RNA interference-mediated osteoprotegerin silencing increases the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio and promotes osteoclastogenesis

Abstract

BACKGROUND

In vivo degradation of bone scaffolds is significantly influenced by osteoclast (OC) activity, which is orchestrated by the interplay between receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). The ratio of RANKL/OPG is a crucial determinant of OC-mediated bone resorption, which plays an integral role in bone remodeling and scaffold degradation. Elevated levels of RANKL relative to OPG enhance osteoclastogenesis, thereby accelerating the degradation process essential for integrating bone scaffolds into the host tissue.

AIM

To elucidate the effects of OPG gene silencing on osteoclastogenesis within rat bone marrow-derived mesenchymal stem cells (BMSCs). By investigating these effects, the study aimed to provide deeper insights into the regulatory mechanisms that influence bone scaffold degradation, potentially leading to improved bone repair and regeneration strategies.

METHODS

We employed recombinant lentiviral plasmids to silence the OPG gene in rat BMSCs to achieve the aims. The efficacy of gene silencing was assessed using quantitative reverse transcription polymerase chain reaction and western blot analysis to measure the expression levels of OPG and RANKL. Tartrate-resistant acid phosphatase staining was utilized to evaluate the formation of OCs. Additionally, co-immunoprecipitation assays were conducted to explore the interactions between RANKL and OPG proteins, further assessing the biochemical pathways involved in osteoclastogenesis.

RESULTS

The silencing of the OPG gene in BMSCs resulted in a significant increase in the RANKL/OPG ratio, evidenced by decreased expression levels of OPG and increased levels of RANKL. Enhanced osteoclastogenesis was observed through tartrate-resistant acid phosphatase staining, which indicated a substantial rise in OC formation in response to the altered RANKL/OPG balance. The co-immunoprecipitation assays provided concrete evidence of the direct interaction between RANKL and OPG proteins, substantiating their pivotal roles in regulating OC activity.

CONCLUSION

The findings from this study underscore the critical role of the RANKL/OPG axis in osteoclastogenesis. Silencing of the OPG gene in BMSCs effectively increases the RANKL/OPG ratio, promoting OC activity and potentially enhancing bone scaffold degradation. This regulatory mechanism offers a promising avenue for modulating bone remodeling processes, which is essential for effective bone repair and the successful integration of bone scaffolds into damaged sites. Future research might focus on optimizing the control of this axis to better facilitate bone tissue engineering and regenerative therapies.

Key Words: Osteoprotegerin; Receptor activator of nuclear factor-kappa B ligand; Bone marrow-derived mesenchymal stem cells; RNA interference; Osteoclast; Bone scaffold

Core Tip: This study reports, for the first time, that enhancing the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) ratio through RNA interference promotes osteoclastogenesis. Our findings reveal a significant upregulation of RANKL mRNA levels after OPG gene silencing. The study demonstrates a significant downregulation of OPG mRNA and protein levels. The increase in the RANKL/OPG ratio significantly promotes osteoclastogenesis. This study provides a new theoretical basis and molecular targets for degrading bone scaffolds and bone tissue repair.

INTRODUCTION

Bone scaffolds play a crucial role in clinical bone tissue repair and are widely used in bone regeneration and repair. Bone scaffolds aid bone tissue regeneration by providing physical support and promoting new bone formation. With advancements in materials science and biotechnology in recent years, novel bone scaffold materials are being developed. However, the degradation properties of bone scaffolds are essential for their proper function, as either too fast or too slow degradation can impact the effectiveness of bone repair[1].

Currently, clinical treatment of fractures primarily relies on the implantation and fixation of exogenous bone scaffold materials. However, this surgical approach requires an additional operation to remove the fixation materials. If porous structures can be developed, allowing bone tissue or stem cells to grow within, bioactive bone scaffolds could degrade after being implanted into the body. At this stage, osteoclasts (OCs) activate and begin regulatory functions, prompting bone growth. Over time, the body would completely absorb the bioactive scaffold, and the bone would largely heal. Such bone materials could save patients half the time and costs, enabling faster recovery without the risk of a second surgery.

OCs play a central role in the degradation of bone scaffold materials, primarily regulating degradation rates through the bone resorption[2]. Studies have demonstrated that the receptor activator of nuclear factor-kappa B ligand (RANKL)/receptor activator of nuclear factor-kappa B (RANK)/osteoprotegerin (OPG) system is crucial for bone remodeling and OC function regulation[3]. OCs, as multinucleated giant cells, are key mediators of bone resorption and remodeling[4]. Their formation and activity are regulated by RANKL and OPG[5,6].

Existing research has established the decisive role of the RANKL/OPG balance in OC differentiation and bone remodeling, forming the theoretical foundation of this signaling axis[7]. RANKL binds to the RANK receptor on OC precursor cells, promoting OC differentiation and maturation[5]. Conversely, OPG acts as a decoy receptor for RANKL, competitively binding and preventing its interaction with RANK, thereby inhibiting osteoclastogenesis and activity[8]. Thus, the RANKL/OPG ratio is a key factor in OC activity regulation[9,10]. Further studies indicate that this signaling axis regulates bone homeostasis and plays a critical role in osteoimmunology[11]. An increased RANKL/OPG ratio is closely associated with enhanced OC activity, suggesting that modulating this ratio could effectively regulate OC function[3,7]. Additionally, under RANKL stimulation, OCs can undergo cyclic renewal through osteomorphs, providing new insights into RANKL-mediated bone resorption mechanisms[12]. Specific peptide interventions have been used to modulate RANKL-mediated OC activation, further verifying the promoting effect of OPG inhibition on osteoclastogenesis[13].

