Sclerostin-silenced human umbilical cord mesenchymal stem cells ameliorate bone metabolism in steroid-induced femoral head necrosis

Abstract

BACKGROUND

Steroid-induced avascular necrosis of the femoral head (SANFH) involves bone metabolism imbalance and lacks effective therapies. Mesenchymal stem cells (MSCs), particularly human umbilical cord MSCs (hUCMSCs), offer promise due to their osteogenic and immunomodulatory potential. Sclerostin (SOST) inhibits bone formation, so we developed a multi-target gene silencing strategy against SOST using RNA interference. We created hUCMSCs with SOST-silenced (sh-hUCMSCs) and compared their therapeutic efficacy with unmodified hUCMSCs in SANFH mice. This study explores a novel approach to enhance osteogenesis and mitigate SANFH progression.

AIM

To assess the effects of sh-hUCMSCs on bone metabolism in SANFH.

METHODS

hUCMSCs were isolated from placental tissue and transfected with SOST-targeting short hairpin RNA plasmids. A SANFH mouse model was established through intraperitoneal injection of lipopolysaccharide (20 μg/kg) followed by intramuscular methylprednisolone administration (40 mg/kg). Mice were randomized into four experimental groups (n = 10/group): Sham control, SANFH (untreated), hUCMSCs-treated, and sh-hUCMSCs-treated. Micro-computed tomography was used to measure bone volume (BV), bone surface area, bone surface/BV ratio, trabecular number, trabecular thickness, and trabecular separation. Quantification of adipocyte area by hematoxylin and eosin staining. Collagen fiber volume was assessed by Masson’s trichrome staining. Serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL), tartrate-resistant acid phosphatase, and the OPG/RANKL ratio were measured by enzyme-linked immunosorbent assay. The expression levels of alkaline phosphatase, OPG, SOST, β-catenin, peroxisome proliferator-activated receptor gamma, and CCAAT/enhancer-binding protein in bone tissue were determined by western blot analysis.

RESULTS

hUCMSCs and sh-hUCMSCs exhibited typical fibroblast-like morphology and high expression of MSC surface markers (CD90, CD73, CD105 > 98%). These cells demonstrated tri-lineage differentiation potential, confirmed by positive Alizarin Red S, Oil Red O, and Alcian Blue staining, and upregulation of lineage-specific genes. After SOST-RNA interference modification, sh-hUCMSCs showed enhanced inhibition of adipogenesis and improved bone formation in a rat model of SANFH. Histological analysis revealed reduced lipid infiltration and empty lacunae in the femoral head of the sh-hUCMSC group. Western blot showed decreased CCAAT/enhancer-binding protein and peroxisome proliferator-activated receptor gamma expression (P < 0.05). Masson staining and micro-computed tomography analysis confirmed significantly increased BV, trabecular number, trabecular thickness, and reduced trabecular separation in the sh-hUCMSC group compared to unmodified MSCs and SANFH groups (P < 0.05). Serum enzyme-linked immunosorbent assay showed higher OPG and lower RANK, RANKL, and tartrate-resistant acid phosphatase levels in the sh-hUCMSCs group. Western blot further confirmed upregulated alkaline phosphatase, OPG, β-catenin, and downregulated SOST expression in sh-hUCMSCs compared to controls (P < 0.05). These results suggest that SOST inhibition enhances the osteogenic potential and therapeutic efficacy of hUCMSCs in SANFH.

CONCLUSION

sh-hUCMSCs alleviate SANFH by activating the Wnt/β-catenin signaling pathway, thereby promoting osteogenic differentiation and suppressing adipogenesis to restore bone metabolic balance.

Keywords: Genetic modification; Human umbilical cord mesenchymal stem cells; Steroid-induced avascular necrosis of the femoral head; Sclerostin; Bone metabolism; Wnt signaling pathway; RNA interference

Core Tip: In this study, we developed a multi-target gene silencing strategy against sclerostin (SOST), a negative regulator of bone formation, using RNA interference technology. We established a cellular model of human umbilical cord mesenchymal stem cells (hUCMSCs) with SOST-silenced (sh-hUCMSCs) with SOST gene silencing and compared the therapeutic effects of hUCMSCs and sh-hUCMSCs in a mouse model of steroid-induced avascular necrosis of the femoral head. Compared with hUCMSCs, sh-hUCMSCs more effectively inhibited adipogenic differentiation, promoted new bone formation, and improved trabecular bone parameters such as bone volume, trabecular number, and thickness, while reducing trabecular separation. Additionally, sh-hUCMSCs modulated the expression of key bone metabolic factors, including increased levels of osteoprotegerin and decreased levels of receptor activator of nuclear factor kappa B ligand and peroxisome proliferator-activated receptor gamma. These results suggest that silencing SOST enhances the osteogenic capacity and regenerative efficacy of hUCMSCs.