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Sildenafil promotes osteogenic differentiation of human mesenchymal stem cells and inhibits bone loss by affecting the TGF-β signaling pathway

Stem Cell Research & Therapy volume 16, Article number: 201 (2025) Cite this article

Abstract

Background

Osteoporosis, a common bone disorder, is primarily managed pharmacologically. However, existing medications are associated with non-trivial side-effects. Sildenafil, which already finds many clinical applications, promotes angiogenesis and cellular differentiation. Osteoporotic patients often exhibit a reduced intraosseous vasculature and impaired cellular differentiation; sildenafil may thus usefully treat osteoporosis.

Methods

Here, the effects of sildenafil on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) were explored, as were the molecular mechanisms in play. We treated hMSCs with varying concentrations of sildenafil and measured cell proliferation and osteogenic differentiation in vitro. We used a mouse model of subcutaneous ectopic osteogenesis to assess sildenafil’s effect on hMSC osteogenic differentiation in vivo. We also explored the effects of sildenafil on bone loss in tail-suspended (TS) and ovariectomized (OVX) mice. Mechanistically, we employed RNA-sequencing to define potentially relevant molecular pathways.

Results

The appropriate concentrations of sildenafil significantly enhanced osteogenic hMSC differentiation; the optimal sildenafil concentration may be 10 mg/L. Sildenafil mitigated osteoporosis in OVX and TS mice. The appropriate concentrations of sildenafil probably promoted hMSC osteogenic differentiation by acting on the transforming growth factor-β (TGF-β) signaling pathway.

Conclusions

In conclusion, sildenafil enhanced hMSC osteogenic differentiation and inhibited bone loss. Sildenafil may usefully treat osteoporosis. Our findings offer new insights into the physiological effects of the medicine.

Graphical Abstract

Background

Osteoporosis is a common bone disorder associated with reduced bone density and quality, and microstructural damage; bones become more fragile and the fracture risk rises. Osteoporosis affects not only patients but also their families and society [1]. The global prevalence of osteoporosis in older adults is 21.7% [2, 3]. The numbers of intraosseous blood vessels decrease in patients with senile and postmenopausal osteoporosis [4]. Such changes in bone structure may be related to impaired osteogenic differentiation capacities of mesenchymal stem cells (MSCs) [5, 6]. Studies have shown that hMSCs derived from osteoporotic patients exhibit reduced osteogenic differentiation potential and decreased mineralization compared to those from healthy donors [7, 8]. MSCs are a widely distributed class of pluripotent stem cells with the ability to self-renew and differentiate into various cell types. Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ASCs) are the two most extensively studied types of MSCs. In recent years, with advancing research on MSC differentiation mechanisms, their potential applications in osteoporosis treatment have gained increasing attention. MSCs play a crucial role in maintaining bone metabolic balance and homeostasis, and alterations in their proliferation and differentiation potential are key pathogenic mechanisms of osteoporosis [9, 10]. Regulating MSC differentiation balance to promote osteogenesis offers a promising therapeutic strategy for osteoporosis and related diseases.

Many osteoporosis management and prevention strategies have been developed [11, 12]. Currently, pharmacotherapy is the simplest, most convenient, and most effective approach; the fracture risk is decreased either by reducing bone resorption or stimulating bone formation. Bisphosphonates find many clinical applications, but the adverse effects include upper gastrointestinal tract symptoms, renal toxicity, and (rarer) complications such as medication-related jaw osteonecrosis [11, 13]. New pharmacological interventions are required.

Any new drug must be evaluated both preclinically and clinically; this is expensive (over 2 billion dollars), risky, and time-consuming (10 to 15 years) [14, 15]. New indications for existing drugs are thus very attractive.

Sildenafil, the small-molecule C22H30N6O4S, is currently primarily used to treat erectile dysfunction but may also aid patients with fatty liver and heart failure [4, 16]. Sildenafil improves vascular function, promotes angiogenesis, and enhances wound-healing [17,18,19,20,21]. As an inhibitor of phosphodiesterase type 5 (PDE5), sildenafil enhances the activity of the nitric oxide/soluble guanylate cyclase/cyclic guanosine monophosphate (NO/sGC/cGMP) pathway that controls cell growth and differentiation and smooth muscle relaxation [16]. NO stimulates both bone regeneration and new blood vessel formation via the NO/sGC/cGMP pathway [22,23,24]. Given the close relationship between angiogenesis, NO activity, and osteogenesis, and that of the reduced bone vasculature and impaired cell differentiation of osteoporosis patients, we hypothesized that sildenafil might effectively treat osteoporosis. We thus explored whether sildenafil enhanced hMSC osteogenic differentiation. To the best of our knowledge, no study has yet addressed this topic. Importantly, sildenafil is an approved drug; sildenafil is safe.

Extensive literature has documented various animal models for osteoporosis research. Primary osteoporosis includes postmenopausal and senile osteoporosis. Ovariectomized (OVX) mouse and rat models are widely used to study postmenopausal osteoporosis [25, 26], while senile osteoporosis models typically involve naturally aging mice or rats [27]. Secondary osteoporosis models include various types, such as weightlessness-induced osteoporosis and glucocorticoid-induced osteoporosis (GIOP). The tail-suspended (TS) mouse model simulates bone loss under microgravity conditions and prolonged bed rest-induced osteoporosis. The GIOP model induces bone loss through long-term administration of glucocorticoids [28, 29]. These models serve as essential tools for investigating the pathophysiology of osteoporosis and developing novel therapeutic strategies. This study selects one model from both primary and secondary osteoporosis for further exploration, utilizing OVX and TS mouse models to simulate postmenopausal and weightlessness-induced osteoporosis, respectively.

We here examine how sildenafil affects hMSC osteogenic differentiation both in vivo and in vitro, and the potential molecular mechanisms in play. Sildenafil at 10 mg/L promoted hMSC osteogenic differentiation both in vitro and in vivo, the latter in a model of ectopic bone formation; and inhibited bone loss in ovariectomized mice and those suspended by their hindlimbs. Sildenafil may modulate the TGF-β signaling pathway. We expand the therapeutic applications of sildenafil; the material may usefully treat osteoporosis.