Advancements in osteoimmunology are driving the development of interventions targeting abnormal osteoclastogenesis[14]. For instance, anti-RANKL antibodies have been clinically validated as an effective treatment for postmenopausal osteoporosis[11,15,16]. Meanwhile, targeted OC drug delivery systems are also considered promising for clinical applications[13].

OCs play a central role in the degradation process of bone scaffolds, regulating the degradation rate of the bone matrix through bone resorption[2]. Therefore, studying the enhancement of OC-mediated bone resorption activity regulation is of great significance for optimizing the performance of bone scaffold materials. OC are multinucleated giant cells responsible for bone resorption and remodeling[4]. Their generation and activity are regulated by RANKL and OPG[5,6]. RANKL binds to the RANK receptor on the surface of OC precursor cells, promoting OC differentiation and maturation[5]. As a decoy receptor for RANKL, OPG binds to RANKL, preventing its binding to the RANK receptor, thereby inhibiting OC generation and activity[8]. Hence, the RANKL/OPG ratio is a key factor in regulating OC activity[9,10]. Studies have found that an increase in the RANKL/OPG ratio is closely related to the gradual enhancement of OC activity, suggesting that regulating the RANKL/OPG ratio may be an effective approach to control OC activity.

RNA interference (RNAi) technology is a technique that silences target gene expression specifically using small interfering RNA or short hairpin RNA (shRNA) and is widely applied in gene function and disease mechanism studies[17,18]. In recent years, RNAi technology has shown great potential in regulating gene expression, exploring gene function, and developing new therapeutic approaches. In this study, we utilized recombinant lentiviral plasmids to introduce shRNA into rat bone marrow-derived mesenchymal stem cells (BMSCs) to specifically interfere with the expression of the OPG gene. Through this method, we could stably and efficiently silence the OPG gene, enabling the study of its effects on the RANKL/OPG ratio and OC generation.

This study investigates the effect of RNAi-mediated silencing of the OPG gene in BMSCs on the RANKL/OPG ratio and OC generation. By modulating this signaling axis, we seek to explore its potential to enhance the degradation properties of OCs on bone scaffold materials, thereby improving bone tissue repair. The findings of this study may provide a theoretical basis for gene-regulated bone tissue engineering and contribute to the development of novel therapeutic strategies. Future research will focus on expanding the application of RNAi technology to regulate other genes and evaluating its effects on different bone scaffolds to further advance bone tissue engineering.

MATERIALS AND METHODS

Isolation and cell culture of BMSCs

BMSCs were isolated from the bone marrow of Sprague-Dawley (SD) rats (Beijing Vital River Laboratory Animal Technology Co., Ltd., Cat. No: 101) and cultured and passaged in vitro using a cell adhesion culture method. One-week-old specific pathogen-free SD rats (weighing 80-100 g) were intraperitoneally injected with 2% pentobarbital sodium and euthanized. The femurs and tibias were collected aseptically, all soft tissues were removed, and the bones were washed three times with phosphate-buffered saline (PBS) and sterilized by soaking in 75% ethanol for 7-10 minutes. The bones were dissected to remove the epiphyses, and bone marrow cells were collected by washing the marrow cavity with culture medium (DMEM/F12; Gibco, Nanjing, China) containing 10% fetal bovine serum (Gibco, Nanning, China) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Cells isolated from each rat were used to prepare single-cell suspensions. The suspensions were centrifuged at 1000 rpm for 5 minutes, and then the cell pellets were re-suspended and seeded into a 25 cm² culture flask. BMSCs were cultured in a 37 °C, 5% CO₂ humidified incubator. Initially, the medium was replaced 24 hours after seeding to remove non-adherent cells. Subsequently, the medium was changed twice a week until confluence was reached, followed by passaging. The 3^rd passage BMSCs were differentiated into adipocytes and osteoblasts (OBs) under specific induction conditions: When the BMSCs reached 80%-90% confluence, they were cultured in induction media containing specific inducers (adipocytes: 1 μM dexamethasone, 0.5 mmol/L isobutyl-1-methylxanthine, 10 μg/mL insulin, 200 μM indomethacin; OBs: 50 μg/mL ascorbic acid, 10 mmol/L β-glycerophosphate, 100 nM dexamethasone) for 21 days, with medium changes every 3 days. Oil Red O and Alizarin Red staining assessed adipogenic and osteogenic differentiation.

Cell morphology was observed daily under an inverted phase-contrast microscope. When cell confluency reached approximately 90%, cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, stained with Giemsa for 2 minutes, rinsed with distilled water, and observed under a microscope equipped with a camera. The cells used in the experiments were from the 3^rd passage. On day 21, Oil Red O staining was used to monitor adipocyte differentiation, and Alizarin Red staining was used to monitor OB differentiation.

Flow cytometry analysis of cultured cells

Flow cytometry was used to identify surface markers of rat BMSCs. E BMSCs were incubated at 4 °C in the dark for 45 minutes with PE-labeled CD45 (12-0461-80), FITC-labeled CD11B (11-0112-82), FITC-labeled CD73 (11-0739-42), and FITC-labeled CD90 (11-0900-81) antibodies (all from eBioscience, San Diego, CA, USA). After incubation, the cells were washed twice with PBS to remove unbound antibodies and resuspended in 500 μL of FACS buffer. Equal amounts of cell preparations were incubated with the corresponding isotype control antibodies to determine nonspecific fluorescence signals. Flow cytometry analysis was performed using a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States), and data were acquired and analyzed using BD Accuri C6 software.

