Combined effects of low temperature, hyperosmolarity and seawater-conditioned pathogens on open fracture healing in a rat model simulating circumpolar environments

Abstract

Objective

To investigate the factors influencing the healing of open fractures in circumpolar latitude region seawater immersion conditions.

Materials and methods

A femoral fracture model was established in ninety 6-to-8-week-old male Sprague-Dawley rats, randomly assigned to five groups (n = 18 per group): (1) fracture only, (2) fracture with circumpolar seawater immersion, (3) fracture with low-temperature isotonic solution immersion, (4) fracture with aseptic circumpolar seawater immersion, and (5) fracture with low-temperature aseptic circumpolar seawater immersion. Fractures were confirmed postoperatively by radiographs on days 7, 21, and 42. Micro-CT and H&E staining were performed on day 42 to assess bone healing. Bacterial cultures from internal fixation devices were analyzed on day 3. Blood samples were collected on days 3, 7, and 14 to assess leukocyte and neutrophil counts, and serum ALP and VEGF levels were measured on days 7, 14, and 21. Pathogenic microorganisms in the seawater were identified by metagenomic analysis. Fracture healing and callus formation rates were compared using the Log-rank test.

Results

X-ray, micro-CT, and histological analyses revealed significantly impaired fracture healing in the group exposed to circumpolar seawater immersion compared to the fracture-only group (P < 0.05). Bacterial colony counts on internal fixation devices were highest in the circumpolar seawater group (P < 0.05). Leukocyte and neutrophil levels were significantly elevated in this group on days 3 and 7 (P < 0.05), with no significant differences observed on day 14 (P > 0.05). Serum ALP and VEGF levels were significantly reduced on days 7, 14, and 21 (P < 0.05), although ALP levels on day 21 showed no significant difference (P > 0.05). Log-rank analysis indicated that the bone union and callus maturation rates were significantly lower in the circumpolar seawater group compared to the other four groups. Metagenomic analysis identified Flavobacterium, Rhodobacter, and Bacteroides as the dominant pathogens in circumpolar seawater.

Conclusions

This study demonstrates that hyperosmolarity, low temperature, and exposure to opportunistic pathogens under circumpolar seawater conditions collectively delay open fracture healing. Among these factors, opportunistic pathogens exert the most significant impact, highlighting microbial contamination as the primary barrier to bone regeneration in such environments and providing direction for future therapeutic strategies.

Clinical trial number

not applicable.

Introduction

Advances in science and technology have drawn more tourists and researchers to circumpolar regions, where chronic sub-zero temperatures, volatile weather, and rugged terrain greatly increase operational hazards [1]. Epidemiological data link higher latitude with greater idiopathic-arthritis incidence, and molecular studies implicate cold-induced protein changes and circadian disruption in polar disease mechanisms [2, 3]. The same glaciated topography and ice-sheet margins that hinder mobility also predispose workers to falls and high-energy open fractures [4, 5]. These wounds are often exposed to cold, hyperosmotic seawater, and emerging evidence shows that combined cryogenic, osmotic, and microbial stressors can suppress osteogenesis and disrupt cytokine signalling, delaying fracture repair [6, 7]. Together, these observations highlight the need for integrated research to guide both clinical management and environmental-safety strategies in high-latitude settings.

Open fractures—defined as full-thickness skin breaches that expose bone to the environment [8, 9] show characteristic epidemiological trends. In long bones, diaphyseal fractures outnumber metaphyseal ones, reflecting the shaft’s greater susceptibility to high-energy trauma [10, 11]. Most cases arise from polytraumatic events such as motor-vehicle collisions and falls from height; low-energy mechanisms account for < 15% of presentations [12, 13]. High-energy open fractures occur predominantly in young men, whereas low-energy injuries are more common in elderly women, a pattern that mirrors age-related bone-density loss and activity profiles [14, 15]. High-velocity trauma initiates a complex pathophysiological cascade involving soft tissue devitalization and microvascular injury, which facilitates polymicrobial contamination through disrupted anatomical barriers [16]. Concurrent hypovolemic shock further impairs perfusion, creating ischemic conditions that promote anaerobic metabolism, tissue necrosis, and invasive infection [17]. Prognostic analyses have identified several comorbidity-related risk factors: diabetes mellitus (via impaired angiogenesis and neutrophil dysfunction) [18–20], HIV/AIDS (due to CD4 + lymphocyte depletion) [21–23], and chronic smoking (through microvascular constriction and oxidative stress) [24–26], all of which are strongly associated with delayed union, infection, and increased sepsis risk.

