Neurochem Res DOI 10.1007/s11064-017-2252-6
ORIGINAL PAPER
Taurine Promotes the Cartilaginous Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro Xiuhua Yao1 · Huiling Huang1 · Zhou Li2 · Xiaohua Liu3 · Weijia Fan1 · Xinping Wang4 · Xuelian Sun3 · Jianmin Zhu3 · Hongrui Zhou2 · Huaying Wei3
Received: 14 November 2016 / Revised: 16 February 2017 / Accepted: 29 March 2017 © Springer Science+Business Media New York 2017
Abstract Taurine has been reported to influence osteogenic differentiation, but the role of taurine on cartilaginous differentiation using human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) remains unclear. In this study, we investigated the effect of taurine (0, 1, 5 and 10 mM) on the proliferation and chondrogenesis of hUC-MSCs by analyzing cell proliferation, accumulation of glycosaminoglycans and expression of cartilage specific mRNA. The results show though taurine did not affected the proliferation of hUC-MSCs, 5 mM of taurine is sufficient to enhanced the accumulation of glycosaminoglycans and up-regulate cartilage specific mRNA expression, namely collagen type II, aggrecan and SOX9. Taurine also inhibits chondrocyte dedifferentiation by reducing expression of collagen type I mRNA. Taken together, our study reveals that taurine promotes and maintains the chondrogenesis of hUC-MSCs.
Keywords Human mesenchymal stem cells · Umbilical cord · Taurine · Chondrogenesis Abbreviations BM-MSCs Bone marrow mesenchymal stem cells CCK-8 Cell counting kit-8 assay cDNA Complementary DNA DAPI 4′,6-Diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium EDTA Ethylene diamine tetraacetic acid FACS Flow cytometry GAG Glycosaminoglycan hUC-MSCs Human umbilical cord-derived mesenchymal stem cells MSCs Mesenchymal stem cells PBS Phosphate-buffered saline TGF The transforming growth factor
Introduction * Huiling Huang
[email protected] 1
Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases, Tianjin Neurosurgical Institute, Tianjin Huanhu Hospital, No. 6, JiZhao Road, Hexi district, Tianjin 300350, People’s Republic of China
2
NewScen Coast Bio-Pharmaceutical Co., Ltd., 65 sixth Ave., TEDA, Tianjin 300457, People’s Republic of China
3
Arthro-Anda Tianjin Biologic Technology Co., Ltd., 2F Building No. 2, Tian Bao Industrial Park, Xi Qi Road, Tianjin Airport Industrial Park, Tianjin 300308, People’s Republic of China
4
Departement of Neurology, Tianjin Huanhu Hospital, No. 6, JiZhao Road, Hexi district, Tianjin 300350, People’s Republic of China
Mesenchymal stem cells (MSCs) are defined by their unique capacity to self-renew, differentiate into one or more cell lineages and tissue types, and functionally repopulate a tissue in vivo [1]. The umbilical cord, which is considered medical waste and is obtained by a painless, simple and safe procedure during delivery, has aroused much attention in recent years as a promising source for MSC isolation [2, 3]. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) share many properties with adult bone marrow mesenchymal stem cells (BM-MSCs), but are generally considered to be a more differentiated population than the latter. In fact, they do not form teratomas upon transplantation [4] and their research does not raise ethical or
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legal issues. These features highlight the intriguing prospect of engineering hUC-MSCs for cell-based therapies. Damaged human articular cartilage have a limited capacity for repair. The resident cartilage cells, the chondrocytes, fail to mount an effective repair process, unable to recruit local sources of progenitor cells at the articular surface [5] and in the synovial lining of the joint cavity [6, 7]. Isolated MSCs from rabbit and human bone marrow were first shown to be capable of in vitro chondrogenic differentiation in micromass pellet cultures using a serum-free, defined culture medium [8, 9]. Critical factors in chondrogenic differentiation of MSCs in vitro appear to be high cell density at initial culture and exposure of the cells to glucocorticoids and members of the transforming growth factor (TGF) family [8–10]. Taurine is a widely distributed organic acid, and is detected in high concentrations in mammalian tissues [11–13]. Taurine has a variety of biological actions such as anti-oxidation, osmoregulation, modulation of ion movement, and conjugation of bile acids [14–16]. Studies show that Taurine increases ALP activity and collagen synthesis in osteoblast-like UMR-106 cells [17], repairs damaged articular cartilage [18], and inhibits development of rheumatoid arthritis [19, 20]. Recently, a study demonstrated that taurine promotes proliferation of chondrocytes [21] and osteogenesis of hMSCs [22]. However, the role of taurine on chondrogenic differentiation of hUC-MSCs remains unclear. In this study, we investigated the effect of taurine on chondrogenic differentiation of hUC-MSCs.
