The Effect of Different Saline Suspension Height on Injured Articular Cartilage During Arthroscopic Surgery

Abstract

Objective

Saline is typically suspended at a certain height to maintain a clear surgical field in arthroscopic surgery. The effects of saline on cartilage have been extensively studied; however, the impact of the pressure generated by saline solution suspended at different heights on injured cartilage is largely unknown. This study investigates suspension-height-dependent cellular responses and tissue damage in traumatized cartilage.

Methods

Osteochondral explants were harvested from porcine stifle joints, then were cut perpendicularly before immersed or irrigated for 2 hours with saline at 4 heights (80/105/130/155 cm). The explants were then transferred to and cultured in chondrogenic medium for 5 days. Chondrocytes viability was subsequently assessed with confocal imaging. Cell response was assessed with expression levels of proapoptosis and proinflammatory genes. Tissue damage was evaluated by secretome analysis of proinflammatory cytokines and extracellular matrix and histological test.

Results

Irrigation exacerbated cut-induced chondrocytes death in superficial zone of cartilage, with mild change on 80 cm, 105 cm, 130 cm and severe damage on 155 cm. Similarly, explants that underwent irrigation with heights of 80 cm to 130 cm exhibited relatively slighter change of gene expression of BAX, BCL2, IL-6 and NOS2 and release of GAG, IL-6 and NO to a comparable extent.

Conclusion

This study provides evidence of damaging effects of irrigation on injured cartilage surface. Suspension heights of 80 cm to 130 cm led to comparable minor cartilage damage.

Keywords

arthroscopy irrigation suspension height cartilage injury

Introduction

Arthroscopy is an indispensable surgical technique in modern orthopedics and sports medicine. As a minimally invasive procedure, it preserves the surrounding joint tissues more effectively, allowing for a shorter recovery time for patients.¹ In arthroscopy, a sufficient surgical vision is crucial for successful outcomes. To maintain a sufficient vision, surgeons typically suspend saline at a certain height as an irrigating solution for continuous joint cavity perfusion, creating sustained pressure to distend the joint.²

As an exogenous substance replacing synovial fluid, the physical and chemical properties of irrigation fluids can influence the viability, oxidative stress, and metabolism of articular cartilage.³ Short-term exposure to irrigation fluids at room temperature or lower inhibited chondrocytes’ metabolic capacity and RNA synthesis.⁴ Low PH of irrigation fluids can increase the oxidative stress of articular cartilage and decrease the metabolic capacity of chondrocytes.⁵ Chondrocytes exposed to a low osmolarity environment, such as when irrigated with isotonic solutions, are more likely to suffer in situ chondrocyte death.⁶

Saline is the most commonly used arthroscopic irrigation fluid in clinical practice. In knee arthroscopy, saline is commonly suspended at a height of 80 cm to maintain the surgical field.² When visibility is compromised, surgeons may increase the height of the saline to improve the view. The change of suspension height will cause the change of irrigating pressure on cartilage. The irrigating pressure of irrigation fluids is the most direct external mechanical stimulation to cartilage during arthroscopy. In arthroscopic knee surgery, the proportion of patients with cartilage injury is up to 61%,⁷ and the sensitivity of tissues and cells to exogenous stimulation will increase after injury.⁸

In vitro studies have shown that mechanical stimuli such as shear force, tensile stimulation, and hydrostatic pressure have direct effects on the phenotype of chondrocytes.⁹ Excessive hydrostatic pressure can lead to chondrocyte degeneration, resulting in the loss of the hyaline cartilage phenotype, and in more severe cases, can cause chondrocyte death.¹⁰ Irrigating with irrigation fluids can exacerbate stress on joint tissues, subjecting articular cartilage to not only mechanical damage due to preexisting chondral defects but also to matrix degradation and joint inflammation.⁶ However, the adverse effects of mechanical stimulation induced by irrigation pressure at elevated arthroscopic saline height on the injured cartilage remain unexplored in macroscopic clinical settings.

This study is based on an ex vivo porcine cartilage injury model and aimed to evaluate its effects on the activity, inflammatory response, and proteoglycan loss in the injured cartilage, thereby providing clinical reference on a safe range of saline suspension heights for maintaining optimal visualization during arthroscopy.