Polar exposure adds unique pathophysiological stressors to open fractures. Cold, hyperosmolar seawater simultaneously triggers vasoconstriction-driven microthrombosis and hypertonicity-induced cellular dehydration, worsening local ischemia [27]. Accelerated glacial melt also releases psychrophilic and extremophilic microbes into coastal waters [28]. These cold-adapted pathogens carry novel virulence factors and antimicrobial-resistance traits that thrive at low temperatures, form robust biofilms, and evade leukocyte responses, thereby intensifying wound infection and delaying tissue regeneration [29]. Such multifactorial environmental–biological interactions complicate fracture management in circumpolar settings and demand modified antimicrobial strategies and targeted physiologic support.

Although pharmacological agents, mechanical compression, and biophysical modalities such as low-intensity pulsed ultrasound can enhance bone repair in conventional environments [30–32], their efficacy under polar situation remains uncertain. Therefore, this study evaluates how immersion in circumpolar seawater—combining low temperature, hyperosmolarity, and pathogen exposure—affects open-fracture healing in a rat model. Clarifying these effects will guide optimized treatment protocols and improve safety for personnel working in high-latitude regions.

Materials and methods

Animal model and fracture grouping

Ninety male Sprague-Dawley rats (6–8 weeks, 230–280 g, RRID: RGD_5508396) were housed under SPF conditions with a 12 h light/dark cycle and ad libitum access to sterilized food and water. After a 7-day acclimation, all animals underwent standardized midshaft femoral osteotomy under isoflurane anesthesia, followed by intramedullary fixation with a 1.2 mm Kirschner wire. Rats were randomly assigned to six groups (n = 18/group): control (BF), polar seawater (BF + PS), low temperature (BF + LT, 4 °C saline), sterilized seawater (BF + AS), combined cold and sterilized seawater (BF + LA), and non-immersed control. Postoperatively, fracture sites were immersed for 10 min in the designated solution (4 °C, salinity 35‰ or saline) using anatomically sealed chambers to prevent systemic hypothermia. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC-2018-R002). All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Navy 905th Hospital, PLA (Ethics Approval No. 2024-CN06), in accordance with the National Guidelines for the Care and Use of Laboratory Animals in China.

X-ray examination

Radiographs were obtained on postoperative days 7, 21, and 42 using a digital system (55 kVp, 2.5 mAs, 100 cm SID) to assess fracture healing. Images were evaluated using the Lane-Sandhu scoring system, covering callus formation, cortical bridging, and bone remodeling. Two blinded radiologists independently scored all images, with high interobserver reliability (ICC > 0.85). Healing progression was tracked by assessing callus density, cortical continuity, and trabecular alignment. Fracture healing progression was systematically quantified through application of the Lane-Sandhu radiographic scoring system (Table 1) [29], a validated multidimensional evaluation framework comprising 11 parameters across three domains: osteogenic activity (callus volume/density), union integrity (cortical continuity, medullary bridging), and remodeling maturity (trabecular alignment, cortical reconstitution).

Table 1.

Lane-Sandhu grading scale

Score	0	1	2	3	4
Bone Formation	No bone formation	Bone formation accounts for 25% of the defect	Bone formation accounts for 50% of the defect	Bone formation accounts for 75% of the defect	Bone formation full detect
Bone Junction	Clear fracture line	——	Fracture line partially present	——	Fracture lines disappear
Bone shaping	No bone plastic pattern was seen	——	Bone marrow cavity formation	——	Cortical bone plasticity

Micro-CT scanning

On postoperative day 42, femurs from randomly selected rats (n = 6/group) were harvested aseptically and scanned by high-resolution micro-CT (SkyScan 1276, ThermoFisher Scientific Co., LTD.; 55 kVp, 200 µA, 18 μm resolution). A standardized region of interest (ROI) extending 3 mm from the fracture interface was analyzed using manufacturer software (CTAn v1.20), quantifying three-dimensional bone morphometric parameters: absolute bone volume (BV, mm³), bone volume fraction (BV/TV, %), and bone mineral density (BMD, mg hydroxyapatite/cm³). Data normalization and statistical comparisons were performed according to standardized protocols for osseous defect analysis.

