Translate this page into:
A bone-targeting delivery platform based on mesoporous silica loaded with piR7472 for the treatment of osteoporosis
⁎Corresponding authors. fasaj2009@163.com (Yong Li), tbo0820@163.com (Bo Tao), billsuntw@163.com (Tianwei Sun)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Promoting osteogenic differentiation and inhibiting osteoclast formation remain significant challenges in the treatment of osteoporosis. With the growing understanding of osteoporosis, increasing literature has highlighted the regulatory role of m6A methylation in this condition. However, there is currently no reliable method to stably regulate cellular m6A methylation levels. Here, we report a novel approach utilizing alendronate (aln)-modified mesoporous silica nanoparticles (MSNs) to deliver sodium bicarbonate and piR7472, modulating cellular behavior. Our experimental results demonstrate that Aln modification enables the nanoparticles to stably target hydroxyapatite, thereby accumulating in osteoporotic regions. Sodium bicarbonate suppresses osteoclastogenesis, while piR7472 enhances m6A methylation, promoting osteogenic differentiation of bone marrow stromal cells (BMSCs). Computed tomography (CT) and hematoxylin and eosin (HE) staining showed that after 2 weeks of treatment with MSNs-Na@piR7472, cortical bone thickened, trabecular bone density increased, collagen fiber thickness improved, and both the number and staining area of osteoclasts were significantly reduced. These findings indicate a marked improvement in osteoporosis.
Keywords
Osteoporosis
Alendronate
Mesoporous silica nanoparticles
m6A methylation
piR7472
1 Introduction
As global technological advancements and improvements in healthcare continue to rise, the number of elderly individuals worldwide is increasing annually, leading to a growing prevalence of age-related diseases, including osteoporosis (Miao et al., 2023). Osteoporosis is a systemic skeletal disorder characterized by decreased bone mass and deterioration of bone microarchitecture, resulting in increased bone fragility and susceptibility to fractures (Cao et al., 2023). Typically associated with aging, bone formation and resorption occur at roughly the same rate (Liao et al., 2024). During adolescence, bone formation significantly outpaces bone resorption, leading to a net gain in bone mass. However, as aging progresses, the rate of bone resorption gradually exceeds that of bone formation, leading to a reduction in bone mass and ultimately the development of osteoporosis (Wan et al., 2018; Liu et al., 2022; Zeng and Xie 2022; Wang et al., 2023; Mohankumar et al., 2024).
Clinically, the primary methods for treating osteoporotic bone defects include oral pharmacotherapy and bone grafting. Among oral medications, bisphosphonates, such as alendronate (aln), are the most widely used (Wang et al., 2023). Their effectiveness in increasing bone mineral density (BMD) and combating osteoporosis is attributed to their affinity for hydroxyapatite, their distribution and persistence within bone tissue, and their ability to inhibit farnesyl pyrophosphate synthase (FPPS) in osteoclasts (Bai et al., 2023). This results in a reduction in bone remodeling rate, decreased bone resorption, and osteoclast apoptosis (Helft et al., 2015). However, these drugs have certain limitations and side effects, such as an increased risk of breast cancer with long-term use and the development of drug resistance. Consequently, developing effective drug delivery platforms and identifying new therapeutic targets remain critical concerns for clinical practitioners (Hwang et al., 2023; Huang et al., 2023). Sodium bicarbonate (NaHCO3) has been widely used to treat severe metabolic acidosis due to its excellent biocompatibility. In the local acidic environment of the osteoclast resorption area, saturated NaHCO3 can be employed to form a protective layer on the bone surface, effectively neutralizing the acidic environment and disrupting osteoclast formation. However, the targeted delivery of sodium bicarbonate to bone tissue has been a challenging issue.
N6-methyladenosine (m6A) is a dynamic methylation modification located at the N6 position of adenosine, widely present in eukaryotic mRNA (Liu et al., 2024; Gu et al., 2024). It plays a crucial role in various mRNA metabolic processes, including splicing, transport, translation, and degradation (Lu et al., 2023). Due to the concentration of m6A modification sites in genes related to development and cell fate regulation, m6A plays a key role in stem cell fate determination, directional differentiation, and gene expression (Gao et al., 2022; Wu et al., 2018). The m6A modification is primarily mediated by methyltransferases such as METTL3 and METTL14 and involves m6A demethylases and recognition proteins (Yang et al., 2023; Wang et al., 2024). For example, literature reports that the reduction of m6A epigenetic modifications in bone marrow mesenchymal stem cells inhibits the translation of Pth1r from early stages in mammals, thereby blocking its anabolic response to PTH during bone accumulation. These functional defects skew the delicate balance of MSC differentiation towards the adipogenic lineage, leading to severe bone loss and excessive accumulation of MAT (marrow adipose tissue), ultimately causing osteoporosis(Wu et al., 2018). Therefore, regulating intracellular m6A modifications may be a feasible therapeutic approach for osteoporosis.