Methods

Isolation of Osteochondral Explants From Porcine Stifle Joints

Porcine were provided by a local abattoir. Female Porcine aged 2 to 3 years were employed for the experiments. The osteochondral explants were harvested within 24 hours after slaughter. In detail, the femoral trochlea was exposed by cautiously dissecting the knee joints, a diamond-coated 5-mm diameter trephine drill was used to harvest cylindrical osteochondral explants. During the process, the cartilage surface was continuously and gently irrigated with sterile phosphate-buffered saline (PBS; Sigma-Aldrich) supplemented with 1% penicillin/streptomycin (1% PS; Gibco). Subsequently, the isolated explants were trimmed with a manual saw to achieve a height of 8 mm and washed with PBS 10% PS. The prepared explants were then transferred to a 24-well plate and incubated overnight in High Glucose Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% PS, at 37°C and 5% CO₂.

Experimental Design

Study scheme is shown in Figure 1 . Medical saline was use as irrigation fluid in this study. Next, explants were incubated in saline for 20 minutes. The explants were collected and damaged with a sharp dermatome blade. The cartilage was first cut along the diameter of chondral surface. Then, the cartilage as well as subchondral bone was cut perpendicularly to the first cut and was split gently. The final sample represented injury limited from cartilage surface to subchondral bone junction.

Figure 1.

Study scheme. Osteochondral explants were harvested from mature porcine, then underwent artificial injuries followed by irrigation by saline from different suspension heights using an irrigation simulator. Created with BioRender.com.

Considering that the irrigation fluid is usually stored at a height of 80 cm before flowing into joint cavity during operations, this study aims at investigating the irrigation pressure of liquids flowing down from heights of 80, 105, 130, 155 cm and their corresponding impacts on cartilage. The suspension height was defined as the vertical distance between explants and the top position of bag-packaged saline. The surface of explants was irrigated by saline at different suspension height for 2 hours in total to replicate the arthroscopic operation. Control explants underwent same procedure with immersion in saline for 2 hours instead of irrigation. Irrigation pressure was recorded after the reading of the gauge stabilized. Chondrogenic culture medium was used to culture the explants. To be specific, serum-free DMEM-HG supplemented with 1% PS, 1% insulin-transferrin-selenium (Gibco), 1% nonessential amino acids (Gibco), 50 mg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), and 10^–7 mol/L dexamethasone (Sigma-Aldrich) was used as the culture medium. Samples were randomly allocated to the groups and timepoints. Finally, all samples were cultured in medium for 5 days or a staining solution for imaging. Medium was changed on days 3 and collected on day 3 and day 5.

Irrigation Simulator Design

An irrigation device consisted of a customized cylindrical container, a pressure gauge, saline, and an infusion support was designed to imitate the irrigation process in arthroscopic operations ( Fig. 2C ). The container with a base a diameter of 5 mm represented cavity joint. Each osteochondral explant was placed in the base of cylindrical container which has 2 openings allowing saline inflow and gauge connection. Suspension height of saline was altered by adjusting the infusion support. Irrigation pressure was detected by a pressure gauge connected to the container and placed above the experimented osteochondral explant.

Figure 2.

Design of irrigating process. The explants were harvested from porcine femoral trochlea (A) and cut perpendicularly (B). (C) The irrigation simulator was composed of a container, a pressure gauge, saline, and infusion support. Scale = 5 mm.

Viability Test

Confocal laser scanning microscopy was used to assess viability of chondrocytes by evaluating live and dead cells by a 2-color fluorescence assay. Calcein, which is retained by live cells, emits a strong green fluorescence (excitation/emission [ex/em]: ~494 nm/~514 nm), while ethidium homodimer-1 (EthHD1) can penetrate cells with compromised membranes, resulting in bright red fluorescence in dead cells (ex/em: ~350 nm/~617 nm). A working solution of 4 mM calcein AM (Sigma-Aldrich) and 2 mM EthHD1 (Sigma-Aldrich) was prepared in sterile PBS just prior to use. The explants were then incubated at 37°C for 1 hour in an upright position, followed by observation using a confocal microscope (LSM 800; Carl Zeiss AG).

