Histopathological Analysis of Adipose-Derived Mesenchymal Stem Cells and Their Osteogenic Derivatives in Promoting Bone Regeneration in Model of Fracture Non-Union

Abstract

Objective. To perform a histopathological analysis of the effects of mesenchymal stem cells (MSCs) and their osteogenically differentiated derivatives (Dif-MSCs) on reparative processes in bone tissue using an experimental model of fracture nonunion. Subjects and Methods. Fifteen male rabbits aged 3 months were randomly assigned to three groups: control with induced femoral nonunion (n=5), nonunion treated with adipose-derived MSCs (n=5), and nonunion treated with Dif-MSCs (n=5). MSCs were isolated from lipoaspirate, cultured to passage 3, and differentiated osteogenically for 4 weeks. A 5-mm bone defect was created surgically, and cells were injected into the defect zone on day 7. Animals were euthanized at 6 weeks post-injection. Bone specimens underwent fixation, decalcification, histological staining (hematoxylin-eosin and Van Gieson), and quantitative analysis of osteoblasts and osteocytes using ImageJ. Statistical analysis included one-way ANOVA and Welch's t-test. Results. Macroscopic and histological evaluation revealed a gradation in regeneration: control group showed predominant fibrous tissue with immature bone and inflammation; MSCs group exhibited fibrocartilaginous callus with immature bone, bone marrow formation, and increased osteoblasts (52.0 ± 16.2 per 10 HPF vs. control 28.6 ± 7.0, p = 0.032); Dif-MSCs group demonstrated mature lamellar bone, organized osteons, mature bone marrow, and higher osteocytes (vs. MSCs, p = 0.006). ANOVA indicated trends in osteoblast increases (p = 0.092) and significant osteocyte variability (p = 0.014). Conclusion. Undifferentiated MSCs enhance early osteogenesis via endochondral ossification, while Dif-MSCs promote advanced maturation through mixed intramembranous and endochondral pathways, suggesting superior efficacy for treating fracture nonunion.