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miR-208a-3p discriminates osteoporosis, predicts fracture, and regulates osteoclast activation through targeting STC1

Journal of Orthopaedic Surgery and Research volume 20, Article number: 98 (2025) Cite this article

Abstract

Background

Osteoporosis (OP) frequently occurs in post-menopausal women, increasing the risk of fracture. Early screening OP could improve the prevention of fractures.This study focused on the significance of miR-208a-3p in diagnosing OP and development regulation, aiming to explore a novel biomarker and therapeutic target for OP.

Methods

The study enrolled a total of 154 post-menopausal women and grouping was performed based on the incidence of OP and fracture. The significance of miR-208a-3p was evaluated from the perspectives of menopausal correlation, OP diagnosis, and fracture prediction. In mechanism, the regulatory effect and mechanism of miR-208a-3p on osteoclast activation was investigated.

Results

miR-208a-3p was menopause-related showing a negative correlation with E2 and positive correlations with FSH and LH. Significant upregulation of miR-208a-3p was observed in post-menopausal women with OP and showed significant diagnostic potential. Increasing miR-208a-3p was positively correlated with bone metabolism markers and negatively correlated with BMD of post-menopausal women with OP. Moreover, miR-208a-3p was also identified as a risk factor for fracture. STC1 was identified as a direct target of miR-208a-3p and was negatively regulated by miR-208a-3p. Silencing miR-208a-3p significantly alleviated macrophage inflammation and osteoblast activation, which was reversed by the knockdown of STC1.

Conclusion

Serum miR-208a-3p served as a diagnostic biomarker for OP and a risk factor for fracture in post-menopausal women. miR-208a-3p regulated macrophage inflammation and further mediated osteoclast activation via targeting STC1.

Introduction

Osteoporosis (OP) is a systemic bone disease characterized by low bone mass and degeneration of the microstructure of bone tissues. In recent years, the incidence of OP has been increasing globally, which seriously threatens human health [1]. The type of OP mainly includes primary OP and secondary OP, involving distinct etiologies. Primary OP is induced by complex factors, which lead to difficult cures and adverse outcomes [2]. By contrast, the etiology of secondary OP is relatively clear and is less difficult to treat [3]. Menopausal women are at high risk of osteoporosis. It has been demonstrated that estrogen could stimulate osteoblasts to make bone matrix and weaken osteoclast activity by directly acting on estrogen receptors. After menopause, due to the declining function of the ovarian, the estrogen level decreases, which cannot effectively suppress osteoclasts and results in rapid decomposition and absorption of bone cells, decreasing bone mass, and accelerating bone loss [4]. As insufficient estrogen has been considered a major cause of OP, estrogen replacement therapy (ERT) has also become the first choice among various therapeutic strategies [5,6,7,8]. Additionally, ERT also showed the advantages of regulating body function, preventing ischemic cardiovascular diseases, and improving reproductive system dysfunction [9]. However, long-term medication of estrogen would increase the risk of breast cancer, and there are many taboos in the application of ERT. Hence, the prevention of menopausal OP is more beneficial than its treatment, and it is also necessary to explore novel therapeutic targets for developing more universal treatments.

With the development of biotechnology, an increasing amount of evidence has revealed the significance of microRNAs (miRNAs) in the onset and development of OP [10, 11]. As miRNAs are stable and easily detected in various body liquids, such as blood, saliva, and urine, they have been considered of great potential in the clinical diagnosis of human diseases [12, 13]. Studies employing sequencing techniques identified several dysregulated miRNAs, which were considered of great predictive potential in OP [14, 15]. There have been several investigations focusing on menopausal OP, which links hormones to bone formation. A previous study revealed the significant upregulation of miR-208a-3p in postmenopausal women, and the expression of miR-208a-3p was higher in OP patients [16]. Additionally, miR-208a-3p was also identified as a functional molecule regulating the differentiation of osteoblasts, which makes it hypothesized as a candidate for the onset and development of menopausal OP [17]. Whether miR-208a-3p could serve as a biomarker for menopausal OP and its specific functional mechanism remains unknown.