Materials and methods

hMSC culture

Human bone marrow-derived MSCs (hBMSCs) and human adipose-derived mesenchymal stem cells (hASCs) (ScienCell Company, USA) were grown at 37 °C. The proliferation medium (PM) was α-minimum essential medium (α-MEM) for hBMSCs and Dulbecco’s modified Eagle’s medium (DMEM) for hASCs, both supplemented with fetal bovine serum (FBS) and penicillin/streptomycin. Each osteogenic medium (OM) was DMEM or α-MEM supplemented with β-glycerophosphate, dexamethasone, L-ascorbic acid, penicillin/streptomycin, and FBS. DMEM (11965092), α-MEM (12571063), the penicillin-streptomycin mixture (15140122), and FBS (A5670701) were purchased from Gibco (Grand Island, USA). β-Glycerophosphate (G9422), dexamethasone (265005), and L-ascorbic acid (A4403) were all purchased from Sigma-Aldrich (USA). The details have been previously described [30].

The sildenafil concentrate

Sildenafil (Y0001578, Sigma-Aldrich, China) was dissolved in PM with 1‰ dimethyl sulfoxide (DMSO) to 100 mg/L and then diluted to 1, 5, 10, 20, and 40 mg/L in PM or OM.

Cell proliferation assay

hBMSCs and hASCs were seeded at a density of 2000 cells per well into 96-well plates. Cell proliferation was assessed on days 0, 1, 3, 5, 7, and 14 (three replicate wells). During detection, the culture medium was removed, and cells were washed three times with PBS. Fresh medium containing 10% (v/v) Cell Counting Kit-8 (CCK-8) was added and incubated at 37 °C for 1 h. The supernatant was then transferred to a new 96-well plate for measurement. Cells were counted using a CCK-8 (Dojindo Laboratories, Japan) and the absorbance at 450 nm was measured to quantify cell proliferation by a microplate reader (ELx800, Biotek, America). The details have been previously described [31].

Scratch assay

hBMSCs and hASCs were cultured in six-well plates to approximately 70% confluence; scratches were created using the tip of a 200-µL pipette, and serum-free media with varying concentrations of sildenafil added. The cells were photographed under an inverted optical microscope (TE2000-U, Nikon, Japan) at 0, 12, and 24 h. Image J software (Open access, USA) was used to measure cell migration, as follows:

Cell migration ratio (%)\(\:=\frac{\text{A}0-\:\text{A}\text{t}}{\text{A}0}\times\:100\%\)

where A0 and At are the respective scratch areas before and after addition of serum-free culture media [32].

Transwell assay

PM (600 µL) was added to the lower chamber and 1 × 105 hBMSCs or hASCs in 200 µL of serum-free medium supplemented or not with various concentrations of sildenafil to the upper chamber. The chambers were separated by a membrane filter with pores 8 μm in diameter (Corning, USA). After 24 h of incubation, the upper chamber (with non-migratory cells) was removed. The membrane was fixed, and stained for 10 min in 0.1% (w/v) crystal violet; then washed, dried, images captured, and migrated cells counted. The details have been previously published [30].

Alkaline phosphatase (ALP) staining and quantification

Cells (20,000) were added to each well of a 12-well plate, grown to 70–80% confluence, and osteogenically induced. Experiments commenced after 7 days of culture in OM with various concentrations of sildenafil. For ALP staining, cells were washed with PBS, fixed with pre-cooled 95% ethanol for at least 15 min, and gently rinsed with PBS. An equal volume of working solution was then added to each well, ensuring complete coverage of the well bottom. For ALP activity quantification, total protein was extracted and its concentration measured before analysis using a commercial assay kit. ALP staining/assessment employed a dedicated kit (Beyotime, China) and a microplate reader (ELx800, Biotek, USA). The details have been published previously [31].

Alizarin red S (ARS) staining and quantification

Cells (20,000) were added to each well of a 12-well plate, grown to 70–80% confluence, and osteogenically induced. After incubation in PM, OM, or OM with various concentrations of sildenafil for 14 days, hBMSCs or hASCs were stained with an ARS solution (Sigma-Aldrich, USA) for 10–20 min and imaged (HP Scanjet G4050; Hewlett-Packard, USA). To quantify staining, mineralized nodules were dissolved in 100 mM cetylpyridinium chloride and absorbances at 490 nm recorded using a spectrophotometer (ELx800; Biotek, USA).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Cells (50,000) were added to each well of a 6-well plate, grown to 70–80% confluence, and osteogenically induced. Total RNAs were prepared from hBMSCs and hASCs cultured for 7 and 14 days. The equipment and methods used to determine total RNA concentrations, reverse transcription to cDNA, and qRT-PCR, have been published previously [31]. The primers are listed in Table 1.

Table 1 The primer sequences
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Subcutaneous cell transplantation into nude mice

Guided by the in vitro results, sildenafil at 10 mg/L was used in vivo. Twelve BALB/C nude mice (female, 6 weeks of age) were randomly divided into two groups of five and given hBMSCs grown in either PM or PM + 10 mg/L sildenafil. hBMSCs that had been cultured for 7 days were mixed with β-tricalcium phosphate (Rebone, China) and then subcutaneously implanted into the dorsa of nude mice. Samples collected after 8 weeks were subjected to Masson staining, hematoxylin and eosin (H&E) staining, and immunohistochemical staining for OCN (osteocalcin) to evaluate osteogenesis [33]. We thus explored whether sildenafil aided the heterotopic osteogenic differentiation of hBMSCs in vivo.