PIWI-interacting RNA (piRNA) is a class of non-coding RNA, 26–31 nucleotides in length, first discovered in mammalian germ cells (Cai et al., 2023). PiRNA forms complexes with members of the Argonaute family, particularly PIWIL proteins, influencing transposon silencing, genome rearrangement, spermatogenesis, protein regulation, epigenetic regulation, and the maintenance of germline stem cells. Recently, an increasing number of studies have shown that piRNAs play an important role in the progression of osteoporosis. Literature reports that PiR-36741 upregulates BMP2 expression by blocking METTL3-mediated m6A methylation of BMP2 mRNA, thereby promoting osteogenic differentiation of BMSC cells. This provides new insights and directions for the clinical treatment of osteoporosis. piRNAs exhibit hydrophilicity and a negative charge, which impede their effective passage through biological membranes. Therefore, developing a delivery platform that is biocompatible, biodegradable, non-immunogenic, and non-toxic is crucial for the clinical application of piRNAs. Chitosan has garnered attention for its excellent biocompatibility and charge properties. Electrostatic loading does not compromise the biological activity of piRNAs and allows for membrane penetration.
Here, we propose a novel method using ALN-modified MSNs for the targeted delivery of sodium bicarbonate and piR7472 to bone tissue. The modification of MSNs with ALN enables the targeted delivery of sodium bicarbonate and piR7472 to osteoporotic areas. The loading of sodium bicarbonate inhibits osteoclastogenesis, while piR7472 enhances m6A methylation, promoting the osteogenic differentiation of BMSCs. In vivo experiments indicate that MSNs-Na@piR7472 exhibit excellent biocompatibility and facilitate BMSC osteogenic differentiation. In vitro studies show that after 2 weeks of treatment with MSNs-Na@piR7472, cortical bone thickness increased, trabecular bone density improved, collagen fiber thickness enhanced, and both the number and staining area of osteoclasts significantly decreased.
2 Materials and methods
2.1 Materials
Cetyltrimethylammonium Bromide (CTAB): Purchased from Sigma-Aldrich. Tetraethyl Orthosilicate (TEOS): Provided by Merck KGaA. 3-Trihydroxysilylpropyl Methylphosphonate: Acquired from Tokyo Chemical Industry Co., Ltd. Alendronate Sodium (Aln): Obtained from Aladdin Reagent. Polyethylene Glycol (PEG, 3500 Da): Supplied by Alfa Aesar. Sodium Bicarbonate: Procured from Sinopharm Chemical Reagent Co., Ltd. Chitosan: Purchased from Sigma-Aldrich. PiR7472: Synthesized by Guangzhou RiboBio Co., Ltd. Other Reagents: Analytical grade reagents were used without further purification.
2.2 Synthesis
2.2.1 Synthesis of mesoporous silica nanoparticles (MSNs)
Mesoporous silica nanoparticles were synthesized following a previously reported method (Pan and Shi 2018). Briefly, 1 g of cetyltrimethylammonium bromide (CTAB) was dissolved in a mixture of 480 mL ultrapure water and 3.5 mL NaOH solution. The solution was heated to 80 °C for 20 min. Subsequently, 4.5 mL of tetraethyl orthosilicate (TEOS, 20.15 mmol) was added dropwise at a rate of 0.33 mL/min. After 30 min, 0.5 mL of 3-trihydroxysilylpropyl methylphosphonate (1.31 mmol) was added for phosphonate modification and the solution was heated for an additional 1.5 h at 80 °C. The resulting solution was centrifuged, washed three times with water, twice with ethanol, and vacuum-dried at room temperature to yield a white powder. The obtained MSNs were added to hydrochloric acid (37 wt%) and ethanol solution (hydrochloric acid:ethanol = 1:9) and stirred for 12 h. The process was repeated three times to remove the CTAB templating agent, and then centrifuged to obtain pure template-free MSNs.
2.2.2 Synthesis of PEG-Alendronate Conjugates
153 mg of polyethylene glycol (PEG, 3500 Da) and 30 mg of alendronate sodium (Aln) were dissolved in 10 mL water. Then, 30 μL triethylamine was added, and the reaction mixture was stirred at 50 °C overnight. The product was dialyzed for 3 days in a dialysis bag, with the solution changed twice daily. The dialyzed product was lyophilized for further use.
