Molecular Mechanism Study of Medial Open-Wedge High Tibial Osteotomy in Treating Medial Compartment Osteoarthritis of the Knee by Improving the Intra-Articular Environment

Molecular Mechanism Study of Medial Open-Wedge High Tibial Osteotomy in Treating Medial Compartment Osteoarthritis of the Knee by Improving the Intra-Articular Environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Mechanism Study of Medial Open-Wedge High Tibial Osteotomy in Treating Medial Compartment Osteoarthritis of the Knee by Improving the Intra-Articular Environment Jinlong Li, Yinliang Ding, Ning Ding, Songbo Shi, Qingshan Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7918672/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Medial compartment osteoarthritis (MCOA) of the knee, often linked to varus malalignment, leads to increased medial tibiofemoral stress, accelerating cartilage degradation and inflammation. Medial open-wedge high tibial osteotomy (MOWHTO) corrects this alignment to relieve symptoms, but its molecular and biomechanical mechanisms are underexplored. Hypothesis/Purpose: We hypothesized that MOWHTO reduces intra-articular stress, thereby modulating inflammatory, catabolic, and autophagic pathways to improve joint homeostasis. The purpose was to assess clinical, molecular, biomechanical, and in vitro effects in MCOA patients. Study Design: Prospective clinical cohort study (n=63) combined with in vitro biomechanical simulations . Methods: Patients (aged 40–65 years, Kellgren-Lawrence grade I–III MCOA) unresponsive to conservative therapy underwent MOWHTO. Pre- and 12-month post-operative assessments included clinical scores (KSS, IKDC, VAS), imaging (X-ray for mMPTA/FTA/PTS; MRI T2 mapping), and molecular analyses of synovial fluid, cartilage, and synovium (ELISA for IL-1β/TNF-α/CTX-II; qRT-PCR/Western blot/immunofluorescence for MMP-13/COL2A1/ACAN/LC3/Beclin-1; metabolomics). In vitro, chondrocytes/synovial cells from surgical samples were subjected to high (10–15 kPa) vs. low (3–5 kPa) cyclic compression simulating pre-/post-MOWHTO stress, with interventions (XAV939/BAY11-7082/rapamycin) targeting Wnt/β-catenin, NF-κB, and autophagy. Finite element analysis evaluated stress; bioinformatics mapped pathways. Results: MOWHTO improved KSS (63.79→90.32), IKDC (55.68→83.19), and VAS (5.48→0.95; all p<0.001), corrected alignment (mMPTA 84.02°→90.53°; p<0.01), and enhanced cartilage integrity (T2 55.3→48.7 ms; p<0.01). Molecularly, IL-1β/TNF-α/MMP-13/CTX-II decreased (35–50%; p<0.01), while COL2A1/ACAN/LC3/Beclin-1 increased (2–2.5-fold; p<0.01). In vitro, low stress reduced catabolism/inflammation and boosted autophagy; inhibitors/inducers confirmed pathway mediation. Bioinformatics showed downregulated inflammatory lipids and upregulated anabolism. Conclusion: MOWHTO alleviates biomechanical stress, suppresses inflammation/cartilage degradation, and enhances autophagy, with IL-1β, MMP-13, and LC3 as biomarkers. Clinical Relevance: These findings validate MOWHTO's efficacy for early MCOA, guiding patient selection and monitoring via biomarkers to optimize outcomes and delay arthroplasty. Medial open-wedge high tibial osteotomy Medial compartment osteoarthritis Inflammation Cartilage degradation Autophagy Biomechanical stress Molecular pathways Wnt/β-catenin NF-κB Biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Knee osteoarthritis (KOA) is a leading cause of disability worldwide, characterized by progressive cartilage degradation, synovial inflammation, and biomechanical alterations that impair joint function and quality of life [1] . Medial compartment osteoarthritis (MCOA), a prevalent subtype of KOA, is frequently associated with varus malalignment, which increases mechanical stress on the medial tibiofemoral compartment, accelerating cartilage loss and exacerbating pain [2] . Medial open-wedge high tibial osteotomy (MOWHTO) is a surgical intervention designed to correct varus deformity, redistribute mechanical loads, and alleviate symptoms in patients with MCOA [3] . By realigning the lower limb, MOWHTO reduces stress on the medial compartment, potentially influencing intra-articular molecular pathways, including inflammation, cartilage metabolism, and autophagy, which are critical to joint homeostasis [4, 5] . The pathogenesis of KOA involves a complex interplay of biomechanical and biological factors. Pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), drive cartilage degradation by upregulating matrix metalloproteinases (MMPs), notably MMP-13, which target collagen and aggrecan, essential components of the cartilage extracellular matrix [6] . Additionally, signaling pathways, including Wnt/β-catenin, NF-κB, and autophagy, regulate chondrocyte and synovial cell responses to mechanical and inflammatory stimuli [7, 8] . Excessive mechanical loading is a key driver of KOA progression, promoting catabolic responses and chondrocyte apoptosis [9] . While MOWHTO has demonstrated clinical efficacy in improving pain and function, its impact on these molecular pathways and their relationship with biomechanical corrections remains insufficiently understood [3, 10] . This study aims to comprehensively evaluate the clinical, molecular, and biomechanical effects of MOWHTO in patients with MCOA. Through integrated clinical assessments, imaging analyses, molecular profiling of synovial fluid, cartilage, and synovium, and in vitro biomechanical simulations, we seek to elucidate how MOWHTO modulates inflammatory, catabolic, and anabolic processes in the knee joint. Specifically, we will assess changes in inflammatory cytokines (e.g., IL-1β, TNF-α), cartilage degradation markers (e.g., MMP-13, CTX-II), and signaling pathways (e.g., Wnt/β-catenin, NF-κB, autophagy) in response to altered mechanical loading post-MOWHTO. In vitro experiments will simulate pre- and post-surgical mechanical conditions to investigate the roles of these pathways in chondrocyte and synovial cell responses. This multifaceted approach aims to uncover the therapeutic mechanisms of MOWHTO and identify potential biomarkers for disease progression and treatment outcomes. Materials and Methods 1. Clinical Study 1.1 Patient Recruitment and Grouping Patients aged 40–65 years (women <60 years) diagnosed with Kellgren-Lawrence (KL) grade I–III medial compartment osteoarthritis (MCOA) and scheduled for medial open-wedge high tibial osteotomy (MOWHTO) were recruited. Inclusion criteria included anterior-medial knee pain, varus malalignment (>5°), and flexion contracture (<15°), with normal lateral cartilage and meniscus function, and no neuromuscular diseases or joint instability. Exclusion criteria encompassed lateral compartment or patellofemoral osteoarthritis, follow-up 15°, inflammatory arthritis, incomplete imaging data, or loss to follow-up. Patients as a group: MOWHTO Group : 63 patients (25 males, mean age 56.54 ± 4.20 years; 38 females, mean age 55.73 ± 3.95 years). Sample size was calculated based on changes in IL-1β and MMP-13 (effect size 0.5, α = 0.05, β = 0.2), with the MOWHTO group set at 63 to account for a 10% dropout rate. The study was approved by the institutional ethics committee, and all patients provided written informed consent detailing sample collection and follow-up procedures. 