Materials and Methods Reagents Taurine, dexamethasone, ascorbic acid-2-phosphate, ITS Liquid Media Supplement, proline, 4′,6-diamidino-2-phenylindole (DAPI) and Ethylene Diamine Tetraacetic Acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phycoerythrin (PE)-conjugated anti-human CD34, PE-conjugated anti-human CD45, PE-conjugated anti-human CD73 and PE-conjugated anti-human CD105 were purchased from BD Biosciences (San Jose, CA, USA). Cell counting kit-8 assay (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan) Sodium pyruvate and M-MLV reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). Human glycosaminoglycan ELISA Kit and TGFβ3 was purchased from R&D Systems (Minneapolis, USA). DNA content quantitation assay kit was purchased from KeyGEN Biotech.CO., LTD. (Nanjing, China). Anti-collagen II antibody (mouse-antihuman) was purchased from Abcam (Cambridge, UK). The high glucose Dulbecco’s modified Eagle’s medium
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(DMEM) and Trypsin were purchased from Gibco (Carlsbad, CA, USA). HP Total RNA Kit was purchased from OMEGA (Norcross, Georgia, USA). Preparation of Taurine Taurine was fully dissolved with DMEM, to the concentration of 1 M. In a sterile environment, the taurine solution was then filtrated through a 0.22 µm filter membrane to obtain an aseptic solution. The taurine solution was then diluted to a final working concentration of 0, 1, 5, and 10 mM, and the action time of taurine was adjusted according to different experimental requirements. Human Umbilical Cord‑Derived Mesenchymal Stem Cells Culture Human umbilical cord-derived mesenchymal stem cells and Mesenchymal Stem Cell Growth Medium were purchased from Union Stemcell & Gene Engineering Co. Ltd. (Tianjin, China). Cultures were maintained in a humid atmosphere of 5% CO2/95% air at 37 °C. When the adherent cells achieved 80–90% confluence, cells were passaged using 0.25% Trypsin/0.02% EDTA at a split ratio of 1:3. Cells at passage 4 were used for further studies, and all experiments were repeated with cells for three times. Flow Cytometric Immunophenotyping The immunophenotyping of the MSCs was analyzed on BD FACSCanto™ flow cytometer (BD Biosciences, San Jose, CA). The cells were trypsinized and washed twice with phosphate-buffered saline (PBS). MSCs were then stained with fluorochrome conjugated monoclonal antibodies against PE-CD34, PE-CD45, PE-CD73 and PE-CD105 for 30 min at 4 °C in the dark. Finally, they were washed twice with PBS and fixed in 500 μl of 1% paraformaldehyde. PEconjugated isotype-matched Abs were used as negative controls. The experiment was repeated three times. Cell Proliferation Cells were seeded in a 96 flat-bottomed well plate at a density of 2 × 104 cells/ml with 100 μl per well and incubated by media containing different concentrations taurine (0, 1, 5 and 10 mM) at 37 °C for 1, 3 and 5 days. Then, cells were treated with the 10 μl/well CCK8 solution for 1 h at 37 °C and the absorbance was measured at 450 nm with a microplate reader. The experiment was repeated three times.