The mice were purchased from the Vital River Corporation (China). All animal experiments were complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. In the animal experiments of this study, the animals were housed under SPF condition with a controlled temperature of 20–26 °C, relative humidity of 40–70%, and a 12/12-hour light/dark cycle. The sample size for each group of mice was determined based on previous studies [31]. The mice were first acclimated for one week, after which those without apparent abnormalities in appearance or behavior were selected. They were then randomly assigned to different groups using a random number generator. Experimental and control groups were randomly assigned. If a mouse died due to surgery, it was excluded from the group, and a new mouse from the same batch was randomly selected for subsequent experiments. All mice were anesthetized with avertin (a 1:1 mixture of tribromoethanol and tert-amyl alcohol), which was diluted to 0.25% with saline before use and administered via intramuscular injection at a dose of 150 mg/kg (‌MA0478‌‌1, meilunbio, China) [34, 35]. Euthanasia of mice was performed by cervical dislocation following deep anesthesia. Data analysts were blinded to the group assignments during analysis.

Intraperitoneal injection of ovariectomized (OVX) mice

Mice were anesthetized with avertin (150 mg/kg), and bilateral ovariectomy was performed through a dorsal approach to remove the ovaries. Sham-operated mice underwent the same procedure without ovary removal. The OVX model induces estrogen deficiency, which mimics the pathophysiological changes observed in postmenopausal osteoporosis [36].

The daily drug dosage for each mouse was 1 mg/kg. The calculation was as follows: an adult mouse weighs approximately 25 g, with a circulating blood volume of about 2.5 mL. Based on the optimal concentration of 10 mg/L for promoting osteogenic differentiation of hMSCs in vitro, the corresponding in vivo dosage was determined [31].

Twenty female SPF C57BL/6 N mice (8 weeks of age) were categorized into four groups (n = 5): Sham + PBS, sham + sildenafil, OVX + PBS, and OVX + sildenafil. Sildenafil was intraperitoneally injected daily for 1 month commencing 3 months after surgery; the control group receiving an equivalent volume of PBS. After 1 further month, femoral osteogenesis was evaluated via H&E staining, Masson staining and micro-CT. Heart, liver, spleen, lung, kidney, and blood samples were subjected to H&E staining and serological analysis.

Intraperitoneal injection of tail-suspended mice

Mice were tail-suspended with the heads tilted downward at 30° and the hind limbs elevated for 14 days. Sham-treated mice were not suspended, mouse movement was not restricted [29]. Twenty female SPF C57BL/6 N mice (8 weeks of age) were divided into four groups (n = 5): Sham + PBS, sham + sildenafil, suspension + PBS, and suspension + sildenafil. The daily sildenafil dosage for each mouse was 1 mg/kg, calculated using the same method as described above. Commencing on day 14 after tail suspension, mice received daily intraperitoneal injections of sildenafil or PBS for 14 days, after which samples were collected. Femora were subjected to H&E staining, Masson staining and micro-CT analysis that evaluated osteogenesis status. Heart, liver, spleen, lung, kidney, and blood samples were taken for H&E staining and serological analysis.

ELISA of serum biomarkers

Blood samples were obtained from the mice of Sect. 2.11 and 2.12 above. Bone alkaline phosphatase (BALP) and procollagen type 1 N-terminal propeptide (P1NP), both of which are indicators of bone formation, were quantitated using ELISA kits (Telenbiotech, China). For detailed experimental procedures, refer to the instruction manual.

Micro-computed tomography (CT)

Mouse femora were fixed in 10% (v/v) formalin for 24 h and then scanned from the proximal end. The scan time was 1,500 ms and the scan resolution 8.82 μm. Data analysis employed an Inveon Research Workplace (Siemens, Germany). This derived the bone mineral density (BMD), bone surface area/bone volume (BS/BV), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N).

RNA-sequencing (RNA-Seq) and statistical analyses

hBMSCs (50,000) were seeded into each well of a six-well plate, grown to 70–80% confluence, osteogenically induced in OM (control) and OM with 10 mg/L sildenafil for 7 days, and RNAs collected. RNA-Seq was performed by Novogene Bioinformatics Technology Co. Ltd. All RNA samples underwent rigorous quality control, primarily using an Agilent 2100 bioanalyzer. High-quality libraries underwent Illumina sequencing. RNA-Seq identifies differences in gene expression via reference genome alignment; quality control; and quantitative, functional enrichment, differential expression, alternative splicing, and variant site analyses.

Western blotting

hBMSCs were induced in OM (control) and OM supplemented with 10 mg/L sildenafil for 7 days and proteins collected. A digital Western blot system (Simple Western Blot; Wes Separation Module, ProteinSimple, USA) was used to measure the expression levels of TGF-β1, TGF-βR2, p-TGF-βR2, Smad2/3 and p-Smad2/3 proteins and the data were analyzed with the aid of Compass software (ProteinSimple) [37]. Detailed experimental procedures can be found in the manufacturer’s instructions. The following antibodies were employed: anti-GAPDH rabbit polyclonal antibody (HX1832, Huaxingbio, China), anti-TGF-β1 antibody (ab215715, abcam, UK), anti-TGF-βR2 antibody (T56879, abmart, China), anti-TGF-βR2 (phosphor-S225) antibody (ab183037, abcam, UK), anti-Smad2 + Smad3 antibody [EPR19557-4] (ab202445, abcam, UK), and anti-Smad2 + Smad3 (phospho T8) antibody (ab254407, abcam, UK).

Statistical analyses

Data were subjected to one-way analysis of variance (ANOVA) using SPSS ver. 24.0 software (IBM, USA). A P-value < 0.05 was considered statistically significant. All results are presented as means ± standard deviations (SDs). The work has been reported in line with the ARRIVE guidelines 2.0.

Results

The appropriate concentrations of sildenafil enhanced the proliferation and migration of hBMSCs and hASCs in vitro

Compared to the PM-alone group, 10 mg/L sildenafil most potently enhanced the proliferation of hBMSCs and hASCs in vitro (Figs. 1B, S1A), as revealed by the CCK-8 data. The scratch and transwell assays similarly showed that 10 mg/L sildenafil most effectively promoted migration of hBMSCs and hASCs in vitro (Figs. 1C-F, S1B-E). When the sidenafil concentration was below or above 10 mg/L, efficacy decreased. In summary, 10 mg/L sildenafil enhanced the proliferation and migration of hBMSCs and hASCs in vitro.