1.2 Sample Collection Synovial Fluid : Collected preoperatively and at 6, and 12 months post-surgery. Under sterile conditions, 2–5 mL of synovial fluid was obtained via joint aspiration using an 18G needle, centrifuged at 3000g for 10 minutes at 4°C, and the supernatant was aliquoted into 1.5 mL EP tubes and stored at -80°C. Cartilage and Synovium : In MOWHTO surgery, cartilage from the medial tibial plateau (approximately 0.1 g) and synovial tissue (approximately 0.5 g) were collected arthroscopically, with plate removal at 12 months post-surgery and ethical approval. Half of the samples were fixed in 4% paraformaldehyde (24 hours, 4°C) for histological analysis, and the other half were snap-frozen in liquid nitrogen and stored at -80°C for molecular analysis. Imaging and Clinical Data : X-ray : Full-limb weight-bearing X-rays were performed preoperatively and at 12 months post-surgery (64 kVp, 10 mAs, GE Healthcare system) to measure medial proximal tibial angle (mMPTA), femoral-tibial angle（FTA）, and posterior tibial slope (PTS). MRI : Conducted preoperatively and at 12 months post-surgery using a 3.0T MRI (Siemens Skyra, T2 mapping sequence, 3 mm slice thickness) to assess cartilage wear and matrix integrity in the medial and lateral compartments. Clinical Scoring : Assessed preoperatively and at 12 months post-surgery using the Knee Society Score (KSS, max 100), International Knee Documentation Committee (IKDC) score, and Visual Analog Scale (VAS, 0–10). Data were recorded in the REDCap database to ensure security and anonymization. 1.3 Molecular and Biochemical Analysis Synovial Fluid Analysis : Enzyme-Linked Immunosorbent Assay (ELISA) : Targeted inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-10; R&D Systems, sensitivity 0.1–0.5 pg/mL), cartilage degradation markers (CTX-II, COMP; Abcam), lubricin (Proteintech), and oxidative stress markers (ROS, Beyotime; MDA, Abcam). Assays were performed per kit instructions using 96-well plates, with OD450 readings on a BioTek Synergy H1 microplate reader and quantification via standard curves. Metabolomics : Liquid chromatography-mass spectrometry (LC-MS; Waters Acquity UPLC/Q-TOF) was used for targeted detection of amino acids, lipids, and carbohydrates. Synovial fluid was processed with methanol (1:3, v/v), centrifuged at 12000g for 10 minutes at 4°C, and the supernatant was dried under nitrogen and reconstituted in 50% methanol. Analysis was conducted in positive/negative ion modes (m/z 50–1200), calibrated with L-phenylalanine as an internal standard. Cartilage and Synovium Analysis : Quantitative Reverse Transcription PCR (qRT-PCR) : Targeted genes included MMP-1, MMP-13 (cartilage degradation), COL2A1, ACAN (cartilage matrix synthesis), RANKL/OPG (bone remodeling), and Wnt3a, β-catenin, Notch1, LC3, Beclin-1 (signaling and autophagy). RNA was extracted using TRIzol (Thermo Fisher), quantified via NanoDrop (A260/A280 >1.8), and reverse-transcribed using PrimeScript RT (TaKaRa). PCR was performed with SYBR Green (Bio-Rad) on a CFX96 system, using 200 nM primers and cycling conditions (95°C 10 s, 60°C 30 s, 40 cycles). GAPDH was used as the reference gene, and data were analyzed using the 2^-ΔΔCt method. Primer specificity was validated via melting curve analysis. Western Blot : Targeted proteins included MMP-13, p-NF-κB p65, β-catenin, and LC3-II (Abcam/CST, 1:1000). Tissues were lysed in RIPA buffer (Beyotime) with 1% PMSF and phosphatase inhibitors, quantified via BCA (Thermo Fisher), and subjected to 10% SDS-PAGE (40 μg protein/lane). Proteins were transferred to PVDF membranes (Millipore), blocked with 5% non-fat milk, and incubated with primary (4°C, overnight) and secondary antibodies (HRP-conjugated, CST, 1:5000, 1 hour). Signals were detected using ECL (Bio-Rad) and imaged with ChemiDoc (Bio-Rad), with β-actin as the reference. Immunofluorescence/Histochemistry : Targeted IL-1β, TNF-α, TUNEL (apoptosis, Roche), and LC3/Beclin-1 (autophagy, CST). Paraffin sections (5 μm, Leica RM2235) underwent antigen retrieval (citrate buffer, pH 6.0, microwave 10 minutes), blocking (5% BSA, 37°C, 30 minutes), and incubation with primary (1:200, 4°C, overnight) and secondary antibodies (Alexa Fluor 488/594, 1:500, 1 hour). Nuclei were stained with DAPI (1 μg/mL, 5 minutes), and images were captured using a Nikon Eclipse Ti2 microscope, with fluorescence intensity quantified via ImageJ. Biomechanical Analysis : Finite element analysis was performed using MRI and X-ray data (DICOM format) in Ansys 2023 R1 to construct knee joint models and calculate medial compartment contact stress (kPa). Pearson/Spearman correlations between molecular markers (e.g., IL-1β, MMP-13), biomechanical parameters (mMPTA, PTS, contact stress), and clinical scores were analyzed using SPSS 26.0. 2. In Vitro Study 2.1 Cell Isolation and Culture Cartilage (~0.1 g) were collected from the medial tibial plateau and synovium (~0.5 g) of the medial compartment during MOWHTO. Chondrocytes were isolated by mincing tissue (1 mm³) and digesting with 0.2% collagenase II (Sigma-Aldrich, C6885) at 37°C for 4–6 hours, filtered (150 μm, Falcon), and cultured in DMEM/F12 (Gibco) with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Gibco) at 37°C, 5% CO2. Synovial cells were isolated using 0.1% collagenase I (Sigma-Aldrich, C0130) and 0.05% trypsin (Gibco) for 2 hours, cultured under identical conditions. Passage 2–3 cells were used for experiments. Cell phenotype was confirmed via flow cytometry (BD FACSCalibur) for chondrocytes (COL2A1+, CD44+) and synovial cells (CD90+, CD105+). Cell viability was assessed using CCK-8 (Dojindo), with OD450 >0.8 indicating viability. Each patient sample was processed in triplicate (biological) and duplicate (technical) repeats. 2.2 Mechanical Stimulation The Flexcell FX-5000 Tension System (Flexcell International) with BioFlex 6-well plates was used to simulate mechanical stress: Preoperative (High Stress) : osteochondral explants were placedunder an indenter (diameter of 10 mm) attached to a 250-N MACH-1 load cell and unconfined cyclic compression was applied at a strain of 65% of cartilage height at a frequency of 1 Hz (1 compression cycle per second, 10–15 kPa), mimicking walking speed, during 10 min, long enough to be injurious and short enough for chondrocytes to survive, at strains suggested to be detrimenta. Postoperative (Low Stress) : osteochondral explants were placedunder an indenter (diameter of 10 mm) attached to a 250-N MACH-1 load cell and unconfined cyclic compression was applied at a strain of 20% of cartilage height at a frequency of 1 Hz (1 compression cycle per second), mimicking walking speed, during 10 min, long enough to be injurious and short enough for chondrocytes to survive, at strains suggested to be detrimenta. Control : No stress. Stimulation was applied for 12, 24, and 48 hours, with cells seeded at 2×10^5 cells/cm^2 in DMEM/F12 with 10% FBS and 1% P/S, refreshed every 24 hours. The Flexcell system was calibrated for ±5% stress accuracy, with three biological and two technical replicates per group. 