Image analysis was conducted using ImageJ (National Institutes of Health). The horizontal distance of the injured sites was recorded along with their respective gray value. Dead cells display number-dependent red fluorescents which increased the gray value of the certain area. The distance from the beginning of the sharp increase in gray value in the image to the end of the sharp drop in gray value is defined as the zone of cell death (ZCD). Comparing the ZCD of different groups allows straightforward viability assessment.

Histology

Full-thickness cartilage slices were carefully separated from the subchondral bone of each explant using a sharp scalpel. The cartilage samples were then fixed in 4% buffered formaldehyde (Formafix AG) for a duration of 48 hours. Subsequently, the samples underwent dehydration through a gradient of ethanol and were embedded in paraffin, then sectioned into 5 mm slices using a microtome (Microm). Hematoxylin-eosin (H&E) staining, toluidine blue O staining, and safranin O/fast green staining was conducted following standardized protocols. Brightfield images were captured using an Olympus BX63 microscope.

RNA Extraction and Quantitative Polymerase Chain Reaction

All explants were collected on day 5 for total RNA extraction and explants that underwent immersion with saline instead of irrigation were used as a normalizing control. In detail, cartilage tissue was separated from the osteochondral bone and total RNA was extracted using TRIzol reagent (15 596 018, Invitrogen, USA), according to the manufacturer’s instructions. Then, the RNA concentration was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized using a commercial kit (R323-01, Vazyme, Nanjing, China). Target gene amplification was performed using by magnifying the diluted complementary DNA of 20 µl with SYBR Green Q-PCR Kit (Q141-03, Vazyme, Nanjing, China) using the Applied Biosystems StepOnePlus Real-Time PCR System (Foster City, CA, USA). The differences in quantification cycle (Cq) values between the target gene and the reference gene were calculated (ΔCq). Data were then normalized to the ΔCq values of the non-exposed control samples from day 0 of the respective animal (ΔΔCq).

Secretome Analysis

Conditioned media were collected on day 5 during culture. The concentrations of sulfated glycosaminoglycans (GAGs), interlukin-6 (IL-6) and nitric oxide (NO) were assessed for the respective samples. The levels of GAGs in the media and GAGs in cartilage tissues were quantified using the dimethyl-methylene blue dye-binding assay to evaluate the release of matrix molecules from the osteochondral explants. NO release was measured with the Griess Reagent Kit for Nitrite Determination (G-7921) following the manufacturer’s protocol. The IL-6 levels in the collected culture media were determined using a Porcine IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Elabscience). Values were determined by a microplate reader (ThermoScientific) and normalized to the corresponding wet weight of the cartilage samples.

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism Version 9.3.1. The normality of the data was evaluated using the Shapiro–Wilk test. The standard deviations among samples were proved equal and an ordinary 1-way analysis of variance (ANOVA) was applied. Then, a post hoc Tukey test was employed to compare the mean values of each group with those of all other groups. A significance threshold of P < 0.05 was established.

Results

Effects of Suspension height on Hydrostatic Pressure

Osteochondral explants were carefully harvested from porcine femoral trochlea which exhibited a healthy, smooth cartilage surface ( Fig. 2A ). The samples were first cut along the diameter on the surface, and then the entire explants were cut and split perpendicularly ( Fig. 2B ). Hydrostatic pressure on cartilage surface caused by saline irrigation was positively correlated with suspension height, ranging from 7.0 kPa at 80 cm to 14.5 kPa at 155 cm ( Table 1 ).

Table 1.

Effects of Suspension Height on Hydrostatic Pressure.

Suspension Height	80 cm	105 cm	130 cm	155 cm
Irrigating Pressure	10.2 kPa	13.5 kPa	16.8 kPa	20.1 kPa

Affected Cell Viability

The cut caused by blade induced cell death alongside the cutting edge as observed in axial view. Injured cartilage without being irrigated showed significantly limited amount and expansion of dead cells which was mostly confined in the top 4 to 6 layers of cells in outer layer, while other explants that underwent irrigation procedure for 2 hours exhibited increased cell death to varying degrees on day 5 ( Fig. 3 ). Comparing to 105 cm or 130 cm, suspension height of 80 cm showed similar distribution of cell death. However, cell viability of suspension height of 130 cm was slightly more impaired, indicated by increased number of dead cells on the edges. Suspension height of 155 cm showed significantly wider spread of cell death region than 80 cm (P < 0.001), 105 cm (P < 0.001) and 130 cm (P < 0.001), which expanded toward deeper zones of cartilage ( Fig. 4 ).

