Non-alcoholic fatty liver disease (NAFLD) is reaching pandemic proportions due to overnutrition. The understanding of advanced stages that recapitulate the human pathology is of great importance to get a better mechanistic insight. We hypothesized that feeding of WT (C57BL) mice with a diet containing a high content of fat (21%), sugar (41.5%) and 1.25% of cholesterol (called from now on high fat, sucrose and cholesterol diet, HFSCD) will reproduce the characteristics of disease severity. Analysis of 16 weeks HFSCD-fed mice demonstrated increased liver weight and plasmatic liver damage markers compared with control diet (CD)-fed mice. HFSCD-fed mice developed greater hepatic triglyceride, cholesterol and NEFA content, inflammation and NAFLD activity score (NAS) indicating an advanced disease. HFSCD-fed mice displayed augmented hepatic total CD3+ T and Th9 lymphocytes, as well as reduced Th2 lymphocytes and CD206 anti-inflammatory macrophages. Moreover, T cells and anti-inflammatory macrophages correlated positively and inversely, respectively, with intrahepatic cholesterol content. Consistently, circulating cytotoxic CD8+ T lymphocytes, Th1, and B cell levels were elevated in HFSCD-fed WT mice. Hepatic and adipose tissue expression analysis demonstrated changes in fibrotic and metabolic genes related with cholesterol, triglycerides, and fatty acid synthesis in HFSCD-fed WT. These mice also exhibited reduced antioxidant capacity and autophagy and elevated ERK signaling pathway activation and CHOP levels. Our results indicate that the feeding with a cholesterol-enriched diet in WT mice produces an advanced NAFLD stage with fibrosis, characterized by deficient autophagy and ER stress along with inflammasome activation partially via ERK pathway activation.

This study evaluated the biological performance in vitro of two 3D-printed hydroxyapatite (HA) and polylactic acid (PLA) composite scaffolds with two different infill densities (50% [HA-PLA50] and 70% [HA-PLA70]). Comparative analysis using MG-63 cell cultures evaluated the following: (1) integrity after exposure to various sterilization methods; (2) cell viability; (3) morphological characteristics; (4) cell proliferation; (5) cytotoxicity; (6) gene expression; and (7) protein synthesis. Ultraviolet radiation was the preferred sterilization method. Both scaffolds maintained adequate cell viability and proliferation over 7 days without significant differences in cytotoxicity. Notably, HA-PLA50 scaffolds demonstrated superior osteogenic potential, showing a significantly higher expression of collagen type I (COL1A1) and an increased synthesis of interleukins 6 and 8 (IL-6, IL-8) compared to HA-PLA70 scaffolds. While both scaffold types supported robust cell growth, the HA-PLA50 formulation exhibited enhanced bioactivity, suggesting a potential advantage for bone tissue engineering applications. These findings provide important insights for optimizing 3D-printed bone graft substitutes.

This study characterized the biological response of MG-63 cells to synthetic, hydroxyapatite scaffolds (HAsint) fabricated via fused filament fabrication. Scaffolds were compared to 2D plate-adherent cultures using six assays: cell morphology and distribution with scanning electron microscopy and confocal laser scanning microscopy; cell proliferation and cytotoxicity via WST-1 tetrazolium assay; relative osteogenic gene expression through reverse-transcription–quantitative polymerase chain reaction, and protein synthesis via multiplex immunoassay. Data were analyzed using one-way ANOVA. Results confirmed high cell viability and uniform distribution on HAsint scaffolds. Proliferation increased significantly over 7 days, though direct cytotoxicity also increased, likely due to the static conditions of the experiment and, subsequently, the high ion reprecipitation from scaffold degradation. Importantly, HAsint scaffolds significantly enhanced osteogenic marker expression of phosphatase alkaline (ALPL), osteopontin (OPN), and osteocalcin (OCN) genes, and elevated concentrations of interleukins (IL)-6, IL-8 and matrix metalloproteinase 1 compared to plate-adherent controls. It can be concluded that 3D-printed HAsint scaffolds support robust osteogenic differentiation and proliferation despite inducing a transient cytotoxic response in vitro. The marked upregulation of key osteogenic genes and proteins confirms the scaffolds’ bioactivity and highlights their potential for bone tissue engineering applications.