Construction of lentiviral vectors and cell transfection

Invitrogen Life Technologies (Carlsbad, CA, United States) synthesized the shRNA coding DNA sequences and constructed them into lentiviral plasmids. The preparation of lentivirus was carried out following the previously described procedure. The sense strand sequence of the valid shRNA chain targeting OPG is 5’-ACTTGCATTATGACCCAGAAA-3’, and the antisense strand is 5’-TTTCTGGGTCATAATGCAAGT-3’. The shRNA was named “shOPG” for the experimental group, while shScr represents scrambled shRNA used for the control group. The control group includes cells not transfected.

During the logarithmic growth phase of the 3^rd passage, rat BMSCs were seeded in a 6-well cell culture plate for 12-16 hours (during this period, cells did not exceed 50% confluence). Subsequently, the cells were treated with shOPG or shScr (scrambled) for 24 hours, then replaced with fresh medium (containing 2 μg/mL puromycin). Untreated cells were used as controls. RNA and protein were isolated on days 3, 7, 14, and 21. Detailed information regarding the shRNA sequences can be found in Table 1.

Table 1 Short hairpin RNA nucleotide sequences used for RNA interference.

Name	shRNA primer sequence (5’-3’)
shOPG (F)	5’-CCGGACTTGCATTATGACCCAGAACTCGAGTTTCTGGGTCATAATGCAAGTTTTTTG-3’
shOPG (R)	5’-AATTCAAAAAACTTGCATTATGACCCAGAAACTCGAGTTTCTGGGTCATAATGCAAGT-3’

shRNA: Short hairpin RNA; OPG: Osteoprotegerin; F: Forward; R: Reverse.

MTT cell proliferation assay

The experimental group seeded 8 × 10³ cells per well into a 96-well culture plate and cultured them in 200 μL of medium per well for 1, 3, 5, 7, and 9 days. 20 μL of 5% MTT solution (Abcam, Cat. No: Ab211091) was added to each well and incubated for 4 hours. After incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well. After shaking for 10 minutes on a microplate reader, the absorbance was measured at a wavelength of 570 nanometers (A value). The time points and absorbance were plotted to create a cell growth curve.

In vitro osteoclastogenesis assay

Non-adherent bone marrow stromal cells were obtained from 4-week-old SD rats. In the co-culture experiment, shOPG, shScr, and control group cells were cultured at a density of 1 × 10⁶ cells/mL in six-well plates, followed by adding 1 mL of medium containing 1 × 10⁶ non-adherent rat bone marrow stromal cells (as OC precursor cells) to each group. The co-culture was maintained for 7 days. The old medium was replaced with a fresh medium every 3 days. TRAP staining was used to quantify the presence of TRAP-positive multinucleated cells (more than three nuclei) through cytochemical staining, thereby measuring OC formation.

Bone resorption assay

Cells from the shOPG, shScr, and control groups are seeded onto bovine bone slices in 6-well plates at 1 × 10⁶ cells/mL density. Each well is supplemented with 1 mL of culture medium containing 1 × 10⁶ non-adherent rat bone marrow stromal cells (serving as OC precursor cells). The co-culture is maintained for 7 days. OCs are detached from the bone slices using mechanical agitation and ultrasonic treatment. After collection, the cells are stained with toluidine blue, and resorption pits are visualized and imaged under a microscope (ZEISS, Jena, Germany). The percentage of resorption pit area is quantified. Three random fields of view are selected from each bone slice for further statistical analysis[19].

RNA isolation and RT-qPCR

According to the manufacturer’s instructions, total RNA was extracted from cells using TRIzol (Takara, Dalian, Liaoning, China). The optical density of RNA was assessed at 260 nanometers (OD₂₆₀) and 280 nanometers (OD₂₈₀) using a Nanodrop 2000 spectrophotometer (Thermo Scientific, MA, United States) to evaluate RNA concentration. The OD₂₆₀:OD₂₈₀ratio of RNA samples was between 1.8-2.0, confirming satisfactory purity. The SYBR^® PrimeScriptTM RT-PCR Kit (Takara) generated cDNA from total RNA following the manufacturer's instructions. SYBR^® Premix Ex Taq (2 ×) (Takara) was utilized for mRNA expression quantification. Lastly, samples were processed and analyzed using the StepOnePlus^™ RT-PCR system (Applied Biosystems, Foster City, CA, United States) with an initial denaturation step at 95 °C for 30 seconds, followed by 40 cycles at 95 °C for 5 seconds, 60 °C for annealing and extension for 30 seconds. Relative mRNA levels were compared using the comparative 2^-ΔΔCt method at 3, 7, 14, and 21 days, with β-actin as the endogenous RT-qPCR control for all groups. Table 2 provides detailed PCR information, including primer sequences. The experiments were independently repeated three times, each with three replicates per experiment.

Table 2 Nucleotide sequences of the primers used for real-time quantitative polymerase chain reaction.

