Pre-Conditioned Human Adipose-Derived Mesenchymal Stem Cells With Hypoxia-Mimetic Cobalt Chloride Improve Bone Healing in a Critical-Size Tibial Defect in an Ovariectomized Rat Model of Osteoporosis Publisher



Gorji Valokola M ; Ahmadi F ; Bayat R ; Haeri S M J ; Fallahnezhad S ; Sahebkar A
Source: Regenerative Engineering and Translational Medicine Published:2026

Abstract

Abstract: Hypoxia-mimetic cobalt chloride (CoCl2) induces favorable effects on human adipose-derived mesenchymal stem cells (hAD-MSCs) function. This study was aimed at the bone-healing effects of CoCl2-preconditioned hAD-MSCs on a critical-size tibial defect (CSTD) in an ovariectomized rat-induced osteoporosis (OP) model. Hypoxia was induced by 100, 200, and 400 µM CoCl2 for 24 and/or 48 h. The cell viability and apoptosis were evaluated using the MTT assay and acridine orange staining, respectively. Adult female Wistar rats were ovariectomized. After 3.5 months, the OP progression was evaluated using CT scanning. Twenty-four rats were then divided into 3 groups: sham (control), normoxia, and hypoxia groups receiving PBS, hAD-MSCs, and CoCl2-exposed hAD-MSCs, respectively, via the tail vein 7 days after CSTD. At four and eight weeks after CSTD surgery, western blot analyses were performed to evaluate OPG, OCN, and Runx2 proteins in local fractures. hAD-MSCs exposure to 100 µM CoCl2 for 48 h had the best therapeutic effect, which increased cell viability, decreased apoptosis, and improved the ossification at the site of a tibia fracture. The Hounsfield Unit (HU) of CSTD in the hypoxia group was significantly higher than in other groups. Seven days after CSTD induction, the injection of 100 µM CoCl2-pretreated hAD-MSCs for 48 h significantly upregulated the expression of the mentioned proteins in the hypoxia group, except for OCN protein. Hypoxia induced by CoCl2 significantly improved the cell viability of hAD-MSCs and the remodeling and healing processes of CSTD in ovariectomized rats by increasing OPG, OCN, and Runx2 proteins. A Lay Summary: This study investigates a new technique to improve bone healing in osteoporosis, a condition where bones become weak and proneto fractures. Researchers concentrated on special cells called human adipose-derived mesenchymal stem cells (hAD-MSCs), which can help bone healing. They exposed these cells to cobalt chloride (CoCl2), a chemical that mimics low oxygen conditions (hypoxia), to improve their healing capacity. When these preconditioned cells were injected into rats with ovariectomy-inducedosteoporosis and critical-size tibial bone defects, the treated cells significantly improved bone healing compared to untreated cells. This approach significantly resulted in increased bone formation and strength by activating important and key proteins involved inbone growth and repair. The findings highlight that using CoCl2-preconditioned hAD-MSCs could be a promising and effective therapy to accelerate bone repair in osteoporotic patients, potentially providing hope for better treatments and outcomes with fewerside effects than existing medications; however, further studies are needed in this field. © The Author(s), under exclusive licence to The Regenerative Engineering Society 2026.