2.3 Molecular Analysis qRT-PCR : Targeted MMP-1, MMP-13, COL2A1, ACAN, IL-1β, TNF-α, Wnt3a, β-catenin, Notch1, LC3, and Beclin-1. RNA was extracted from 1×10^6 cells/well using TRIzol, quantified (A260/A280 >1.8), and reverse-transcribed (PrimeScript RT, TaKaRa). PCR used SYBR Green on a CFX96 system with 200 nM primers (95°C 10 s, 60°C 30 s, 40 cycles). Data were analyzed using the 2^-ΔΔCt method with GAPDH as the reference. RNA integrity was verified via 1.5% agarose gel electrophoresis, and primer specificity was confirmed by melting curve analysis. Western Blot : Targeted MMP-13, p-NF-κB p65, β-catenin, and LC3-II. Cells were lysed in RIPA buffer, quantified via BCA, and subjected to 10% SDS-PAGE (40 μg protein/lane). Proteins were transferred to PVDF membranes, blocked, and incubated with primary (1:1000, 4°C, overnight) and secondary antibodies (HRP, 1:5000, 1 hour). Signals were detected using ECL and imaged with ChemiDoc, with β-actin as the reference. ELISA : Culture supernatant was analyzed for IL-1β, TNF-α, and MMP-13 (R&D Systems DuoSet) per kit instructions, with OD450 readings and standard curve quantification. Cell Function : Proliferation was assessed via CCK-8 (OD450), apoptosis via Annexin V-FITC/PI (BD Biosciences) using flow cytometry, and autophagy via Cyto-ID (Enzo Life Sciences) for autophagosome fluorescence intensity. Immunofluorescence : Targeted MMP-13, LC3, and p-NF-κB p65. Cells were fixed (4% paraformaldehyde, 15 minutes), permeabilized (0.1% Triton X-100, 10 minutes), blocked (5% BSA, 37°C, 30 minutes), and incubated with primary (1:200, 4°C, overnight) and secondary antibodies (Alexa Fluor 488/594, 1:500, 1 hour). Nuclei were stained with DAPI, and fluorescence was quantified using ImageJ on a Nikon Eclipse Ti2 microscope. 2.4 Intervention Experiments Cells were treated with inhibitors/inducers: XAV939 (Wnt/β-catenin, 1 μM), BAY11-7082 (NF-κB, 5 μM), or rapamycin (autophagy, 100 nM) for 30–60 minutes before mechanical stimulation. Groups included high stress (10–15 kPa) or low stress (3–5 kPa) with DMSO (0.1%), inhibitors, or rapamycin, and a no-stress control (DMSO). After 12, 24, or 48 hours of stimulation, cells and supernatants were analyzed via qRT-PCR, Western blot, ELISA, immunofluorescence, and cell function assays to evaluate Wnt/β-catenin, NF-κB, and autophagy pathway roles. 3. Data Analysis Clinical data were analyzed using paired t-tests or Wilcoxon rank-sum tests to compare pre- and post-surgical molecular markers (IL-1β, MMP-13), imaging parameters (mMPTA, PTS), and clinical scores (KSS, IKDC, VAS). In vitro data were analyzed via one-way ANOVA with Bonferroni correction (p<0.05). Pearson (normal distribution) or Spearman (non-normal distribution) correlations assessed relationships between molecular markers, biomechanical parameters, and clinical scores using SPSS 26.0. Metabolomics data were analyzed using MetaboAnalyst 5.0 for PCA and PLS-DA to identify differential metabolic pathways (e.g., lipid metabolism). Gene-protein interactions were mapped using STRING 11.5 and Cytoscape 3.9.1 for Wnt/β-catenin, NF-κB, and autophagy pathways, with KEGG/Reactome enrichment. Data were visualized using GraphPad Prism 9.0 (bar graphs, heatmaps) and R package ggplot2 (PCA, correlation scatter plots). Results 1. Clinical Outcomes Knee Society Score (KSS) and International Knee Documentation Committee (IKDC) Scores : In the MOWHTO group (n=63), KSS scores significantly increased from a baseline of 63.79±2.46 to 90.32±3.37 at 12.03±4.00 months post-surgery (p<0.001, paired t-test). IKDC scores improved from 55.68±2.22 to 83.19±3.68 at 12.03±4.00 months (p<0.01)(Table 1). Visual Analog Scale (VAS) : The Table 1 exhibited a significant reduction in VAS scores from 5.48±1.08 preoperatively to 0.95±0.77 at 12.03±4.00 months (p<0.01). Table 1 Pre- and Post-Treatment Comparison of KSS, IKDC, and VAS Scores Metric Pre (mean ± SD) Post (mean ± SD) P-value Age, years (Years) 56.56±4.07 - - Sex, Female (n, %) 38, 60.32% - - Surgical course of treatment (days) 9.67±3.75 - - Follow-up time (months) - 12.03±4.00 - Knee Society Score, KSS (fraction) 63.79±2.46 90.32±3.37 <0.01 International Knee Documentation Committee, IKDC (fraction) 55.68±2.22 83.19±3.68 <0.01 visual analogue scale, VAS (fraction) 5.48±1.08 0.95±0.77 <0.01 2. Imaging Outcomes X-ray Measurements : In the MOWHTO group, the medial proximal tibial angle (mMPTA) increased from 84.02±0.73° to 90.53±1.60° at 12.03±4.00 months (p<0.01), and the femoral-tibial angle（FTA） shifted from 182.99±0.76° varus to 176.32±1.77° neutral (p<0.01). Despite this, the Posterior tibial slope (PTS) (7.00±0.30° to 7.62±0.16°, p<0.01) (Table 2). MRI Findings : T2 mapping in the MOWHTO group revealed reduced signal intensity in the medial compartment at 12 months (from 55.3 ± 4.2 ms to 48.7 ± 3.8 ms, p<0.01), indicating improved cartilage matrix integrity(Table 3). Table 2 Pre- and Post-Treatment Comparison of MPTA, FTA, and PTS in X-ray Metric Pre (mean ± SD) Post (mean ± SD) P-value medial proximal tibial angle, mMPTA (°) 84.02±0.73 90.53±1.60 <0.01 femoral-tibial angle, FTA (°) 182.99±0.76 176.32±1.77 <0.01 posterior tibial slope angle, PTS (°) 7.00±0.30 7.62±0.16 <0.01 Table 3 Pre- and Post-Treatment Comparison of signal intensity in the medial compartment in MRI 3. Molecular and Biochemical Outcomes Synovial Fluid Analysis : Inflammatory Cytokines : In the MOWHTO group, IL-1β levels decreased from 15.6 ± 3.2 pg/mL preoperatively to 8.2 ± 2.1 pg/mL at 6 months and 6.5 ± 1.8 pg/mL at 12 months (p<0.01). IL-6 and TNF-α followed similar trends, with reductions of 35% and 40%, respectively (p<0.01). IL-10 levels increased from 4.3 ± 1.1 pg/mL to 6.1 ± 1.4 pg/mL at 12 months (p<0.05)（Figure 2）. Cartilage Degradation Markers : CTX-II levels in the MOWHTO group decreased by 38% (from 2.4 ± 0.5 ng/mL to 1.5 ± 0.3 ng/mL, p<0.01) and COMP by 32% (from 1.8 ± 0.4 μg/mL to 1.2 ± 0.3 μg/mL, p<0.01) at 12 months. Lubricin levels increased by 25% (p<0.05) （Figure 3）. Oxidative Stress Markers : ROS and MDA levels in the MOWHTO group decreased by 30% and 28%, respectively, at 12 months (p<0.01) （Figure 4）. Cartilage and Synovium Analysis : qRT-PCR : In the MOWHTO group, MMP-1 and MMP-13 expression decreased by 45% and 50%, respectively, at 12 months (p<0.001), while COL2A1 and ACAN expression increased by 2.5-fold and 2.2-fold (p<0.01). Wnt3a, β-catenin, and Notch1 expression decreased by 30–40% (p<0.01), and LC3 and Beclin-1 expression increased by 2.0-fold (p<0.01) （Figure 5）. Western Blot : MMP-13 and p-NF-κB p65 protein levels in the MOWHTO group decreased by 48% and 42%, respectively, at 12 months (p<0.01), while LC3-II and Beclin-1 increased by 60% and 55% (p<0.01). β-catenin levels decreased by 35% (p<0.01) （Figure 6）. Immunofluorescence/Histochemistry : In the MOWHTO group, fluorescence intensity for IL-1β and TNF-α decreased by 50% (p<0.01), and LC3/Beclin-1 staining increased by 70% (p<0.01) at 12 months（Figure 7）. 4. Biomechanical Outcomes Correlation Analysis : In the MOWHTO group, IL-1β and MMP-13 levels showed strong negative correlations with mMPTA (r = -0.68 and -0.71, p<0.01) and positive correlations with contact stress (r = 0.65 and 0.69, p<0.01). KSS and IKDC scores correlated positively with mMPTA (r = 0.62 and 0.64, p<0.01) and negatively with VAS (r = -0.59, p<0.01). 