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Cell Cycle
Detection of GAG by ELISA
Cells were seeded in a six flat-bottomed well plate at density 6 × 104/ml/well and incubated with media containing different concentrations taurine (0, 1, 5 and 10 mM) at 37 °C for 24, 48 and 72 h. Cell cycle analysis was done using the DNA content quantitation assay kit according to manufacturer’s instruction. Briefly, cells were harvested and washed in PBS. Fixed in cold 75% ethanol and for overnight at 4 °C. The cells were washed in PBS and added 100 µl of RNase A. After incubation at 37 °C for 30 min, 400 µl propidium iodide was added and incubated at 4 °C for 30 min. After staining, cell cycle was immediately analyzed on a on BD FACSCanto™ flow cytometer (BD Biosciences, San Jose, CA). The assays were carries out in triplicate.
Chondrogenic differentiation potential was evaluated by a glycosaminoglycan assay. After cultured for 7, and 14 days, the culture media were collected from flask cultures for measurement of total GAG content using a Human glycosaminoglycan ELISA Kit according to manufacturer’s instructions. Briefly, 40 μl of sample dilution and 10 μl of sample buffer was added in reaction plate for gently mixing, and then incubated at 37 °C for 30 min. After washing for five times with washing buffer, the plate was dried. Subsequently, HRP-Conjugate reagent (50 μl) was added in each well, and the plate was left 30 min at 37 °C for the reaction. The plate was washed, dried, and then placed in the dark for 15 min after addition of substrate working solution. Finally, after adding 50 μl of stop solution, the spectrometric absorbance of 450 nm was read using a microplate reader (Molecular Devices, CA, USA).
Chondrogenic Differentiation Isolated hUC-MSCs were resuspended in chondrogenic culture medium consisting of high glucose Dulbecco’s modified Eagle’s medium containing 100 µg/ml sodium pyruvate, 10 ng/ml TGFβ3, 100 nM dexamethasone, 1× ITS + 1 premix, 40 µg/ml proline, and 25 µg/ml ascorbate2-phosphate [23, 24]. For chondrogenesis in pellet cultures to observe cells after cartilage induced into spheroids, aliquots of hUC-MSC (5 × 105) were spun in 50-ml polypropylene tubes (1 ml of medium, 240 g, 5 min) [8, 9, 23]. For chondrogenesis in flask cultures, isolated hUC-MSCs were resuspended in Mesenchymal Stem Cell Growth Medium and seed in T-25 cm2 tissue culture flasks. They were changed to chondrogenic culture medium till monolayer cells spread to >90% of the tissue culture flasks and contained different concentrations taurine (0, 1, 5 and 10 mM). Chondrogenic differentiation of umbilical cord mesenchymal cells was incubated with serum-free medium supplemented for 14 days and replaced twice a week for up to 14 days. Histological Staining The pellets were washed with PBS and fixed in 4% paraformaldehyde and processed into paraffin wax. Sections cut at 3 µm were blocked with 5% BSA at room temperature for 1 h, probed with antibody raised against collagen type II and incubated overnight at 4 °C. The sections were washed, followed by a second incubation with PE-labeled anti-mouse IgG for another 2 h at room temperature. Nuclei were stained with DAPI. The sections were washed, viewed under an DM RXA2 microscope (Leica, Germany), and photographed by DFC280 (Leica, Germany).
