Y34 - Development of Decellularized Lung Scaffolds With Sex as a Biological Variable

Introduction: : The lung is a structurally and biochemically complex organ, and replicating its microenvironment in vitro remains a major challenge in tissue engineering. This is particularly important in the study of lung mechanobiology, where extracellular matrix (ECM) composition and mechanics guide cell behavior and responses. While current scaffold models often utilize single-protein constructs, these lack the intricate biomechanical cues of native tissue. Decellularization offers a promising solution by preserving ECM composition while removing immunogenic cellular elements, enabling the fabrication of physiologically relevant scaffolds.
Sex-based biological differences in ECM structure and disease outcomes have gained attention, yet remain underexplored in the lung. Studies in other tissues have identified significant sex-related differences in ECM proteins and remodeling, impacting cellular function and therapeutic response. However, the lung ECM has not been characterized in this context. Our study addresses this gap by investigating whether ECM composition in decellularized lung tissue varies by sex in porcine models. We hypothesize that sex-specific differences in ECM protein content may influence cellular responses, making it critical to incorporate sex as a biological variable in lung scaffold design.

Materials and

Methods: : Lungs from healthy male and female pigs were harvested and decellularized using a detergent-enzymatic protocol optimized to minimize residual DNA content while preserving ECM integrity. Decellularization efficiency was confirmed via DAPI staining, DNA quantification, and gel electrophoresis. Biochemical characterization of the decellularized ECM (dECM) included quantification of collagen, elastin, and sulfated glycosaminoglycans (sGAG) using standard colorimetric assays. The ultrastructure of the scaffolds was visualized using scanning electron microscopy (SEM), while stiffness was assessed using atomic force microscopy (AFM). Student t-tests were used for statistical comparison between male and female derived dECM.
To evaluate functional biocompatibility, a preliminary cytotoxicity study was performed by seeding fibroblasts onto the dECM, and cell viability was assessed using live-dead staining. Additionally, we examined whether sex based differences in ECM composition are consistent across multiple decellularization protocols. These findings will be extended to tissues derived from other animal species in future work. This multi-modal approach enabled us to assess both compositional and mechanical sex-based differences and to evaluate their reproducibility and biological impact on cell behavior.

Results, Conclusions, and Discussions:: Our decellularization protocol effectively removed >95% of cellular material in both male and female lungs, with residual DNA levels under 50 ng/mg wet weight, confirming established decellularization standards. Biochemical assays revealed no significant sex-based differences in total protein and collagen content. However, male-derived dECM showed significantly higher levels of elastin and sGAG, indicating subtle but functionally relevant compositional differences. Mechanical characterization revealed a tenfold increase in stiffness between native and decellularized tissues. Notably, while native male lung tissue exhibited higher baseline stiffness compared to female tissue, this sex-based difference was eliminated following decellularization, suggesting the mechanical profile of dECM is more homogenized than that of native tissue. Cytotoxicity testing using fibroblasts demonstrated excellent biocompatibility of the dECM scaffolds, with no observable impact on cell viability.
These findings provide the first experimental evidence of sex-specific differences in lung ECM composition and highlight the importance of including sex as a biological variable in scaffold-based lung models. Differences in ECM proteins like elastin and sGAGs could influence key cellular processes such as contraction, migration, and matrix remodeling - critical factors in both normal lung physiology and pathological conditions like fibrosis. Moreover, we assessed whether these sex-related trends persist across alternative decellularization methods, and preliminary results suggest that certain protein differences are conserved, while others are method-dependent. These insights have direct implications for standardizing scaffold preparation methods in both research and clinical applications.
Our study lays essential groundwork for more physiologically relevant and inclusive models of lung tissue. Future directions include evaluating how these sex-specific ECM cues influence cell signaling pathways, inflammatory responses, and regenerative outcomes, particularly in co-culture or dynamic bioreactor systems. Incorporating sex-based ECM variability will ultimately enhance the predictive accuracy of in vitro lung models and contribute to the design of sex-informed therapeutic strategies.

Acknowledgements and/or References (Optional)::