ABCB5⁺ mesenchymal stem cells enable high-ratio cellular augmentation of bone grafts without loss of osteogenic function.

Autologous bone grafting remains the standard for reconstruction of bone defects but is constrained by limited graft volume and loss of osteogenic capacity when expanded with acellular materials. We hypothesized that allogeneic ABCB5⁺ mesenchymal stem cells (MSCs) can serve as a biologic cell source to augment bone marrow-derived MSC (BM-MSC) populations without impairing osteogenesis. Primary human BM-MSCs from five donors were combined with GMP-manufactured ABCB5⁺ MSCs at graded replacement ratios (0-95%) and cultured under osteogenic conditions for 21 days. Osteogenic potential was evaluated using complementary assays of mineralization, biochemical activity, and matrix composition. Robust osteogenesis was maintained across all groups, with cultures containing 50% ABCB5⁺ MSCs demonstrating comparable functional osteogenic activity to BM-MSC controls, including similar 99mTc-HDP uptake (2.96 vs. 2.68 MBq) and calcium-phosphate ratios (1.2 vs. 1.1). Higher substitution levels showed a progressive decline in osteogenic output, consistent with dilution of BM-MSC-driven effects. These findings establish a quantitative threshold for biologic augmentation and demonstrate that substantial replacement of BM-MSCs is feasible without loss of function. This approach may enable cell-based expansion of bone grafts and supports further evaluation in translational in vivo models.