2.2.3 Preparation of MSNs-Na
1.5 mg of phosphonic acid (PA) and 1.5 mg of PEG were dissolved in 1 mL of toluene. To this mixture, 0.5 mL of dry toluene containing 1 μL 3-triethoxysilylpropyl succinic anhydride (SATES) was added dropwise. Catalytic amounts of 4-dimethylaminopyridine (DMAP) dissolved in 1 mL toluene were added to the solution, and the reaction was allowed to proceed overnight. Subsequently, 1 mL of the solution was added dropwise to 5 mL of toluene containing 50 mg of previously dispersed MSNs, with vigorous stirring for 24 h. The functionalized MSNs were collected by centrifugation, washed twice with toluene and ethanol, and vacuum-dried for 16 h
2.2.4 Chitosan modification and piR7472 loading
The synthesized MSNs-Na were dispersed in an aqueous solution. To this suspension, 2 mL of 2 % chitosan solution was added dropwise and reacted for 6 h. piR7472 was then added to the solution and stirred for 4 h to obtain MSNs-Na@piR7472.
2.3 Characterization
The morphology of nanoparticles was characterized using scanning electron microscopy (SEM). The zeta potential of the nanoparticles was measured to assess surface charge.
2.4 N6-methyladenosine (m6A) methylation levels and osteogenesis-related gene expression
BMSC cells were co-cultured with different groups of materials for 2 or 3 weeks. After the culture period, m6A methylation levels and osteogenesis-related gene expression in each group were quantified using qRT-PCR.
2.5 m6A methylation detection
The m6A RNA methylation level in total RNA was quantified using an m6A RNA methylation quantification kit (colorimetric method), following the manufacturer's instructions. Each assay utilized 200 ng of total RNA.
2.6 In vitro biocompatibility of nanoparticles
BMSC and RAW264.7 cells were seeded at 1.0 × 105 cells/mL into 24-well plates. PBS (10 μL) and PBS containing nanoparticles from four groups (Blank, MSNs, MSNs-Na, and MSNs-Na@piR7472) were added to the wells. After 1, 3, 5, and 7 days, 10 μL of CCK-8 working solution was added to each well. After a 2-h incubation at 37 °C in a 5 % CO2 atmosphere, optical density (OD) at 450 nm was measured. Cell viability was also assessed using a Calcein-AM/PI dual staining kit after 1 and 3 days of culture.
2.7 Cellular uptake of nanoparticles
For obtained FITC-labeled MSNs-Na@piR7472, MSNs-Na@piR7472 and FITC were mixed in ethanol solution protected from light for 24 h. Unlabeled FITC was removed by centrifugation and washed with water to obtain FITC-labelled MSNs-Na@piR7472. BMSCs were seeded at 1.0 × 105 cells/mL into confocal dishes. After 12 h, 10 μL of FITC-labeled MSNs-Na@piR7472 solution was added, and nuclei were stained with DAPI. Fluorescence images were captured using a laser confocal microscope to evaluate nanoparticle uptake and distribution.
2.8 ARS and ALP staining
BMSCs were seeded at 1.0 × 105 cells/mL into 24-well plates and cultured under standard conditions. After 24 h, PBS, MSNs, MSNs-Na, and MSNs-Na@piR7472 were added to the wells and cultured with medium changes every 3 days. After 2 weeks, alkaline phosphatase (ALP) staining was performed to observe ALP protein content qualitatively. Alizarin Red S (ARS) staining was used to detect calcium mineralization, with observations made using an inverted fluorescence microscope.
2.9 qRT-PCR
Total cellular RNA was extracted using TRIzol reagent. cDNA was synthesized using the GoScript Reverse Transcription Mix (Promega, USA). qRT-PCR analysis was conducted using GoTaq qPCR Master Mix on a Roche LC480II system.
2.10 Establishment of an animal model of osteoporosis
An osteoporotic rat model was established as previously reported. Briefly, rats were anesthetized via intraperitoneal injection of sodium pentobarbital (Kirillova et al., 2018). After anesthesia, the rats were placed in the supine position on the surgical board. The back was shaved, and a 0.5–1 cm incision was made along the midline of the back. The skin and muscle were separated to expose the muscle layer. The muscle was incised 1 cm below the spine to reveal the adipose tissue surrounding the ovary and the uterine horn connected to the ovary. The adipose tissue was gently clamped and the ovary was excised. The uterine horn was ligated at both the upper and lower parts of the fallopian tube. The adipose tissue was pushed back into the abdominal cavity, and the peritoneum and skin were sutured.
2.11 In vivo therapeutic efficacy for osteoporosis
Post-ovariectomy, rats were randomly divided into 5 groups. Each group received weekly injections of respective nanoparticles for 8 weeks. After 8 weeks of treatment, the femurs of the rats were analyzed by micro-CT. Three rats from each group were randomly selected and euthanized by overexposure to high-concentration isoflurane. The femurs were dissected, soft tissues were removed, and the femur specimens were immersed in 4 % (w/v) paraformaldehyde. The specimens were subjected to micro-CT scanning and quantitative analysis, examining bone microstructural parameters such as bone volume/total volume (BV/TV), bone surface/total volume (BS/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) in the region of interest (ROI).