Fig. 1
figure 1

The appropriate concentrations of sildenafil enhanced the proliferation and migration of hBMSCs in vitro. A. Chemical structure of sildenafil; B. hBMSC growth curve derived using the CCK-8 assay; C, D. The transwell assay was employed to investigate how sildenafil at different concentrations affected hBMSC migration; E, F. The morphologies and quantitative analyses of the scratch assay migration areas used to explore the effects of sildenafil at different concentrations on hBMSC migration. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the PM group. N ≥ 3. Transwell assay image [scale bar]: 100 μm. Scratch assay image [scale bar]: 500 μm. hBMSCs, human bone marrow-derived mesenchymal stem cells

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The appropriate concentrations of sildenafil enhanced the osteogenic differentiation of hBMSCs and hASCs in vitro

Quantification of the ALP activity and ALP staining of hBMSCs indicated that sildenafil at 5, 10, and 20 mg/L promoted osteogenic differentiation in vitro; sildenafil at 10 mg/L exerted the greatest effects. ARS staining and quantification were performed after 14 days of culture. The results were consistent with those for ALP activity and ALP staining (Fig. 2A-D). After 7 days of osteogenic induction, qRT-PCR showed that 10 mg/L sildenafil most significantly (compared to other levels) enhanced the expression of osteogenic genes (RUNX2, ALP) (Fig. 2E). Similarly, after 14 days, sildenafil at 10 mg/L optimally promoted RUNX2 and BGLAP expression in hBMSCs (Fig. 2F). The sildenafil-induced enhancement of hBMSC osteogenic differentiation in vitro fell at sildenafil concentrations below or over 10 mg/L. The hASC data were similar to those of hBMSCs. ALP staining and quantification, and ARS staining and quantification, indicated that 10 mg/L sildenafil most effectively enhanced osteogenic differentiation (Fig. S2A-D). qRT-PCR similarly showed that sildenafil at 10 mg/L exhibited the strongest effect (Fig. S2E, F). In summary, 10 mg/L sildenafil optimally promoted the osteogenic differentiation of hBMSCs and hASCs in vitro.

Fig. 2
figure 2

The appropriate concentrations of sildenafil enhanced osteogenic differentiation of hBMSCs in vitro. A. ALP staining revealing the effects of sildenafil at different concentrations on the osteogenic differentiation of hBMSCs; B. ARS staining revealing the effects of sildenafil at different concentrations on the osteogenic differentiation of hBMSCs; C. Quantification of ALP staining; D. Quantification of ARS staining; E. The relative levels of mRNAs encoding RUNX2 and ALP in hBMSCs as revealed by qRT-PCR after 7 days of osteogenic induction; F. The relative levels of mRNAs encoding RUNX2 and BGLAP in hBMSCs after 14 days of osteogenic induction. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the OM group. hBMSCs, human bone marrow-derived mesenchymal stem cells; ALP, alkaline phosphatase; ARS, alizarin red S; RUNX2, runt-related transcription factor 2; BGLAP, bone gamma-carboxyglutamate protein; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; OM, osteogenic medium

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Sildenafil at 10 mg/l promoted osteogenic differentiation of hBMSCs in vivo

The above in vitro experiments revealed that 10 mg/L sildenafil optimally promoted the proliferation and migration of hMSCs. More importantly, sildenafil at 10 mg/L optimally enhanced osteogenic differentiation in vitro. Therefore, sildenafil at 10 mg/L was used in the subsequent in vivo experiments.

H&E staining showed that the PM + sildenafil group exhibited more new bone formation than did the PM group (Fig. 3A, D). Masson staining indicated that the PM + sildenafil group exhibited increased collagen formation (Fig. 3B, E). Furthermore, the results of OCN immunohistochemical staining demonstrated that the PM + sildenafil group exhibited more brown-stained tissue, indicating a higher expression of OCN compared to the PM group (Fig. 3C, F). Thus, 10 mg/L sildenafil enhanced the osteogenic differentiation of hBMSCs in vivo and promoted ectopic bone formation.

Fig. 3
figure 3

Sildenafil at 10 mg/L promoted osteogenic differentiation of hBMSCs in the subcutaneous ectopic osteogenesis model. A. H&E staining of the PM and the PM + sildenafil groups; B. Masson trichrome staining of the PM and the PM + sildenafil groups; C. Immunohistochemical staining for OCN of the PM and the PM + sildenafil groups; D. Statistical analysis of the heterotopic bone area/tissue area (%) in the H&E staining of the PM and PM + sildenafil groups; E. Statistical analysis of the collagen area/tissue area (%) in the Masson staining of the PM and PM + sildenafil groups; F. Statistical analysis of the relative IOD in the immunohistochemical staining for OCN of the PM and PM + sildenafil groups. [scale bars]: 500 μm (10x) and 100 μm (40x). hBMSCs, human bone marrow-derived mesenchymal stem cells; H&E, hematoxylin and eosin; PM, proliferation medium; OCN, osteocalcin

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Sildenafil at 10 mg/l inhibited bone loss in OVX mice

Micro-CT revealed that the surgical group injected with PBS exhibited more significant bone loss than did the sham surgical group; the OVX mouse model was successfully established. Gross micro-CT images of femora showed that OVX mice injected with sildenafil exhibited denser bone and more trabeculae than did the OVX + PBS group (Fig. 4A). Sildenafil injection significantly increased the Tb.Th, BV/TV, BMD, and Tb.N values compared to those of the OVX + PBS group, and decreased BS/BV and Tb.Sp (Fig. 4B-G). Thus, sildenafil (compared to PBS) reduced bone loss. ELISAs showed that sildenafil (compared to PBS) increased the serum BALP and P1NP levels; sildenafil promoted bone formation and prevented bone loss in OVX mice (Fig. 4H, I).

