Sclerostin silencing in human umbilical cord mesenchymal stem cells enhances bone regeneration via Wnt pathway activation

Abstract

Steroid-induced avascular necrosis of the femoral head is a debilitating condition caused by prolonged glucocorticoid exposure, leading to bone death and disrupted metabolism. Current treatment options are limited, especially in late-stage disease, emphasizing the urgent need for novel regenerative therapies. This study by Lv et al explores the therapeutic potential of human umbilical cord mesenchymal stem cells (MSCs) genetically modified to silence sclerostin, a known inhibitor of bone formation via the Wnt/β-catenin pathway. The authors engineered sh-human umbilical cord MSCs and demonstrated their enhanced osteogenic differentiation and suppression of adipogenesis compared to unmodified MSCs in a steroid-induced rat model of femoral head necrosis. Significant improvements were observed in bone microarchitecture, biochemical markers of bone metabolism, and histological parameters. This investigation suggests that sclerostin silencing enhances MSC efficacy by activating Wnt signaling, thereby offering a promising regenerative strategy for steroid-induced avascular necrosis of the femoral head that targets the core imbalance in bone remodeling.

Key Words: Sclerostin; Mesenchymal stem cells; Wnt signaling pathway; Osteogenesis; Avascular necrosis; Glucocorticoid-induced bone disease

Core Tip: Silencing sclerostin in human umbilical cord mesenchymal stem cells enhances their bone-regenerative potential by activating the Wnt/β-catenin signaling pathway, providing a novel therapeutic approach for steroid-induced avascular necrosis of the femoral head. In the study by Lv et al, sh-human umbilical cord mesenchymal stem cells demonstrated better osteogenesis, suppressed adipogenesis, and improved bone architecture in a steroid-induced rat model compared to unmodified cells.

INTRODUCTION

Steroid-induced avascular necrosis of the femoral head (SANFH) is a serious complication from glucocorticoid therapy, where bone death occurs due to impaired blood supply within the hip joint’s ball (femoral head)[1,2]. Corticosteroids, commonly used during organ transplantation and inflammatory disorders management, increase pressure inside the confined space of the femoral head, causing damage to delicate blood vessels[3]. This elevated pressure leads to ischemia, a restriction in blood flow, depriving bone tissue of oxygen and nutrients[4]. The resulting lack of blood supply causes osteonecrosis i.e., death of bone cells and marrow, progressively weakening the bone structure. Clinically, steroid-induced avascular necrosis (AVN) presents with groin pain that worsens with weight-bearing, leading to joint dysfunction and eventual collapse of the femoral head. Late-stage disease often requires hip replacement surgery. Early detection via magnetic resonance imaging is critical to managing AVN before irreversible damage. Mechanistically, steroids cause bone marrow edema, which increases intraosseous pressure, compressing blood vessels and triggering ischemia. Bone cells die due to insufficient oxygen, and over time, the weakened bone collapses, causing joint destruction and osteoarthritis. This condition underscores the importance of cautious steroid use and vigilant monitoring for early signs of AVN to prevent progression and preserve joint function. The pathogenesis of SANFH is multifactorial and incompletely understood, but major contributory mechanisms include increased intraosseous pressure from excessive fat deposition, dysregulated lipid metabolism, direct damage to vascular endothelial cells, reduced collagen synthesis impairing capillary growth, oxidative stress, diminished angiogenesis, increased apoptosis of bone and endothelial cells, and intramedullary hemorrhage[2,5,6]. Glucocorticoids stimulate marrow adipogenesis, causing fat accumulation in bone sinusoids that creates a tamponade effect, compressing the microvasculature and provoking ischemia. This results in vascular congestion, impaired nutrient delivery, and secondary bone and marrow necrosis. Steroids also directly injure the endothelium, exacerbate hypercoagulability, and reduce production of vascular endothelial growth factor[7], thus compromising new vessel formation and further hampering blood flow. Additionally, oxidative stress induced by steroids accelerates osteocyte apoptosis, decreasing bone resilience. The cumulative outcome is reduced perfusion to the femoral head’s critical watershed zones, leading to progressive osteocyte and marrow necrosis, destabilization of trabecular architecture, and eventual collapse of the femoral head. It primarily affects young and middle-aged adults (20-50 years) and is associated with a high disability rate, often leading to secondary arthritis and hip collapse. SANFH imposes a significant burden on patients, their families, and healthcare systems worldwide.