Gene	Primer sequence (5’-3’)	PCR product (bp)
RANKL	5’-TTACCTGTACGCCAACATTTGC-3’ (F)	283
	5’-AAGTACGTCGCATCTTGATCC-3’ (R)
OPG	5’-TGTTCTGGTGGACAGTTTGC-3’ (F)	167
	5’-AGAGGTCAATGTCTTGGAT-3’ (R)
CTSK	5’-ATATGTGCAGCAGAATGGAGG-3’ (F)	195
	5’-CTTGCATCGATGGACACAGAG-3’ (R)
TRAP	5’-CCACAACCTGCAGTATCTTC-3’ (F)	200
	5’-CCACATACGTGATGCTCATTTC-3’ (R)
MMP-9	5’-TTGCCTGCAAAGTTGAACTCAG-3’ (F)	264
	5’-CAAGCGAGTAACGCTCTGG-3’ (R)
CTR	5’-AGCTGGTGTAATGTCCTATCAG-3’ (F)	236
	5’-CAATCAGAGCAGCAATTGACATGG-3’ (R)
β-actin	5’-GGAGATTACTGCCCTGGCTCCTA-3’ (F)	150
	5’-GACTCATCGTACTCCTGCTTGCTG-3’ (R)

PCR: Polymerase chain reaction; RANKL: Receptor activator of nuclear factor-kappa B ligand; OPG: Osteoprotegerin; CTSK: Cathepsin K; TRAP: Tartrate-resistant acid phosphatase; MMP-9: Matrix metalloproteinase-9; CTR: Receptors for calcitonin; F: Forward; R: Reverse.

Protein extraction and western blot analysis

Culture cells in a six-well culture plate with DMEM/F12 containing 10% fetal bovine serum until reaching nearly 90% confluence. Subsequently, the cells were washed twice with ice-cold PBS and harvested using cell RIPA buffer (Fudebio-tech, Hangzhou, China). After centrifugation at 14000 × g and 4 °C for 15 minutes, collect the supernatant and determine the total protein concentration using a BCA protein assay kit (Fudebio-tech, Hangzhou, China) following the manufacturer’s instructions. Prepare a 10% sodium-dodecyl sulfate gel electrophoresis gel, loading 10 mg of cell protein per well. Transfer the separated proteins to a polyvinylidene fluoride membrane (Abcam, ab133411) and incubate with mouse monoclonal antibodies against RANKL (SC 52950) and OPG (SC 390518) from Santa Cruz Biotechnology, CA, United States, and β-actin (CB100997M) mouse monoclonal antibody from California Bioscience, California, United States (diluted at 1:1000) following the manufacturer’s instructions, together (diluted at 1:200). Scan the membrane with a fluorescent secondary antibody [Goat anti-Mouse immunoglobulin G (IgG); Abcam] using the dual-color infrared imaging system (Odyssey, LI-COR, NE, United States) and quantify the protein bands’ grayscale using image processing software (Bio-Rad, Hercules, CA, United States).

CoIP

CoIP is a commonly used experimental technique for detecting protein-protein interactions in vivo. Firstly, cells are lysed using RIPA lysis buffer (Thermo Fisher Scientific, Cat. No. 89900), and the lysates are centrifuged at 15000 × g for 10 minutes at 4 °C to collect the supernatant, referred to as the Input. The Input represents the BMSC protein lysates before antibody addition or immunoprecipitation. Next, specific antibodies against RANKL (R&D Systems, Cat. No. AF462) or OPG (R&D Systems, Cat. No. AF805), as well as control IgG, are pre-incubated with protein A/G agarose beads (Santa Cruz Biotechnology, Cat. No. sc-2003) to form antibody-protein A/G bead complexes. Then, the cell lysate supernatants are mixed with the antibody-protein A/G bead complexes and incubated overnight at 4 °C to allow the antibodies to capture the target proteins and their interacting partners. The residues are washed multiple times with wash buffer (Thermo Fisher Scientific, Cat. No: 28360) to remove non-specifically bound proteins and impurities. Finally, the target protein complexes (RANKL, OPG, and IgG) are eluted from the beads using elution buffer, separated by sodium-dodecyl sulfate gel electrophoresis electrophoresis (Bio-Rad, Cat. No: 456-1093), transferred to a polyvinylidene fluoride membrane (Millipore, Cat. No: IPVH00010), and detected using western blot to analyze the target proteins (such as RANKL and OPG) and their interacting proteins. This method successfully validates the protein-protein interaction between RANKL and OPG, providing a reference for studying other protein interactions.

Statistical analysis

Statistical analysis was conducted using SPSS 17.0 software. Data are expressed as the mean ± SD of all experiments. Significant differences between the study groups were determined using statistical methods such as one-way analysis of variance (ANOVA) or Student’s t-test as appropriate. A P-value less than 0.05 was considered statistically significant.

RESULTS

Identification and differentiation capacity of BMSCs

The morphological and phenotypic characteristics of third-generation rat BMSCs were identified. Giemsa staining revealed a typical spindle-shaped morphology (Figure 1A). Under specific induction conditions, these cells were successfully differentiated into adipocytes (Figure 1B) and OBs (Figure 1C), confirmed by Oil Red O and Alizarin Red staining, respectively. Flow cytometry analysis showed that over 90% of the cells expressed the mesenchymal stem cell markers CD73 (91.0%) and CD90 (94.4%) (Figure 1D and E), while the expression of hematopoietic markers CD11B (0.58%) and CD45 (0.043%) was negligible (Figure 1F and G).

