Hydrogel samples were fixed overnight in 4% paraformaldehyde at 4°C, dehydrated in a graded series of ethanol, crystalized in a graded series of xylene, and embedded in paraffin. Histological sections (7 μm) were stained for type I collagen using the VECTASTAIN ABC Kit and the DAB Substrate Kit (Vector Laboratories, Burlingame, California, USA). Briefly, hyaluronidase (0.5 g liter−1) was applied to the rehydrated samples to predigest them at 37°C for 30 min. The samples were then incubated with 0.5 N acetic acid for 4 hours at 4°C to induce swelling, followed by incubation at 4°C overnight with a primary antibody directed against type I collagen (anti–type I collagen, diluted 1:200; sc-59772, Santa Cruz Biotechnology). Immunofluorescence staining for the osteogenic-related proteins (YAP and RUNX2) and cellular contraction–related proteins (RhoA) was conducted as previously reported (9). The calcification of phosphates and calcium ions was identified with von Kossa staining and Alizarin Red S staining, respectively. Briefly, von Kossa stain was applied to the rehydrated sections, as previously reported. To stain with Alizarin Red S, 1 ml of 0.5% (w/v) Alizarin Red S solution (Sigma) was applied to each hydrogel sample; the samples were then incubated for 5 min at room temperature before being washed, dehydrated, cleared, and sealed. Images were captured using a bright-field microscope (Nikon). The quantification was conducted by using the ImageJ software (NIH). First, we adjusted the color images of immunohistochemistry (IHC) staining to the 8-bit grayscale images, and then we selected regions of interests of identical size that best represent the overall staining intensity of the samples in the IHC staining results. The average grayscale values of the region of interest taken from three parallel samples in each group were determined and compared to get the quantification results. The reported data of biochemical assays are pooled results from three experiments.

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