Due to the decreasing bone mineral density and the slowing bone formation, OP has become the most common cause of fracture [18]. The risk prediction of fracture in OP patients is critical for the formulation of therapeutic and nursing strategies [19, 20]. Both the onset and OP and the development of fracture are associated with the activation of osteoclasts [21]. Therefore, the regulatory effect and mechanism of miR-208a-3p on the function of osteoclasts was the key to completely understanding the function of miR-208a-3p in menopausal OP. miRNAs could bind to the 3’UTR of the downstream targets and therefore regulate their expression and function at the posttranscriptional level. Among the target genes of miR-208a-3p, STC1 was an energy metabolism-related gene in osteocytes, which could regulate osteoclasts and mediate bone loss under various environments [22,23,24]. However, its significance in osteoporosis and its involvement in the functional role of miR-208a-3p is unclear.

This study assessed the potential of miR-208a-3p in screening menopausal OP and predicting the risk of fracture with a clinical trial. The regulatory mechanism of miR-208a-3p in the development of OP was evaluated in osteoclasts.

Materials and methods

Clinical data

The study enrolled 154 menopausal women aged 40–60 years and 132 non-menopausal women who received physical examinations in The Affiliated Hospital of Youjiang Medical University for Nationalities from 2021 to 2022. According to the results of physical examination, menopausal women with abnormal bone mineral density detected by the X-ray bone densitometer (Luna, USA) were diagnosed with OP. Individuals with one of the following terms were excluded: (1) malignancies; (2) severe mental diseases; (3) cardiovascular cerebrovascular, liver, kidney, hematopoietic system dysfunction, and other serious primary diseases; (4) received surgical operation within 1 year; (5) patients received long-term hormone therapy; (6) patients suffered fracture before menopause.

The occurrence of vertebral fracture in enrolled subjects after admission were diagnosed by radiography examination and were grade according to Genant classification.

All participants possess completed clinical records, and the baseline information was collected and confirmed to be matched between the two groups. This study had been approved by the Ethics Committee of The Affiliated Hospital of Youjiang Medical University for Nationalities, and informed consent was obtained from all participants.

Sample collection and preparation

Fasting venous blood was collected from all study subjects into anti-coagulation tubes and centrifugated at 300 r/min for 5 min to isolate serum. Serum samples were collected and stored at -80 °C until the following analyses.

Cell culture and osteoclast inducement

The human monocyte cell line, THP-1 cell, was obtained from ATCC and maintained in RPMI1640 culture medium supplemented with 10% FBS. Cell culture was conducted at 37 °C with 5% CO2.

After reaching the fusion of 80–90%, THP-1 cells were treated with 200 ng/mL Phobolol 12-myristate 13-acetate (PMA, Sigma Aldrich, USA) inducing it differentiate to into macrophage. Osteoclast inducement was performed with 25 ng/mL macrophage colony-stimulating factor (M-CSF, Sigma Aldrich, USA) and 30 ng/mL Receptor Activator of Nuclear Factor-κB Ligand (RANKL, Sigma Aldrich, USA).

Cell transfection

Cells were transfected with miR-208a-3p inhibitor, small interference RNA of STC1 (si-STC1), or negative controls (inhibitor NC or siRNA NC) with the help of Lipofectamine 3000 (Invitrogen, USA) at room temperature. Transfected cells were maintained at 37 °C with 5% CO2, and the culture medium was replaced with a fresh completed culture medium after 6 h of cell transfection. Transfection efficiency was assessed after 48 h of cell transfection.

Real-time quantitative PCR

Total RNA was extracted from serum and cells using Trizol reagent (Invitrogen, USA) and assessed by OD260/280 and OD260/230 ratios. Isolated RNA was reverse-transcribed into cDNA using an RT kit (Thermo Fisher, USA), and then PCR amplification was conducted on the ABI-7300 system (Applied Biosystem, USA) with the help of SYBR Premix Ex Taq II (Takara Biotechnology, USA). The relative expression levels were calculated with the 2− deltdeltCT method. GAPDH and cel-miR-39 were employed as internal references. The primers have been summarized in Table S1.