2.12 HE staining
Samples were decalcified in a slow decalcification solution for 15 days. After dehydration and paraffin embedding, 5 μm thick sections were prepared. Histological analysis was conducted using hematoxylin and eosin (H&E) and Masson's trichrome staining. Bone resorption was analyzed by tartrate-resistant acid phosphatase (TRAP) staining.
2.13 Statistical analysis
We used FlowJo 7.6 software to analyze the data obtained from flow cytometry. We report the data as mean values, either with or without the standard error of the mean. Thereafter, we performed the statistical analysis using a one-way ANOVA with the assistance of GraphPad Prism software. We used the symbols *, **, and *** to indicate statistical significance levels. These symbols indicate different levels of p < 0.05, p < 0.01, and p < 0.001, respectively.
3 Results and discussions
3.1 Preparation and characterization of MSNs-Na@piR7472
The preparation process of MSNs-Na@piR7472 is depicted in Fig. 1A. Through PEG-active ester, alendronate (aln) was conjugated with MSNs to enable the nanoparticles to target hydroxyapatite, allowing them to accumulate in osteoporotic regions (Zhang et al., 2023). Scanning electron microscopy (SEM) was used to examine the morphology of the nanoparticles in different groups (Fig. 1B). The particle size of the nanoparticles increased progressively with the loading of sodium bicarbonate and the modification with chitosan (Fig. 1C). Additionally, after the chitosan modification, the Zeta potential of the nanoparticles shifted from negative to positive. Because of chitosan is rich in positive charge (Feng et al., 2014). This cation-rich environment lays the groundwork for the subsequent loading of piR-7472.
Preparation and Characterization of MSNs-Na@piR7472. (A) Flowchart of MSNs-Na@piR7472 preparation. (B) Scanning electron microscope characterization of the morphology of each nanoparticle group. (C) Particle size and Zeta potential analysis of the nanoparticles. Sale bar, 100 nm.
3.2 In vitro biocompatibility and cellular uptake
Good biocompatibility is a prerequisite for further application of nanoparticles. Previous experiments have demonstrated that piR-7472 can promote osteogenic differentiation of BMSC cells by regulating m6A methylation, while aln and sodium bicarbonate can inhibit osteoclasts. Therefore, we assessed the biocompatibility of the nanoparticles co-cultured with BMSC and RAW264.7 cells using CCK-8 and LIVE/DEAD staining assays. As shown in Fig. 2A, the OD values of each cell group showed no significant difference compared to the control group on days 1, 3, 5, and 7 (p > 0.05), indicating no cytotoxicity of the nanomaterials. Additionally, LIVE/DEAD staining was performed on days 1 and 3. As illustrated in Fig. 2B and C, BMSC and RAW264.7 cells stained green at all time points, indicating good biocompatibility of the nanoparticles.
Biocompatibility of Nanoparticles. (A) Proliferation ability of BMSC and RAW264.7 cells co-cultured with various nanoparticle groups detected by CCK-8 assay. Images of live/dead staining of (B) BMSC and (C) RAW264.7 after 1 and 3 days (green: live cells; red: dead cells). Sale bar, 100um. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Aln can bind to mineral substances such as hydroxyapatite and has been widely used in the design of osteoporotic-targeted drugs. To evaluate the cellular targeting of MSNs-Na@piR7472 in vivo, FITC-labeled MSNs-Na@piR7472 were co-cultured with BMSC cells and observed via confocal microscopy. As shown in Fig. 3, the fluorescence intensity within cells increased with the co-culturing time, indicating that the cells continuously internalized the nanoparticles. This provides the necessary condition for piR-7472-mediated m6A methylation modification.
Cellular Uptake of Nanoparticles. Live cell imaging of FITC-labeled nanoparticles detected by CLSM. Scale bar, 25 μm.
3.3 In vitro osteogenic promotion
It has been reported that low concentrations of bisphosphonates can act on osteoblasts to promote their proliferation, although there are no studies on whether piR-7472 can enhance osteogenesis. To evaluate the effect of MSNs-Na@piR7472 on BMSC osteogenic differentiation, BMSC cells were co-cultured with PBS, MSNs, MSNs-Na, and MSNs-Na@piR7472 for 14 days, followed by ALP and ARS staining. Alkaline phosphatase (ALP) is an enzyme present on the cell membrane of osteoblasts, and its activity can be used to determine whether cells have undergone osteogenic differentiation. Alizarin Red S (ARS) stains calcium deposits and is used to analyze cell mineralization. As shown in Fig. 4A, the color of the MSNs-Na group was more pronounced than that of the control and MSNs groups, while the most distinct ALP staining was observed in the MSNs-Na@piR7472 group. Similarly, the most prominent calcium nodules were observed in the MSNs-Na@piR7472 group (Fig. 4B), indicating a significant in vitro osteogenic promoting effect of MSNs-Na@piR7472.