Quantitative Real‑Time PCR Cells were collected from flask cultures at 14 days after chondrogenic differentiation. Total cellular RNA was extracted with HP Total RNA Kit and first-strand Complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase according to the manufacturer’s instructions. cDNA was generated on a Roche real-time PCR system. The mRNA expression levels were estimated by real-time quantitative PCR with Light Cycler Fast Start DNA Master SYBR Green I and Lightcycler 480 instrument (Roche, Denmark). The hUC-MSCs were cultured with Mesenchymal Stem Cell Growth Medium at the same time as the control group. Primer sequences were as follows: human collagen II sense: 5′-TCACGTACACTGCCC TGAAG-3′, antisense: 5′-TGCAACGGATTGTGTTGT TT-3′; huaman SOX 9 sense: 5′-AGGAAGTCGGTGAAG AACGG-3′, antisense: 5′-CGCCTTGAAGATGGCGTT G-3′; huaman aggrecan sense: 5′-CTACACGCTACACCC TCGAC-3′, antisense: 5′-ACGTCCTCACACCAGGAA AC-3′; huaman collagen X sense: 5′-CGCTGAACGATA CCAAATGCC-3′, antisense: 5′-TTCCCTACAGCTGAT GGTCC-3′; huaman collagen IA sense: 5′-GCAGCCCTG GTGAAAATGGA-3′, antisense: 5′-CAGCACCAGTAG CACCATCA-3′; huaman TauT (taurine transporter) sense: 5′-TGGGACTCTACACCCATCTT-3′, antisense: 5′-GCT TCAGCCAGTCTCCTTTAT-3′; β-actin sense: 5′-CTG GCACCACACCTTCTAC-3′, antisense: 5′-TGGATAGCA ACGTACATGGC-3′. The mRNA expression levels of each gene were calculated by the 2−ΔCt method, in which ΔCt represents the difference between the cycle threshold of the target gene and Ct of the housekeeping gene (ΔCt = Ct of target gene − Ct of the housekeeping gene). For comparison, cells in the hUC-MSCs group were assigned an
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arbitrary value of one. Data are represented as 2−ΔΔCt. Each sample was repeated three times. Statistical Analysis Data are reported as means ± standard deviation (SD). Differences were analyzed by the two-tailed Student’s t test for comparisons of paired and unpaired data between groups, by one-way analysis of variance (ANOVA) for comparison between various groups. Statistical analyses were performed with the SPSS software (version 17.0, SPSS). Values of P < 0.05 were considered statistically significant.
Results Characterization of hUC‑MSCs
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analysis revealed the hUC-MSCs were positive for human MSC markers CD73 and CD105, but negative for hematopoietic lineage markers CD34 and CD45 (Fig. 1b). Effect of Taurine on Proliferation of hUC‑MSCs The effect of taurine on cell proliferation was investigated at concentrations ranging from 1 to 10 mM after 1, 3 and 5 days of culture. The CCK8 result showed that proliferation of hUC-MSCs was not affected after exposure to taurine at concentrations of 1, 5 and 10 mM for 1–5 days (Fig. 2). In addition, we observed cell cycle using flow cytometry to calculate percentage of G0/G1, S, and G 2/M to the distribution of the activity of proliferation (Fig. 3a). After 24–72 h of cultures, the taurine groups did not show significant G0/G1, S or G2/M increased compared with 0 mM group (Fig. 3b).
To determine whether the hUC-MSCs we used have stem cell properties, we examined their morphology and surface markers. By the 4th passage of cells, hUC-MSCs exhibited a fibroblast-like phenotype (Fig. 1a), and Flow cytometric
Chondrogenic Differentiation of hUC‑MSCs
Fig. 1 Characteristics of hUC-MSCs. a Cells were used at passage 4 of hUC-MSCs. The cells had a fibroblast-like morphology. Bar 100 µm. b Flow cytometric analysis of surface-marker expression
on hUC-MSCs at passage 4. c The percentages of positive cells were expressed as the mean ± SD. The results were repeated three times
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hUC-MSCs developed chondrogenic characteristics after 14 days with serum-free chondrogenic differentiation
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Fig. 2 Effect of taurine on the proliferation of hUC-MSCs The cells were treated with taurine at the concentrations of 0, 1, 5 and 10 mM for 1, 3 and 5 days, and the cell proliferation was performed using CCK8 assay. Each group compared with the 0 mM group at the corresponding time point. The results were repeated three times. Data are expressed as the mean ± SD