Conventional pharmacotherapies are largely ineffective at reversing advanced SANFH, necessitating hip replacement in many cases. Mesenchymal stem cells (MSCs), especially those derived from human umbilical cord tissue (hUCMSCs), have gained attention for their regenerative and immunomodulatory capabilities[8]. However, modulation of these cells’ bone-forming efficiency remains a challenge. Sclerostin (SOST), a glycoprotein secreted by osteocytes, antagonizes the Wnt/β-catenin signaling pathway, thereby suppressing osteoblast differentiation and bone formation. Targeting SOST through gene silencing could potentiate MSC-mediated bone repair. While hUCMSCs are a promising therapeutic vehicle, their bone-forming efficacy is naturally limited by inhibitory pathways within the bone microenvironment. Therefore, strategies to enhance their osteogenic potential are critically needed. Lv et al[9] addressed this gap by exploring SOST silencing to potentiate hUCMSC-mediated bone regeneration. This editorial discusses Lv et al’s study[9] that investigates SOST-silenced hUCMSCs as a novel cellular therapy to restore bone metabolic balance in SANFH.

RESEARCH RATIONALE

SANFH incidence is rising globally due to widespread glucocorticoid use for autoimmune and inflammatory diseases. MSCs are promising candidates for cell-based therapy due to their multipotency and paracrine effects promoting osteogenesis and angiogenesis. hUCMSCs provide several clinical advantages, including ethical accessibility, low immunogenicity, and abundant supply[8,10,11]. However, MSCs’ efficacy is often limited by inherent regulatory pathways that restrict osteoblast differentiation. The negative regulator SOST inhibits osteoblast proliferation by blocking Wnt signaling, a pathway critical to bone metabolism[12-14]. Thus, SOST appears to have a detrimental role in the development and progression of SANFH by suppressing bone-building processes and blood vessel formation[13]. Therefore, inhibiting SOST presents a potential therapeutic strategy for treating this debilitating condition. Prior models demonstrate increased bone mass and strength in SOST knockout animals[15]. By silencing SOST in hUCMSCs, the authors hypothesized an enhancement in osteogenic potential and suppression of adipogenesis that together could restore bone homeostasis in SANFH.

STRENGTHS OF THE STUDY

Lv et al[9] employed a comprehensive approach combining molecular biology, cell culture, animal modeling, and multiple histological and biochemical assays. The authors employed a multi-target RNA interference approach, wherein several distinct shRNAs targeting different sites on the SOST mRNA were designed and tested to achieve efficient gene silencing. This strategy was validated by sequencing, fluorescence, and protein/mRNA expression analysis in hUCMSCs, ensuring effective genetic modification. The SANFH rat model was robustly established involving lipopolysaccharide and methylprednisolone injections, with histological confirmation of osteonecrosis. Treatment groups allowed direct comparison between unmodified MSCs and SOST-silenced MSCs. The use of micro-computed tomography provided a detailed quantitative analysis of bone microarchitecture. Furthermore, enzyme-linked immunosorbent assay and western blotting measured circulating and local regulatory factors central to bone remodeling, offering mechanistic insights.

The key findings of the study demonstrate that these modified stem cells maintained their typical fibroblast-like morphology and multilineage differentiation potential, as confirmed by flow cytometry and staining assays. Importantly, SOST expression was significantly reduced in sh-hUCMSCs, as validated by both western blot and quantitative real-time reverse-transcription polymerase chain reaction analyses, without impairing the cells’ proliferative capacity, indicating the absence of cytotoxic effects due to gene silencing. The gene-silenced MSCs displayed enhanced osteogenic differentiation, marked by the upregulation of key osteogenic markers including alkaline phosphatase, osteoprotegerin (OPG), and runt-related transcription factor 2, while adipogenic markers such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding proteins were significantly downregulated. This shift in differentiation was consistently observed both in vitro and in vivo. Histological examinations revealed diminished adipocyte infiltration and increased deposition of new bone collagen in femoral heads treated with sh-hUCMSCs, reflecting a favorable remodeling environment.

Moreover, micro-computed tomography analyses uncovered marked improvements in bone microarchitecture in mice treated with sh-hUCMSCs. Specifically, there were significant increases in bone volume, trabecular number, and trabecular thickness, accompanied by reductions in trabecular spacing compared to unmodified MSC treatments. These structural improvements are critical for bone mechanical strength and functional recovery. Finally, systemic and local bone metabolic marker assessments by enzyme-linked immunosorbent assay and western blot further supported the therapeutic efficacy of SOST silencing. Serum levels showed elevated OPG and decreased receptor activator of nuclear factor kappa B (RANK) ligand, RANK, and tartrate-resistant acid phosphatase, signifying a restored balance that favors bone formation over resorption. Correspondingly, bone tissue exhibited increased β-catenin expression, indicating activation of the canonical Wnt/β-catenin signaling pathway, and a significant reduction in SOST protein levels. Collectively, these results highlight that SOST silencing enhances the osteogenic potential and bone regenerative capacity of hUCMSCs by promoting Wnt pathway activation, suppressing adipogenesis, and restoring bone metabolic homeostasis in a steroid-induced femoral head necrosis model.