Figure 1 Cultured bone marrow-derived mesenchymal stem cells micrographs and flow cytometry analysis. A: Microscopic image of bone marrow-derived mesenchymal stem cells (BMSCs) after Giemsa staining (scale bar = 200 μm); B: Microscopic image of BMSCs stained with Oil Red O on day 21 (scale bar = 200 μm); C: Microscopic image of BMSCs stained with Alizarin Red on day 21 (scale bar = 200 μm); D and E: Flow cytometry analysis of CD73 (D) and CD90 (E) expression in cultured BMSCs; F and G: Flow cytometry analysis of CD11B (F) and CD45 (G) expression in cultured BMSCs. The results show that over 95% of cultured BMSCs express the mesenchymal stem cell markers CD73 and CD90, while less than 2% of BMSCs express the hematopoietic stem cell markers CD11B and CD45.

Silencing the OPG gene does not significantly affect the proliferation of BMSCs

In this study, we evaluated the effect of OPG gene silencing on the proliferation of BMSCs using the MTT cell proliferation assay. The experimental results showed that the growth of cells in the shOPG, shScr, and control groups was similar throughout the experiment. Specifically, all groups of cells were in an adaptation period during the first three days after seeding, followed by rapid growth, reaching a peak proliferation on the 7^th day. Subsequently, the proliferation rate of the cells gradually stabilized (Figure 2). At each time point from day 1 to day 9, the absorbance values (A values) of the three groups of cells were measured to assess their proliferation activity. The results indicated that the proliferation curve of the shOPG group almost overlapped with that of the shScr group and the control group, suggesting that the shOPG treatment did not significantly affect the proliferation capacity of BMSCs. Specific statistical data showed no significant differences in absorbance values between the groups on day 1, day 3, day 5, day 7, and day 9 (P > 0.05). These results indicate that although the OPG gene in BMSCs was successfully silenced using RNAi technology, this gene silencing did not significantly affect the proliferation activity of the cells.

Figure 2 MTT assay to detect changes in cell proliferation in each group. The cell proliferation growth curves for each group were plotted with time on the X-axis and absorbance (A value) on the Y-axis. The experimental group, “shOPG”, represents osteoprotegerin gene-silenced bone marrow-derived mesenchymal stem cells (BMSCs) transfected with shOPG. The control group, “shScr”, consists of BMSCs transfected with a scrambling vector, while the “control” group includes untreated BMSCs.

The shOPG expression vector effectively inhibits OPG gene expression and regulates the expression of the RANKL gene

To evaluate the effect of silencing the OPG gene using RNAi technology, we transduced BMSCs with a lentiviral vector encoding shRNA targeting OPG (OPG shRNA). We then assessed the mRNA expression levels of OPG and RANKL genes at different time points post-transfection using RT-qPCR. We compared the shOPG group with a mock group (shScr) and a control group (non-transfected) to validate the specificity and efficiency of gene silencing.

At days 3, 7, 14, and 21 post-transfection, we measured the expression levels of OPG mRNA in the cells of each group. The results showed that the OPG mRNA expression in the shOPG group was significantly lower than in the control group at all time points post-transfection. On day 3 post-transfection, the OPG mRNA expression in the shOPG group was only 23% of the control group; on days 7 and 14, it was 37% and 54% of the control group, respectively; and on day 21, it recovered to 74% of the control group (Figure 3A). In comparison, the expression of OPG mRNA in the shScr group did not show significant differences from the control group at all time points, indicating that the gene silencing effect of shOPG on the OPG gene is highly specific, with the inhibition reaching its peak at day 3 post-transfection and gradually diminishing thereafter.

Figure 3 The mRNA levels of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin genes in each group. A and B: On the 3^rd, 7^th, 14^th, and 21^st day post-transfection, real-time quantitative polymerase chain reaction was used to evaluate the mRNA levels of osteoprotegerin (OPG) (A) and receptor activator of nuclear factor-kappa B ligand (RANKL) (B) in each group. The experimental group, “shOPG”, represents OPG gene-silenced bone marrow-derived mesenchymal stem cells (BMSCs) transfected with shOPG, while the control groups, “shScr” and “control”, represent BMSCs transfected with a scramble vector and untreated BMSCs, respectively. Compared to the shScr and control groups, RANKL was upregulated in shOPG-transfected BMSCs, particularly on day 3. Conversely, OPG expression was downregulated in shOPG-transfected BMSCs compared to the shScr and control groups (mean ± SEM, n = 3 experiments). ^aP < 0.05; ^bP < 0.01. BMSC: Bone marrow-derived mesenchymal stem cell; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-kappa B ligand.

Furthermore, we also measured the mRNA expression levels of RANKL in the cells of each group. The results showed that the RANKL mRNA in the shOPG group was significantly upregulated at day 3 post-transfection, reaching 390% of the control group; on days 7 and 14, the expression levels were 135% and 97% of the control group, respectively; by day 21, the expression level recovered to 84% of the control group (Figure 3B). It indicates that in BMSCs transduced with shOPG, the expression of RANKL mRNA peaked briefly at day 3 post-transfection and gradually returned to normal levels. In contrast, the expression of RANKL mRNA in the shScr group was 212% of the control group on day 3 but did not differ significantly from the control group at other time points.

