was to research the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs. Specifically, we found that curcumin had no effect on chondrogenesis of MSCs. But chondrocyte hypertrophy of MSCs was inhibited by curcumin. Additional, several crucial genes of signaling pathway and transcription factors were examined to explore the mechanism of the above interesting phenomenon.
Curcumin was purchased from Sigma Corporation of America which was dissolved in dimethyl sulfoxide (DMSO). Besides, the experimental concentration of DMSO is 0.1%. C3H10T1/2 mesenchymal stem cells were purchased from the American Type Culture Collection (ATC C, Manassas, VA, U.S.A.). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Hyclone, Logan, UT, U.S.A.) containing 10% fetal bovine serum (FBS) (Hyclone) and 1% Penicillin–streptomycin solution (Beyotime, Shanghai, China) at 37°C with 5% CO2. The culture medium was replaced every 2–3 d until the cells reached 90% confluence. Cells were passaged by 0.25% trypsin (Hyclone) for 2 min at room temperature. The second generation cells were used for the following experiments.
In Vitro Cell Proliferation AssayC3H10T1/2 cells were seeded (2×103 per well) into 96-well plates (Corning, NY, U.S.A.) in triplicates and allowed to adhere overnight. On the following day, the medium was replaced by fresh medium with curcumin (Sigma, Shanghai, China). Cultures were incubated for 24, 48 and 72 h. Then, 10-µL Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Japan) reagents were added to each well and incubated for an additional 4 h. The absorbance was read at the wavelength of 450 nm in an automated plate reader. Wells containing the CCK8 reagents without cells were used as the blank control. Cell proliferation was assessed by the absorbance values according to the manufacturer’s protocol.
Chondrogenic Differentiation AssayC3H10T1/2 cells were trypsinized by 0.25% trypsin and modulated at a density of 105 cells/mL. Two milliliters of the suspension was placed into the center of each well on a 6-well plate (Corning). After incubation for 24 h at 37°C and 5% CO2, the medium was replaced by 2 mL chondrogenic differentiation medium (Hyclone). The chondrogenic differentiation medium composed of dexamethasone (100 nmol/L), ascorbate (50 µg/mL), Insulin-Transferrin-Selenium (ITS)+Supplement, proline (40 µg/mL) and transforming growth factor (TGF)-β3 (10 ng/mL) was replaced every 3 d.
Hypertrophic Differentiation AssayC3H10T1/2 cells were treated by chondrogenic differentiation medium as previously described for 14 d. Then the cells were inducted by hypertrophic differentiation medium composed of dexamethasone (1 nmol/L), ascorbate (50 µg/mL), ITS+Supplement, proline (40 µg/mL) and triiodothyronine (T3, 100 ng/mL) for another 14 d. Each medium was replaced every 3 d.
Quantitative RT-PCR AnalysisTotal RNA was isolated using Trizol reagent (Life Technologies, NY, U.S.A.). Single-stranded cDNA was prepared from 1 µg of total RNA using reverse transcriptase with oligo-dT primer according the manufacturer’s instructions (TaKaRa, Liaoning, Dalian, China). Two microlitres of each cDNA was subjected to PCR amplification using specific primers with detailed information in Table 1. The cycling conditions were set as 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s. The relative mRNA level was calculated by the normalization to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
| Genes | Forward | Reverse | Tm (°C) |
|---|---|---|---|
| Gli2 | 5′-GTCACCAAGAAACAGCGTAA-3′ | 5′-GATGGCTTTGATATGTAGGC-3′ | 60 |
| Gli3 | 5′-ACAGCTCTACGGCGACTG-3′ | 5′-GCATAGTGATTGCGTTTC-3′ | 63 |
| Col2a1 | 5′-TGGTGGAGCAGCAAGAGC-3′ | 5′-TGGACAGTAGACGGAGGAAA-3′ | 61 |
| Sox9 | 5′-CCCAGCGAACGCACATCA-3′ | 5′-TGGTCAGCGTAGTCGTATT-3′ | 61 |
| GAPDH | 5′-GTTGTCTCCTGCGACTTCA-3′ | 5′-GGTGGTCCAGGGTTTCTTA-3′ | 62 |
