Evaluation of mesenchymal stem cells’ bone repair capacity via osteogenic lineage differentiation in experimental nonunion fractures

Abstract Background Nonunion of fractures is a major challenge in orthopedics and traumatology, especially with increasing high-energy injuries. Adipose-derived mesenchymal stem cells (ASCs) are a readily accessible source with strong osteogenic potential. This study compared the bone regenerative efficacy of undifferentiated ASCs versus their osteogenically pre-differentiated derivatives in a critical-size femoral nonunion model. Methods Eighteen male New Zealand White rabbits ( n = 18), aged 3 months and weighing 2.94 ± 0.10 kg, were included. MSCs were isolated enzymatically from abdominal adipose tissue of three donor rabbits. At passage 3, cells were cultured either in basal medium (undifferentiated cohort) or osteogenic medium containing β-glycerophosphate, dexamethasone, and ascorbic acid for 4 weeks (differentiated cohort). 5-bromo-2′-deoxyuridine solution (BrdU) labeling was performed. In this randomized controlled in vivo study, a 5-mm mid-femoral defect was created and stabilized with angular-stable plating. In this randomized, controlled in vivo experimental study, on postoperative day 7 the animals were allocated to three groups ( n = 5 per group): control (defect alone), undifferentiated mesenchymal stem cells, and differentiated mesenchymal stem cells. Radiographic assessment of bridging callus and cortical thickness index was performed at 4 and 6 weeks; serum total protein, calcium, and alkaline phosphatase were measured. Results In vitro , osteogenic differentiation induced progressive mineralization (Alizarin Red staining) and a statistically significant 34% reduction in BrdU corrected total cell fluorescence (CTCF) ( p = 0.0473). In vivo, both cell-augmented groups accelerated fracture consolidation versus controls. The differentiated cohort achieved 88% bridging callus at 6 weeks (versus 80% in the undifferentiated group), with superior improvements in bridged cortices (+ 9.4%), bridging callus coverage (+ 10.0%), and distal gap closure (–31.0%) at 6 weeks, plus more pronounced cortical remodeling (average CTI decrease − 6.2% at 4 weeks). Serum alkaline phosphatase (ALP) declined by 53.4% (undifferentiated) and 63.6% (differentiated) versus control (both p < 0.001); plasma calcium was 16.8% higher in the undifferentiated group. Immunofluorescence showed 19% higher BrdU-positive cell density and 12% higher CTCF in undifferentiated cells. Conclusions Osteogenically pre-differentiated ASCs demonstrated enhanced bone regenerative capacity compared with undifferentiated ASCs. These findings indicate that osteogenic pre-differentiation augments the therapeutic potential of ASCs and support their further evaluation in multimodal strategies for fracture nonunion.