Hyaluronic acid enhances the effect of the PAMPS/PDMAAm double-network hydrogel on chondrogenic differentiation of ATDC5 cells

The PAMPS/PDMAAm DN gel was synthesized using the previously reported two-step sequential polymerization method [11]. 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) (Toagosei Co. Ltd., Japan) and N,N’-dimethyl acrylamide (DMAAm) (KOHJIN Co., Ltd., Tokyo, Japan) were used as purchased. Briefly, PAMPS hydrogel was obtained by radical polymerization using N,N^’-methylenebisacrylamide (MBAA) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) as a cross-linker and 2-oxoglutaric acid (Wako Pure Chemical Industries, Ltd, Osaka, Japan) as an initiator. The monomer concentration was 1 mol/l for PAMPS, 4 mol% for the cross-linker, and 0.1 mol% for the initiator. Aqueous solution containing a monomer, cross-linker, and the initiator was injected into a cell consisting of a pair of glass plates separated by a silicone rubber. The cell was irradiated with ultraviolet (UV) light (wave length 365 nm) for about 8 hours under argon gas atmosphere. The DN gel was synthesized by the sequential network formation technique (two-step method). The PAMPS hydrogel (1^st network) was immersed in an aqueous solution of 2 mol/L DMAAm, containing 0.1 mol% MBAA, and 0.1 mol% 2-oxoglutaric acid for one day until reaching the equilibrium. The 2^nd network (PDMAAm) was subsequently polymerized in the presence of the PAMPS hydrogel by irradiating UV for 8 hours between two plates of glasses under argon gas atmosphere. After polymerization, the PAMPS/PDMAAm DN gel was immersed in 0.9 % NaCl solution for 1 week and the water was changed twice daily to remove any un-reacted materials. After sterilizing by autoclaving (120 degrees Celsius, 20 min), gel disks were punched out of a gel plate with a hole puncher having a diameter of 1.5 cm. The gel disks were then placed in a 24-well polystyrene (PS) tissue culture dish for cell culture.

The ATDC5 cell line was obtained from the RIKEN cell bank (Tsukuba, Japan).The cells were cultured in the maintenance medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (Life Technologies, GIBCO, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum, 10 mg/ml human transferrin (Roche Applied Science, Indianapolis, IN, USA) and 3 × 10⁻⁸ M sodium selenite (Sigma–Aldrich, St. Louis, MO, USA). The ATDC5 cells were seeded at a cell density of 5 × 10⁴ cells/cm², and cultured on the PAMPS/PDMAAm DN gel and the PS dish surface at 37°C under 5% CO2 for 21 days. The PS dish was used as the control. The maintenance medium was changed twice each week without damaging the gels.

This study was composed of 2 sub-studies. The first sub-study was conducted to test the hypothesis that the PAMPS/PDMAAm DN gel may induce chondrogenic differentiation of ATDC5 cells even in a maintenance medium without insulin. We compared the ATDC5 cells cultured on the DN gel with the cells cultured on the PS dish (n = 6, each), using the maintenance medium. In the second sub-study, we tested the hypothesis that supplementation of hyaluronic acid into the maintenance medium may enhance the chondrogenic differentiation effect of the PAMPS/PDMAAm DN gel on the ATDC5 cells. We prepared 3 types of the maintenance medium containing hyaluronic acid (ARTZ, Seikagaku Co., Tokyo, Japan) at the concentration of 0.01, 0.1, or 1 mg/mL, respectively. The hyaluronic acid had a molecular weight of approximately 800 kDa. The ATDC5 cells cultured in the 3 hyaluronic acid-supplemented media were compared with the cells cultured in the maintenance medium without hyaluronic acid (n = 6, each). In each sub-study, we performed real-time polymerase chain reaction (PCR) analyses to evaluate expression of type-2 collagen, aggrecan, and Sox9 mRNA in the cultured ATDC5 cells at 7 and 21 days of culture. We also carried out immunocytochemical examination to assess expression of type-2 collagen at the same periods (n = 2, each).

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline without calcium (PBS(−)), and permeabilized with 0.1% Triton X-100 in PBS, followed by pretreatment to block nonspecific reactions with 5% nonimmune goat serum in PBS(−). For collagen staining, the primary immunoreaction was carried out with a mouse monoclonal antibody against type-2 collagen (Abcam Inc., Cambridge, MA, USA). The secondary immunoreaction was carried out with Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) in 1% nonimmune goat serum in PBS, followed by rinsing with PBS(−). For cell nucleus staining, cells were incubated on 1 μg/ml Hoechst 33258 (Dojindo, Kumamoto, Japan) for 1 min, followed by rinsing with PBS(−). Fluorescent images were recorded with a fluorescence microscope.

All data were described as the mean and standard deviation values. A commercially available software program (StatView 5.0, SAS Institute Inc., Cary, NC, USA) was used for statistical calculation. To test the first hypothesis, we compared each gene expression in the maintenance medium without hyaluronic acid between cultures on the PS dish and the DN gel at each period, using unpaired t-test. To test the second hypothesis, we compared each gene expression among the cells cultured on the DN gel in the maintenance medium with or without hyaluronic acid, using two-way analysis of variance (ANOVA). This was followed by Dunnett’s test for post-hoc multiple comparisons at each period if ANOVA detected statistical significance. The significance level was set at p = 0.05 in each comparison.

At 7 days, mRNA expression of type-2 collagen and aggrecan was significantly greater in culture on the DN gel than in culture on the PS dish (p < 0.0001 and p < 0.0001, respectively) (Figure 1). At 21 days, mRNA expression of type-2 collagen and aggrecan was significantly greater in culture on the DN gel than in culture on the PS dish (p = 0.0008 and p = 0.0008, respectively) (Figure 1). There were no significant differences in mRNA expression of Sox9 between the 2 culture conditions at each period. In immunocytochemical examination performed at 21 days, the cells cultured on the DN gel surface formed nodules and type-2 collagen was expressed in the nodules (Figure 2), while we did not find nodule formation or type-2 collagen expression on the PS dish.

Concerning expression of type-2 collagen mRNA, the ANOVA demonstrated significant effects of the degrees of hyaluronic acid concentration (p = 0.0088) and the time (p = 0.0027) (Figure 3). Expression of type-2 collagen mRNA increased, depending on the concentration of hyaluronic acid. Hyaluronic acid supplementation of a high concentration (1.0 mg/mL) significantly enhanced expression of type-2 collagen mRNA in comparison with culture without hyaluronic acid supplementation at 21 days, while we did not find any effect of hyaluronic acid supplementation at 7 days. In immunocytochemical examination at 21 days, type-2 collagen was markedly expressed in nodules cultured on the DN gel surface in the medium supplemented by the high concentration (1.0 mg/mL) of hyaluronic acid (Figure 4).Regarding mRNA expression of aggrecan, the ANOVA also showed a significant effects of the degrees of hyaluronic acid concentration (p = 0.0411) and the time (p = 0.0380) (Figure 3). Expression of aggrecan mRNA increased, depending on the concentration of hyaluronic acid. Hyaluronic acid supplementation of a high concentration (1.0 mg/mL) significantly enhanced expression of aggrecan mRNA in comparison with culture without hyaluronic acid supplementation at 21 days, while we did not find any effect of hyaluronic acid supplementation at 7 days.Concerning mRNA expression of Sox9, the ANOVA did not indicate any significance (Figure 3). In addition, supplementation of hyaluronic acid into the medium did not provide any significant effects on the ATDC5 cells cultured on the PS dish.