Fig. 4
figure 4

Sildenafil at 10 mg/L alleviated osteoporosis in OVX mice. A. Micro-CT images, H&E staining and Masson staining of the sham + PBS, sham + sildenafil, OVX + PBS, and OVX + sildenafil groups; B-G. The BMD, BV/TV, Tb.N, Tb.Th, BS/BV, and Tb.Sp values of the of sham + PBS, sham + sildenafil, OVX + PBS, and OVX + sildenafil groups; H, I. The expression levels of the bone formation-related serum markers BALP and P1NP in the sham + PBS, sham + sildenafil, OVX + PBS, and OVX + sildenafil groups. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001. n ≥ 3. H&E staining image [scale bar]: 500 μm. OVX, ovariectomized; micro-CT, micro-computed tomography; PBS, phosphate balanced solution; H&E, hematoxylin and eosin; BMD, bone mineral density; BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; BS/BV, bone surface area/bone volume; Tb.Sp, trabecular separation; BALP, bone alkaline phosphatase; P1NP, procollagen type 1 N-terminal propeptide

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Femoral H&E staining and Masson staining indicated that the OVX + sildenafil group exhibited more new bone formation than the OVX + PBS group (Fig. 4A). H&E staining of liver, heart, kidney, lung, and spleen samples from all four groups revealed no significant toxicity; sildenafil at 10 mg/L exhibited good biocompatibility in vivo (Figure S3) and effectively mitigated bone loss in OVX mice.

Sildenafil at 10 mg/l suppressed bone loss in TS mice

To investigate whether sildenafil mitigated bone loss under weightless conditions, we tail-suspended mice to simulate weightlessness. Micro-CT revealed that bone loss was greater in the suspension + PBS group than the sham + PBS group; the TS model was successfully established. The micro-CT data were similar to those of Sect. 3.4, thus confirming significantly less bone loss in the suspension + sildenafil group compared to the suspension + PBS group. The former group exhibited a denser bone structure and more trabeculae than the latter group (Fig. 5A). Abdominal injection of sildenafil into TS mice significantly increased the femoral Tb.Th, BV/TV, Tb.N, and BMD values, and decreased the Tb.Sp and BS/BV (Fig. 5B-G). The serum levels of BALP and P1NP were higher in the suspension + sildenafil group than in the suspension + PBS group (Fig. 5H, I). H&E staining similarly demonstrated reduced bone loss in the group injected with sildenafil, compared to PBS (Fig. 5A). H&E staining of spleen, liver, heart, kidney, and lung sections from all groups revealed no significant toxicity (Fig. S4). In summary, 10 mg/L sildenafil effectively inhibited bone loss in TS mice.

Fig. 5
figure 5

Sildenafil at 10 mg/L alleviated osteoporosis in TS mice. A. Micro-CT images, H&E staining and Masson staining of the sham + PBS, sham + sildenafil, suspension + PBS, and suspension + sildenafil groups; B-G. The BMD, BV/TV, Tb.N, Tb.Th, BS/BV, and Tb.Sp values of the sham + PBS, sham + sildenafil, suspension + PBS, and suspension + sildenafil groups; H, I. The expression levels of the bone formation-related serum markers BALP and P1NP in the sham + PBS, sham + sildenafil, suspension + PBS, and suspension + sildenafil groups. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001. n ≥ 3. H&E staining image [scale bar]: 500 μm. TS, tail-suspended; micro-CT, micro-computed tomography; PBS, phosphate balanced solution; H&E, hematoxylin and eosin; BMD, bone mineral density; BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; BS/BV, bone surface area/bone volume; Tb.Sp, trabecular separation; BALP, anti-bone alkaline phosphatase; P1NP, type I N terminal propeptide

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The appropriate concentrations of sildenafil promoted osteogenic differentiation of hBMSCs by modulating the TGF-β signaling pathway.

hBMSCs (a control OM group and a test OM + sildenafil group) were used to explore how sildenafil enhanced osteogenic differentiation of hBMSCs. The Venn diagram of co-expressed genes revealed 10,154 such genes between the two groups (Fig. 6A). The volcano plot of differentially expressed genes showed that, compared to the OM group, the OM + sildenafil group exhibited 2,048 upregulated and 1,734 downregulated genes (Fig. 6B). Gene Ontology (GO) enrichment maps revealed the differential enrichment of genes involved in relevant activities (Fig. 6C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment scatter-plot illustrated the most significantly enriched pathways (Fig. 6D). Quality control plots are provided in the supplementary materials (Fig. S5-7), demonstrating the reliability of our results of RNA-Seq. Of these, the TGF-β signaling pathway exhibited notable between-group difference and thus a close association with osteogenesis. The levels of mRNAs encoding TGF-βR1, TGF-βR2, TGF-β1, and TGF-β2 (key components of TGF-β signaling) were significantly higher in the OM + sildenafil group than in the OM group (Fig. 6E-H). At the protein level, the levels of TGF-β1, p-Smad2/3/Smad2/3, and p-TGF-βR2/TGF-βR2 were significantly higher in the OM + sildenafil group than in the OM group (Fig. 6I-L). We thus (preliminarily) suggest that the appropriate concentrations of sildenafil may enhance the osteogenic differentiation of hBMSCs by influencing the TGF-β signaling pathway.