Microscopic Images of ALP Staining and Alizarin Red Staining. (A) ALP staining. (B) Alizarin Red staining. Scale bar, 100um. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Furthermore, we analyzed mRNA levels of each group via qRT-PCR. As shown in Fig. 5A–5, the expressions of ALP, Runx2, and VEGF were significantly increased in the MSNs-Na and MSNs-Na@piR7472 groups compared to the control group (p < 0.001), suggesting that the nanoparticles markedly promote osteogenesis. Immunofluorescence staining for Runx2 expression was also performed. As shown in Fig. 5D and F, there was no significant difference in Runx2 expression between the control and MSNs groups, while the fluorescence intensity of Runx2 significantly increased in the MSNs-Na and MSNs-Na@piR7472 groups.
In Vitro Osteogenic Promotion of MSNs-Na@piR7472. PCR detection of osteogenic-related genes (A) ALP, (B) Runx2, (C) VEGF in BMSCs. (D) Immunofluorescence analysis of osteogenic-related protein localization in BMSCs (Runx-2 protein: green, BMP2 protein: red, nucleus: blue). (E) Quantitative analysis of immunofluorescence intensity. (F) Effect of nanoparticles on RAW264.7 osteoclast differentiation. Scale bar, 50um. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
RAW264.7 cells are precursor cells of osteoclasts, capable of differentiating into osteoclasts under the influence of RANKL (Guan et al., 2024). Bisphosphonates, widely used clinically as anti-osteoporosis drugs, inhibit the formation and activity of osteoclasts. Our in vitro experiments confirmed that multifunctional polymers exhibited good affinity with cell membranes, enhancing the cellular uptake of drugs and improving their efficacy. To further validate this characteristic, we explored the effect of multifunctional polymers on osteoclast formation and activity in vitro. As shown in Fig. 5F, TRAP expression significantly decreased in the MSNs-Na and MSNs-Na@piR7472 groups, indicating that the nanoparticles inhibited osteoclast formation. In addition, RAWA264.7 may promote osteogenesis by upregulating the typical gene expression of pro-inflammatory cytokines (IL-18, IL-6, IL-1β, OSM) and downregulating the protein expression of inflammatory factor IκB under the stimulation of MSN nanoparticles (Shi et al., 2017).
3.4 In vivo osteogenesis
The aforementioned experiments demonstrated that our designed MSNs-Na@piR7472 nanoparticles can target the osteoporotic microenvironment, promoting BMSC osteogenic differentiation and inhibiting osteoclasts through the sustained release of sodium bicarbonate and piR7472. We then established an ovariectomy (OVX)-induced osteoporosis animal model to evaluate the in vivo efficacy of the nanoparticles (Fig. 6A). The therapeutic effects of the nanoparticles were assessed by detecting the expression of VEGF, Runx2, and RANKL genes after 2 or 3 weeks of treatment.
In Vivo Osteogenic Effect of Nanoparticles. (A) Schematic of in vivo detection process. (B) m6A, (C) Vascular Endothelial Growth Factor (VEGF), (D) Runx2, (E) Receptor Activator of Nuclear Factor κB Ligand (RANKL) expression. *p < 0.05, **p < 0.01, ***p < 0.001.
As previously reported, piR-7472 can regulate m6A methylation levels in cells, thereby modulating the expression of ALP, Runx2, and VEGF. VEGF is involved in the regulation of bone formation (osteogenesis) and bone resorption, affecting bone health and disease status (Zeng et al., 2020), along with promoting angiogenesis. Runx2 is a characteristic transcription factor of osteoblasts responsible for the activation of bone matrix genes such as type I collagen, osteocalcin, and ALP (van der Poest Clement et al., 2002). It initiates the differentiation program of osteoblasts, enabling the synthesis and mineralization of bone matrix. As shown in Fig. 6C and D, the expression of VEGF and Runx2 significantly decreased in the OVX and MSNs groups. After treatment, these indicators markedly improved, indicating that MSNs-Na@piR7472 may enhance bone formation (osteogenesis) and bone resorption through angiogenesis and osteoblast differentiation. The upregulation of Runx2 expression can promote the proliferation and differentiation of osteoblasts, contributing to an increase in bone density. RANKL, expressed by osteoblasts and bone marrow stromal cells, promotes the differentiation and maturation of osteoclasts by binding to RANK on osteoclast precursors (Rifas et al., 2003). As shown in Fig. 6E, consistent with previous results, the treatment groups significantly inhibited osteoclast formation.
