TNF-α driven inflammation in bone tissues using an anatomical- and bio-integrated hydroxyapatite-enriched spongy-like hydrogel 3D model

Inflammatory bone diseases like osteoporosis affect over 200 million people globally, yet the mechanisms by which acute inflammation progresses to chronic remain poorly understood. To address this, we developed an in vitro 3D cortical-sponge bone model of acute inflammation by introducing TNF-α. The model consists of GG-HAp spongy-like hydrogels with osteoblasts from human mesenchymal stem cells (HBM-MSCs) in an outer compartment, and human dermal microvascular endothelial cells plus supporting HBM-MSCs in an inner compartment, mimicking cortical and trabecular bone, respectively. Acute inflammation was induced by TNF-α supplementation (1, 10, or 100 ng/mL) over 7 days, and its impact on vascular assembly, osteogenesis, and inflammation was evaluated via RT-PCR, luminex and ELISA. TNF-α did not affect cell viability and minimally affected angiogenic factors release. However, 100 ng/mL of TNF-α significantly reduced type I collagen, specifically to 53 % at 3 days and to 82 % at 7 days. Pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, MCP-1, and M-CSF) gene expression and protein release were upregulated in a dose-dependent manner. For instance, 100 ng/mL of TNF-α increased IL-6 gene expression by 2.96 ± 2.38-fold at 3 days and 5.69 ± 3.09-fold at 7 days. Interestingly, 100 ng/mL of TNF-α declined IL-1β, IL-6 and TNF-α protein release from 3 to 7 days, indicating a proper resolution of the acute inflammatory response, mimicking the healing phase in bone repair. Overall, this model replicates acute inflammatory events in bone tissue, providing a quantitative platform to study TNF-α-driven bone dynamics and to evaluate targeted interventions, including anti-TNF-α therapies for osteoporosis.