Morphological Features of Bone Tissue Restoration with Local Use of Cdc42-Targeted Mesenchymal Stem Cells

The aim was to evaluate morphological effect of Cdc42 inhibition in mesenchymal stem cells (MSCs) on the restoration process bone defects during the late post-traumatic period of 24-months male rats with ulnar fractures. Material and methods. The experiment involved adult Wistar rats (males, weighing 400–500 grams, aged 24 months; n=40) undergoing ulnar diaphysis osteotomy. After the injury, the animals were randomized into four groups (10 rats per group). Group I animals served as controls and received no treatment. Group II animals were administered aged MSCs (cells isolated from 24-month-old rats). Group III received aged MSCs modified with the small molecule CASIN, while Group IV received aged MSCs transfected with miRNA (Cdc42 knockdown). Observations were completed 4 and 6 months post-injury. The cell dose was identical across all experimental groups – 1×106 cells in 200 µL of sodium phosphate buffer. The cell transplantation procedure was performed once, 24 hours after the fracture. Paraffin sections from the fracture site were stained using hematoxylin & eosin and the Van Gieson method. Morphometric analysis was conducted using ImageJ 1.53 with the StarDist plugin, and statistical hypothesis testing was performed using non-parametric methods in Statistica 8.0 software. Results. In all animals, foci with varying degrees of maturation of the cellular and extracellular matrix of the hard callus formed at the fracture site. A comparison of ulnar defect healing in the control group (which did not receive additional osteogenesis sources) and in the three groups with varying degrees of Cdc42 activity in transplanted MSCs revealed statistically significant differences in the number of cells within key regenerating pools. At 4 and 6 months post-injury, the most pronounced restoration of chondroblast and osteoblast precursors, as well as chondrocytes and osteocytes, occurred when using MSCs transfected with miRNA. This was likely due to maximal suppression of Cdc42 activity in MSCs and partial inhibition of their aging in the regeneration zones of the ulnar bone, facilitating further osteogenesis stimulation. Conclusion. Targeted inhibition and suppression of Cdc42 activity before the transplantation of MSCs derived from adipose tissue significantly improve the formation of immature callus and its transformation into mature tubular bone in aged animals. These results confirm the potential and feasibility of using Cdc42 targeting as combination therapy for fractures in elderly individuals.

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