| Hey1 | 5′-GAATGCCTGGCCGAAGTT-3′ | 5′-CCGCTGGGATGCGTAGTT-3′ | 62 |
| Col10a1 | 5′-CTTTCTGGGATGCCGCTTGT-3′ | 5′-GGGTCGTAATGCTGCTGCCTA-3′ | 61 |
| Runx2 | 5′-CCAACTTCCTGTGCTCCGTG-3′ | 5′-ATAACAGCGGAGGCATTTCG-3′ | 63 |
| Mmp13 | 5′-TTGATGCCATTACCAGTCTCCG-3′ | 5′-CACGGGATGGATGTTCATATGC-3′ | 61 |
The cells were extracted with lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.2% Aprotinin (Beyotime). After we measured the protein concentration, the equal protein samples were mixed with 5× sample buffer (Beyotime) and boiled. The samples were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred on polyvinylidene difluoride (PVDF) membrane (Millipore, Hong Kong, China) by using the semi-dry transfer method. After blocking in 10% nonfat dried milk in Tris-buffered saline and Tween 20 (TBST) for 2 h, the blots were incubated with primary antibodies including Col2a1 (rabbit polyclonal 1 : 500, Abcam), Sox9 (rabbit polyclonal 1 : 500, Abcam), Runx2 (rabbit polyclonal 1 : 500, Abcam), Col10a1 (rabbit polyclonal 1 : 500, Abcam), Gli2 (rabbit polyclonal 1 : 500, Bioss), Gli3 (rabbit polyclonal 1 : 500, Bioss), Notch intracellular domain (NICD) (rabbit polyclonal 1 : 500, Bioss), Hey1 (rabbit polyclonal 1 : 500, Bioss) and GAPDH (rabbit polyclonal 1 : 1000, Abcam) at 4°C overnight. After washing by TBST, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1 : 2000, Santa Cruz) at room temperature for 1 h. Blots against GAPDH served as loading control.
Glycosaminoglycan (GAG) Synthesis Analysis by Toluidine Blue StainingTo demonstrate the deposition of cartilage matrix proteoglycans, representative cultures were collected at day 28 and sulfated cartilage glycosaminoglycans (GAGs) were measured by Toluidine blue (Beyotime) staining. The cells were fixed in 4% Paraformaldehyde for 15 min and stained with Toluidine blue for 5 min. The mean density was normalized to total cell number.
ImmunohistochemistryThe cells were fixed in 4% Paraformaldehyde for 15 min. This was followed by washing in phosphate-buffered saline (PBS) and treated with H2O2 (ZSGB-BIO, Peking, China) for 10 min to inactivate endogenous peroxidase. After treatment with normal goat serum (ZSGB-BIO) at room temperature for 15 min, cells were incubated with primary antibodies including Runx2 (rabbit polyclonal 1 : 150, Abcam) and Col10a1 (rabbit polyclonal 1 : 150, Abcam) at 4°C overnight. After washing, the cells were incubated with biotinylated goat anti-rabbit (ZSGB-BIO) secondary antibodies for 30 min, followed by washing and incubation with horseradish peroxidase (HRP) (ZSGB-BIO) for 15 min. The area of the immunocomplex was visualized by chromogen 3,3′-diaminobenzidine (DAB) for 5 min. The cells were investigated under the Olympus microscope. Image-Pro plus 6.0 software was used for image analysis.
ImmunofluorescenceCells were washed with PBS, fixed with 4% Paraformaldehyde for 15 min, rinsed with PBS, and permeabilised with 0.2% Triton X-100, 1% bovine serum albumin (BSA) for 1 h. Cells were incubated with primary antibodies including Runx2 (rabbit polyclonal 1 : 150, Abcam) and Col10a1 (rabbit polyclonal 1 : 150, Abcam) at 4°C overnight, then washed three times with PBS, and incubated with the appropriate Cy3-conjugated secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 : 100, Life Technologies) for 15 min. Fluorescence was detected using Olympus microscope. Image-Pro plus 6.0 software was used for image analysis.
StatisticsAll data are representative of at least three experiments of similar results performed in triplicate unless otherwise indicated. Data are expressed as mean±standard error of the mean (S.E.M.). One-way ANOVA followed by Student–Newman–Keuls post hoc tests was used to determine the significance of difference between results, with * p<0.05, ** p<0.01 and *** p<0.001 being regarded as significant.