3.5 Micro-CT analysis of bone morphology
To more accurately and comprehensively analyze changes in bone morphology parameters in mice, we performed Micro-CT scanning of the spine and tibia after 8 weeks of treatment (Xie et al., 2022). As shown in Fig. 7A, in the coronal and sagittal CT images of the femur, we found that the cortical bone of the femur in the control group was still thick, with densely arranged trabeculae and a small bone marrow cavity without osteoporotic manifestations. In the OVX group, the cortical bone became thinner with increased porosity, bone resorption pits filled the structure, and trabeculae became sparse and thin. After 8 weeks of treatment, the cortical thickness of each treatment group showed some improvement compared to the OVX group, but remained lower than that of the control group. In the 3D images, the OVX group showed very little bone tissue, mostly confined to the cortical bone edge, with discontinuous, slender, sparse trabeculae and significantly reduced bone mass. The microstructure of bone tissue in each treatment group showed varying degrees of improvement compared to the OVX group, with increased bone mass.
In Vivo Osteogenic Effect of Nanoparticles. (A) Three-dimensional reconstruction images of the distal femur in different groups. Parameters analyzed include (B) BMD (Bone Mineral Density), (C) BMC (Bone Mineral Content), (D) BVF (Bone Volume Fraction), (E) BV/TV (Bone Volume/Total Volume), (F) BS/BV (Bone Surface Area/Bone Volume), (G) SMI (Structure Model Index), (H) Tb.Th (Trabecular Thickness), (I) Tb.N (Trabecular Number), and (J) Tb.Sp (Trabecular Separation). *p < 0.05, **p < 0.01, ***p < 0.001.
Currently, dual-energy X-ray absorptiometry (DXA) is widely used for diagnosing osteoporosis and is considered the gold standard for diagnosis and monitoring treatment efficacy. Bone Mineral Density (BMD) refers to the amount of minerals (mainly calcium and phosphorus) per unit volume or unit area of bone. Bone Mineral Content (BMC) is the total amount of minerals in the entire bone. Bone Volume Fraction (BVF) is the proportion of bone tissue within a specific volume, representing the density of bone tissue in the trabecular bone structure (Pinna et al., 2021). As shown in Fig. 7B–D, BMD, BMC, and BVF significantly decreased in the OVX group. There was no significant difference between the MSNs group and the OVX group, while these parameters significantly increased in the treatment group.
To quantitatively analyze the therapeutic effect of multifunctional polymers on osteoporosis, we used six bone morphometric parameters BV/TV, BS/BV, SMI, Tb.Th, Tb.N, and Tb.Sp—to analyze the microstructure of the femur (Fig. 7E–J). Bone volume (BV) and tissue volume (TV) were used to evaluate basic bone morphology (Fig. 7E). Compared to the control group, the BV/TV of the OVX group decreased from 0.82 to 0.58, with no significant difference in BV/TV between the OVX and MSNs groups (p > 0.05). The BV/TV of the MSNs-Na and MSNs-Na@piR7472 treatment groups significantly increased.
Bone surface (BS) is another common parameter used to assess bone morphology (Vater et al., 2010). As shown in Fig. 7F, the BS/BV of the OVX group was the highest, while the BS/BV of the MSNs-Na and MSNs-Na@piR7472 treatment groups significantly decreased.
The structure model index (SMI) is used to assess trabecular bone structure. As shown in Fig. 7G, following successful OVX modeling, the SMI index increased from −7.5 to −2.3. After 2 weeks of MSNs-Na@piR7472 treatment, the SMI decreased to −6.8, indicating significant improvement.
Elevated SMI values indicate poorer bone structure, often associated with osteoporosis (Kirillova et al., 2021). In this study, after ovariectomy (OVX) in mice, the SMI increased from −8 to −1. Subsequent treatment with our system or PTH resulted in reductions to −6 and −2, respectively (Fig. 7G). Our system demonstrated superior outcomes compared to PTH management, highlighting the potential of this new approach.
Additionally, CT 3D reconstruction was employed to measure trabecular morphology. Average trabecular thickness (Tb.Th) and trabecular number (Tb.N) significantly decreased after OVX modeling but increased notably following treatment with Ns-Na and MSNs-Na@piR7472. Conversely, average trabecular separation (Tb.Sp) increased from 0.14 post-modeling to 0.35 mm but significantly decreased post-treatment, with the MSNs-Na@piR7472 group showing a recovery to 0.16, not significantly different from the Control group (p > 0.05).
These findings underscore the efficacy of MSNs-Na@piR7472 in restoring trabecular bone structure, suggesting its potential as a therapeutic intervention for osteoporosis.
3.6 Histopathological analysis of mouse femurs after 8 weeks of treatment
Histopathological analysis of mouse femurs was conducted to further verify the therapeutic effects of multifunctional polymers on osteoporosis, as depicted in Fig. 8A. Hematoxylin and eosin (H&E) staining images showed distinct differences among the groups. In the Control group, femoral trabeculae were dense, well-organized, and abundant, without signs of osteoporosis. In contrast, the OVX group exhibited sparse, thin, and reduced trabeculae, accompanied by multiple resorption pits, widened marrow cavity spacing, disrupted bone microstructure, and the most severe osteoporosis. The MSNs group showed no significant difference compared to the OVX group, while the MSNs-Na and MSNs-Na@piR7472 treatment groups demonstrated noticeable improvements.
