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Stem cell spheroids have been widely investigated to accelerate bone tissue regeneration. However, the directed differentiation of stem cells into osteoblastic lineage and the prevention of cells from damage by reactive oxygen species (ROS) remain challenge. Here, we developed osteoinductive and ROS scavenging extracellular matrix-mimicking synthetic fibers based on epigallocatechin gallate (EGCG) coating. They were then utilized to fabricate engineered spheroids with human adipose-derived stem cells (hADSCs) for bone tissue regeneration. The EGCG-mineral fibers (EMF) effectively conferred osteoinductive and ROS scavenging signals on the hADSCs within spheroids, demonstrating relative upregulation of antioxidant genes (SOD-1 (25.8 ± 2.1) and GPX-1 (3.3 ± 0.1) and greater level of expression of osteogenic markers, runt-related transcription factor (5.8 ± 0.1) and osteopontin (5.9 ± 0.1), compared to hADSCs in the spheroids without EMF. The in vitro overexpression of osteogenic genes from hADSCs was achieved from absence of osteogenic supplements. Furthermore, in vivo transplantation of hADSCs spheroids with the EMF significantly promoted calvarial bone regeneration (48.39 ± 9.24%) compared to that from defect only (17.38 ± 6.63%), suggesting that the stem cell spheroid biofabrication system with our novel mineralization method described here is a promising tool for bone tissue regeneration.

Three-dimensional (3D)-printed scaffolds have proved to be effective tools for delivering growth factors and cells in bone-tissue engineering. However, delivering spheroids that enhance cellular function remains challenging because the spheroids tend to suffer from low viability, which limits bone regeneration in vivo. Here, we describe a 3D-printed polycaprolactone micro-chamber that can deliver human adipose-derived stem cell spheroids. An in vitro culture of cells from spheroids in the micro-chamber exhibited greater viability and proliferation compared with cells cultured without the chamber. We coated the surface of the chamber with 500 ng of platelet-derived growth factors (PDGFs), and immobilized 50 ng of bone morphogenetic protein 2 (BMP-2) on fragmented fibers, which were incorporated within the spheroids as a new platform for a dual-growth-factor delivery system. The PDGF detached from the chamber within 8 h and the remains were retained on the surface of chamber while the BMP-2 was entrapped by the spheroid. In vitro osteogenic differentiation of the cells from the spheroids in the micro-chamber with dual growth factors enhanced alkaline phosphatase and collagen type 1A expression by factors of 126.7 ± 19.6 and 89.7 ± 0.3, respectively, compared with expression in a micro-chamber with no growth factors. In vivo transplantation of the chambers with dual growth factors into mouse calvarial defects resulted in a 77.0 ± 15.9% of regenerated bone area, while the chamber without growth factors and a defect-only group achieved 7.6 ± 3.9% and 5.0 ± 1.9% of regenerated bone areas, respectively. These findings indicate that a spheroid-loaded micro-chamber supplied with dual growth factors can serve as an effective protein-delivery platform that increases stem-cell functioning and bone regeneration.