HE staining

Femoral specimens were fixed in 4% paraformaldehyde solution for 72 h, decalcified in 10% EDTA, dehydrated through graded ethanol, and embedded in paraffin blocks. Section (10 μm) were prepared using a paraffin slicer and stained with Harris hematoxylin and eosin Y (H-E dye solution, item No. G1003,RRID: SCR-019021). Slides were examined under an optical microscope (Nikon) to evaluate fracture healing histology.

Internal fixation device bacterial count

At 72 h post-fracture, Kirschner wires were aseptically removed and processed. Bacterial suspensions were cultured on LB medium and blood agar plates incubated at 25 ± 0.5 °C for 24 h. Colony forming units (CFUs) were quantified using standard protocols.

Blood routine examinations

Blood samples were collected via retro-orbital plexus under isoflurane anesthesia at days 3, 7, and 14 post-op. Samples were anticoagulated with EDTA and analyzed within 2 h using an automated hematology analyzer (Sysmex XN-1000). All sampling adhered to NC3Rs guidelines.

ELISA

Serum was isolated by centrifugation from blood collected at days 7, 14, and 21. ALP and VEGF concentrations were measured using ELISA kits (VEGF Elisa Kit: Thermo Fisher, BMS619, RRID: SCR-023950;ALP Elisa Kit: Sigma-Aldrich, MAK440, RRID: AB-2921305) with absorbance read on an enzyme marker (Thermo) and plates washed with an automated plate washer (Thermo).

Identification of conditioned pathogens

Antarctic seawater samples were systematically collected from four georeferenced stations (73°E, 63.5°W; 73°E, 64.5°W; 73°E, 65.5°W; 73°E, 66.5°W; designated PZB-1 to PZB-4) within the upper 5-meter photic zone. At each station, triplicate 3-liter seawater aliquots were obtained using a peristaltic pump filtration system equipped with 0.22 μm polyethersulfone membranes (Merck Millipore, SLGP033RB, RRID: SCR-023955), yielding 12 cryopreserved (-20 °C) microbial biomass samples. Seawater samples were collected from circumpolar latitude sites (73°E, 65.5°W within 1 km). Microbial biomass was filtered using 0.22 μm membranes and DNA sequenced on Illumina NovaSeq. Taxonomic and virulence analyses were performed using Kraken2, VFDB, and CARD databases.

Statistical analysis

Data analysis was conducted using GraphPad Prism 9.5 and R v4.3.2. Statistical tests included ANOVA with Tukey’s correction and Kaplan–Meier survival analysis. Significance was set at p < 0.05 after FDR correction.

Results

Construction of rat femur fracture model was successful

Postoperative radiographs confirmed clear visualization of the fracture line, stable intramedullary fixation, and no evidence of iatrogenic joint damage, thereby validating the successful establishment of the femoral fracture model in accordance with standard orthopedic criteria (Fig. 1, Table 2).

Fig. 1.

Construction of the rat femoral fracture model

Table 2.

P values of serological index detection in femur fracture model rats

Groups		BF		BF + LT		BF + AS		BF + LA
		WBC	Gran%	WBC	Gran%	WBC	Gran%	WBC	Gran%
Compared with BF + PS group (P value)	3d	0.001	< 0.0001	< 0.0001	< 0.0001	0.0029	< 0.0001	0.0002	< 0.0001
	7d	0.0002	< 0.0001	0.0006	0.0004	0.0006	0.0002	0.0001	0.0006
	14d	0.7069	0.4814	0.6864	0.1132	0.9891	0.4769	0.8620	0.0698

X-ray demonstrated that circumpolar seawater exposure impairs open fracture healing

Serial radiographic assessments revealed distinct time-dependent differences in bone regeneration among the experimental groups. On postoperative day 7, all cohorts demonstrated adequate fracture alignment and early periosteal callus formation, with no significant differences in Lane-Sandhu scores (p = 0.5698). By day 21, control rats exhibited substantial callus bridging and mineralization, while the polar seawater immersion group showed markedly delayed healing, with visible fracture lines persisting (p = 0.004). At day 42, radiographic union was observed in 85% of control animals, compared to only 42% in the polar seawater group (log-rank p = 0.003). The low-temperature isotonic saline and sterile seawater groups demonstrated moderate delays in healing, whereas combined exposure to cold sterile seawater significantly impaired osteogenesis (p < 0.001), as evidenced by diminished trabecular continuity and incomplete cortical bridging. These results underscore the synergistic inhibitory effects of hyperosmotic and hypothermic stressors on fracture repair (Fig. 2, Table 3).

