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 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 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 h. The resulting dUECM was characterized using DNA and glycosaminoglycan (GAG) quantification, Picrosirius Red-polarized light microscopy, histology, scanning electron microscopy, FTIR, Raman spectroscopy, and thermogravimetric analysis. To prepare the ink, dUECM powder was enzymatically digested with pepsin and blended with 2% and 3% alginate. Constructs were fabricated using extrusion-based 3D printing and assessed for filament fidelity, swelling, degradation, and mechanical properties. Biocompatibility was evaluated using hTERT-HM myometrial cells through MTT assays, Live/Dead staining, and alpha-SMA immunohistochemistry. The optimal protocol (1% Triton X-100 + 1% SDS for 48 h) reduced DNA to 51.3 +/- 9 ng/mg while retaining high GAGs (54.9 +/- 7.6 ug/mg). Preservation of the ECM structure was confirmed by spectroscopy. The 3% Alg + 1.5% dUECM hydrogel exhibited suitable printability (1.5 +/- 0.2), swelling (47 +/- 12%), degradation resistance (94 +/- 18% mass retention), and mechanical strength (323 to 175 kPa over 14 days), 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.