Figure 3.

Affected cell viability of immersed or irrigated osteochondral explants after 5 days of chondrogenic culturing. The merged confocal fluorescent image (A), staining for live cells (B), and staining for dead cells (C) of chondrocytes immersed or irrigated with saline with different suspension heights demonstrated impaired cell viability caused by irrigation. Scale = 100 μm.

Figure 4.

Analysis of zone of cell death. The zone of cell death of chondrocytes immersed (A) or irrigated with saline with suspension heights of 80 cm (B), 105 cm (C), 130 cm (D), and 155 cm (E) showed diverse cell viability. (F) Distance of ZCD illustrated suspension height-dependent harmful effects on chondrocytes. Scale = 50 μm. *P < 0.05. ns = nonsignificant.

Altered Gene Expression Profile Induced by Irrigation

To further determine the response of chondrocytes to irrigation, qPCR of apoptosis-related genes was performed. Consistent with the result of cell death analysis, explants showed altered expression level of proapoptosis genes BAX ( Fig. 5A ) and BCL2 ( Fig. 5B ). Suspension height of 80 cm and 105 cm exhibited similar expression level of BAX comparing to immersion group, while 130 cm (P = 0.016) and 155 cm (P = 0.013) exhibited upregulation. Irrigation also led to down-regulation of BCL2, with mild change in range of 80 cm to 130 cm and drastic change in 155 cm. Expression levels of inflammation-related genes NOS2 ( Fig. 5C ) and IL-6 ( Fig. 5D ) were also evaluated to illustrate altered phenotype of chondrocytes. Samples with different suspension height exhibited a proinflammatory profile compared to control group, while group 155 cm demonstrated a significantly higher expression than group 80 cm, 105 cm, and 130 cm.

Figure 5.

Altered gene expression profile after 5 days of chondrogenic culturing. Expression levels of BAX (A), BCL2 (B), IL-6 (C), and NOS2 (D) were affected by suspension heights of saline. *P < 0.05. ns = nonsignificant.

Loss of Extracellular Matrix

Histological test was performed on osteochondral explants cultured in chondrogenic medium for 5 days. H&E staining demonstrated that cartilage with a suspension height of 80 cm exhibited well-arranged chondrocytes with an elongated and parallel morphology in superficial zone and a typical round shape in middle and deeper zone, similar to the control group. However, other groups exhibited compromised cell morphology as well as a less smooth cartilage surface ( Fig. 6A ). Safranin O/fast green staining and toluidine blue O staining indicated that as saline irrigation of suspension height over 80 cm would result in a greater loss of extracellular matrix such as aggrecan (GAG) (Fig. 6B and C). It is worth mentioning that suspension height of 155 cm led to a significant drop of matrix protection. The compromised synthesis of GAG mainly occurred in superficial zone (Fig. 6B and C).

Figure 6.

Histological analysis of osteochondral explants on day 5. H&E staining (A), safranin O/fast green staining (B) and toluidine blue O staining (C) showed extracellular matrix loss induced by saline irrigation. Scale = 100 μm.

Secretome Analysis

The result of secretome analysis was consistent with histological test. The irrigation procedure resulted in decreased GAG in cartilage tissue (80 cm: P = 0.011; 105 cm: P = 0.002; 130 cm: P < 0.001; 150 cm: P < 0.001) ( Fig. 7A ) and increased release of GAG into the culture medium (80 cm: P < 0.001; 105 cm: P < 0.001; 130 cm: P < 0.001; 150 cm: P < 0.001) ( Fig. 7B ), indicating damage of extracellular matrix. It also led to a proinflammatory secretory profile of chondrocytes, confirmed by the excessive IL-6 (105 cm: P = 0.033; 130 cm: P < 0.001; 150 cm: P < 0.001) ( Fig. 7C ) and NO (80 cm: P = 0.004; 105 cm: P = 0.004; 130 cm: P < 0.001; 150 cm: P < 0.001) ( Fig. 7D ) release. Among them, suspension height of 80 cm, 105 cm, and 130 cm shared similar levels of GAGs, NO and IL-6, while 155 cm resulted in a drastic change of such profile.