Dual-luciferase reporter assay

The targets of miR-208a-3p were predicted from miRDB, Target Scan, miRWalk, and Starbase databases. The intersection of the four databases was obtained through Venn plotting.

The binding sites between miR-208a-3p and STC1 were cloned into the pGL3 vector (Promega, USA), and cells were co-transfected with miR-208a-3p mimic, inhibitor, and negative controls (mimic NC and inhibitor NC). The luciferase activity of STC1 was detected with the dual-luciferase reporter kit on the Dual-luciferase reporter system (Promega, USA) with Renila as the internal reference.

Statistical analyses

SPSS26.0 software and GraphPad Prism 9.0 were employed for data analyses. Difference comparison was conducted with student t-test, one-way ANOVA, and Chi-square test. Logistic regression analysis was performed to identify risk factors for menopausal OP and fracture. A receiver operating curve (ROC) was employed to assess the discriminating potential of miR-208a-3p in menopausal OP and fracture. Correlation analysis was performed with Pearson correlation analysis to evaluate the predictive value of miR-208a-3p in disease development and severity. P < 0.05 indicates statistical significance.

Results

miR-208a-3p is menopause-related and correlated with hormone levels

Among enrolled study subjects, post-menopausal women showed lower E2 levels and higher FSH and LH levels relative to pre-menopausal women (P < 0.0001). The baseline information, except for age, was matched between the two groups (P > 0.05, Table 1).

Table 1 Baseline information of study subjects
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Significant upregulation of serum miR-208a-3p was observed in post-menopausal women (Fig. 1a). Moreover, miR-208a-3p was negatively correlated with the level of E2 (r = -0.862, Fig. 1b) in post-menopausal women and positively correlated with FSH (r = 0.820, Fig. 1c) and LH (r = 0.831, Fig. 1d), indicating the menopause-related feature of miR-208a-3p.

Fig. 1
figure 1

Significance of miR-218a-3p in post-menopausal women. (a). comparison of serum miR-208a-3p level between pre- and post-menopausal women. b-e. Correlation of serum miR-208a-3p with the levels of E2 (b), FSH (c), LH (d), and BMD (e) in post-menopausal women. ****P < 0.0001

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miR-208a-3p served as an OP-related biomarker in post-menopausal women

Post-menopausal women showed decreasing BMD negatively correlated with serum miR-208a-3p levels (Table 1; Fig. 1e, r = -0.893), and there were 95 individuals diagnosed with OP accounting for the incidence rate of 61.69%. Post-menopausal women with OP showed a higher serum miR-208a-3p level relative to women without OP (Fig. 2a). Serum miR-208a-3p (OR = 14.558, 95% CI = 3.116–31.678) was identified as a risk factor for the incidence of OP in post-menopausal women together with BMD (OR = 0.343, 95% CI = 0.129–0.913, Table 2). Consistently, miR-208a-3p showed significant diagnostic significance in OP with a sensitivity and specificity of 72.63% and 91.53%, respectively (AUC = 0.861, Fig. 2b). Additionally, miR-208a-3p was closely correlated with the bone metabolism of post-menopausal OP patients. A significant positive correlation was observed between serum miR-208a-3p and OC (r = 0.788, Fig. 2c), ICTP (r = 0.835, Fig. 2d), and TRACP (r = 0.861, Fig. 2e). BMD was negatively correlated with serum miR-208a-3p (r = -0.801, Fig. 2f).