Fig. 6
figure 6

Sildenafil may promote osteogenic differentiation of hBMSCs by modulating the TGF-β signaling pathway. A. The Venn diagram of co-expressed genes in the OM and OM + sildenafil groups; B. The volcano plot of differentially expressed genes in the OM and OM + sildenafil groups; C. GO gene enrichment maps; D. The KEGG enrichment scatter plot; E-H. qRT-PCR analysis of the relative levels of mRNAs encoding TGF-β1, TGF-β2, TGF-βR1, and TGF-βR2 in hBMSCs; I. Western blots revealing the expression levels of TGF-β1, TGF-βR2, p-TGF-βR2, Smad2/3, p-Smad2/3, and GAPDH. Full-length blots are presented in Supplementary Figures (Fig. S8-12), the samples derived from the same experiment and that blots were processed in parallel; J-L. Quantification of TGF-β1, p-TGF-βR2/ TGF-βR2, and p-Smad2/3/ Smad2/3 expression. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to OM. N ≥ 3. hBMSCs, human bone marrow-derived mesenchymal stem cells; OM, osteogenic medium; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; TGF-β1, transforming growth factor-β1; TGF-β2, transforming growth factor-β2; TGF-βR1, transforming growth factor-β type I receptor; TGF-βR2, transforming growth factor-β type II receptor; p-TGF-βR2, phospho- transforming growth factor-β type II receptor; p-Smad2/3, phospho-Smad2/3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OM, osteogenic media

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Fig. 7
figure 7

Sildenafil promoted osteogenic differentiation of hBMSCs by activating the TGF-β signaling pathway. A. ALP staining revealing the effects of sildenafil and the TGF-β pathway inhibitor SB431542 on the osteogenic differentiation of hBMSCs; B. ARS staining revealing the effects of sildenafil and the TGF-β pathway inhibitor SB431542 on the osteogenic differentiation of hBMSCs; C. Quantification of ALP staining; D. Quantification of ARS staining; E. The relative levels of mRNAs encoding RUNX2 and ALP in hBMSCs as revealed by qRT-PCR after 7 days of osteogenic induction; F. The relative levels of mRNAs encoding RUNX2 and BGLAP in hBMSCs after 14 days of osteogenic induction. The data are means ± standard deviations. *p < 0.05, **p < 0.01, and ***p < 0.001. hBMSCs, human bone marrow-derived mesenchymal stem cells; ALP, alkaline phosphatase; ARS, alizarin red S; RUNX2, runt-related transcription factor 2; BGLAP, bone gamma-carboxyglutamate protein; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; OM, osteogenic medium; TGF-β, transforming growth factor-β

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To further validate the role of the TGF-β signaling pathway, we conducted additional experiments using the TGF-β inhibitor SB431542 [38, 39]. hBMSCs were divided into four groups: PM, OM, OM + sildenafil, and OM + sildenafil + SB431542 groups. ALP staining and activity quantification, as well as ARS staining and quantification, demonstrated that the osteogenic differentiation of hBMSCs was enhanced in the OM + sildenafil group compared to the OM group. However, the OM + sildenafil + SB431542 group showed reduced osteogenic differentiation of hBMSCs compared to the OM + sildenafil group (Fig. 7A-D). The results of qRT-PCR showed that after 7 days of osteogenic induction, the expression of RUNX2 and ALP were significantly reduced in the OM + sildenafil + SB431542 group compared to the OM + sildenafil group. Similarly, after 14 days of osteogenic induction, the mRNA expression levels of RUNX2 and BGLAP were significantly reduced in the OM + sildenafil + SB431542 group compared to the OM + sildenafil group (Fig. 7E-F). These findings indicate that inhibiting the TGF-β signaling pathway weakens the pro-osteogenic effect of sildenafil on hBMSCs, further confirming that sildenafil promotes osteogenic differentiation of hBMSCs through the TGF-β signaling pathway. However, further investigation is required.

Discussion

We found that the appropriate concentrations of sildenafil (optimally 10 mg/L) enhanced the migration and proliferation of hMSCs in vitro and promoted osteogenic differentiation of hMSCs both in vivo and in vitro. In vivo, sildenafil mitigated bone loss in both TS and OVX mice. Sildenafil may facilitate osteogenic differentiation of hBMSCs by influencing the TGF-β signaling pathway (Fig. 8). Sildenafil has been FDA-approved to treat various conditions, and is affordable and safe [40]. The fact that sildenafil inhibits bone loss suggests that sildenafil may usefully treat osteoporosis.

Fig. 8
figure 8

Sildenafil promotes osteogenic differentiation of hMSCs and suppresses bone loss by affecting TGF-β signaling. The appropriate concentrations of sildenafil enhanced the proliferation and migration of hMSCs in vitro, and promoted osteogenic differentiation of hMSCs both in vitro and in vivo. In vivo, 10 mg/L sildenafil reduced bone loss in both OVX and TS mice. Sildenafil may promote osteogenic differentiation of hBMSCs by influencing the TGF-β signaling pathway. hMSCs, human mesenchymal stem cells; TGF-β, transforming growth factor-β; OVX, ovariectomized; TS, tail-suspended; hBMSCs, human bone marrow-derived mesenchymal stem cells

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The appropriate concentrations of sildenafil enhanced the proliferation and migration of hBMSCs and hASCs in vitro

We found that the appropriate concentrations of sildenafil promoted the proliferation and migration of hBMSCs; this has seldom been reported. Previous research indicated that sildenafil enhanced migration of human umbilical vein endothelial cells by acting on a cGMP-dependent pathway that influences cell growth and differentiation [41], suggesting that sildenafil might similarly enhance the proliferation and migration of hBMSCs. We confirmed that 10 mg/L sildenafil optimally enhanced the proliferation and migration of hBMSCs in vitro. It was earlier shown that sildenafil decreased reactive oxygen species levels and reversed DNA damage in mouse bone marrow cells [42]. Sildenafil also reduced endothelial cell apoptosis [43, 44].

We simultaneously investigated hASCs and hBMSCs. Both hASCs and hBMSCs possess osteogenic differentiation potential in vitro. hASCs are also important in terms of bone regeneration. Moreover, compared to hBMSCs, hASCs are more readily accessible and cause less damage to the body [45, 46]. We found that the effects of sildenafil on hASC proliferation and migration were similar to those on hBMSCs. In summary, 10 mg/L sildenafil enhanced the proliferation and migration of hBMSCs and hASCs in vitro.

The appropriate concentrations of sildenafil promoted osteogenic differentiation of hMSCs in vitro

We found that the appropriate concentrations of sildenafil enhanced the osteogenic differentiation of hBMSCs and hASCs in vitro; this is novel. Previous studies indicated that various PDE5 inhibitors, such as sildenafil, enhanced the osteogenic differentiation of neonatal mouse calvarial cells (MCO). However, the sildenafil concentrations employed were not specified, and MSCs were not tested [47]. We found that sildenafil at 10 mg/L optimally promoted osteogenic differentiation of hMSCs in vitro; this laid the groundwork for subsequent in vivo experiments.