MSCs were treated with basal culture medium for 24 and 72 h with different concentrations of curcumin followed by CCK8 assays. The chemical formula of curcumin is shown (Fig. 1A). The results showed that concentration of curcumin lower than 1 µM had little effect on cell proliferation (Figs. 1B, C). Curcumin concentration of 1 µM was regarded safe and used for following experiments. After induction by chondrogenic medium containing TGF-β3 for 14 d, the cells were collected to evaluate the expression of chondrogenic genes through RT-PCR. The results showed that expressions of Sox9 and Col2a1 were increased on day 14 compared with day 0 (Fig. S1A). Following curcumin treatment for 3, 7 and 14 d, RT-PCR analysis showed chondrogenesis markers including Sox9 and Col2a1 were not changed compared to the control group (0 µM) (Fig. S1B). On the protein level, treatment of MSCs with or without curcumin did not significantly affect the expression of Sox9 and Col2a1 (Figs. S1C, D). Our data demonstrated that curcumin had no effect on chondrogenesis at the concentration of 1 µM.
Curcumin concentration of 1 µM was regarded safe and used for following experiments. (A) Chemical formula of curcumin. (B) CCK8 analysis of cell viability of MSCs treated with different concentrations of curcumin for 24 h. (C) CCK8 analysis of cell viability of MSCs treated with different concentrations of curcumin for 72 h.
After inducted by chondrogenic differentiation medium containing TGF-β3 for 14 d without curcumin, MSCs were inducted by hypertrophic medium containing T3 for another 14 d. From RT-PCR, we found that chondrogenic markers such as Sox9 and Col2a1 were decreased while hypertrophic markers including Runx2 and Col10a1 were increased on day 28 compared with day 14 (Fig. S2A). According to this in vitro culture system, following curcumin treatment, Toluidine blue staining at 28 d showed that the experimental groups had higher intensity than control groups (Fig. 2C). We next performed immunofluorescence to examine expression of Col10a1 and Runx2. The integrated optical density of groups treated with curcumin on day 28 was lower than control groups (Figs. 2A, B). Besides, we performed immunohistochemistry to test expression of Col10a1 and Runx2. The integrated optical density of groups treated with curcumin on days 21 and 28 were lower than control groups (Figs. S2B, C). Through qRT-PCR, upon curcumin treatment, the expression of hypertropyic markers including Mmp13, Runx2 and Col10a1 were decreased on days 21 and 28 (Fig. 3A). Through Western blot, we found that the expression of Mmp13, Col10a1 and Runx2 were decreased on days 21 and 28 compared with control groups (Fig. 3B). Thus, these data demonstrated curcumin inhibited chondrocyte hypertrophy of MSCs.
MSCs were induced with chondrogenic medium for 14 d and then treated with hypertrophic medium for another 14 d. (A) Representative immunofluorescence images of Col10a1 and Runx2 from MSCs in different groups (day 28). Scale bar represents 200 µm. (B) Quantification of mean intensity of Col10a1 and Runx2 in (A). (C) Representative toluidine blue staining images of MSCs in different groups (day 28). Quantification of mean intensity was shown on the right.
(A) Relative mRNA expression levels of Mmp13, Runx2 and Col10a1. (B) Representative Western blot images of Mmp13, Col10a1, Runx2 and GAPDH from MSCs in different groups (days 21 and 28).
Curcumin had an obvious inhibitive effect on T3 induced chondrocyte hypertrophy of MSCs. We explored the mechanism through qRT-PCR to screen the expression of altered transcriptional factors and key molecules in canonical pathways related with chondrocyte hypertrophic differentiation. The results revealed that following curcumin treatment, the mRNA levels of Gli2 was decreased on day 21. Meanwhile expression of Gli3 was not changed on day 21 and was increased on day 28 by the treatment of curcumin. Besides, Hey1 expression was decreased on days 21 and 28 compared with control groups (Fig. 4A). Then we verified the results of qRT-PCR by Western blot. On the protein level, after curcumin treatment, Gli2 expression was decreased and Gli3 expression was increased on days 21 and 28 compared with control group. In addition, for Notch signaling, expressions of NICD and Hey1 were decreased following curcumin treatment on days 21 and 28 (Fig. 4B). These data demonstrated that curcumin inhibited chondrocyte hypertrophy of MSCs trough inhibition of IHH signaling and Notch signaling.
