One-step decellularization of porcine uterine tissue for developing alginate–decellularized uterine ECM hydrogel for uterine tissue engineering

Decellularized uterine extracellular matrix (dUECM) is promising for uterine tissue engineering because of its inherent bioactivity and structural complexity. However, transforming dUECM into porous, functional 3D constructs remains a significant challenging. This study aimed to (1) synthesize dUECM using a modified decellularization protocol and formulate it into a hydrogel ink and (2) fabricate 3D-printed constructs from this ink to assess their capacity to support human uterine myometrial cell growth in vitro. Porcine uterine tissues were decellularized using 1% Triton™ X-100 with varying concentrations of sodium dodecyl sulfate (SDS) (0.1-1.5%) for 48-72 hours. The resulting dUECM was characterized using DNA and glycosaminoglycan (GAG) quantification, picrosirius red-polarized light microscopy, routine histology, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, and thermogravimetric analysis (TGA). To prepare the ink, dUECM powder was enzymatically digested with pepsin and subsequently blended with 2% and 3% alginate to obtain a printable hydrogel formulation. Constructs were fabricated using extrusion-based 3D printing and assessed for filament fidelity, swelling, degradation behavior, and mechanical properties. Biocompatibility was evaluated using hTERT-HM myometrial cells through MTT metabolic assays, Live/Dead staining, and immunohistochemical (IHC) α-SMA staining. The optimal protocol (1% Triton™ X-100 + 1% SDS for 48 h) reduced DNA to 51.3 ± 9 ng/mg while retaining a high level of GAGs (54.9 ± 7.6 µg/mg). Preservation of the ECM structure was confirmed by spectroscopy analyses. The 3% Alg + 1.5% dUECM hydrogel exhibited suitable printability (1.5 ± 0.2), swelling capacity (47 ± 12%), degradation resistance (94 ± 18% mass retention), and mechanical strength (decreasing from 323 kPa to 175 kPa over 14 days), along with high viability and proliferation (258 ± 13%). The developed dUECM-based hydrogel supports 3D bioprinting with strong mechanical and biological performance, offering a promising platform for uterine tissue engineering.