Osteogenic differentiation is a continuous process, with RUNX2 and ALP highly expressed in the early stages, while BGLAP is predominantly expressed in the late stages. Studies have shown that ALP and RUNX2 exhibit higher expression on day 7, whereas BGLAP expression peaks at day 14 [48, 49]. Therefore, based on previous literature [31], we selected different genes for analysis at days 7 and 14. Further studies can explore the expression patterns and dynamic changes of osteogenesis-related genes at various time points.

The appropriate concentrations of sildenafil promoted osteogenesis in vivo

We used a nude mouse model of subcutaneous ectopic osteogenesis, and OVX and TS mouse models, to show that 10 mg/L sildenafil promoted osteogenesis in vivo. This suggested that sildenafil might usefully treat both primary and secondary osteoporosis [50]. The animal models used simulate both forms of osteoporosis. The OVX model mimics osteoporosis caused by post-menopausal estrogen deficiency; this form of primary osteoporosis is very common. In recent years, as space exploration has advanced, bone loss caused by weightlessness has garnered increasing attention. The TS mouse model simulates such bone loss (secondary osteoporosis). Meanwhile, in the context of global population aging, osteoporosis caused by mechanical unloading, as represented by the TS mouse model (e.g., prolonged bed rest), is becoming increasingly prevalent [51]. Sildenafil alleviated bone loss in both the OVX and TS animal models; this is clinically relevant.

Previous studies have loaded sildenafil and phenytoin onto poly (lactic acid) bilayer nanofibrous scaffolds for orthopedic regeneration, primarily utilizing sildenafil to enhance vascularization within regenerating tissues and improve blood supply. These scaffolds demonstrated the ability to promote vascular repair and eventual healing in fracture models, but the focus was not on sildenafil’s role in promoting bone regeneration. In contrast, our study emphasized the effects of sildenafil on the osteogenic differentiation of hMSCs and its potential to improve osteoporosis [52]. Another study combined sildenafil with keratin-based nanofibers and merwinite nanoparticles for bone tissue regeneration, but similarly highlighted sildenafil’s role in promoting vascularization. Besides, this study lacked osteogenesis-related in vivo experiments, which distinguishes it from the focus of our research [53]. Recent studies have suggested that sildenafil mitigates bisphosphonate-induced osteonecrosis of the rat jaw and promotes healing in rat mandibular fracture models [54,55,56]. The aforementioned studies were limited to in vivo experiments and lacked in vitro investigations. Additionally, they did not directly apply to osteoporosis animal models or ectopic bone formation models but rather focused on the pro-osteogenic effects in a specific disease context. In contrast, our study focuses on the effects of sildenafil on the osteogenic differentiation of hMSCs, its therapeutic potential for osteoporosis, and the exploration of its possible mechanisms. In summary, sildenafil promotes the osteogenic differentiation of hBMSCs in vivo and may usefully treat osteoporosis. Sildenafil, as a clinically widely used drug, has demonstrated a relatively well-established safety profile despite side effects such as headache and dyspepsia. Our animal experiments further confirmed its biosafety.

Sildenafil promoted osteogenic differentiation of hBMSCs by affecting the TGF-β pathway

It remains unclear how sildenafil enhances the osteogenic differentiation of hBMSCs. It is essential to understand the mechanism(s) in play. We performed RNA-Seq transcriptomic analysis to this end; sildenafil modulated the TGF-β pathway signaling. Sildenafil activated that pathway in hBMSCs; the pathway critically regulates various biological processes including bone metabolism [57]. Salidroside alleviated inhibition of osteogenic differentiation by acting on the TGF-β signaling pathway [58]. Regulation of TGF-β signaling enhanced the osteogenic differentiation of BMSCs [59]. We suggest that sildenafil may enhance the osteogenic differentiation of hBMSCs by influencing TGF-β signaling; this is novel. However, more work is required.

Additionally, NO can enhance the activity of angiogenic factors such as vascular endothelial growth factor (VEGF) and promote endothelial cell growth and migration by activating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. In the sequencing results, we observed that both the VEGF and PI3K/AKT signaling pathways were upregulated in the OM + sildenafil group compared to the OM group. This further suggests a potential role for NO, although further experimental validation is needed. This provides additional possibilities for exploring the mechanisms.

Limitations and prospects

We found that the appropriate concentrations of sildenafil promoted the osteogenic differentiation of hMSCs and inhibited bone loss. However, our work has certain limitations. First, the optimal sildenafil concentration was 5–20 mg/L; we used 10 mg/L, but the precise optimal concentration remains to be determined. On account of experimental budget and time constraints, as well as the principles of the 3R (Replacement, Reduction, Refinement) in animal experimentation, we aimed to minimize animal use and selected the most appropriate concentration for in vivo experiments based on in vitro results (10 mg/L). However, due to differences between in vivo and in vitro conditions, further studies can incorporate gradient experiments to explore the optimal in vivo dosage. Second, we did not explore why higher concentrations inhibited osteogenesis; future studies should evaluate toxicity and other explanations. Transcriptome data revealed upregulation of the chemical carcinogenesis pathway, Parkinson’s disease pathway, and ErbB signaling pathway, potentially indicating treatment-related side effects. The RNA sequencing results were obtained from in vitro cell experiments, which serve as a preliminary reference and may differ from the actual situation in vivo, so further in vivo studies are needed to investigate its safety more comprehensively. Besides, more in vivo and in vitro experiments are required to clarify how TGF-β signaling affects sildenafil-induced osteogenic differentiation of hBMSCs. Additionally, further studies can be conducted to validate the effects and underlying mechanisms of sildenafil on the osteogenic differentiation of hASCs in vivo. Furthermore, additional research is needed to explore the therapeutic effects of sildenafil in other osteoporosis models, such as the senile mouse model and the GIOP model, to expand its potential treatment applications. Finally, large-animal experiments are required if sildenafil is to be used to treat osteoporosis.