Histological staining of nanoparticle therapeutic effects in vivo. (A) H&E staining. (B) Masson's trichrome staining. (C) TRAP staining.
Masson's trichrome staining (Fig. 8B) revealed that collagen fibers in the Control group were tightly packed and clustered. With worsening osteoporosis, collagen content notably decreased in the OVX group. After 8 weeks of treatment, collagen fiber quantity in the MSNs-Na and MSNs-Na@piR7472 groups was significantly higher compared to the OVX group.
Osteoclasts are critical effector cells in bone resorption, and tartrate-resistant acid phosphatase (TRAP) serves as a marker enzyme for osteoclasts. Enhanced osteoclast activity leads to increased TRAP secretion, indicated by darker staining in TRAP-stained sections. As shown in the enlarged TRAP staining image (Fig. 8C), the OVX group exhibited the highest number of intensely red-stained osteoclasts, predominantly located within resorption pits at the metaphysis. In contrast, TRAP staining intensity and distribution area were reduced in the MSNs-Na and MSNs-Na@piR7472 treatment groups, indicating inhibited osteoclast activity.
Histopathological analysis of mouse femurs at the distal end after 8 weeks of treatment demonstrated improved bone microstructure across various treatment groups. This improvement included increased cortical thickness, enhanced trabecular number, increased collagen fiber thickness, and reduced number and staining area of osteoclasts compared to the OVX group.
4 Conclusion
The MSNs-Na@piR7472 nanoparticles designed in this study effectively targeted bone tissue sites and regulated cellular m6A methylation levels through piR7472 release, thereby promoting osteogenic differentiation. Additionally, sodium bicarbonate inhibited osteoclast formation, preventing and ameliorating osteoporosis progression. Therefore, MSNs-Na@piR7472 nanoparticles exhibit promising therapeutic effects against osteoporosis and merit further investigation and application.
Funding
This research was funded by Tianjin Key Medical Discipline(Specialty) Construction Project, grant number TJYXZDXK-064B; Tianjin Municipal Science and Technology Project, grant number 22JCZDJC00250; Hunan Provincial Natural Science Foundation of China, grant number: 2024JJ7474. Shaoyang Science and Technology Project, grant number 2023NS2015.
CRediT authorship contribution statement
Yubin Long: Conceptualization, Writing – original draft, Funding acquisition. Yuan Ma: Data curation, Formal analysis. Houzhi Yang: Writing – original draft, Investigation, Methodology. Xiangbin Wang: Data curation, Methodology, Project administration. Jigeng Fan: Project administration, Resources, Software. Yong Li: Supervision, Validation, Visualization. Bo Tao: Formal analysis, Data curation, Conceptualization. Tianwei Sun: Validation, Visualization, Writing – review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- Osteoporosis remission via an anti-inflammaging effect by icariin activated autophagy. Biomaterials. 2023;297
- [CrossRef] [Google Scholar]
- Functional micro-RNA drugs acting as a fate manipulator in the regulation of osteoblastic death. Nanoscale. 2023;15:12840-12852.
- [CrossRef] [Google Scholar]
- Silk-based hydrogel incorporated with metal-organic framework nanozymes for enhanced osteochondral regeneration. Bioact. Mater.. 2023;20:221-242.
- [CrossRef] [Google Scholar]
- Effect of pH-responsive alginate/chitosan multilayers coating on delivery efficiency, cellular uptake and biodistribution of mesoporous silica nanoparticles based nanocarriers. ACS Appl. Mater. Interfaces. 2014;6:8447-8460.
- [CrossRef] [Google Scholar]
- RNA interference-based osteoanabolic therapy for osteoporosis by a bone-formation surface targeting delivery system. Biochem. Biophys. Res. Commun.. 2022;601:86-92.
- [CrossRef] [Google Scholar]
- The essential roles of m6A modification in osteogenesis and common bone diseases. Genes Dis.. 2024;11:335-345.
- [CrossRef] [Google Scholar]
- Magnetic aggregation-induced bone-targeting nanocarrier with effects of Piezo1 activation and osteogenic-angiogenic coupling for osteoporotic bone repair. Adv. Mater.. 2024;36:e2312081
- [CrossRef] [Google Scholar]
- GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c+MHCII+ macrophages and dendritic cells. Immunity. 2015;42:1197-1211.
- [CrossRef] [Google Scholar]
- Muscle-derived extracellular vesicles improve disuse-induced osteoporosis by rebalancing bone formation and bone resorption. Acta Biomater.. 2023;157:609-624.