Cells were induced with chondrogenic medium for 14 d and then treated with hypertrophic medium for another 7 and 14 d. (A) Relative mRNA expression levels of Gli2, Gli3 and Hey1 from MSCs in different groups (days 14, 21 and 28). (B) Representative Western blot images of Gli2, Gli3, NICD, Hey1 and GAPDH from MSCs in different groups (days 21 and 28).
Curcumin has been considered as a multifunctional agent and proved to have protective effects on nervous system,16) circulatory system,17) digestive system,18,19) and tumor metastasis.20,21) Given those factors, we assume that curcumin might also have effect on chondrocyte metabolism. A previous study showed that curcumin could ameliorate glucocorticoid-induced osteoporosis by protecting osteoblasts from apoptosis through activation of the ERK pathway.22) It has been demonstrated that curcumin combined with piperine suppressed the osteoclastogenesis.23)
In this study, we found that curcumin had no effect on TGF-β3 induced chondrogenesis of MSCs. Several chondrogenic genes such as Sox9 and Col2a1 were not changed on the molecular level.24) However, curcumin inhibited chondrocyte hypertrophy of MSCs. The expression of specific genes related to chondrocyte hypertrophy including Runx2, Mmp13 and Col10a1 were inhibited by curcumin.25) A recent study has found that resveratrol inhibited chondrocyte hypertrophy through suppressing the expression of Mmp13 in human articular chondrocytes.26) Interestingly, both curcumin and resveratrol are part of antioxidant and anti-inflammatory agents.27) However, resveratrol belongs to a natural polyphenolic substance while curcumin is a diketone compound. Therefore the mechanisms of inhibiting chondrocyte hypertrophy may be different.
To investigate the underlining mechanisms about the inhibitory effect of curcumin on chondrocyte hypertrophy, we screened several crucial genes of signaling pathway and transcription factors and focused on two important signaling pathways, IHH and Notch. IHH signaling and Notch signaling promotes chondrocyte hypertrophic differentiation.28–30) IHH binding to the receptor Patched homolog 1 (PTCH1) activates signaling through Smoothened (SMO), thereby inhibiting the generation of Gli3 and promoting the generation of Gli2.31) Activated Gli transcription factors enter nucleus to regulate expression of genes related to chondrocyte hypertrophy. In Notch signaling, following binding to their ligands, Jagged (JAG) or Delta-like (DLL), Notch receptors are cleaved by the g-secretase complex, leading to release of the NICD from the plasma membrane. NICD interacts with RBPJk and together they activate downstream target genes, including Hes and Hey family transcription factors, ultimately leading to regulate chondrocyte hypertrophy.31)
In MSCs, the expression of NICD and Hey1 was decreased following curcumin treatment on days 21 and 28. Besides, curcumin inhibited Gli2 expression on days 21 and 28 while promoted Gli3 expression on day 28. So we figured out that curcumin down-regulating IHH signaling by suppressing Gli2 expression at early stages of chondrocyte hypertrophy and increasing Gli3 expression at late stages. Hence, we drew a conclusion curcumin inhibited chondrocyte hypertrophy through down-regulating IHH signaling and Notch signaling.
Tissue engineering technique provides a novel method for repairing cartilage injury caused by OA. Maintaining the balance between anabolic and catabolic of extracellular matrix in regenerated cartilage is a key element for the best outcome. Moreover, inhibiting of chondrocyte hypertrophy and maintaining chondrocyte phenotype are considerable. The effects of curcumin on chondrocyte hypertrophy might provide us a new insight. Curcumin can reduce degradation of cartilage matrix to balance anabolic and catabolic cellular activity of repair cells. These data indicate the potential clinical application of curcumin in modulating cartilage homeostasis and repairing cartilage.
This work was funded by Grants from the Nature Science Foundation of China (81271980, 81571893), the National High-tech R&D Program of China (863 Program, 2015AA020315), and the National Key Technology Research and Development Program of China (2017YFC1103300).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
