👤 Nima Rahbar

Lyophilization of protein solutions, such as silk fibroin (silk), produces porous scaffolds useful for tissue engineering (TE). The impact of modifying lyophilization primary drying parameters on scaffold properties has not yet been explored previously. In this work, changes to primary drying duration and temperature were investigated using 3%, 6%, 9%, and 12% (w/v) silk solutions, via protocols labeled as Long Hold, Slow Ramp, and Standard. The 9% and 12% scaffolds were not successfully fabricated using the Standard protocol, while the Long Hold and Slow Ramp protocols resulted in scaffolds from all silk solution concentrations. Scaffolds fabricated using the Long Hold protocol had higher Young's moduli, smaller pore Feret diameters, and faster degradation. To investigate the utility of the different lyophilized scaffolds for in vitro cell culturing, the HepaRG liver cell line was cultured in the 3% to 12% scaffolds fabricated using the Long Hold protocol. The HepaRG cells grown in 3% scaffolds initially had greater lipid accumulation and metabolic activity than the other groups, although these differences were no longer apparent by Day 28. The deoxyribonucleic acid content of the HepaRG cells grown in 3% scaffold group was also initially significantly higher than the other groups. Significant differences in gene expression by 9% scaffolded HepaRG cells (CK19, HNFα) were seen on Day 14 while significant differences by 12% scaffolded HepaRG cells (ALB, APOA4) were seen on Day 28. Overall, modifying the primary drying parameters and silk concentration resulted in lyophilized scaffolds with tunable properties useful for TE applications. Show less

The Modern Western Diet has been associated with the rise in metabolic and inflammatory diseases, including obesity, diabetes, and cardiovascular disease. This has been attributed, in part, to the increase in dietary omega-6 polyunsaturated fatty acid (PUFA) consumption, specifically linoleic acid (LA), arachidonic acid (ARA), and their subsequent metabolism to pro-inflammatory metabolites which may be driving human disease. Conversion of dietary LA to ARA is regulated by genetic variants near and within the fatty acid desaturase (FADS) haplotype block, most notably single nucleotide polymorphism rs174537 is strongly associated with FADS1 activity and expression. This variant and others within high linkage disequilibrium may potentially explain the diversity in both diet and inflammatory mediators that drive chronic inflammatory disease in human populations. Mechanistic exploration into this phenomenon using human hepatocytes is limited by current two-dimensional culture models that poorly replicate in vivo functionality. Therefore, we aimed to develop and characterize a three-dimensional hepatic construct for the study of human PUFA metabolism. Primary human hepatocytes cultured in 3D hydrogels were characterized for their capacity to represent basic lipid processing functions, including lipid esterification, de novo lipogenesis, and cholesterol efflux. They were then exposed to control and LA-enriched media and reproducibly displayed allele-specific metabolic activity of FADS1, based on genotype at rs174537. Hepatocytes derived from individuals homozygous with the minor allele at rs174537 (i.e., TT) displayed the slowest metabolic conversion of LA to ARA and significantly reduced FADS1 and FADS2 expression. These results support the feasibility of using 3D human hepatic cultures for the study of human PUFA and lipid metabolism and relevant gene-diet interactions, thereby enabling future nutrition targets in humans. Show less

Genetic variants near and within the fatty acid desaturase (FADS) cluster are associated with polyunsaturated fatty acid (PUFA) biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM) associations (p = 2.69×10-29) between a single nucleotide polymorphism (SNP) rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans) were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86%) exhibited high levels of methylation with the greatest variability observed at three key regulatory regions-the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05) ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs. Show less