- [CrossRef] [Google Scholar]
- Yam-derived exosome-like nanovesicles stimulate osteoblast formation and prevent osteoporosis in mice. J. Control. Release. 2023;355:184-198.
- [CrossRef] [Google Scholar]
- Bioinspired mineral-organic bioresorbable bone adhesive. Adv. Healthc. Mater.. 2018;7:e1800467
- [CrossRef] [Google Scholar]
- Reinforcement and fatigue of a bioinspired mineral-organic bioresorbable bone adhesive. Adv. Healthc. Mater.. 2021;10:e2001058
- [CrossRef] [Google Scholar]
- Cell membrane coated nanoparticles: cutting-edge drug delivery systems for osteoporosis therapy. Nanoscale. 2024;16:8236-8255.
- [CrossRef] [Google Scholar]
- Engineered bacterial extracellular vesicles for osteoporosis therapy. Chem. Eng. J.. 2022;450:138309
- [Google Scholar]
- Delivery of m7G methylated Runx2 mRNA by bone-targeted lipid nanoparticle promotes osteoblastic bone formation in senile osteoporosis. Nano Today. 2024;54
- [CrossRef] [Google Scholar]
- Human urine-derived stem cell exosomes delivered via injectable GelMA templated hydrogel accelerate bone regeneration. Mater. Today Bio. 2023;19
- [CrossRef] [Google Scholar]
- Black phosphorus nanosheets-enabled DNA hydrogel integrating 3D-printed scaffold for promoting vascularized bone regeneration. Bioact. Mater.. 2023;21:97-109.
- [CrossRef] [Google Scholar]
- Process parameters optimization of EDM for hybrid aluminum MMC using hybrid optimization technique. Heliyon.. 2024;10:e35555
- [CrossRef] [Google Scholar]
- Chemical design of nuclear-targeting mesoporous silica nanoparticles for intra-nuclear drug delivery. Chin. J. Chem.. 2018;36:481-486.
- [CrossRef] [Google Scholar]
- Nanoceria provides antioxidant and osteogenic properties to mesoporous silica nanoparticles for osteoporosis treatment. Acta Biomater.. 2021;122:365-376.
- [CrossRef] [Google Scholar]
- Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts. J. Cell. Biochem.. 2003;88:650-659.
- [CrossRef] [Google Scholar]
- Europium-doped mesoporous silica nanosphere as an immune-modulating osteogenesis/angiogenesis agent. Biomaterials. 2017;144:176-187.
- [CrossRef] [Google Scholar]
- Alendronate in the prevention of bone loss after a fracture of the lower leg. J. Bone Miner. Res.. 2002;17:2247-2255.
- [CrossRef] [Google Scholar]
- Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells. J. Biomed. Mater. Res. A. 2010;92:1452-1460.
- [CrossRef] [Google Scholar]
- A magnetic-field guided interface coassembly approach to magnetic mesoporous silica nanochains for osteoclast-targeted inhibition and heterogeneous nanocatalysis. Adv. Mater.. 2018;30:e1707515
- [CrossRef] [Google Scholar]
- Bioinspired drug-delivery system emulating the natural bone healing cascade for diabetic periodontal bone regeneration. Bioact. Mater.. 2023;21:324-339.
- [CrossRef] [Google Scholar]
- METTL3-mediated m6A modification increases Hspa1a stability to inhibit osteoblast aging. Cell Death Discov.. 2024;10:155.
- [CrossRef] [Google Scholar]
- Flourishing antibacterial strategies for osteomyelitis therapy. Adv. Sci. (Weinh.). 2023;10:e2206154
- [CrossRef] [Google Scholar]
- Mettl3-mediated m(6)A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis. Nat. Commun.. 2018;9:4772
- [CrossRef] [Google Scholar]
- piRNA-14633 promotes cervical cancer cell malignancy in a METTL14-dependent m6A RNA methylation manner. J. Transl. Med.. 2022;20:51.
- [CrossRef] [Google Scholar]
- Liposomal α-cyperone targeting bone resorption surfaces suppresses osteoclast differentiation and osteoporosis progression via the PI3K/Akt axis. Nano Res.. 2023;17:2949-2959.
- [CrossRef] [Google Scholar]
- Mesenchymal stem cell-derived extracellular vesicles: a possible therapeutic strategy for orthopaedic diseases: a narrative review. Biomater Transl.. 2022;3:175-187.
- [CrossRef] [Google Scholar]
- Sustained delivery of alendronate by engineered collagen scaffold for the repair of osteoporotic bone defects and resistance to bone loss. J. Biomed. Mater. Res. A. 2020;108:2460-2472.
- [CrossRef] [Google Scholar]
- Recapitulation of growth factor-enriched microenvironment via BMP receptor activating hydrogel. Bioact. Mater.. 2023;20:638-650.
- [CrossRef] [Google Scholar]