Preparation of agarose gel wells
First, 1% agarose gel was prepared in phosphate-buffered saline (PBS) and heated in microwave oven. The soluble gel was poured into a 6-cm cell culture plate after a specific cylinder-shaped mold (diameter = 10 mm) was put on the plate to form the wells in the gel. After gelation at room temperature for about 1 h, the mold was taken away, and the gel well plates were ready for cell culture (Fig. 1).
Isolation of chondrocytes and cell seeding
Cartilage was harvested from bovine femoral condyles provided by HK Scan (Outokumpu, Finland) and Strömdahla (Nordmaling, Sweden) abattoirs. The chondrocytes were isolated by overnight collagenase digestion in high glucose DMEM supplemented with fetal bovine serum, penicillin, streptomycin, L-glutamine and L-ascorbic acid 2-phosphate trisodium salt, as previously described .
The next day, six million chondrocytes were seeded into the agarose gel wells in HyStem™ or HydroMatrix™ scaffolds, or as a control without the scaffold. Chondrocytes seeded in the HyStem™ scaffolds were prepared by mixing six million chondrocytes into 100 µl of the HyStem™ solution and adding 25 µl of the crosslinking solution of the kit to start the gelation process. The suspension was immediately gently pipetted into the gel well, and placed into a humidified incubator for 20 min. After the gelation, 5 ml of high glucose hypertonic (390 mOsm) DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 50 µg/ml L-ascorbic acid 2-phosphate trisodium salt was added into plate.
The HydroMatrix™ solution was kept on ice before initiating the gelation, then six million chondrocytes were mixed into the product (100 µl, final concentration 0.25%) and 5 ml of the hypertonic high glucose medium, supplemented as described above, was gently poured into the plate. The sample was then placed into the humidified incubator to start the gelation process and cultivation.
The scaffold-free controls, prepared by suspending six million chondrocytes in 100 µl of the above-described high glucose hypertonic DMEM into the agarose wells, were placed into incubator for 1 h, and then 5 ml of the high glucose hypertonic DMEM was very carefully added to the plate. The samples were grown for 1, 3 and 6 weeks. After each time point, the samples were collected for histological staining, PG analyses and gene expression analyses. Each tissue was also photographed for macroscopical evaluation. The experiments were repeated for four times performing four different chondrocyte isolations from four different animals. For gene expression sample collection, the experiments were repeated for six times.
At the end of each culture period, one sample from each treatment and control was cut into two halves: one for the PG analyses, while the other half was fixed in 4% paraformaldehyde and processed for the histological analyses. The samples reserved for the PG analyses were weighed in a pre-weighed tubes to obtain the wet weight of the sample, and then frozen in −70 °C for the further analyses.
The histological sections of the samples were stained with Toluidine Blue, and type II collagen was immunostained with anti-type II collagen mouse monoclonal antibody E8 [6, 13]. Envision+ System-HRP kit (Dako, Glostrup, Denmark) was used for detection. The stained sections were then imaged using a light microscope (Carl Zeiss Axioimager M2, Thornwood, NY, USA). The thicknesses of the neotissues were measured from the same sections using the same device using a standard scale bar for calibration.
Analysis of the proteoglycans
The PGs were extracted with 4 M guanidinium hydrochloride as described in our previous studies [6, 8]. The extracted PGs were precipitated in 70% ethanol, dissolved in water, and glycosaminoglycan (GAG) contents were quantitated using 1,9-dimethylmethylene blue (Sigma-Aldrich) assay . Chondroitin sulfate from shark (Sigma) was used as a standard. Separation of the extracted PGs in a 1.2% agarose gel and staining with Toluidine Blue was performed as previously described .
Gene expression analyses
The gene expression analyses were performed as described in our previous study . Briefly, TRI reagent (Molecular Research Center, Cincinnati, OH, USA) was used to extract the total RNA from each sample, and reverse transcription was performed according to the kit’s instructions (Verso cDNA Synthesis Kit, Thermo Scientific, Waltham, MA, USA). The quantitative RT-PCR was accomplished by using MX3000P Real Time PCR System (Stratagene, La Jolla, CA, USA) as previously described  using the primers optimized for aggrecan , procollagen α2(I) , procollagen α1(II)  and Sox9 . Ribosomal protein large P0 (RPLP0) was used as a house-keeping gene .
One-way analysis of variance with the Bonferroni correction was used to analyze the statistically significant differences in the determined parameters between the different time points, and scaffold vs. scaffold-free cultures. A difference was interpreted to be statistically significant when the P-value was less than 0.05.