Self-assembled hADSCs/hNSCs spheroids combined with 3D printed M-shaped GelMA/Pu Scaffolds: Creating histologically biomimetic engineered cartilage that meets the characteristics of alar cartilage.

Abstract

Traditional cartilage grafts for nasal alar retraction repair often lack sufficient elasticity and histocompatibility, leading to suboptimal clinical outcomes. To address this challenge, we developed a biomimetic engineered cartilage scaffold that combines self-assembled human adipose-derived stem cell (hADSC)/human nasal septal chondrocyte (hNSC) spheroids with a 3D-printed M-shaped polyurethane (Pu)/gelatin methacryloyl (GelMA) hybrid scaffold, aiming to replicate the extracellular matrix (ECM) components and mechanical properties of native alar cartilage. The M-shaped scaffold was designed based on clinical CT reconstruction and fabricated via 3D printing using Pu for optimal elasticity. hADSCs and hNSCs were co-cultured at varying ratios (0:5-5:0) and loaded as spheroids into GelMA-coated scaffolds. ECM components (Col-1, Col-2, ACAN, Elastin) were analyzed via RT-qPCR and immunohistochemistry. Subcutaneous (nude mice) and in-situ nasal (sheep) implantation models evaluated chondrogenic performance, ECM deposition and structural integration over 1-2 months. Through comparative analysis of extracellular matrix (ECM) components among human alar cartilage, nasal septal cartilage, auricular cartilage, and costal cartilage, we identified the target ECM profile for biomimetic alar cartilage engineering. It was verified that self - assembled spheroids have stronger chondrogenic abilities. Among them, the ECM secretion of 80 % hADSCs/20 % hNSCs spheroids was closest to that of alar cartilage. In vitro and in vivo experiments on the M-shaped spheroids-laden Pu/GelMA hybrid scaffolds showed that the secretion levels of key ECM proteins were similar to those of natural alar cartilage. 3D surface imaging confirmed that the scaffold could provide precise alar - nasal tip contour subcutaneously, and no contracture was observed. MRI confirmed the stability of the scaffold in nude rats and sheep, as well as its seamless integration with host nasal tissues. By prioritizing ECM component regulation through cell ratio optimization and spheroid culture, this study establishes a histologically biomimetic scaffold that faithfully recapitulates the biochemical and structural complexity of alar cartilage. The approach offers a promising alternative to traditional grafts for nasal reconstruction.