The study was approved by the Ethical Review Committee of 455th hospital of PLA (Shanghai, China). After obtaining informed consents from the mothers and family, the umbilical cords were harvested from the full-term natural delivery infants. WJ-MSC were isolated and cultured by Shanghai Omnicells Biotechnology Co., Ltd. (Shanghai, China) as described (22): Umbilical cords were washed with sterile phosphate-buffered saline (PBS) three times and diced into pieces, and the blood vessels in umbilical cords were dissected. Wharton's jelly was cut into small pieces and digested in DMEM/Hams's F-12 (1:1 vol/vol) culture medium containing 10% FBS, collagenase type II (1 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin-B (2.5 µg/ml) (all from Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a shaker for 4–6 h, and then centrifuged at 500 × g for 15 min. The cell pellet was resuspended in the above culture medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, Inc., Minneapolis, MN, USA), and cells were subcultured using 0.05% trypsin/0.02% EDTA (Thermo Fisher Scientific, Inc.) on reaching 80–90% confluence.

The cartilage samples were obtained from the knee joints of OA patients (n=12, KL grade >2) undergoing joint replacement surgery, and chondrocytes were harvested by enzymatic digestion, chondrocytes were cultured in low glucose DMEM at 37°C with 5% CO₂ as previously described (23).

In the present study, the cells were divided into 2 groups: Experimental group (co-culture group) and control group. In proliferation assay, the medium in experimental group was changed with the supernatant from hUC-MSCs, while the medium in control group was changed with normal medium. In other experiments, OA chondrocytes and hUC-MSC were incubated in a noncontact co-culture system: Chondrocytes were cultured in the bottom well, and hUC-MSCs were cultured in a Transwell insert (Transwell; Corning Costar, Corning, NY, USA) in co-culture group; while chondrocytes were cultured alone in the bottom well in control group.

The expression of hUC-MSC surface markers was tested by using flow cytometry as described previously (24). hUC-MSCs were isolated and harvested by using 0.1% trypsin-EDTA treatment, and washed with PBS. Then the cells were incubated with the following antibodies: Rabbit polyclonal to CD44 (ab157107; 1/500 dilution), rabbit polyclonal to CD73 (ab175396; 1/500 dilution), rabbit monoclonal to CD90 (ab92574; 1/1,000 dilution), mouse monoclonal to CD105 (ab11414; 1/1,000 dilution), rabbit monoclonal to CD34 (ab81289; 1/1,000 dilution), rabbit polyclonal to CD45 (ab10559; 1/1,000 dilution), rabbit polyclonal to CD106 (ab134047; 1/1,000 dilution), rabbit polyclonal to CD133 (ab16518; 1/1,000 dilution) (all from Abcam, Cambridge, MA, USA), in the dark for 30 min at room temperature, then conjugated with either fluorescein PE or FITC [Goat Anti-Rabbit IgG H&L (FITC) (ab6717) or Goat Anti-Mouse IgG Secondary Antibody (PE) LS-C60691; 1/1,000 dilution; LifeSpan BioSciences, Seattle, WA, USA]. The labeled cells were washed and tested by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA).

A 6 well-plate was cultured with 5×10⁴ cells/well. At 48 h, the medium was replaced by adipogenic medium (high glucose DMEM containing 10% FBS, 500 µM isobutylmethylxanthine, 5 µg/ml insulin, 200 µg/ml ascorpate-2-phosphate, 100 U/ml penicillin, 100 U/ml streptomycin, 1 µM dexamethasone), osteogenic medium (glucose DMEM containing 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbate-2-phosphate), and chondogenic medium (high glucose DMEM containing 10% FBS, 10 ng/ml TGF-β1, 100 nM dexamethasone, 50 ng/ml ascorbate-2-phosphate, 1 mM sodium pyruvate). All the reagents were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

The induction medium was replaced every 2 days for 3 weeks, control group were cultured without induction. After 3 weeks, RNA extractions and RT-PCR were performed to evaluate the cell differentiation.