Conclusion

The appropriate concentrations (optimally 10 mg/L) of sildenafil enhanced hMSC proliferation, migration, and osteogenic differentiation in vitro. In vivo, 10 mg/L sildenafil promoted hBMSC osteogenic differentiation in the nude mouse model of subcutaneous heterotopic osteogenesis and prevented bone loss in TS and OVX mice. Mechanistically, sildenafil may enhance the osteogenic differentiation of hBMSCs by modulating the TGF-β signaling pathway. The appropriate concentrations of sildenafil promoted the osteogenic differentiation of hMSCs, enhancing our understanding of the physiological effects of sildenafil and broadening the possible applications to management of osteoporosis.

Data availability

The RNA-seq data are available at the National Center for Biotechnology Information (PRJNA1243296).

Abbreviations

α-MEM:

α-Minimum Essential Medium

ALP:

Alkaline Phosphatase

ARS:

Alizarin Red S

BALP:

Bone Alkaline Phosphatase

BGLAP:

Bone Gamma-Carboxyglutamate Protein

BMD:

Bone Mineral Density

BS/BV:

Bone Surface Area/Bone Volume

BV/TV:

Trabecular Bone Volume/Tissue Volume

DMEM:

Dulbecco’s Modified Eagle’s Medium

DMSO:

Dimethyl Sulfoxide

GAPDH:

Glyceraldehyde-3-Phosphate Dehydrogenase

FBS:

Fetal Bovine Serum

GO:

Gene Ontology

H&E:

Hematoxylin and Eosin

hASCs:

Human Adipose-Derived Mesenchymal Stem Cells

hBMSCs:

Human Bone Marrow-Derived Mesenchymal Stem Cells

hMSCs:

Human Mesenchymal Stem Cells

KEGG:

Kyoto Encyclopedia of Genes and Genomes

Micro-CT:

Micro-Computed Tomography

MCO:

Mouse Calvarial Cells

OCN:

Osteocalcin

OM:

Osteogenic Medium

OVX:

Ovariectomized

P1NP:

Procollagen Type 1 N-Terminal Propeptide

PBS:

Phosphate Balanced Solution

PDE5:

Phosphodiesterase Type 5

PM:

Proliferation Medium

p-Smad2/3:

Phospho-Smad2/3

p-TGF-βR2:

Phospho- Transforming Growth Factor-β type II receptor

qRT-PCR:

quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

RNA-Seq:

RNA-Sequencing

RUNX2:

Runt-Related Transcription Factor 2

Tb.N:

Trabecular Number

Tb.Sp:

Trabecular Separation

Tb.Th:

Trabecular Thickness

TGF-β:

Transforming Growth Factor-β

TGF-β1:

Transforming Growth Factor-β1

TGF-β2:

Transforming Growth Factor-β2

TGF-βR1:

Transforming Growth Factor-β type I Receptor

TGF-βR2:

Transforming Growth Factor-β type II receptor

TS:

Tail-Suspended

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Acknowledgements

The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by the National Science Foundation of China [grant numbers 82170929, 82370924]; the Youth Research Fund of Peking University School and Hospital of Stomatology [grant number PKUSS20230101]; and the Beijing Natural Science Foundation-Haidian Original Innovation Joint Fund Project [grant number L222030, L222090, L222145].

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Author notes

  1. Menglong Hu and Likun Wu contributed equally to this work.

Authors and Affiliations

  1. Department of Prosthodontics, Peking University School and Hospital of StomatologyPeking University School and Hospital of Stomatology, Beijing, 100081, China

    Menglong Hu, Likun Wu, Erfan Wei, Xingtong Pan, Qiyue Zhu, Xv Xiuyun, Xinyi Dong & Yunsong Liu

  2. The Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, 100081, China

    Letian Lv & Hao Liu

  3. National Center of Stomatology, National Clinical Research Center for Oral Diseases, National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing Key Laboratory of Digital Stomatology, Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health, NMPA Key Laboratory for Dental Materials, Beijing, 100081, China

    Menglong Hu, Likun Wu, Erfan Wei, Xingtong Pan, Qiyue Zhu, Xv Xiuyun, Letian Lv, Xinyi Dong, Hao Liu & Yunsong Liu

  4. Peking University School and Hospital of StomatologyPeking University School and Hospital of Stomatology, No.22, Zhongguancun South Avenue, Haidian District, Beijing, 100081, China

    Hao Liu & Yunsong Liu

Authors
  1. Menglong Hu
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  2. Likun Wu
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  3. Erfan Wei
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  9. Hao Liu
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Contributions

Menglong Hu and Likun Wu primarily conducted the experiments and data analysis. Erfan Wei, Xingtong Pan, and Qiyue Zhu assisted in some experimental procedures. Likun Wu drafted the initial manuscript, with Menglong Hu providing revisions. Xv Xiuyun, Letian Lv, and Xinyi Dong reviewed the manuscript. Hao Liu and Yunsong Liu took primary responsibility for conceptual design and funding support. All authors have approved the final submitted version.

Corresponding authors

Correspondence to Hao Liu or Yunsong Liu.

Ethics declarations

Ethics approval and consent to participate

The study titled “The Effects and Mechanisms of Sildenafil in the Treatment of Osteoporosis” was approved by the Laboratory Animal Welfare and Ethics Committee of the Biomedical Ethics Committee at Peking University (Date: 16. 02. 2023, No. LA2023198). The specific animal experimental protocol for this study was pre-designed prior to the research. All surgeries were performed under anesthesia, and all efforts were made to minimize animal suffering. hBMSCs and hASCs were obtained from ScienCell Company (USA). ScienCell Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent.

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Not applicable.

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Hu, M., Wu, L., Wei, E. et al. Sildenafil promotes osteogenic differentiation of human mesenchymal stem cells and inhibits bone loss by affecting the TGF-β signaling pathway. Stem Cell Res Ther 16, 201 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04320-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04320-7

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512