Figure 7.

Secretome analysis of immersed of irrigated osteochondral explants on day 5. GAG contents (A) preserved in explants along with GAG (B), IL-6 (C) and NO (D) release into medium indicated irrigation-dependent extracellular loss. *P < 0.05. ns = nonsignificant.

Discussion

In arthroscopic operations, saline is commonly used for irrigation to provide a clear view of cartilage surface.¹¹ To our knowledge, there is no documented consensus on suspension heights of saline. Based on our experience in clinical practice, irrigation height in range of approximately 80 to 130 cm is relatively safe without significant cartilage damage.

In this study, saline was placed at different heights of 80 cm, 105 cm, 130 cm, and 155 cm above porcine osteochondral explants before irrigating the cartilage. During the procedure, the saline was in contact with the cartilage surface with diverse pressure and caused dissimilar cell viability and loss of extracellular matrix, confirmed by cell death analysis, histological test, secretome analysis, and qPCR. As far as we could tell, this is the first study to demonstrate the impact of suspension heights of saline on cartilage.

Visualization in arthroscopic operations is crucial for accurate procedures. Techniques that improve visualization include correct portals, tourniquet, drug-diluted irrigation, and irrigation pump system.^12
-14 Studies have shown that irrigation fluid with epinephrine could improve surgeon-rated visualization in shoulder arthroscopy¹⁵ and could subsequently shorten operation time up to 15 minutes¹⁶ without significant effect of the addition of epinephrine on heart rate and blood pressure in all repairing procedures. Other measures such as intravenous tranexamic acid (TXA) have also been proved to be beneficial for visual field clarity with shortened operation time and decrease in total amount of irrigation fluid.¹⁷ However, careless use of the auxiliary drugs could lead to local and systemic complications.¹² Irrigation systems could pump flowing liquid through a joint, which not only enlarges the joint space but also removes tissue debris and thus creating a clear operating field.² Two kinds of irrigation systems are commonly used. The gravity flow system (GFS) requires manual adjustments throughout the operation, such as changing the suspension height of the irrigation fluid, to control the amount of inflow and outflow while automated pump systems (APSs) could maintain the intra-articular pressure at adequate levels automatically(60-80 mm Hg equivalent to 8-10.67 kPa).¹³ The typical perfusion pressure in APS devices approximates the hydrostatic pressure generated by an 80 cm suspended saline. However, APS could result in significant fluid retention indicated by greater weight gain as well as a greater mean deltoid diameter increase due to its relatively higher irrigation pressure.² In summary, surgeons need to strike a balance between clear visualization and minor surgical complications in all kinds of strategies.

The irrigation procedure should be carefully controlled as chondrocytes are sensitive to even slight changes of environmental conditions such as pH, osmolarity, oxygen saturation, and temperature.^1,6,18
-21 Different kinds of irrigation fluids, such as normal saline (sodium chloride 0.9%), Ringer’s solution, lactated Ringer’s solution, and Hartmann’s solution^4,22,23 could also lead to dissimilar cell death possibly due to their different ionic components such as calcium which could induce response to mechanical stimulus of chondrocytes through mechanosensitive ion channels.²⁴ It is suggested that traditional irrigation procedure could cause significant cell death compared to simple immersion.²⁵ Moreover, it could also lead to depletion of resident synovial fluid MSCs, which are crucial cell sources for chondral repair.²⁶ In our study, we proved that viability and ECM synthesis capacity of chondrocytes was impaired by saline irrigation. Interestingly, the general response of chondrocytes to irrigation seemed to be stable at a suspension height of 80 cm to 130 cm. After exceeding the limit of 130 cm, the cartilage exhibited drastic impairment of nearly 3-fold of distance of ZCD and substantial ECM loss. It suggests that although irrigation is inevitable in today’s arthroscopic operations, there is still a way to manage the harm to a minimal degree by limiting the suspension heights. The commonly adopted suspension height is approximately 80 cm in operations. Increased height could possibly flow into the cavity with a higher speed, taking away trimmed tissue and blood more rapidly and thus providing more satisfactory visualization. Taking that into consideration, further research is needed to strike a balance between cartilage protection and adequate visualization.