Fig. 2.

Radiographic assessment of femoral fracture healing in rats. (A) Representative X-ray images showing the progression of femoral fracture healing across groups at postoperative days 7, 21, and 42. (B) Time-course trends of Lane–Sandhu radiographic scores for each group, illustrating differences in callus formation and fracture union over time

Table 3.

P values of VEGF and ALP in femur fracture model rats

Groups		BF		BF + LT		BF + AS		BF + LA
		VEGF	ALP	VEGF	ALP	VEGF	ALP	VEGF	ALP
Compared with BF + PS group (P value)	7d	< 0.0001	< 0.0001	0.0002	0.0225	0.0124	< 0.0001	0.0038	0.0045
	14d	< 0.0001	< 0.0001	< 0.0001	0.0003	0.0264	0.0003	0.0008	0.0443
	21d	< 0.0001	0.4749	0.0138	0.4901	0.0488	0.4233	0.0026	0.8916

Micro-CT analysis demonstrated that circumpolar seawater exposure impairs open fracture healing

Quantitative micro-CT revealed significant differences in bone regeneration among the experimental groups. Rats exposed to polar seawater exhibited markedly reduced bone volume (BV, p < 0.001), bone mineral density (BMD, p = 0.002), and bone volume fraction (BV/TV, p < 0.001) compared to controls, the hypothermic isotonic group, and the sterile seawater group. Although the combined hypothermic–sterile seawater group showed modest improvement over the polar seawater group (p = 0.015), all measured parameters remained significantly lower than those of the control group (p < 0.01).

Three-dimensional micro-CT reconstructions supported these findings: the polar seawater group demonstrated incomplete trabecular bridging and disrupted cortical continuity, in contrast to the control group, which displayed mature callus formation and well-defined cortical remodeling (p < 0.001). Intermediate healing patterns were observed in the hypothermic isotonic and sterile seawater groups, consistent with their partial impairment relative to the polar seawater-exposed cohort (Fig. 3).

Fig. 3.

Micro-CT examination of femoral fracture in rats. A: 3DMicro-CT B-D: Differences in BV, BV/TV and BMD in rat models. ^*P<0.05, ^**P<0.01

Histological analysis revealed variable fracture healing patterns across treatment groups

Hematoxylin and eosin (H&E) staining showed distinct differences in tissue repair among groups. In the polar seawater group, specimens demonstrated extensive fibroplasia, reduced neovascularization, and dense infiltration of neutrophils and lymphocytes (arrows), along with focal hemorrhage and minimal osteoid formation. In contrast, control specimens exhibited mature trabecular bone, well-developed neovasculature, and minimal inflammatory cell infiltration.

The hypothermic isotonic, sterile seawater, and combined hypothermic–sterile groups showed intermediate histological features, including moderate osteogenesis, active angiogenesis, and decreased granulocytic infiltration compared to the polar seawater group, though residual lymphocytic infiltration was still observed. These findings indicate that seawater exposure—especially with pathogenic and cold stressors—delays osseous repair relative to controls (Fig. 4).

Fig. 4.

H-E staining results of femur tissue section in femur fracture model rats

Bacterial count of femoral internal fixation device in rats showed fracture related infection

The results of coating plate count showed that the number of colonies on the internal fixation device in the circumpolar latitude seawater group was significantly higher than that in the control group(p < 0.001), the low temperature isosmotic group(p = 0.0017), the sterile circumpolar latitude seawater group(p = 0.0066) and the composite group on the third day after surgery(p = 0.007), suggesting that the osseous union rates of femur in the circumpolar latitude seawater group may be slow due to bacterial infection (Fig. 5).

Fig. 5.

