Spatiotemporal control of autonomous adipogenesis of pre-adipocyte spheroids by bioactive nanofibers and soft hydrogel microenvironments

Despite significant clinical utility, current soft tissue reconstruction modalities employing enriched grafts or liposuction impose considerable limitations including volume reduction and donor-site morbidity. Here, we present a biomimetic approach for engineering 3D adipose tissue through strategic integration of pre-adipocyte (3T3-L1 cells)/nanofiber composite spheroids within mechanically optimized hydrogel matrices. The nanofibers (IM/F@IS) enabling the simultaneous delivery of indomethacin and insulin were prepared such that when incorporated into 3T3-L1 spheroids, they significantly enhanced adipogenic differentiation (an increase in the gene expression of FABP4 and adiponectin by 6.1 ± 0.2 and 11.2 ± 1.4 times, respectively) without exogenous differentiation supplements. Following encapsulation in UV-crosslinked gelatin methacryloyl (GelMA) hydrogels, cells from composite spheroids exhibited robust proliferation, migration, and maturation into functional adipocytes with substantial triglyceride accumulation and homogeneous lipid droplet distribution. Notably, these engineered constructs maintained structural integrity with minimal contraction following subcutaneous implantation in mice. We also confirmed that softer hydrogels significantly enhanced cell sprouting and expression of matrix remodeling proteins, collectively improving adipogenic differentiation of 3T3-L1 cells within the hydrogel. This approach addresses the critical challenge of creating physiologically relevant adipose constructs with predefined dimensions by combining pre-adipocyte spheroids incorporating adipo-inductive fibers and GelMA hydrogels.