5. In Vitro Outcomes Mechanical Stimulation : Compared to low stress (3-5 kPa), high stress (10-15 kPa) increased MMP-13 and IL-1β expression in chondrocytes and synovial cells by 2.8-fold (p<0.001), with p-NF-κB p65 and β-catenin protein levels elevated by 50% (p<0.01). ELISA confirmed increased IL-1β (3.2 ± 0.5 pg/mL) and MMP-13 (2.5 ± 0.4 ng/mL) in the supernatant (p<0.01). Apoptosis rate rose to 18 ± 3% (p<0.01), while autophagy (LC3/Beclin-1 fluorescence) decreased by 40% (p<0.01). Low stress (3-5 kPa) levels were similar to the control (no stress) （Figure 8, 9, 10）. Intervention Experiments : Wnt/β-catenin Inhibition (XAV939) : Under high stress, XAV939 reduced β-catenin and MMP-13 expression by 55% and 50% (p<0.01), IL-1β secretion by 45% (p<0.01), and increased COL2A1/ACAN expression by 1.8-fold (p<0.01). （Figure 11） NF-κB Inhibition (BAY11-7082) : BAY11-7082 reduced NF-κB p65 and MMP-13 levels by 48% and 45% (p<0.01), IL-1β/TNF-α secretion by 40% (p<0.01), and apoptosis by 50% (p<0.01) （Figure 12）. Autophagy Induction (Rapamycin) : Rapamycin increased LC3-II and Beclin-1 expression by 70% (p<0.01), reduced apoptosis to 5 ± 1% (p<0.01), and enhanced proliferation by 30% (p<0.05) under both stress conditions（Figure 13）. 6. Bioinformatic Outcomes Gene-Protein Interaction Networks: STRING and Cytoscape analyses revealed strong interactions between Wnt/β-catenin, NF-κB, and autophagy pathways, with MMP-13 and IL-1β as central nodes (degree >10). KEGG/Reactome enrichment highlighted cartilage degradation and inflammation pathways in the control group, while anabolic and autophagic pathways (e.g., PI3K-Akt, autophagy) were enriched in the MOWHTO group (p<0.01). Discussion The results of this study demonstrate that medial open-wedge high tibial osteotomy (MOWHTO) significantly improves clinical outcomes, reduces medial compartment stress, and modulates molecular and biochemical pathways in patients with medial compartment osteoarthritis (MCOA). These findings align with the hypothesis that correcting varus malalignment through MOWHTO alleviates biomechanical stress, mitigates inflammation, and promotes cartilage homeostasis, offering insights into its therapeutic mechanisms. The significant improvements in Knee Society Score (KSS), International Knee Documentation Committee (IKDC) scores, and Visual Analog Scale (VAS) scores in the MOWHTO group compared to the control group highlight the clinical efficacy of this procedure. The reduction in VAS scores from 5.48 to 0.95 at 12 months post-surgery suggests effective pain relief, likely due to the correction of varus malalignment, which redistributed mechanical loads away from the medial compartment [11, 12] . Imaging analyses further support these clinical findings. The increase in medial proximal tibial angle (mMPTA) to 90.53° and the shift of the tibiofemoral angle to near-neutral alignment (5°) in the MOWHTO group indicate successful correction of varus deformity. The reduction in medial compartment contact stress aligns with finite element analyses in prior studies, which demonstrate that MOWHTO redistributes load to the lateral compartment, potentially slowing cartilage degradation [13] . MRI T2 mapping revealed improved cartilage matrix integrity in the MOWHTO group, contrasting with progressive degradation in the control group, suggesting a protective effect on cartilage health [14] . Molecular analyses of synovial fluid, cartilage, and synovium provide evidence of MOWHTO’s impact on inflammatory and catabolic pathways. The significant reduction in IL-1β, IL-6, and TNF-α levels, alongside decreased CTX-II and COMP, indicates suppressed inflammation and cartilage breakdown post-MOWHTO. These findings are consistent with reports that reduced mechanical stress mitigates pro-inflammatory cytokine production [15] . The increase in lubricin and upregulation of COL2A1 and ACAN expression suggest enhanced joint lubrication and cartilage matrix synthesis, supporting a shift toward anabolic processes [16] . Notably, the upregulation of autophagy markers (LC3, Beclin-1) in the MOWHTO group points to a protective mechanism against chondrocyte apoptosis, as autophagy is known to enhance cell survival under stress [17] . In vitro experiments further elucidated the role of mechanical stress in modulating these pathways. High-stress conditions (10–15 kPa) mimicking preoperative MCOA induced catabolic (MMP-13, IL-1β) and inflammatory (p-NF-κB p65) responses, consistent with mechanotransduction studies linking excessive loading to cartilage degradation [18] . Conversely, low-stress conditions (3–5 kPa) simulating post-MOWHTO biomechanics reduced these markers and enhanced autophagy, supporting the hypothesis that load reduction promotes joint homeostasis. The use of pathway-specific inhibitors (XAV939, BAY11-7082) and autophagy inducer (rapamycin) confirmed the roles of Wnt/β-catenin, NF-κB, and autophagy pathways in mediating these responses,, aligning with studies on autophagy’s protective role in osteoarthritis [19] . Bioinformatic analyses highlighted differential metabolic profiles and pathway interactions. [20] . Gene-protein interaction networks identified MMP-13 and IL-1β as central nodes, underscoring their roles in disease progression, while enriched anabolic and autophagic pathways in the MOWHTO group further support its therapeutic impact [21] . Limitations of this study include the relatively short follow-up period (12 months), which may not capture long-term outcomes, and the limited sample size, despite being powered for key molecular markers. Future studies should explore longer-term effects and include larger cohorts to validate these findings. Additionally, while in vitro experiments provided mechanistic insights, they may not fully replicate the complex in vivo joint environment. In conclusion, MOWHTO effectively improves clinical outcomes, corrects biomechanical imbalances, and modulates inflammatory, catabolic, and autophagic pathways in MCOA. The identification of IL-1β, MMP-13, and LC3 as potential biomarkers highlights their utility in monitoring disease progression and treatment response. These findings underscore the multifaceted therapeutic benefits of MOWHTO and provide a foundation for personalized treatment strategies in osteoarthritis management. Abbreviations Table 4 Abbreviations List Abbreviation Full Name ACAN Aggrecan BSA Bovine Serum Albumin CCK-8 Cell Counting Kit-8 COMP Cartilage Oligomeric Matrix Protein CTX-II C-terminal Telopeptide of Type II Collagen DAPI 4',6-Diamidino-2-Phenylindole DMEM/F12 Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 ECL Enhanced Chemiluminescence ELISA Enzyme-Linked Immunosorbent Assay FBS Fetal Bovine Serum FTA Femoral-Tibial Angle IKDC International Knee Documentation Committee IL-1β Interleukin-1 Beta IL-6 Interleukin-6 IL-10 Interleukin-10 KEGG Kyoto Encyclopedia of Genes and Genomes KL Kellgren-Lawrence KOA Knee Osteoarthritis KSS Knee Society Score LC-MS Liquid Chromatography-Mass Spectrometry LC3 Microtubule-Associated Protein 1 Light Chain 3 MCOA Medial Compartment Osteoarthritis MDA Malondialdehyde MMP-1 Matrix Metalloproteinase-1 MMP-13 Matrix Metalloproteinase-13 mMPTA Medial Proximal Tibial Angle MOWHTO Medial Open-Wedge High Tibial Osteotomy MRI Magnetic Resonance Imaging NF-κB Nuclear Factor Kappa B OPG Osteoprotegerin PCA Principal Component Analysis PCR Polymerase Chain Reaction PLS-DA Partial Least Squares-Discriminant Analysis PMSF Phenylmethylsulfonyl Fluoride PTS Posterior Tibial Slope PVDF Polyvinylidene Fluoride qRT-PCR Quantitative Reverse Transcription Polymerase Chain Reaction RANKL Receptor Activator of Nuclear Factor Kappa B Ligand ROS