Bacterial colonization on the surface of internal fixation devices. A: Representative images of bacterial growth from fixation devices cultured on LB agar and blood agar plates. The proximal segment of the Kirschner wire was rolled directly onto the media. B–C: Quantification of colony-forming units (CFUs) across experimental groups. *P < 0.01, **P < 0.001 compared with control group

Serological markers reflect early inflammatory response and fracture healing dynamics

On postoperative days 3 and 7, the circumpolar seawater group exhibited significantly elevated white blood cell (WBC) counts and neutrophil percentages compared to the control, hypothermic-isotonic, sterile seawater, and combined treatment groups. By day 14, these differences were no longer statistically significant, indicating that the systemic inflammatory response had subsided and converged across groups. These findings suggest that exposure to circumpolar seawater induces an early, heightened inflammatory reaction that may contribute to delayed fracture healing (Fig. 6).

Fig. 6.

Results of serological index detection in femur fracture model rats. A-F: Differences in WBC and Gran% in rat models

Serum ALP and VEGF levels indicate delayed fracture healing under seawater exposure

ELISA results showed that serum levels of vascular endothelial growth factor (VEGF) and alkaline phosphatase (ALP) were significantly lower in the circumpolar seawater group compared to the control group during the first and second postoperative weeks (p < 0.05). By the third week, no significant differences in ALP or VEGF levels were observed among groups, indicating a convergence in systemic bone healing markers over time. These findings suggest that circumpolar seawater exposure impairs early-stage osteogenesis and angiogenesis during fracture healing (Fig. 7).

Fig. 7.

VEGF and ALP levels were detected by ELISA in rat model of femoral fracture. A-F: Differences in ALP and VEGF in rat models, ^*P<0.05, ^**P<0.01

Osseous union and callus maturation rates indicate impaired fracture healing with circumpolar seawater exposure

The mapping calculation results showed that the bone osseous union rates of the circumpolar latitude seawater group in Fig. 8A was lower than that of the other four groups, and the P value of all groups in Table 4 was less than 0.05 compared with the circumpolar latitude seawater group, so the bone osseous union rates and healing effect of the circumpolar latitude seawater group were the lowest. In Fig. 8B, the rate of callus formation in the circumpolar latitude seawater group was lower than that in the other four groups, and the P value of each group in Table 5 was less than 0.05 compared with the circumpolar latitude seawater group. Therefore, the rate of callus formation in the circumpolar latitude seawater group was the lowest and the formation time was the longest (Fig. 8).

Fig. 8.

Curve of osseous union rates and callus maturation rates of femur fracture in five groups. A-B: Trends of osseous union rates and callus maturation rates in rat models

Table 4.

Description and comparison of healing in five groups of rats

Groups	Total cases	Healing cases (%)	Median healing time (day)	Compared with BF + PS group (P value)
BF + PS	18	4(22.2)	NA	-
BF + AS	18	9(50.0)	28	0.0038
BF + LT	18	14(77.7)	35	<0.0001
BF + LA	18	14(77.7)	35	<0.0001
BF	18	12(66.6)	28	<0.0001

Table 5.

Description and comparison of callus formation in five groups of rats

Groups	Total cases	Cases of callus formation (%)	Median callus formation time (day)	Compared with BF + PS group (P value)
BF + PS	18	9(50.0)	21	-
BF + AS	18	15(83.3)	14	0.0179
BF + LT	18	17(94.4)	14	0.0059
BF + LA	18	17(94.4)	14	0.0011
BF	18	18(100.0)	14	0.0011

Flavobacteriaceae bacterium, rhodobacteraceae bacterium and Bacteroidetes bacterium are the main conditioned pathogens affecting fracture healing in circumpolar latitude seawater

Sample correlation analysis and cluster analysis showed that the other samples had high correlation and similarity except PBZ-3-3 samples (Fig. 9A and B). Through the analysis of different taxonomic levels of Marine microorganisms, the results showed that at the species level, the microorganisms with high species richness were pelagic bacteria, Spirillum Marine, harbor coccus, flavobacterium, Rhodobacter, abundans, flavobacterium, gammoproteus, thiococcus and Bacteroides (Fig. 9C-Fig. 9E). Heat maps and clustering showed that the abundance of pelagic bacterium, portococcus, flavobacterium, rhodobacter and Bacteroides were high in most samples (Fig. 9F). Species Alpha and Beta diversity analysis showed that the microbial diversity of the four randomly selected regions was basically the same, and there was no significant difference (Fig. 9G-Fig. 9J). PCA and PCoA showed that the sample groups in the four randomly selected regions had good biological duplication, and the microbial diversity was basically the same. The selected samples could represent the overall level of microbial species distribution in circumpolar latitude seawater (Fig. 9K and L).