The shOPG expression vector effectively inhibits OPG gene expression and regulates the expression of the RANKL gene

To further validate the effects of shOPG on silencing the OPG gene, we performed western blot analysis to examine the expression levels of OPG and RANKL proteins in cells from different groups at different time points post-transfection (3^rd, 7^th, 14^th, and 21^st days) (Figure 4). The results showed that the expression of OPG protein in the shOPG group significantly decreased to 31% of the control group on the 3^rd day post-transfection, gradually increasing to 45% and 68% of the control group on the 7^th and 14^th days, respectively, and eventually returning to 87% on the 21^st day. In contrast, the expression of OPG protein in the shScr group did not show significant differences from the control group at all time points (Figure 4A). These results indicate that the inhibitory effect of shOPG on OPG protein expression was most significant on the 3^rd day post-transfection, gradually weakening but still maintaining a significant inhibitory effect thereafter. Additionally, we also measured the expression levels of RANKL protein in the cells from each group. The results demonstrated no significant differences in the expression levels of RANKL protein among the shOPG group, shScr group, and control group at all time points post-transfection (Figure 4B). It indicates that while shOPG significantly inhibited the expression of OPG protein, it did not affect the expression level of RANKL protein.

Figure 4 Protein levels of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in each group. A and B: The western blot results of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) protein expression at different time points (3^rd, 7^th, 14^th, and 21^st days) after transfection with shScr or shOPG (A) and their quantitative analysis (B). The experimental group, “shOPG”, represents OPG gene-silenced bone marrow-derived mesenchymal stem cells (BMSCs) transfected with shOPG, while the control groups, “shScr” and “control”, represent BMSCs transfected with a scramble vector and untreated BMSCs, respectively. The data represent results from three independent experiments. Changes in RANKL and OPG protein levels in shOPG-transfected BMSCs were observed compared to the shScr and control groups (mean ± SEM, n = 3 experiments). ^aP < 0.05; ^bP < 0.01. BMSC: Bone marrow-derived mesenchymal stem cell; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-kappa B ligand.

ShOPG significantly increases the RANKL/OPG ratio

We evaluated the impact of shOPG on the RANKL/OPG ratio, analyzing it at both mRNA and protein levels. At the mRNA level, the RANKL/OPG ratio in the shOPG group significantly increased on the 3^rd, 7^th, and 14^th days post-transfection (Figure 5A). Specifically, the RANKL/OPG ratio in the shOPG group was nearly 20-fold higher than that in the control groups on the 3^rd day; on the 7^th and 14^th days, while the ratio decreased slightly, it remained significantly higher than the control group. However, by the 21^st day, the RANKL/OPG ratio decreased to a level similar to the control group. At the protein level, the RANKL/OPG ratio in the shOPG group also significantly increased on the 3^rd, 7^th, and 14^th days post-transfection (Figure 5B). On the 3^rd day, the ratio in the shOPG group increased by 3.7-fold; on the 7^th day, the ratio increased by 2.5-fold. Although the ratio decreased on the 7^th and 14^th days, it remained higher than the control group at these time points. These results indicate that shOPG significantly elevated the RANKL/OPG ratio in the first 14 days post-transfection, with this elevation observed at both the mRNA and protein levels, and the ratio returned to normal levels by the 21^st day. The silencing of OPG increased the RANKL/OPG ratio and promoted the generation of OC-like cells.

Figure 5 Receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio of shOPG and shScr groups in mRNA and protein levels. A and B: Time-dependent changes in the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) mRNA (A) and protein (B) ratio following transfection with shScr or shOPG. The control group, “shScr”, represents bone marrow-derived mesenchymal stem cells transfected with a scramble vector, while the experimental group, “shOPG”, represents bone marrow-derived mesenchymal stem cells transfected with shOPG. Comparisons of RANKL/OPG mRNA and protein levels were conducted on the 3^rd, 7^th, 14^th, and 21^st days post-transfection between the “shOPG” and “shScr” groups. A comparison was made on the 3^rd, 7^th, 14^th, and 21^st days post-transduction with the control group. ^aP < 0.05; ^bP < 0.01 compared to the control group. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-kappa B ligand.

Silencing the OPG gene promotes OC formation and bone resorption significantly

To assess the impact of OPG gene silencing on osteoclastogenesis, we co-cultured BMSCs transfected with shOPG with non-adherent bone marrow stromal cells and evaluated the generation of TRAP-positive multinucleated cells. The experimental results showed that, compared to the shScr and the control groups, the shOPG group exhibited a significant increase in TRAP-positive multinucleated cells (Figure 6A and B). Specifically, during the co-culture process with shOPG BMSCs, where the OPG gene was knocked out, there was a significant increase in TRAP-positive multinucleated cells in non-adherent bone marrow stromal cells. In contrast, there was no significant difference in the co-culture results between the shScr and the control groups (P > 0.05), further confirming the specific effect of shOPG. These results suggest inhibiting the OPG gene in BMSCs can significantly enhance OC generation by increasing the RANKL/OPG ratio.

Figure 6 Representative regions of tartrate-resistant acid phosphatase-positive multinucleated cells in osteoclast generation experiment. A and B: Results of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed after 7 days of co-culture between the experimental group (shOPG bone marrow-derived mesenchymal stem cells) and non-adherent rat bone marrow stromal cells are shown in (A), along with the quantitative statistical analysis (B). TRAP-positive multinucleated cells (indicated by arrows; scale bar = 200 μm); C: Quantitative reverse transcription polymerase chain reaction analysis of mRNA levels of osteoclast marker genes cathepsin K, TRAP, matrix metalloproteinase-9, and receptors for calcitonin; D: Toluidine blue staining showing the proportion of resorption pit areas in co-cultured cells for each group, scale bar = 25 μm. Notably, the shOPG group exhibited significantly stronger osteoclast formation capability compared to the “shScr” and “control” groups. ^aP < 0.05. TRAP: Tartrate-resistant acid phosphatase.