Fig. 2
figure 2

Significance of miR-208a-3p in menopausal OP. a-b. Comparison of serum miR-208a-3p between post-menopausal women with or without OP (a) and its diagnostic value (b). c-f. Correlation of serum miR-208 with OC (c), ICTP (d), TRACP (e), and BMD (f). ****P < 0.0001

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Table 2 Logistic regression analysis evaluating risk factors for OP in menopausal women
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miR-208a-3p predicted the risk of fracture in post-menopausal OP patients

Among enrolled post-menopausal OP patients, there were 35 patients who developed vertebral fractures, accounting for an incidence rate of 36.84%. Patients with vertebral fractures were composed of 13 of Genant Grade 1, 16 of Genant Grade 2, and 3 of Genant Grade 3. Increasing serum miR-208a-3p was observed in patients who experienced fracture (Fig. 3a), and it was identified as a risk factor for fracture with the OR value of 3.533 (95% CI = 1.356–9.201), as well as decreasing BMD (OR = 0.299, 95% CI = 0.105–0.847, Fig. 3b).

Fig. 3
figure 3

Significance of miR-208a-3p in predicting the risk of fracture in post-menopausal women with OP. (a). Comparison of serum miR-208a-3p between OP patients developing fracture or not. (b). Forest plot of logistic regression analysis identifying risk factors for fracture. ****P < 0.0001

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miR-208a-3p regulates osteoclast activation and inflammation in macrophage

In PMA-induced macrophage, significant upregulation of miR-208a-3p was observed, which was suppressed by the transfection of its inhibitor (Fig. 4a). PMA stimulated inflammation, behaving as the increasing levels of IL-1β, IL-6, TNF-α, and IFN-γ (Fig. 4b). While the knockdown of miR-208a-3p significantly suppressed the inflammation in PMA-induced macrophage (Fig. 4b).

Fig. 4
figure 4

Function of miR-208a-3p in macrophage inflammation and osteoclast activation. a-b. Expression of miR-208a-3p in PMA-induced macrophage (a) and its regulatory effect of inflammatory cytokines. c-d. Expression of miR-208a-3p in M-CSF + RANKL-induced osteoclast (c) and its regulatory effect on osteoclast activation (d). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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In M-CSF and RANKL co-treated THP-1 cells, miR-208a-3p was also significantly upregulated (Fig. 4c), and an increasing osteoclast biomarker was observed, including NFATc1, c-Fos, and TRAP (Fig. 4d). Silencing miR-208a-3p reversed its increasing expression level and hindered the activation of osteoclast (Fig. 4c and d).

STC1 was targeted by miR-208a-3p and mediated the function of miR-208a-3p

The downstream targets of miR-208a-3p were predicted from four public databases, and a total of 11 targets were enriched in the intersection (Figure S1a). Among the enriched targets, only STC1 was negatively regulated by miR-208a-3p (Figure S1b), and the other 10 targets, including CHD9 (Figure S1c), ETS1 (Figure S1d), RAB21 (Figure S1e), USP47 (Figure S1f), MED13 (Figure S1g), PTPN4 (Figure S1h), PTPRG (Figure S1i), FAXC (Figure S1j), CDH2 (Figure S1k), and SOX5 (Figure S1l) showed no significant dysregulation by miR-208a-3p overexpression or knockdown. Hence, STC1 was hypothesized to be involved in the regulatory effect of miR-208a-3p on macrophage and osteoclasts.

STC1-silencing macrophage (Fig. 5a) and differential osteoclast (Fig. 5b) were established, and the luciferase activity of STC1 was negatively regulated by miR-208a-3p in both two cells (Fig. 5c and d). The knockdown of STC1 could alleviate the inhibition of inflammation (Fig. 5e) and osteoclast activation (Fig. 5f) by miR-208a-3p silencing, demonstrating its involvement.