Fig. 9.

Identification of pathogenic bacteria in circumpolar latitude seawater. A: Heat map of correlation coefficient between samples. B: Sample similarity clustering diagram. C-E: Columnar analysis of species composition at different taxonomic levels. F: Microbial species composition clustering heat map analysis. G-J: Analysis and comparison of species Alpha diversity. K and L: Analysis and comparison of species Beta diversity. M: VFDB and PHI annotation scores of Top10 abundant microorganisms

Discussion

Seawater immersion imposes pronounced hyperosmotic stress on fractures, driving cellular dehydration and disrupting cytokine signalling pathways that are essential for bone repair [33, 34]. Clinically, such exposure suppresses angiogenesis, inhibits matrix mineralisation, and ultimately delays callus maturation [35]. Circumpolar waters intensify these effects by adding sustained hypothermia (≤ 4 °C) and harbouring psychrophilic microbes uniquely adapted to cold environments [36]. Metagenomic surveys have identified cryotolerant pathogens in these waters—even at temperate latitudes—that produce cold-active virulence factors, including thermolabile exotoxins and antifreeze glycoproteins [37]. Here, we evaluate how hyperosmolarity, hypothermia, and polar-specific microbiota interact to impede fracture healing, providing mechanistic insights to guide the design of next-generation antimicrobial biomaterials for polar trauma management.

This study confirm that both low temperature and hyperosmolarity—hallmarks of the circumpolar environment—independently slow femoral-fracture healing. Cold-induced vasoconstriction and osmotic dehydration reduce local perfusion, suppress angiogenesis, and limit the delivery of nutrients and oxygen to the fracture site. This hypoperfusion impairs cell proliferation, differentiation, and matrix deposition, while also hindering the clearance of necrotic tissue and pathogens. Consistent with these effects, serum vascular endothelial growth factor (VEGF), a key driver of neovascularisation, and alkaline phosphatase (ALP), an enzyme critical for mineralisation, were both significantly reduced under either stressor [38, 39]. Log-rank analysis demonstrated that low temperature and hyperosmolarity each exerted a significant adverse effect on union rates (p < 0.05), but their combination yielded no additional synergy, indicating that either factor alone is sufficient to reach a ceiling of physiological disruption. These findings highlight the need to address thermal and osmotic stress separately when optimising fracture-healing strategies in polar settings.

Conditioned seawater pathogens exert a greater inhibitory effect on fracture repair than either cold or hyperosmolarity alone. Unlike ubiquitous bacteria, these psychrophilic organisms are pathogenic only in circumpolar seawater. Their colonisation of the fracture site disrupts the local microenvironment, amplifies inflammation, and directly damages bone and soft tissue, thereby blocking normal regeneration [40, 41]. Bioinformatic profiling identified three dominant, highly virulent taxa—Flavobacterium, Rhodobacter, and Bacteroides—providing clear targets for antimicrobial intervention. Although our metagenomic analysis of seawater provides a comprehensive profile of microbial exposure, we acknowledge that the clinical relevance of some species may vary. However, the predominant isolates, including Vibrio alginolyticus and Shewanella putrefaciens, have been repeatedly implicated in marine wound infections, particularly in polar or brackish water exposures, supporting the model’s translational relevance.

In summary, this study shows that all circumpolar factors investigated—low temperature, hyperosmolarity, and polar-specific pathogens—retard fracture healing, but seawater-conditioned microbes are the principal drivers of this delay. Future work should elucidate the cold-adaptation mechanisms of these pathogens (e.g., CsdA helicase variants) and develop temperature-responsive antibacterial scaffolds that deliver sustained local therapy under polar conditions.

Author contributions

Concept and design: Tianming Xu and Yangkai Wang; data collection and analysis: Shuo Sun, Hui Wang and Bin Han; drafting of the article: Shuo Sun; critical revision of the article for important intellectual: Hui Wang and Yangkai Wang; study supervision: Hongri Wu and Tianming Xu. All the authors approved the final article.

Funding

This work was supported by Shanghai Natural Science Foundation Project (Grant No.22ZR1476600), Naval Medical University Research Project (Grant No.H0603), Changzheng Hospital Foundation Program(2024DZXYY-209).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.