Optimum Magnesium Enhances Osteogenesis of Adipose-Derived Mesenchymal Stem Cells Implanted in Tricalcium...

Autologous iliac crest bone graft is widely used for spinal fusion, yet its application is limited by donor-site morbidity and finite graft volume. In this study, we developed a highly porous scaffold of magnesium (Mg) combined with β-tricalcium phosphate (β-TCP) and gelatin (Mg/gelatin/β-TCP), seeded with adipose-derived mesenchymal stem cells (AdMSCs), and evaluated the effect of Mg content on cell-material responses and bone formation. Scaffolds containing 0, 0.5, 1, and 2% Mg (expressed as w/v, g of Mg per 100 mL of gelatin solution) were fabricated and tested for Mg release and biological performance. In vitro, AdMSC viability, proliferation, and cytotoxicity were assessed using LIVE/DEAD staining, Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, and osteogenic differentiation by alkaline phosphatase (ALP) and osteocalcin immunofluorescence. In vivo, a rat L4-L5 posterolateral fusion model was used with micro-computed tomography (micro-CT) and histology at 2, 4, and 8 weeks. All groups maintained high cell viability with only a small fraction of dead cells; however, at 2% Mg, the surviving cells showed reduced proliferation and elevated LDH release, indicating that a high Mg burden and the accompanying alkaline microenvironment suppress cellular function without causing wholesale cell death. The 1% Mg scaffold produced the strongest early osteogenic signal (peak ALP) while also supporting matrix mineralization and yielded the most favorable micro-CT bone formation, approaching autograft. These results demonstrate that the 1% Mg-modified scaffold maximizes AdMSC osteogenesis without the cytotoxicity seen at 2% Mg, offering a promising alternative for bone tissue engineering and spinal fusion applications.