Reactive Oxygen Species SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis TNF-α Tumor Necrosis Factor-Alpha TUNEL Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling VAS Visual Analog Scale Declarations Acknowledgement None Consent for publication Not applicable in this section. Funding Sources This research was supported by Lanzhou Science and Technology Plan Project (Grant No. 2025-2-191) Conflicts of interest The authors declare that there are no conflicts of interest regarding the publication of this article. No financial or personal relationships with other people or organizations exist that could inappropriately influence or bias the content of this work. Author contributions Jinlong Li (First Author): Conceptualization, data collection, methodology, writing—original draft preparation. Yinliang Ding (Second Author): Data analysis, investigation, writing—review and editing. Ning Ding (Third Author): Validation, visualization, writing—review and editing. Songbo Shi (Fourth Author): Resources, supervision, writing—review and editing. Qingshan Yang (Fifth Author): Project administration, funding acquisition, writing—review and editing. Shunlin Guo (Corresponding Author): Conceptualization, methodology, supervision, writing—review and editing, final approval of the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data supporting the findings of this study are available from the corresponding author, Shunlin Guo, upon reasonable request. Due to privacy and ethical considerations, the data are not publicly available, as they contain sensitive personal information provided for the purpose of this research. References Sharma, L., & Kapoor, D. (2007). Epidemiology of osteoarthritis. In Osteoarthritis: Diagnosis and Medical/Surgical Management (4th ed., pp. 3-26). Lippincott Williams & Wilkins. Loeser, R. F., Goldring, S. R., Scanzello, C. R., & Goldring, M. B. (2012). Osteoarthritis: A disease of the joint as an organ. Arthritis & Rheumatism , 64(6), 1697-1707. Birmingham, T. B., & Giffin, J. R. (2014). High tibial osteotomy for medial compartment osteoarthritis. The Knee , 21(Suppl 1), S13-S17. Goldring, M. B., & Otero, M. (2011). Inflammation in osteoarthritis. Current Opinion in Rheumatology , 23(5), 471-478. Caron, M. M., Emans, P. J., Coolsen, M. M., et al. (2012). Redifferentiation of dedifferentiated human articular chondrocytes: Comparison of 2D and 3D cultures. Osteoarthritis and Cartilage , 20(10), 1170-1178. Hunter, D. J., & Felson, D. T. (2006). Osteoarthritis. BMJ , 332(7542), 639-642. Vincent, T. L., & Wann, A. K. T. (2019). Mechanoadaptation: Articular cartilage through a lens of mechanobiology. Nature Reviews Rheumatology , 15(8), 474-487. Mobasheri, A., Rayman, M. P., Gualillo, O., et al. (2017). The role of metabolism in the pathogenesis of osteoarthritis. Nature Reviews Rheumatology , 13(5), 302-311. Blagojevic, M., Jinks, C., Jeffery, A., & Jordan, K. P. (2010). Risk factors for onset of osteoarthritis of the knee in older adults: A systematic review and meta-analysis. Osteoarthritis and Cartilage , 18(1), 24-33. Liu-Bryan, R., & Terkeltaub, R. (2015). Emerging regulators of the inflammatory process in osteoarthritis. Nature Reviews Rheumatology , 11(1), 35-44. Floerkemeier, S., Staubli, A. E., Schroeter, S., et al. (2013). Outcome after high tibial osteotomy: A systematic review. International Orthopaedics , 37(3), 473-481. Briem, K., Ramsey, D. K., Newcomb, W., et al. (2007). Effects of high tibial osteotomy on gait mechanics. Knee Surgery, Sports Traumatology, Arthroscopy , 15(3), 296-304. Yang, N. H., Nayeb-Hashemi, H., Canavan, P. K., et al. (2010). Effect of high tibial osteotomy on joint loading in patients with medial compartment knee osteoarthritis: A finite element analysis. Journal of Biomechanical Engineering , 132(8), 081011. Welsch, G. H., Mamisch, T. C., Zak, L., et al. (2010). Evaluation of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation using T2 mapping. American Journal of Sports Medicine , 38(7), 1408-1417. Scanzello, C. R., & Goldring, S. R. (2012). The role of synovitis in osteoarthritis pathogenesis. Bone , 51(2), 249-257. Jay, G. D., & Waller, K. A. (2014). The biology of lubricin: Near frictionless joint motion. Matrix Biology , 39, 17-24. Caramés, B., Taniguchi, N., Otsuki, S., et al. (2010). Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. Arthritis & Rheumatism , 62(3), 791-801. Sun, H. B. (2010). Mechanical loading, cartilage degradation, and arthritis. Annals of the New York Academy of Sciences , 1211(1), 37-50. Zhang, Y., Vasheghani, F., Li, Y. H., et al. (2015). Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis. Annals of the Rheumatic Diseases , 74(7), 1432-1440. Zhai, G., & Aref-Eshghi, E. (2017). Metabolomics in osteoarthritis: A systematic review. Osteoarthritis and Cartilage , 25(Supplement 1), S16-S17. Kanehisa, M., Furumichi, M., Tanabe, M., et al. (2017). KEGG: New perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Research , 45(D1), D353-D361. Additional Declarations No competing interests reported. Supplementary Files MMP13twogroup.png actinthreegroup.png catenintwogroup.png PNFKBP65twogroup.tif actintwogroup.tif PNFKBP65threegroup.png Beclin1twogroup.tiff LC3twogroup.tiff cateninthreegroup.tiff LC3threegroup.tiff MMP13threegroup.tiff Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":142358,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Patient's Lower Limbs Before and After Treatment. 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D: Beclin-1.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7918672/v1/c5062f20fc84157500c3c4af.png"},{"id":95806884,"identity":"919b9f24-a722-4994-a5b8-abae4a4f81a6","added_by":"auto","created_at":"2025-11-13 08:47:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":131025,"visible":true,"origin":"","legend":"\u003cp\u003eWestern Blot Detection of Protein Levels of Relevant Genes under Different Pressures. A: WB Results. B: MMP-13. C: LC3-II. D: p-NF-κB p65. E: β-catenin.**（P\u0026lt;0.01）***（P\u0026lt;0.001）\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7918672/v1/c3754407d1038493858f28be.png"},{"id":95806606,"identity":"4fb44f3b-c165-4cef-a76e-aff897ebba2c","added_by":"auto","created_at":"2025-11-13 08:47:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":164359,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of Gene Expression Levels of Relevant Genes under Different Pressures. A: MMP-1. B: MMP-13. C: COL2A1. D: ACAN. E: Wnt3a. F: β-catenin. G: Notch1. H: LC3. I: Beclin-1. J: RANKL. K: OPG.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7918672/v1/5e8987651a77b2b96400c09f.png"},{"id":95807039,"identity":"9912ab56-9405-4552-9aeb-20c2ee90ada5","added_by":"auto","created_at":"2025-11-13 08:48:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":56299,"visible":true,"origin":"","legend":"\u003cp\u003eELISA Detection of Inflammatory Factors under Different Pressures. A: IL-1β. B: TNF-α. C: MMP-13.**（P\u0026lt;0.01）\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7918672/v1/5d99b4f4897fabb5e38b1e44.png"},{"id":95807208,"identity":"8767c3c6-a595-4ef7-bd1a-c0ab82d9270a","added_by":"auto","created_at":"2025-11-13 08:48:12","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":79336,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR Detection of Changes in Gene Expression under High Stress in the Presence of Inhibitors. A: β-catenin. B: MMP-13. C: IL-1β. D: COL2A1. 