Under physiological conditions, hydrostatic pressure of immobilized joint cavity is determined by the components and flow of synovial fluid.²⁷ During operations, synovial fluid is replaced by saline and the flow is artificially controlled. Therefore, we speculated that by adjusting suspension height, hydrostatic pressure was indirectly affected and contributed to chondrocytes response. Our study shows that increased suspension height is related to higher kPa-scaled hydrostatic pressure and undesirable cell death. In contradiction to our findings, a study reported that primary condylar chondrocytes exposed to hydrostatic compressive forces of 50 to 250 kPa for 2 hours resulted in enhanced cell viability and reduced apoptosis.²⁸ One possible explanation is that chondrocytes cultured in 2D plane and 3D matrix respond differently to altered environmental conditions.²⁹ Meanwhile, irrigation is a complex procedure that induces comprehensive changes. Other factors, such as shear forces³⁰ caused by surface rinsing, should also be taken into consideration in future studies.

Most studies aim at exploring the impact of hydrostatic forces induced by joint movement rather than irrigation; therefore, the experiments were performed based on MPa-scaled pressure.^10,31
-34 The influence of extrovert hydrostatic forces is on chondrocytes is still controversial due to the differences in species of cartilage source and strategies for applying external forces. The chondro-protective effect was observed in human chondrocytes responding to 5 MPa and murine chondrocytes responding to 3 MPa, with enhanced expression of chondro-inductive genes including SOX9 and ACAN and inhibition expression of osteoarthritis-related genes.^10,32 However, impaired cell metabolism, cell morphology and increased apoptosis was observed by forcing 50 MPa and 200 MPa hydrostatic pressure on chondrocytes.^31,33 In general, it seems that hydrostatic pressure in physiological range is beneficial while excessive force is detrimental. The excessive mechanical loading induces gremlin-1 and activates nuclear factor-κB signaling, which results in induction of catabolic enzymes and matrix degradation.³⁴ It is still not clear how kPa-scaled hydrostatic pressure activates mechanical signaling pathway that leads to cell death and loss of ECM. In this study, significant injury was observed in superficial zone of cartilage, which could be the result of direct contact of saline and cartilage surface or up-regulated gremlin-1 of damaged chondrocytes in the superficial zone.³⁴ Detailed mechanism should be illustrated in future studies to discover strategies for protecting cartilage during irrigation. Herein, we revealed the hydrostatic pressure changes with suspension height. It is crucial to clarify that hydrostatic pressure of physiological range is varied in species. Studies that looked into impacts of mechanical loading should be aware that disputed results could be resulted from neglect of species differences.

Certain limitations should be considered in this study. First, we adopted porcine osteochondral explants instead of human cartilage in this study. Although the general structure of large animal model is similar to that of human, it is important to replicate the realistic effects on the human body, especially in experiments that involve improvement of details in operations. Our study could be an initial exploration of adjustment of suspension heights; further experimental validation is needed on human specimens. Moreover, we focused on the cellular behavior after 5 days of culturing. To fully understand the entire process of cartilage responding to altered irrigation conditions, there should be a longer observation period. Finally, while our study investigated perfusion pressure in arthroscopic saline irrigation, the hydrodynamic effects of irrigation flow-induced shear forces on injured cartilage remain unexamined, thus constituting a focus for our subsequent research.

Conclusion

Irrigation on cartilage surface with suspension heights of 80 cm or more results in height-dependent chondrocytes death and loss of cartilage matrix in a porcine cartilage injury model, confirmed by a proinflammatory and matrix degradative secretome, gene expression profile, and histological analysis after 5 days of ex vivo culture. This study underlines the significance of adjusting the height of irrigation fluids during operations, encouraging further investigations into finding irrigation procedure with minimal harm on cartilage.

Footnotes

Acknowledgements

The authors are grateful for the support from the all staffs of Beijing Key Laboratory of Sports Injuries.

ORCID iD

Weili Shi

Ethical Considerations

The osteochondral explants in this study were harvested from porcine provided by a local abattoir.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by the National Natural Science Foundation of China (General Program) (no. 82272462); Institutional Innovation Transformation Funds for Science and Technology Research and Development (BYSYZHKC2022109); Institutional Clinical Key Project and Talent Project Category B (BYSYZD2021021); and Capital’s Funds for Health Improvement and Research (2024-4-40920).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data will be made available on request.

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