MECHANISTIC INSIGHTS AND CLINICAL IMPLICATIONS OF THE STUDY

The findings of Lv et al[9] suggest that SOST silencing in hUCMSCs may influence the Wnt/β-catenin signaling axis, as reflected by elevated β-catenin expression in bone tissue samples post-treatment. This study elucidates the mechanism whereby SOST silencing activates the Wnt/β-catenin signaling axis, leading to enhanced osteoblast differentiation and proliferation, and concurrent suppression of osteoclastogenesis via modulation of the OPG/RANK ligand/RANK axis. By repressing adipogenic differentiation, these modified MSCs prevent marrow fat expansion that exacerbates trabecular weakening. Clinically, sh-hUCMSCs could offer a cell-based regenerative therapy addressing SANFH’s underlying pathophysiology rather than merely symptomatic relief or structural replacement. Gene silencing techniques enhance MSCs’ therapeutic efficacy, highlighting the potential of precision genetic modification in stem cell therapy (Figure 1). These observed improvements in osteogenesis and inhibition of adipogenesis imply a potential role of Wnt pathway modulation, but further targeted studies are required to validate this mechanistic link.

Figure 1 Mechanistic overview illustrating the pathogenesis of steroid-induced avascular necrosis of the femoral head and the potential therapeutic mechanism of sclerostin-silenced human umbilical cord mesenchymal stem cells. The left panel depicts the pathogenesis caused by prolonged steroid exposure whereas the right panel illustrates that steroids decrease the activity of key antioxidant enzymes, leading to excessive oxidative stress and upregulation of osteoclast activity markers. These combined effects disrupt bone remodeling and weaken trabecular architecture, resulting in osteonecrosis of the femoral head. Administration of sclerostin-silenced human umbilical cord mesenchymal stem cells counteracts these effects. hUCMSCs: Human umbilical cord mesenchymal stem cells; γ-GCSc: Gamma-glutamylcysteine synthase; SOD1: Superoxide dismutase 1; HO-1: Heme oxygenase-1; RANKL: Receptor activator of nuclear factor kappa B ligand; OPG: Osteoprotegerin; CTSK: Cathepsin K; TRAP: Tartrate-resistant acid phosphatase.

LIMITATIONS AND FUTURE DIRECTIONS

While the findings of the present study are highly promising, several important limitations must be addressed to fully validate the therapeutic potential and safety of SOST-silenced MSCs in bone regeneration. Long-term safety, off-target effects, and immunogenic potential of gene-modified MSCs require thorough evaluation in larger preclinical models. It is crucial to assess possible off-target genetic modifications, as well as the immunogenicity of gene-edited MSCs, which may not manifest until extended post-transplant surveillance in larger and more diverse animal models. Another important limitation of the current study, as also acknowledged by Lv et al[9], is the lack of direct mechanistic evidence confirming the activation of Wnt/β-catenin signaling following SOST silencing in hUCMSCs. Future investigations should employ targeted molecular analyses such as reporter assays, phosphorylation studies of β-catenin, and Wnt target gene profiling in isolated SOST-silenced hUCMSCs to substantiate this proposed pathway interaction. Further, the interaction between SOST inhibition and chondrogenic or cartilage remodeling pathways remains poorly understood; exploring this molecular interplay could help optimize regenerative outcomes for osteochondral lesions and extend the clinical applicability of this approach. Combinatorial strategies such as synergizing sh-hUCMSCs with local delivery of pro-angiogenic factors or biomaterial scaffolds may enhance cellular engraftment and bone healing, and these should be rigorously explored in preclinical platforms. Before clinical translation, it will be imperative to determine optimal cell dosing, refine administration routes, and establish patient selection benchmarks to maximize therapeutic success while minimizing risk. An additional translational challenge lies in the large-scale manufacturing and regulatory approval of clinical-grade genetically modified stem cells, which remains a significant hurdle between preclinical success and clinical application. Finally, since aberrant SOST expression and Wnt signaling are implicated in a spectrum of skeletal pathologies beyond osteonecrosis, investigating SOST silencing for conditions like osteoporosis, osteoarthritis, and non-union fractures could greatly broaden the future impact of this cellular engineering strategy.

CONCLUSION

Lv et al’s study[9] presents compelling evidence that silencing SOST in hUCMSCs robustly improves bone regeneration and metabolic balance in a steroid-induced femoral head necrosis model. This strategy leverages the intrinsic osteogenic potential of MSCs while overcoming inhibitory pathways through RNA interference, thereby activating Wnt/β-catenin signaling to drive bone repair. Such advancements epitomize the shift towards genetically enhanced stem cell therapies in orthopedics, which could revolutionize treatment paradigms for SANFH and related skeletal disorders. Further research and clinical translation hold promise for meaningful improvements in patients afflicted by this challenging condition.