medium. Chondrogenesis was further validated by a type II collagen-rich extracellular matrix (Fig. 4a). Expression of collage type II was detected in both hUC-MSCs and differentiated chondrogenic cells. However, compared with hUC-MSCs, real-time PCR results revealed that the differentiated chondrogenic cells have significantly increased the mRNA expression of collagen type II (Fig. 4b), signifying differentiation into the cartilage lineage. Taurine Promotes Chondrogenic Differentiation of hUC‑MSCs Proteoglycans are important components of extracellular matrices of cartilage, while GAGs constitute a major component of proteoglycans. During hUC-MSC chondrogenesis, the concentration of GAG in culture rapidly increased from 7 days to 14 days in all experimental groups (Fig. 4c). However, taurine-treated culture media showed significantly higher GAG content in culture than that of control at corresponding time points. The maximal increased was observed at 5 mM with no additional enhancement in 10 mM. Coinciding with GAG concentration in culture, the expression of collagen type II mRNA was also significantly increased by taurine treatment at concentrations of 5 and 10 mM (Fig. 5a). Aggrecan is cartilage-specific proteoglycan present in early chondrogenesis, and SOX9 is a chondrogenic transcription factor and plays a major role in chondrogenesis. As shown in Fig. 5, the expression levels of aggrecan (b) and SOX9 (c) mRNAs were significantly increased by taurine at concentrations of 5 and 10 mM, with the peak of expression at 5 mM. Collagen type X is associated with hypertrophic chondrocytes and its expression precedes the onset of endochondral ossification. In our study, the expression of collagen
type X mRNA remained very low in all groups (Fig. 5d). Collagen type I is a well-known marker for chondrocyte dedifferentiation. The addition of taurine in media decreased the expression of collagen type I mRNA when compared to 0 mM group. Furthermore, the ratio of collagen type II versus collagen type I mRNA expression levels was higher in taurine-treated cells when compared to control (Fig. 5g). Taken together, these results show that taurine enhances chondrogenesis and inhibit dedifferentiation of chondrocytes, and do so most effectively at 5mM concentration. Interestingly, taurine treatment at 5 and 10 mM also increased the expression of the taurine transporter, TauT (Fig. 5f), activating a positive feedback loop that promotes taurine uptake in cells.
Discussion In this study, we focused on the effect of taurine on viability of hUC-MSCs and chondrogenic differentiation. The present data demonstrated that taurine promotes the accumulation of GAG and chondrogenic differentiation of hUCMSCs in culture. MSCs have the potential to differentiate into muscle cells adipocytes, osteocytes and chondrocytes in culture. The fibroblast-like hUC-s (Fig. 1a) express high levels of markers CD73 and CD105, and are devoid of hematopoietic lineage markers CD34 and CD45 (Fig. 1b). Recently studies reported that taurine plays a role in promoting cell proliferation in neural stem/progenitor cells and human osteoblasts [25–28]. Consistent with the results of Zhou et al. [22], we did not observe any effects of taurine on hUC-proliferation by cell proliferation measurements (Fig. 2), cell cycle analysis (Fig. 3). The cell cycle is a series of events that occurs in a cell leading to its division and replication and is a critical process for determining cell proliferation and senescence [29]. In this study, taurine treated hUC-s exhibited similar G0/G1 phase as well as similar S and G2/M phase when cell cycle was examined, howing that hUC-s under taurine treatment maintained the same replicating and mitotic activity. Taurine is involved in cartilage physiopathology and was reported to promote the expression of connective tissue growth factor that possesses the ability to repair damaged articular cartilage [18, 30]. Several studies also demonstrated that taurine and its derivatives could inhibit development of rheumatoid arthritis [19, 20, 31]. All lines of evidence suggest that taurine may affect cartilage growth. Our study found that after 14 days to the cartilage cell differentiation culture, immunofluorescence test found cartilage cells specific collagen type II widely expressed in the cytoplasm and the extracellular matrix (Fig. 4a), and the real-time PCR experiments also showed that the level of
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Fig. 3 Effect of taurine on the distribution of the percentage of cells at each cell cycle stage. a Representative flow cytometry results of the cells were treated with taurine at different concentrations for 24,
48 and 72 h days. b The percentage of cells at each cell cycle stage as determined by flow cytometry analysis. The results were repeated three times. Data are expressed as the mean ± SD
collagen type II mRNA was significantly higher compared with the hUC-MSCs group (Fig. 4b). It’s suggested that hUC-MSCs could be induced to differentiated into cartilage cells.