Additionally, we further validated the levels of OC marker genes cathepsin K (CTSK), TRAP, matrix metalloproteinase-9, and receptors for calcitonin through quantitative reverse transcription polymerase chain reaction experiments. The results showed that during co-culture with OPG-silenced shOPG BMSCs, the CTSK, TRAP, matrix metalloproteinase-9, and receptors for calcitonin mRNA levels were significantly elevated in non-adherent bone marrow stromal cells (Figure 6C). Simultaneously, bone resorption assays revealed that during co-culture with OPG-silenced shOPG BMSCs, bone resorption by non-adherent bone marrow stromal cells was significantly increased, whereas no significant differences were observed in the shScr and control groups (P > 0.05). These findings further confirmed that OPG gene silencing promotes OC-mediated bone resorption (Figure 6D).

Validation of protein-protein interaction between RANKL and OPG

The CoIP experiment results showed that RANKL and OPG exhibited clear protein bands at 55 and 35 kDa, indicating that RANKL can directly bind to OPG. In the negative control group, no significant signals were observed, validating the specificity of the experimental results (Figure 7). These findings reveal a direct protein-protein interaction between RANKL and OPG, supporting their important bone metabolism and immune regulation functions.

Figure 7 Co-immunoprecipitation results of the interaction between receptor activator of nuclear factor-kappa B ligand and osteoprotegerin. This figure demonstrates the protein-protein interaction between receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in co-immunoprecipitation experiments. In the input samples (input, the supernatant obtained from centrifuged bone marrow-derived mesenchymal stem cell protein lysates without antibody addition or immunoprecipitation), distinct protein bands were observed at 55 kDa and 35 kDa, corresponding to OPG and RANKL, respectively. Significant bands were also detected at 55 kDa and 35 kDa in the immunoprecipitation samples using RANKL antibody (RANKL) and OPG antibody (OPG), indicating a direct protein-protein interaction between RANKL and OPG. No significant signals were observed in the negative control group (immunoglobulin G), confirming the specificity of the experimental results. n = 3. RANKL: Receptor activator of nuclear factor-kappa B ligand; OPG: Osteoprotegerin; IgG: Immunoglobulin G.

DISCUSSION

The bone scaffold is crucial in bone tissue repair, especially in treating fractures, bone defect repair, and bone regeneration[20]. However, the degradation characteristics of the bone scaffold directly impact its clinical effectiveness[21,22]. OCs play a key role in remodeling and repairing bone tissue by absorbing and degrading the scaffold[2]. The generation and activity of OCs are regulated by various factors, among which the ratio of RANKL and OPG is a critical regulatory factor[3,12,23]. Previous studies have indicated that an increase in the RANKL/OPG ratio is closely associated with enhanced OC activity[9,13].

This study employed RNAi technology to achieve stable silencing of the OPG gene in BMSCs using a recombinant lentiviral plasmid vector for efficient shRNA delivery. RT-qPCR and western blot analyses confirmed effective OPG silencing, significantly reducing its mRNA and protein levels. TRAP staining revealed increased OC numbers following OPG downregulation, suggesting its inhibitory role in OC differentiation. Additionally, CoIP assays validated the protein interaction between RANKL and OPG, consistent with previous studies, indicating that OPG may indirectly influence osteoclastogenesis by modulating RANKL availability. Although OPG silencing led to an upregulation of RANKL mRNA, no significant change was observed at the protein level, suggesting that the increased RANKL/OPG ratio primarily resulted from reduced OPG-mediated inhibition rather than direct RANKL upregulation. These findings provide a foundation for further investigation into the regulatory role of OPG in OC function and offer a potential strategy for modulating bone scaffold degradation and bone tissue repair.

This study demonstrates significant innovation in applying RNAi compared with previous studies[24,25]. While RNAi has been widely used for gene expression regulation and disease mechanism research[26-28] and has been reported in osteogenesis studies[25], specific silencing of the OPG gene remains limited. Previous studies primarily targeted genes such as S100 calcium-binding protein A4[29], guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1[30], and Noggin[31], whereas precise regulation of OPG in osteoclastogenesis has not been thoroughly explored. By optimizing the RNAi vector system and employing a recombinant lentiviral plasmid vector for efficient shRNA delivery, this study successfully achieved stable OPG silencing in BMSCs. This strategy enhances the stability and specificity of gene silencing and ensures experimental reproducibility and reliability.

In terms of RANKL/OPG ratio modulation, this study differs from previous research in key aspects. Prior studies have primarily focused on the independent roles of RANKL or OPG in osteoclastogenesis and activity regulation, whereas this study specifically examines the overall impact of RANKL/OPG ratio changes on OC formation[3,32-34]. The findings provide new evidence supporting RNAi-mediated OPG inhibition to promote osteoclastogenesis by increasing the RANKL/OPG ratio. Compared with previous studies[35-37], this study further confirms the critical role of RANKL/OPG ratio regulation in osteoclastogenesis.

Additionally, while previous research has explored various regulatory factors in osteoclastogenesis, such as gene expression[38], signaling pathways[39], and microenvironmental changes, systematic investigations on RNAi-mediated OPG suppression and its direct impact on osteoclastogenesis remain limited. By stably silencing OPG through RNAi, this study significantly reduced OPG mRNA and protein levels while maintaining stable RANKL expression, thereby specifically increasing the RANKL/OPG ratio and promoting osteoclastogenesis. This mechanism aligns with prior findings on the RANKL/OPG axis in OC regulation[40-43]. Although RNAi has been explored in bone regeneration[25], its application in enhancing OC activity to optimize bone scaffold degradation remains underdeveloped. This study confirms that OPG silencing increases the RANKL/OPG ratio, enhancing osteoclastogenesis and accelerating scaffold degradation, providing a new strategy for developing highly degradable bone scaffolds. Furthermore, this approach offers potential for designing personalized bone repair materials, such as RNAi-based precision-engineered scaffolds, contributing to the clinical translation of RNAi technology in bone tissue repair.

