Scheme 1. Schematic illustration of preparation of cell-laden gelatin solution-embedded GelMA hydrogel.

Fig. 1. Viscosity curves of gelatin aqueous solutions having a concentration of 5 (w/v)%, 10 (w/v)% and 15 (w/v)% and serum-free cell culture medium at 37 °C.

Fig. 2. Phase-contrast micrographs of biphasic hydrogels embedded with cell-laden gelatin microcubes (a, c) and cell-laden GelMA hydrogels (b, d) without gelatin microcubes immediately after preparation. Dexamethasone and TGF-β3 were added in the hydrogels shown in (c) and (d), but not added in hydrogels shown in (a) and (b) 5 (w/v)%, 10 (w/v)% and 15 (w/v)% indicate concentrations of gelatin solutions. GelMA indicates cell-laden GelMA hydrogels.

Fig. 3. Live/dead staining of hMSCs in biphasic hydrogels embedded with cell-laden gelatin microcubes and cell-laden GelMA hydrogels immediately after preparation (a) and after 21 days of culture (b). Dexamethasone and TGF-β3 were not added to the hydrogels. Low, Mid and High indicate the viscosity of gelatin solution at a concentration of 5 (w/v)%, 10 (w/v)% and 15 (w/v)%. GelMA indicates cell-laden GelMA hydrogels. Live cells were stained green, while dead cells red. Scale bar: 250 μm.

Fig. 4. Live/dead staining of hMSCs in biphasic hydrogels embedded with cell-laden gelatin microcubes and cell-laden GelMA hydrogels under the presence of dexamethasone and TGF-β3 immediately after preparation (a) and after 21 days of culture (b). Low, Mid and High indicate the viscosity of gelatin solution at a concentration of 5 (w/v)%, 10 (w/v)% and 15 (w/v)%. GelMA indicates cell-laden GelMA hydrogels. Live cells were stained green, while dead cells red. Scale bar: 250 μm.

Fig. 5. Quantification of DNA amount (a, d), sGAG content (b, e) and normalized sGAG/DNA ratio (c, f) in biphasic hydrogels embedded with cell-laden gelatin microcubes and cell-laden GelMA hydrogels without (a-c) and with (d-f) dexamethasone and TGF-β3. Low, Mid and High indicate the viscosity of gelatin solution at a concentration of 5 (w/v)%, 10 (w/v)% and 15 (w/v)%. GelMA indicates cell-laden GelMA hydrogels. Data are shown as mean ± SD, n = 3. *, p < 0.05, **, p < 0.01 and ***, p < 0.001.

Fig. 6. Quantification of gene expression of type Ⅰ collagen (a, c), type II collagen (d) and aggrecan (b, e) of hMSCs in biphasic hydrogels embedded with cell-laden gelatin microcubes and cell-laden GelMA hydrogels without (a, b) and with (c-e) dexamethasone and TGF-β3. Low, Mid and High indicate the viscosity of gelatin solution at a concentration of 5 (w/v)%, 10 (w/v)% and 15 (w/v)%. GelMA indicates cell-laden GelMA hydrogels. Data are normalized by gene expression levels measured in P5 hMSCs. Data are shown as mean ± SD, n = 3. *, p < 0.05, **, p < 0.01 and ***, p < 0.001.