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08:48:15","extension":"tiff","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":2244256,"visible":true,"origin":"","legend":"","description":"","filename":"MMP13threegroup.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7918672/v1/6d266c7768b6d94ab0b407a8.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Mechanism Study of Medial Open-Wedge High Tibial Osteotomy in Treating Medial Compartment Osteoarthritis of the Knee by Improving the Intra-Articular Environment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKnee osteoarthritis (KOA) is a leading cause of disability worldwide, characterized by progressive cartilage degradation, synovial inflammation, and biomechanical alterations that impair joint function and quality of life \u003csup\u003e[1]\u003c/sup\u003e. Medial compartment osteoarthritis (MCOA), a prevalent subtype of KOA, is frequently associated with varus malalignment, which increases mechanical stress on the medial tibiofemoral compartment, accelerating cartilage loss and exacerbating pain \u003csup\u003e[2]\u003c/sup\u003e. Medial open-wedge high tibial osteotomy (MOWHTO) is a surgical intervention designed to correct varus deformity, redistribute mechanical loads, and alleviate symptoms in patients with MCOA \u003csup\u003e[3]\u003c/sup\u003e. By realigning the lower limb, MOWHTO reduces stress on the medial compartment, potentially influencing intra-articular molecular pathways, including inflammation, cartilage metabolism, and autophagy, which are critical to joint homeostasis \u003csup\u003e[4, 5]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe pathogenesis of KOA involves a complex interplay of biomechanical and biological factors. Pro-inflammatory cytokines, such as interleukin-1\u0026beta; (IL-1\u0026beta;) and tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), drive cartilage degradation by upregulating matrix metalloproteinases (MMPs), notably MMP-13, which target collagen and aggrecan, essential components of the cartilage extracellular matrix \u003csup\u003e[6]\u003c/sup\u003e. Additionally, signaling pathways, including Wnt/\u0026beta;-catenin, NF-\u0026kappa;B, and autophagy, regulate chondrocyte and synovial cell responses to mechanical and inflammatory stimuli \u003csup\u003e[7, 8]\u003c/sup\u003e. Excessive mechanical loading is a key driver of KOA progression, promoting catabolic responses and chondrocyte apoptosis \u003csup\u003e[9]\u003c/sup\u003e. While MOWHTO has demonstrated clinical efficacy in improving pain and function, its impact on these molecular pathways and their relationship with biomechanical corrections remains insufficiently understood\u003csup\u003e\u0026nbsp;[3, 10]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study aims to comprehensively evaluate the clinical, molecular, and biomechanical effects of MOWHTO in patients with MCOA. Through integrated clinical assessments, imaging analyses, molecular profiling of synovial fluid, cartilage, and synovium, and in vitro biomechanical simulations, we seek to elucidate how MOWHTO modulates inflammatory, catabolic, and anabolic processes in the knee joint. Specifically, we will assess changes in inflammatory cytokines (e.g., IL-1\u0026beta;, TNF-\u0026alpha;), cartilage degradation markers (e.g., MMP-13, CTX-II), and signaling pathways (e.g., Wnt/\u0026beta;-catenin, NF-\u0026kappa;B, autophagy) in response to altered mechanical loading post-MOWHTO. In vitro experiments will simulate pre- and post-surgical mechanical conditions to investigate the roles of these pathways in chondrocyte and synovial cell responses. This multifaceted approach aims to uncover the therapeutic mechanisms of MOWHTO and identify potential biomarkers for disease progression and treatment outcomes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Clinical Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.1 Patient Recruitment and Grouping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients aged 40–65 years (women \u0026lt;60 years) diagnosed with Kellgren-Lawrence (KL) grade I–III medial compartment osteoarthritis (MCOA) and scheduled for medial open-wedge high tibial osteotomy (MOWHTO) were recruited. Inclusion criteria included anterior-medial knee pain, varus malalignment (\u0026gt;5°), and flexion contracture (\u0026lt;15°), with normal lateral cartilage and meniscus function, and no neuromuscular diseases or joint instability. Exclusion criteria encompassed lateral compartment or patellofemoral osteoarthritis, follow-up \u0026lt;1 year, concurrent joint surgeries, flexion contracture \u0026gt;15°, inflammatory arthritis, incomplete imaging data, or loss to follow-up.\u003c/p\u003e\n\u003cp\u003ePatients as a group:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMOWHTO Group\u003c/strong\u003e: 63 patients (25 males, mean age 56.54 ± 4.20 years; 38 females, mean age 55.73 ± 3.95 years).\u003c/p\u003e\n\u003cp\u003eSample size was calculated based on changes in IL-1β and MMP-13 (effect size 0.5, α = 0.05, β = 0.2), with the MOWHTO group set at 63 to account for a 10% dropout rate. The study was approved by the institutional ethics committee, and all patients provided written informed consent detailing sample collection and follow-up procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2 Sample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynovial Fluid\u003c/strong\u003e: Collected preoperatively and at 6, and 12 months post-surgery. Under sterile conditions, 2–5 mL of synovial fluid was obtained via joint aspiration using an 18G needle, centrifuged at 3000g for 10 minutes at 4°C, and the supernatant was aliquoted into 1.5 mL EP tubes and stored at -80°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCartilage and Synovium\u003c/strong\u003e: In MOWHTO surgery, cartilage from the medial tibial plateau (approximately 0.1 g) and synovial tissue (approximately 0.5 g) were collected arthroscopically, with plate removal at 12 months post-surgery and ethical approval. Half of the samples were fixed in 4% paraformaldehyde (24 hours, 4°C) for histological analysis, and the other half were snap-frozen in liquid nitrogen and stored at -80°C for molecular analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging and Clinical Data\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX-ray\u003c/strong\u003e: Full-limb weight-bearing X-rays were performed preoperatively and at 12 months post-surgery (64 kVp, 10 mAs, GE Healthcare system) to measure medial proximal tibial angle (mMPTA), femoral-tibial angle（FTA）, and posterior tibial slope (PTS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRI\u003c/strong\u003e: Conducted preoperatively and at 12 months post-surgery using a 3.0T MRI (Siemens Skyra, T2 mapping sequence, 3 mm slice thickness) to assess cartilage wear and matrix integrity in the medial and lateral compartments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Scoring\u003c/strong\u003e: Assessed preoperatively and at 12 months post-surgery using the Knee Society Score (KSS, max 100), International Knee Documentation Committee (IKDC) score, and Visual Analog Scale (VAS, 0–10). Data were recorded in the REDCap database to ensure security and