Development and in vitro Characterization of a Novel Bioactive Hydrogel for Bioprinting Uterine Constructs

Abstract: Background and Aim: Decellularized uterine extracellular matrix (dUECM) offers a promising bioactive scaffold for uterine tissue engineering, but its application in 3D constructs has been limited by fabrication challenges. This study aimed to develop a printable, bioactive hydrogel from dUECM suitable for 3D bioprinting and to evaluate its ability to support human uterine myometrial cell growth in vitro. Materials and Methods: Porcine uterine tissues were decellularized using 1 percent Triton X-100 and 0.1 to 1.5 percent SDS for 48 to 72 hours. The resulting dUECM was assessed via histology, DNA and GAG quantification, scanning electron microscopy, FTIR, Raman spectroscopy, and thermogravimetric analysis. Selected dUECM samples were digested with pepsin and blended with 2 or 3 percent alginate to create bioinks. Constructs were printed using extrusion-based bioprinting and evaluated for swelling, degradation, mechanical properties, and printability. Biocompatibility was tested by seeding hTERT-HM cells onto cast hydrogels and performing MTT and Live/Dead assays over seven days. Results: The optimal protocol (1 percent Triton X-100 and 1 percent SDS for 48 hours) reduced DNA to 51.3 ng per mg, while retaining 54.9 micrograms per mg of GAGs. FTIR and Raman spectroscopy confirmed collagen preservation, while thermal stability was moderately reduced. The 3 percent alginate with 1.5 percent dUECM formulation showed superior printability, swelling stability, degradation resistance, and mechanical strength. Cell proliferation reached 258 percent by day 7, significantly outperforming alginate alone. Conclusion: The optimized dUECM hydrogel supports printability, mechanical performance, and cell viability, offering a robust platform for uterine tissue engineering.