This study introduces methodological innovations compared with previous research. A recombinant lentiviral plasmid vector delivered shRNA into rat BMSCs, effectively silencing OPG expression. Unlike other studies, this study validated the reliability and specificity of gene silencing through RT-qPCR and western blot, assessed OC formation via TRAP staining, and examined the RANKL-OPG interaction using co-immunoprecipitation[44-46]. TRAP, a classic OC marker, was used to evaluate osteoclastogenesis, while additional markers such as cathepsin K and Occlusion of Stomatal Pore 1 will be explored in future studies for a more comprehensive assessment of OC formation and differentiation.

This research provides new scientific evidence for promoting osteoclastogenesis by regulating the RANKL/OPG ratio, which holds significant application value in the degradation of bone scaffold and bone tissue repair. By controlling the expression of the OPG gene, the degradation rate of the bone scaffold can be precisely controlled, thereby improving the efficiency and effectiveness of bone regeneration. Moreover, this study’s findings have potential clinical implications in treating bone-related diseases such as osteoporosis, offering new theoretical support and molecular targets for related therapeutic strategies.

Although this study demonstrates that RNAi-mediated OPG silencing promotes the differentiation of non-adherent bone marrow stromal cells into OCs, its main limitation is that the experiments were conducted in vitro, lacking in vivo evidence for validation. Currently, many studies have employed vesicles or exosomes derived from BMSCs in experiments on bone formation and regeneration[47,48]. The RNAi mechanism discovered in this study, which suppresses OPG gene expression, may be integrated into in vivo studies using BMSC-derived exosome or vesicle technology. This approach could even be a significant direction for future clinical application research. Additionally, this study used only a rat model, and the generalizability of the findings has not been validated in other animal models.

Further research must validate the findings across different animal models and clinical samples. Multi-faceted data collection, including studies on various animal models, fracture types, materials, and intervention durations, should be conducted to ensure the reliability and reproducibility of the results. Future research should focus on in vivo experiments to verify the efficacy and safety of RNAi technology in regulating osteoclastogenesis. Expanding into multiple directions, such as RNAi regulation of ABCC1 and CDC37 expression to promote OC differentiation or RNAi intervention in the RANKL signaling pathway to regulate OC function, could provide further insights. Moreover, exploring the effects of different doses and time points of RNAi on the RANKL/OPG ratio and osteoclastogenesis could help optimize intervention strategies.

Related applications, such as C-176, are already reported, which inhibits the STING-mediated nuclear factor-kappa B pathway activation and can downregulate OC formation. C-176, as a STING inhibitor, also promotes M1 macrophage polarization to the M2 phenotype, suggesting that the cGAS-STING pathway may suppress osteoclastogenesis and differentiation by inhibiting M1 macrophage polarization[49-51]. Further research should explore the potential application of this mechanism in other bone-related diseases and develop corresponding therapeutic approaches to provide more options for clinical treatment.

Currently, the clinical treatment of fractures primarily relies on the implantation and fixation of exogenous bone scaffold materials. However, this approach necessitates a second surgery to remove the fixation materials. If porous-structured materials can be developed to allow bone tissue or stem cells to grow within, bioactive bone scaffolds could begin degrading within 1-2 months after implantation, during which OCs play a critical role in activation and regulation, promoting bone growth. Within 5-6 months, the body would completely absorb the scaffold, and the bone would largely regenerate. OCs are central to the degradation of bone scaffold materials, as they regulate the degradation rate through bone resorption processes[2]. It could save patients half the time and costs, facilitating faster recovery. Therefore, researching ways to enhance the regulation of osteoclastic bone resorption activity is crucial for optimizing the performance of bone scaffold materials.

CONCLUSION

This study utilized recombinant lentiviral plasmids to interfere with the expression of the OPG gene in BMSCs in rats. It was observed that the mRNA level of RANKL was significantly upregulated while the mRNA and protein levels of OPG were downregulated in the BMSCs, leading to an increased ratio of RANKL/OPG and promotion of osteoclastogenesis. It indicates that inhibiting the expression of the OPG gene in BMSCs through RNAi can effectively regulate OC generation and activity, revealing the RANKL/OPG ratio’s critical role in bone resorption (Figure 8).

Figure 8 Molecular mechanism of osteoprotegerin suppression in enhancing osteoclastogenesis via modulation of the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio. BMSC: Bone marrow-derived mesenchymal stem cell; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-kappa B ligand; shRNA: Short hairpin RNA; TRAP: Tartrate-resistant acid phosphatase.

In conclusion, our study revealed the effects of RNAi on the RANKL/OPG ratio and osteoclastogenesis. These novel findings hold significant potential for advancing bone tissue engineering and regenerative medicine. They are particularly important for the degradation of bone scaffold materials and bone tissue repair, highlighting the critical role of RANKL/OPG regulation in osteoclastogenesis and scaffold degradation. This research provides a new direction for the clinical development of advanced scaffold materials and therapeutic strategies.