The special staining was further performed to show morphological changes of differentiated cells. After 3 weeks of adipogenic induction, cells were fixed in 4% paraformaldehyde at room temperature for 30 min, and washed with deionized water and then with 60% isopropanol, followed by staining with 0.5% Oil red O (Sigma-Aldrich; Merck KGaA) in isopropanol (wt/vol) for 30 min, and excess stain was removed and the cells were washed 3 times with deionized water.

For osteogenesis, cells were fixed in 4% paraformaldehyde at room temperature for 30 min, and washed with deionized water and stained with Alizarin Red Solution (no. 0223, Alizarin Red S staining kit; ScienCell Research Laboratories (San Diego, CA, USA) at room temperature for 15 min following the manufacturer protocol, and excess stain was removed and the cells were washed 3 times with deionized water.

For chondrogenisis, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then were stained with 1% Toluidine Blue Solution (sc-206058; Santa Cruz Biotechnology, Heidelberg, Germany), for 30 min, and excess stain was removed and the cells were washed 3 times with deionized water.

The total RNA was isolated from samples by using TRI Reagent^® RNA Isolation Reagent (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions and complementary DNA (cDNA) was prepared by using a First-Strand cDNA Synthesis kit (Pharmacia LKB, Uppsala, Sweden). Real-time quantitative PCR was performed using the TaqMan system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and Real-time PCR amplification and product detection were performed using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) (25).

Each reaction is consisted of 10 µl SYBR Green (Applied Biosystems; Thermo Fisher Scientific, Inc.), 6 µl molecular grade water, 1 µl each forward primer and 1 µl reverse primer and 2 µl cDNA (Table I). The amplification was performed under the certain conditions: 10 min at 95°C, followed by 50 cycles, 10 sec at 95°C and 60 sec at 60°C. q-PCR was performed under standard conditions and all experiments were performed in triplicate. The quantitative gene expression was normalized to GAPDH, and the relative expression was determined by using the ΔΔCt method.

The Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was used to evaluate the cell proliferation. Chondrocytes from OA patients at passage 2 were isolated and harvested by using 0.1% trypsin-EDTA and plated into 96-well plates at a density of 10³ cells/well. The plates were divided into 2 groups: Experimental (co-culture group) and control groups. The medium in experimental group was changed with the supernatant from hUC-MSCs, while the medium in control group was changed with normal medium. At day 1, 3, 5, 7, the medium was change with 100 µl of fresh medium containing 10 µl CCK-8 solution for 4 h in a 37°C incubator. The OD at 570 nm was measured, and the assay was repeated in a triplicate.

The protein expression was accessed by using western blotting. Cells were prepared in RIPA buffer containing a protease inhibitor cocktail, and then centrifuged at 12,000 × g for 10 min at 4°C, and the protein concentration was tested using the BCA protein assay kit (cat. no. 23225; Applied Biosystems; Thermo Fisher Scientific, Inc.). The certain protein was electrophoresis on a 10% SDS-PAGE gels and electrotransferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). And then block with 5% non-fat milk (Sangon Biotech Co., Ltd., Shanghai, China) in TBS for 1 h at room temperature room, the membrane was incubated with first antibodies (mouse monoclonal to Cox-2, cat. no. sc-19999, dilution 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) (mouse monoclonal to collagen X, cat. no. ab49945, dilution 1:1,000; Abcam) (mouse monoclonal to MMP13, cat. no. sc-101564, dilution 1:1,000; Santa Cruz Biotechnology, Inc.) (anti-β-actin antibody, cat. no. ab6276, dilution 1:1,000; Abcam) for 1 h. Then wash with TBST 3 times, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (anti-mouse IgG, HRP-linked antibody, cat. no. 7076, dilution 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at RT. Then wash with TBST 3 times, a the ECL Plus detection kit (cat. no. 32132; Thermo Fisher Scientific, Inc.) was used to visualize the immunoreactive according to the manufacturer's instructions, and the relative protein expression was calculated by using ImageJ software (version 1.46; Wayne Rasband National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunosorbent assay (ELISA) was performed to measure TNF-α, IL-1β, IL-6, IL-10 in supernatant. Briefly, antibodies specific for TNF-α (cat. no. SMTA00B), IL-1β (cat. no. SLB50), IL-6 (cat. no. S6050), and IL-10 (cat. no. S1000B) (all from R&D Systems, Inc.) was immobilized onto-96-well microtiter plates, and remove unbound antibody and add a blocking reagent. Then the sample were incubated with the solid phase antibodies, and washing unbound molecules away, followed by adding a detection antibody specific for TNF-α, IL-1β, IL-6, IL-10. The incubation and washing was followed by adding HRP-conjugated anti-mouse immunoglobulin. The plate was washed and a TMB substrate solution (Zymed Laboratories, San Francisco, CA, USA) was added for 30 min, and measured using a Beckman Coulter DU 800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA) at 450 nm.