Fig. 5
figure 5

Involvement of STC1 in the effect of miR-208a-3p on macrophage inflammation and osteoclast activation. a-d. Regulation of STC1 by miR-208a-3p in PMA-induced macrophage (a and c) and M-CSF + RANKL-induced osteoclast (b and d) using PCR (a and b) and luciferase reporter assay (c and d). e-f. Involvement of STC1 in the regulation of macrophage inflammation (e) and osteoclast activation (f) by miR-208a-3p. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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Discussion

Due to the bone metabolism disorder and decreased bone mineral density, OP has become a common symptom of menopausal women. Affected by age, hormones, and other factors, the clinical diagnosis of OP based on BMD is limited to the subjects, and the need for universal and specific diagnosis for OP in menopausal women has attracted special attention. miR-208a-3p was previously identified as an OP-related miRNA, which was reported to be dysregulated in OP and mediate the differentiation of osteoblasts [16, 17, 25]. Herein, significant upregulation of miR-208a-3p was observed in post-menopausal women, showing a close correlation with abnormal hormone levels and reducing BMD of post-menopausal women. Decreasing E2 and increasing FSH and LS levels are major causes influencing bone metabolism-inducing OP in post-menopausal women, which showed a close correlation with BMD [26]. Hence, miR-208a-3p was hypothesized as a potential biomarker for post-menopausal OP. As expected, post-menopausal women developing OP showed a higher serum miR-208a-3p, which was identified as a risk factor for OP. Additionally, miR-208a-3p was revealed to show a close correlation with bone metabolism and BMD, which is consistent with its diagnostic significance in OP. Moreover, over 1/3 of enrolled menopausal OP patients experienced fracture in the present study, and serum miR-208a-3p was identified as a risk factor for the incidence of fracture in menopausal OP patients. Based on these findings, miR-208a-3p can serve as an indicator for the onset and development of OP in post-menopausal women.

Considering the pathological mechanism of OP progression, the regulation of macrophage inflammation and osteoclast activation by miR-208a and its underlying mechanism were focused on. The polarization of macrophages would induce inflammation, which further activates the osteoclast, promoting the development of OP [27]. The secretion of inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, could mediate the differentiation of osteoclasts and suppress the differentiation of osteoblasts [28,29,30]. Herein, THP-1 cells were induced to macrophage by PMA, which showed significantly increasing miR-208a-3p levels and significant inflammation. Moreover, silencing miR-208a-3p was found to suppress PMA-induced inflammation. Inflammation also plays a critical role in the activation of osteoclasts. TNF-α, IFN-γ, and other cytokines could indirectly target osteoblast and promote the secretion of RANKL and therefore promote osteoclast activation [31,32,33]. Therefore, the regulation of macrophage inflammation by miR-208a-3p might further affect the activation of osteoclasts, which was further evaluated.

M-CSF is a glycoprotein, that plays a critical role in the growth and survival of osteoclast precursor cells. It has been demonstrated that M-CSF could enhance the activity of osteoclasts and has been considered an essential growth factor [34]. The combination of RANKL with RANK is the key to activating the downstream signaling of osteoclast differentiation [35]. Hence, the co-inducement of M-CSF and RANKL has been commonly employed for the stimulation of macrophage [36, 37]. NFATc1, TRAP, and c-Fos are critical typical transcription factors during the differentiation of osteoclasts. NFATc1 is a critical transcription factor for the differentiation of osteoclasts and regulates the expression of TRAP, CathK, and calcitonin. It was reported that the knockout of NFATc1 would impede the differentiation of osteoclasts [38, 39]. TRAP is highly expressed in osteoclasts and has been accepted as a specific marker for the activity of osteoclasts, of which the expression level is positive with the differentiation of osteoclasts. c-Fos plays an essential role in bone development due to its significant regulatory effect of osteoclasts differentiation. Therefore, TRAP, c-Fos and NFATc1 have been widely employed in evaluating the differentiation of osteoclasts, which were also evaluated in the present study. Herein, the knockdown of miR-208a-3p significantly suppresses the expression of these three markers, indicating the regulation of osteoclast activation by miR-208a-3p. However, bone resorption assay is a more intuitional way to indicate osteoclast activity and reveal the interaction between macrophage and osteoclast, which should be considered in future investigations.