GAGs constitute a major component of proteoglycans, which are important components of extracellular matrices of cartilage [32]. Our study shown that taurine could obviously promote the contents of GAG in cultured
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Fig. 4 Chondrogenic differentiation of hUC-MSCs. a Sections from pellets were examined for the presence of collagen type II (red fluorescence) during the chondrogenic culture medium. Nuclei were stained with DAPI (blue fluorescence). Bar 40 µm. b RNA samples collected from hUC-MSC cultures and at days 14 of chondrogenic culture were examined by real-time PCR for collagen type II mRNA
levels, ***P < 0.001. c Effect of taurine on the contents of GAG in hUC-MSCs during chondrogenic culture, *P < 0.05, **P < 0.01, ***P < 0.001. Data were expressed as the mean ± SD. The results were repeated three times. GAG glycosaminoglycan. (Color figure online)
chondrocytes as shown by ELISA assay (Fig. 4c). GAGs play an important role in maintaining cartilage load-bearing capacity, due to the large number of water molecules generate the expansion pressure and make the cartilage flexible [33, 34]. The events of chondrogenesis is marked by the increased expression of the major chondrogenic transcription factor SOX9 [35, 36], cartilage specific marker collagen type II [21] and cartilage-specific proteoglycan aggrecan [34]. In our study, the taurine of 5mM concentration was able to maximally increase the level of collagen type II (Fig. 5a) aggrecan and SOX9 (Fig. 5c) mRNA during differentiation of hUC-MSCs into cartilage, suggesting that taurine is able to promote chondrogenesis of hUC-MSCs in vitro. Collagen type X is associated with hypertrophic chondrocytes and its expression precedes the onset of endochondral ossification. In our study, the expression of collagen type X mRNA was very low in all groups (Fig. 5d), demonstrating that taurine treatment have no adverse effects on differentiated chondrocytes.
Collagen type I is a critical marker for chondrocyte dedifferentiation, and collagen type II to type I ratio is a key measurement of chondrocyte commitment and differentiation [37]. Taurine treatment decreased the expression of collagen type I mRNA (Fig. 5e) and increased the ratio of mRNA levels of collagen type II to collagen type I. This suggests that taurine treatment can inhibit dedifferentiation and promote more robust differentiation of chondrocytes. Cartilage tissue is a special structure composed of a network of collagen and proteoglycans, allowing a unique osmotic environment for chondrocytes. The presence of taurine in culture could partially rescue cell death caused by hypertonic conditions [38], this suggest taurine could play an important role in osmotic regulation of chondrocytes. The increase in the expression levels of the taurine transporter, TauT after taurine treatment (Fig. 5f) indicates a positive feedback loop that sustains taurine presence in cells during chondrocyte differentiation. Interestingly, Taurine at appropriate concentrations ranging from 100 to 500 µM stimulated the proliferation of P5 neural progenitor
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Neurochem Res ◂Fig. 5 Taurine promoted chondrogenic differentiation of hUC-
MSCs. Effect of taurine on the mRNA expression of collagen II (a), aggrecan (b), SOX9 (c), collagen X (d), collagen I (e), TauT (f) and the ratio of collagen II/collagen I (g) in hUC-MSCs treated with the chondrogenic culture medium for 14 days. The mRNA expression was detected by real-time PCR. Data were expressed as the mean ± SD. Compared with 0 mM group, *P < 0.05, **P < 0.01, ***P < 0.001, a < 0.05, b < 0.05, c < 0.001, d < 0.05. The results were repeated three times
cells for 2 days [39], and from 1 to 10 mM promoted mineralization of hMSCs [22] respectively. These results suggests that the effects of taurine can be tissue dependent in terms of effect and sensitivity. The increased collagen type II expression in taurine-treated groups indicated that taurine could stimulate chondrogenic differentiation of hUC-MSCs. In conclusion, we demonstrated that taurine could effectively promote chondrogenic differentiation, enhance secretion and synthesis of GAG, up-regulating expression level of chondrocyte specific mRNA. The most profound response was observed with the 5 mM of taurine in the chondrogenic differentiation culture. Taurine is very safe in terms of tissue toxicity and is used as a supplement in milk. Thus, taurine might be a useful pro-chondrogenic agent in the treatment of cartilage repair and further studies are needed to explore the underlying mechanism of taurine in chondrogenic differentiation. Acknowledgements This work was supported by National Natural Science Fund of China (81571216), National High Technology Research and Development Program of China (2014AA020703), Tianjin Municipal science and technology application of basic and advanced technology of key projects (11JCZDJC19400, 16JCZDJC35500). Compliance with Ethical Standards Conflict of interest No personal, institutional or corporate financial conflict are involved in the production and publication of this information.
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