Fig. 3.

Biocompatibility and osteoinductive properties of the engineered BME. a) Schematic and representative fluorescence images showing individual culture of MC3T3 (mCherry), MSC (GFP), and C4-2B (BFP) cells in dECM hydrogels. Representative x-y plane projections at days 1, 3, 7, 14, and 21. b) Cell proliferation of MC3T3, MSC, and C4-2B in dECM hydrogels over 21 days (n = 3). c) Schematic and fluorescence images showing MC3T3 attachment to the CPC scaffold and subsequent co-culture with MSCs. d) Proliferation dynamics of MC3T3 and MSCs in the hydrogel-based BME model over 14 days (n = 3), quantified as fluorescence intensity normalized to Day 1.e) ALP staining at day 7. f) ARS at day 14. g) Expression of osteogenic genes at day 7. h) Images of the BME model 7 days after implantation and upon retrieval at day 28. i, j, k) Histological and immunohistochemistry analysis of retrieved tissues: H&E staining (i), Masson trichrome staining (j), and immunostaining of osteogenic markers (k). Statistical analysis was performed using one-way ANOVA (n = 3). Significance is indicated as: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, respectively.