All the data are expressed as mean ± standard deviation. a one-way ANOVA with Tukey's HSD post hoc test was performed to comparise differences between groups. All the statistical evaluations were performed by using the SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). P-values <0.05 were considered to indiacate statistically significant difference.

Flow cytometry assay demonstrated that hUC-MSC at passage 3 show a high expression (>95%) for the mesenchymal stem positive markers CD44 (95.6%), CD73 (97.1%), CD90 (96.3%), CD105 (97.6), while the negative expression of CD34 (0.5%), CD45 (3.5%), CD106 (0.3%), and CD133 (0.4%) was observed (Fig. 1A-H), and the histogram gives a clear picture of the expression ratio (Fig. 1I and J) suggesting our results was in agreement with previous studies (21).

The multi-differentiation (chondrogenic, osteogenic, adipogenic differentiation) assay was used to evaluate the differentiation potential of hUC-MSC. After 3 weeks of induction toward the multi-lineage differentiation, the RNA was extracted, and quantitative RT-PCR was performed to evaluate the cell differentiation. The result indicated that compared with the cells in the control group, cells in the induction group have high expression of chondrogenic genes (aggrecan, collagen II, sox-9), osteogenic genes (OCN, ALP, RUNX2), and adipogenic genes (adipoq, aP2, PPARr) following 3 weeks of chondrogenic, osteogenic, and adipogenic differentiation (P<0.05) (Fig. 2).

For special staining, Oil Red O-positive staining of lipid droplets were observed in adipogenic differentiation after 3 weeks of adipogenic induction. On day 21 of osteogenic differentiation, cells were detected with positive Alizarin Red staining. After 3 weeks of chondrogenic differentiation, cells showed remarkable changes in cellular morphology and were stain positive for toluidine blue (Fig. 2).

Chondrocytes harvested from OA patients seeded at a density of 1,000 cells/wells were cultured in the supernatant from hUC-MSC. From day 3, the cells cultured in the supernatant from hUC-MSC demonstrated significant higher proliferation rates than control group (P<0.05), suggested that the secretion of hUC-MSC enhanced the chondrocytes proliferation (Fig. 3).

The quantitative RT-PCR assay was performed to evaluate the mRNA of hUC-MSC and chondrocytes in coculture system. As shown in Fig. 4, hUC-MSC demonstrated a significant increased mRNA expression of aggrecan, sox-9, collagen II, compared to the control group (P<0.05), indicating chondrocytes promoted chondrogenic differentiation of hUC-MSC (Fig. 4).

After coculture with hUC-MSC, chondrocytes from OA patients showed a significant decreased mRNA expression of cox2, collagen X, MMP13, compared to the control group (P<0.05), which suggested hUC-MSC inhibited inflammatory activity in OA chondrocytes (Fig. 5).

As shown in Fig. 6, the western blot analysis indicated that the protein expression of COX-2, collagen 10A1 and MMP13 in coculture system significantly decreased in comparison to that in control group determined by western blot analysis (P<0.05), suggested that hUC-MSC inhibit the expression inflammatory related protein in OA chondrocytes (Fig. 6).

The supernatant was also collected for ELISA assay, and the result showed that the levels of TNF-α, IL-1β, IL-6, IL-10 were: 22.38±3.12, 13.23±2.32, 16.53±1.92, and 20.89±3.54 pg/ml in the supernatant of co-culture system, respectively which were significantly lower than those in the control group: 32.28±4.21, 21.37±3.31, 26.75±3.24, and 31.23±4.53 pg/ml (P<0.05) (Fig. 7).