In mechanisms, miRNAs could target downstream genes, regulating their expression and influencing their function. Here, STC1 was predicted as a downstream target of miR-208a-3p, which was negatively regulated by miR-208a-3p in M-CSF and RANKL co-induced osteoclasts. STC1 has been reported to be widely expressed in various kinds of cells, such as tumor cells and osteoblasts and also showed hormone-related characteristics [40,41,42,43]. Moreover, STC1 was previously identified as a bone loss- and BMD-related gene. It was reported that STC1 is involved in calcium and phosphate homeostasis and regulate the osteoblast development [23, 24, 41]. Additionally, STC1 was also revealed to mediate bone formation, which implies its potential in mediating osteoclasts [44,45,46,47]. On the other hand, STC1 showed widely regulatory effects on inflammation and oxidative stress in other functional cells, such as intestinal epithelial cells, microglia myocardial cells, and fibroblast-like synovial cells, which further affected the onset and development of related diseases [48,49,50]. In the present study, silencing STC1 was observed to reverse the protective effect of miR-208a-3p on inflammation and osteoclast activation. Although the oxidative stress has not been evaluated in this study, the regulation of inflammation and osteoclast activation was considered the major mechanism underlying the miR-208a-3p/STC1 axis. According to the above findings, miR-208a-3p was hypothesized to regulate inflammation in macrophages and activation of osteoclasts by modulating STC1, and therefore modulate the development of OP in post-menopausal women.

In conclusion, miR-208a-3p is a hormone-related miRNA serving as a biomarker for OP in post-menopausal women and predicting the risk of fracture. Silencing miR-208a-3p inhibited the inflammation in macrophage via targeting STC1 and therefore impeded the activation of osteoclasts.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ERT:

Estrogen replacement therapy

miRNAs:

microRNAs

OP:

Osteoporosis

ROC:

Receiver operating curve

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Acknowledgements

Not applicable.

Funding

The study was supported by Natural Science Foundation of Shandong Province (ZR2023QH395).

Author information

Author notes

  1. Hongbing Qian and Fei Jia contributed equally to this work.

Authors and Affiliations

  1. Spine Surgery, Kunshan Hospital of Traditional Chinese Medicine, Kunshan, 215300, China

    Hongbing Qian

  2. Department of Spine Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250000, China

    Fei Jia

  3. Department of Rehabilitation, The Affiliated Hospital of Youjiang Medical University for Nationalities, No.18, Zhongshan 2nd Road, Baise, 533000, Guangxi Zhuang Autonomous Region, China

    Huiling Qin

  4. Guangxi Key Laboratory for Preclinical and Translational Research on Bone and Joint Degenerative Diseases, Baise, 541000, Guangxi, China

    Huiling Qin

Authors
  1. Hongbing Qian
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Contributions

HB Q and F J wrote the manuscript. HL Q conducted the experiment and analyzed the data. HB Q and F J wrote the manuscript. HL Q revised the manuscript. All authors reviewed and approved for publication.

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Correspondence to Huiling Qin.

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Ethics approval and consent to participate

The study protocol was approved by The Ethics Committee of The Affiliated Hospital of Youjiang Medical University for Nationalities. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration and later versions.

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All patients provided written informed consent.

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The authors declare no competing interests.

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Supplementary Material 1

13018_2025_5512_MOESM2_ESM.tif

Fig. S1: Validation of downstream targets of miR-208a-3p. (a). Venn plot of predicting targets from Starbase, miRWalk, Targetscan, and miRDB databases. b-l. Regulatory effect of miR-208a-3p on the expression of STC1 (b), CHD9 (c), ETS1 (d), RAB21 (e), USP47 (f), MED13 (g), PTPN4 (h), PTPRG (i), FAXC (j), CDH2(k), and SOX5 (l). nsP > 0.05, **P < 0.01

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Qian, H., Jia, F. & Qin, H. miR-208a-3p discriminates osteoporosis, predicts fracture, and regulates osteoclast activation through targeting STC1. J Orthop Surg Res 20, 98 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13018-025-05512-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13018-025-05512-w

Keywords

Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X