Assistant Professor University of Michigan-Dearborn Wixom, Michigan, United States
Introduction: : The extracellular matrix (ECM) of the lung provides essential structural and biochemical support to surrounding cells, influencing tissue development, remodeling, and regeneration. Decellularized human lung ECM is increasingly used to create scaffolds that mimic native environments for in vitro studies and tissue engineering applications. However, the potential impact of biological sex on ECM composition is poorly understood. Understanding how sex influences lung ECM is necessary for improving the clinical relevance of engineered lung tissues. If male and female lungs differ significantly in their biochemical composition, then ignoring these variables could lead to inconsistent results in applications such as hydrogel development, disease modeling, and therapeutic response. This study aims to optimize a decellularization protocol for human lung tissue and characterize sex-based differences in key ECM components. This data will serve as an initial step towards developing sex-specfic or sex-inclusive biomaterials for lung tissue engineering.
Materials and
Methods: : An optimized decellularization protocol for human lung tissue was developed by comparing detergent concentrations. Tissues were treated with either 0.1% or 0.5% sodium dodecyl sulfate (SDS), and the 0.1% SDS condition was selected for final use based on effective DNA removal and improved protein retention. Lung tissues from three male and three female donors were processed using this protocol, which included an initial treatment of 24 hours in 0.1% Triton X-100, followed by 24 hours in 0.1% SDS and a final 24-hour wash in phosphate-buffered saline (PBS). Following decellularization, extracellular matrix (ECM) content was biochemically quantified. Residual double-stranded DNA was measured using the GeneJET Genomic DNA Purification Kit and Nanodrop spectrophotometry. Total protein was assessed via the Pierce™ BCA Protein Assay. Elastin content was quantified using the Fastin™ Elastin Assay, sulfated glycosaminoglycans (sGAGs) with the Blyscan™ sGAG Assay, and insoluble collagen via the Sircol™ Insoluble Collagen Assay. For all assays, sample values were normalized to input wet tissue mass and reported in ng/mg. For each donor, native and decellularized tissues were analyzed in triplicate. Data were grouped by sex (male vs. female) and by treatment (native vs. decellularized) for comparative analysis. Prior to statistical testing, datasets were assessed for normality using the Shapiro-Wilk test and screened for outliers. Welch’s unpaired two-tailed t-tests were used to determine statistical significance (p < 0.05).
Results, Conclusions, and Discussions:: To evaluate sex-based biochemical differences in native and decellularized human lung ECM, there were five key biochemical assays performed: DNA (1A), total protein (1B), elastin (1C), sulfated glycosaminoglycans (sGAGs, 1D), and insoluble collagen (1E). Results revealed significant sex-specific differences in ECM composition prior to and following decellularization using the optimized 0.1% SDS protocol. DNA quantification (1A) confirmed effective decellularization, with all decellularized samples meeting criteria for DNA removal ( < 50 ng/mg). Females retained slightly higher residual DNA than males, though all remained well within acceptable thresholds.
Total protein content (1B) showed statistically significant differences between male native (MN) and female native (FN) samples (*p < 0.05), with males exhibiting higher protein abundance. This differed post-decellularization (Md vs. Fd), indicating that female lung ECM may retain more protein following SDS treatment. Looking into specific proteins, sGAG content (1D) displayed significant differences between MN vs. FN (*p < 0.05), indicating potential sex-related differences in glycoprotein abundance. Additionally, insoluble collagen content was significantly higher in female decellularized ECM (Fd) compared to male decellularized ECM (Md), suggesting that female lungs may retain more structural ECM proteins post-decellularization. This could reflect differences in collagen crosslinking or organization between sexes that make collagen in female tissue more resistant to solubilization and degradation.
These findings highlight that sex is a critical biological variable in ECM composition and decellularization outcomes. Future directions include using LC-MS/MS-based mass spectrometry for comprehensive profiling of ECM proteins. Additionally, decellularized ECM from male and female tissues will be used to form hydrogel scaffolds, which will then be mechanically and biochemically characterized for future studies.