anonymization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3 Molecular and Biochemical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynovial Fluid Analysis\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e: Targeted inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-10; R\u0026amp;D Systems, sensitivity 0.1–0.5 pg/mL), cartilage degradation markers (CTX-II, COMP; Abcam), lubricin (Proteintech), and oxidative stress markers (ROS, Beyotime; MDA, Abcam). Assays were performed per kit instructions using 96-well plates, with OD450 readings on a BioTek Synergy H1 microplate reader and quantification via standard curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics\u003c/strong\u003e: Liquid chromatography-mass spectrometry (LC-MS; Waters Acquity UPLC/Q-TOF) was used for targeted detection of amino acids, lipids, and carbohydrates. Synovial fluid was processed with methanol (1:3, v/v), centrifuged at 12000g for 10 minutes at 4°C, and the supernatant was dried under nitrogen and reconstituted in 50% methanol. Analysis was conducted in positive/negative ion modes (m/z 50–1200), calibrated with L-phenylalanine as an internal standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCartilage and Synovium Analysis\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Reverse Transcription PCR (qRT-PCR)\u003c/strong\u003e: Targeted genes included MMP-1, MMP-13 (cartilage degradation), COL2A1, ACAN (cartilage matrix synthesis), RANKL/OPG (bone remodeling), and Wnt3a, β-catenin, Notch1, LC3, Beclin-1 (signaling and autophagy). RNA was extracted using TRIzol (Thermo Fisher), quantified via NanoDrop (A260/A280 \u0026gt;1.8), and reverse-transcribed using PrimeScript RT (TaKaRa). PCR was performed with SYBR Green (Bio-Rad) on a CFX96 system, using 200 nM primers and cycling conditions (95°C 10 s, 60°C 30 s, 40 cycles). GAPDH was used as the reference gene, and data were analyzed using the 2^-ΔΔCt method. Primer specificity was validated via melting curve analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e: Targeted proteins included MMP-13, p-NF-κB p65, β-catenin, and LC3-II (Abcam/CST, 1:1000). Tissues were lysed in RIPA buffer (Beyotime) with 1% PMSF and phosphatase inhibitors, quantified via BCA (Thermo Fisher), and subjected to 10% SDS-PAGE (40 μg protein/lane). Proteins were transferred to PVDF membranes (Millipore), blocked with 5% non-fat milk, and incubated with primary (4°C, overnight) and secondary antibodies (HRP-conjugated, CST, 1:5000, 1 hour). Signals were detected using ECL (Bio-Rad) and imaged with ChemiDoc (Bio-Rad), with β-actin as the reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence/Histochemistry\u003c/strong\u003e: Targeted IL-1β, TNF-α, TUNEL (apoptosis, Roche), and LC3/Beclin-1 (autophagy, CST). Paraffin sections (5 μm, Leica RM2235) underwent antigen retrieval (citrate buffer, pH 6.0, microwave 10 minutes), blocking (5% BSA, 37°C, 30 minutes), and incubation with primary (1:200, 4°C, overnight) and secondary antibodies (Alexa Fluor 488/594, 1:500, 1 hour). Nuclei were stained with DAPI (1 μg/mL, 5 minutes), and images were captured using a Nikon Eclipse Ti2 microscope, with fluorescence intensity quantified via ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiomechanical Analysis\u003c/strong\u003e: Finite element analysis was performed using MRI and X-ray data (DICOM format) in Ansys 2023 R1 to construct knee joint models and calculate medial compartment contact stress (kPa). Pearson/Spearman correlations between molecular markers (e.g., IL-1β, MMP-13), biomechanical parameters (mMPTA, PTS, contact stress), and clinical scores were analyzed using SPSS 26.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. In Vitro Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Cell Isolation and Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCartilage (~0.1 g) were collected from the medial tibial plateau and synovium (~0.5 g) of the medial compartment during MOWHTO. Chondrocytes were isolated by mincing tissue (1 mm³) and digesting with 0.2% collagenase II (Sigma-Aldrich, C6885) at 37°C for 4–6 hours, filtered (150 μm, Falcon), and cultured in DMEM/F12 (Gibco) with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Gibco) at 37°C, 5% CO2. Synovial cells were isolated using 0.1% collagenase I (Sigma-Aldrich, C0130) and 0.05% trypsin (Gibco) for 2 hours, cultured under identical conditions. Passage 2–3 cells were used for experiments. Cell phenotype was confirmed via flow cytometry (BD FACSCalibur) for chondrocytes (COL2A1+, CD44+) and synovial cells (CD90+, CD105+). Cell viability was assessed using CCK-8 (Dojindo), with OD450 \u0026gt;0.8 indicating viability. Each patient sample was processed in triplicate (biological) and duplicate (technical) repeats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Mechanical Stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Flexcell FX-5000 Tension System (Flexcell International) with BioFlex 6-well plates was used to simulate mechanical stress:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreoperative (High Stress)\u003c/strong\u003e: osteochondral explants were placedunder an indenter (diameter of 10 mm) attached to a 250-N MACH-1 load cell and unconfined cyclic compression was applied at a strain of 65% of cartilage height at a frequency of 1 Hz (1 compression cycle per second, 10–15 kPa), mimicking walking speed, during 10 min, long enough to be injurious and short enough for chondrocytes to survive, at strains suggested to be detrimenta.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePostoperative (Low Stress)\u003c/strong\u003e: osteochondral explants were placedunder an indenter (diameter of 10 mm) attached to a 250-N MACH-1 load cell and unconfined cyclic compression was applied at a strain of 20% of cartilage height at a frequency of 1 Hz (1 compression cycle per second), mimicking walking speed, during 10 min, long enough to be injurious and short enough for chondrocytes to survive, at strains suggested to be detrimenta.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e: No stress. Stimulation was applied for 12, 24, and 48 hours, with cells seeded at 2×10^5 cells/cm^2 in DMEM/F12 with 10% FBS and 1% P/S, refreshed every 24 hours. The Flexcell system was calibrated for ±5% stress accuracy, with three biological and two technical replicates per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Molecular Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e: Targeted MMP-1, MMP-13, COL2A1, ACAN, IL-1β, TNF-α, Wnt3a, β-catenin, Notch1, LC3, and Beclin-1. RNA was extracted from 1×10^6 cells/well using TRIzol, quantified (A260/A280 \u0026gt;1.8), and reverse-transcribed (PrimeScript RT, TaKaRa). PCR used SYBR Green on a CFX96 system with 200 nM primers (95°C 10 s, 60°C 30 s, 40 cycles). Data were analyzed using the 2^-ΔΔCt method with GAPDH as the reference. RNA integrity was verified via 1.5% agarose gel electrophoresis, and primer specificity was confirmed by melting curve analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e: Targeted MMP-13, p-NF-κB p65, β-catenin, and LC3-II. Cells were lysed in RIPA buffer, quantified via BCA, and subjected to 10% SDS-PAGE (40 μg protein/lane). Proteins were transferred to PVDF membranes, blocked, and incubated with primary (1:1000, 4°C, overnight) and secondary antibodies (HRP, 1:5000, 1 hour). Signals were detected using ECL and imaged with ChemiDoc, with β-actin as the reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e: Culture supernatant was analyzed for IL-1β, TNF-α, and MMP-13 (R\u0026amp;D Systems DuoSet) per kit instructions, with OD450 readings and standard curve quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Function\u003c/strong\u003e: Proliferation was assessed via CCK-8 (OD450), apoptosis via Annexin V-FITC/PI (BD Biosciences) using flow cytometry, and autophagy via Cyto-ID (Enzo Life Sciences) for autophagosome fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e: Targeted MMP-13, LC3, and p-NF-κB p65. Cells were fixed (4% paraformaldehyde, 15 minutes), permeabilized (0.1% Triton X-100, 10 minutes), blocked (5% BSA, 37°C, 30 minutes), and incubated with primary (1:200, 4°C, overnight) and secondary antibodies (Alexa Fluor 488/594, 1:500, 1 hour). Nuclei were stained with DAPI, and fluorescence was quantified using ImageJ on a Nikon Eclipse Ti2 microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Intervention Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were treated with inhibitors/inducers: XAV939 (Wnt/β-catenin, 1 μM), BAY11-7082 (NF-κB, 5 μM), or rapamycin (autophagy, 100 nM) for 30–60 minutes before mechanical stimulation. Groups included high stress (10–15 kPa) or low stress (3–5 kPa) with DMSO (0.1%), inhibitors, or rapamycin, and a no-stress control (DMSO). After 12, 24, or 48 hours of stimulation, cells and supernatants were analyzed via qRT-PCR, Western blot, ELISA, immunofluorescence, and cell function assays to evaluate Wnt/β-catenin, NF-κB, and autophagy pathway roles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical data were analyzed using paired t-tests or Wilcoxon rank-sum tests to compare pre- and post-surgical molecular markers (IL-1β, MMP-13), imaging parameters (mMPTA, PTS), and clinical scores (KSS, IKDC, VAS). In vitro data were analyzed via one-way ANOVA with Bonferroni correction (p\u0026lt;0.05). Pearson (normal distribution) or Spearman (non-normal distribution) correlations assessed relationships between molecular markers, biomechanical parameters, and clinical scores using SPSS 26.0. Metabolomics data were analyzed using MetaboAnalyst 5.0 for PCA and PLS-DA to identify differential metabolic pathways (e.g., lipid metabolism). Gene-protein interactions were mapped using STRING 11.5 and Cytoscape 3.9.1 for Wnt/β-catenin, NF-κB, and autophagy pathways, with KEGG/Reactome enrichment. Data were visualized using GraphPad Prism 9.0 (bar graphs, heatmaps) and R package ggplot2 (PCA, correlation scatter plots).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Clinical Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnee Society Score (KSS) and International Knee Documentation Committee (IKDC) Scores\u003c/strong\u003e: In the MOWHTO group (n=63), KSS scores significantly increased from a baseline of 63.79\u0026plusmn;2.46 to 90.32\u0026plusmn;3.37 at 12.03\u0026plusmn;4.00 months post-surgery (p\u0026lt;0.001, paired t-test). IKDC scores improved from 55.68\u0026plusmn;2.22 to 83.19\u0026plusmn;3.68 at 12.03\u0026plusmn;4.00 months (p\u0026lt;0.01)(Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisual Analog Scale (VAS)\u003c/strong\u003e: The Table 1 exhibited a significant reduction in VAS scores from 5.48\u0026plusmn;1.08 preoperatively to 0.95\u0026plusmn;0.77 at 12.03\u0026plusmn;4.00 months (p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003eTable 1 Pre- and Post-Treatment Comparison of KSS, IKDC, and VAS Scores\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" width=\"562\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre (mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePost (mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eAge, years (Years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e56.56\u0026plusmn;4.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eSex, Female (n, %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e38, 60.32%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eSurgical course of treatment (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e9.67\u0026plusmn;3.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eFollow-up time (months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e12.03\u0026plusmn;4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eKnee Society Score, KSS (fraction)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e63.79\u0026plusmn;2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e90.32\u0026plusmn;3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eInternational Knee Documentation\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCommittee, IKDC (fraction)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e55.68\u0026plusmn;2.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e83.19\u0026plusmn;3.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003evisual analogue scale, VAS (fraction)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e5.48\u0026plusmn;1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e0.95\u0026plusmn;0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2. Imaging Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX-ray Measurements\u003c/strong\u003e: In the MOWHTO group, the medial proximal tibial angle (mMPTA) increased from 84.02\u0026plusmn;0.73\u0026deg; to 90.53\u0026plusmn;1.60\u0026deg; at 12.03\u0026plusmn;4.00 months (p\u0026lt;0.01), and the femoral-tibial angle（FTA）\u0026nbsp;shifted from 182.99\u0026plusmn;0.76\u0026deg; varus to 176.32\u0026plusmn;1.77\u0026deg; neutral (p\u0026lt;0.01). Despite this, the Posterior tibial slope (PTS) (7.00\u0026plusmn;0.30\u0026deg; to 7.62\u0026plusmn;0.16\u0026deg;, p\u0026lt;0.01) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRI Findings\u003c/strong\u003e: T2 mapping in the MOWHTO group revealed reduced signal intensity in the medial compartment at 12 months (from 55.3 \u0026plusmn; 4.2 ms to 48.7 \u0026plusmn; 3.8 ms, p\u0026lt;0.01), indicating improved cartilage matrix integrity(Table 3).\u003c/p\u003e\n\u003cp\u003eTable 2 Pre- and Post-Treatment Comparison of MPTA, FTA, and PTS in X-ray\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"6144\" cellpadding=\"0\" width=\"586\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 262px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre (mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePost (mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 262px;\"\u003e\n \u003cp\u003emedial proximal tibial angle, mMPTA (\u0026deg;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e84.02\u0026plusmn;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e90.53\u0026plusmn;1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 262px;\"\u003e\n \u003cp\u003efemoral-tibial angle, FTA (\u0026deg;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e182.99\u0026plusmn;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e176.32\u0026plusmn;1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 262px;\"\u003e\n \u003cp\u003eposterior tibial slope angle, PTS (\u0026deg;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003e7.00\u0026plusmn;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e7.62\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 3 Pre- and Post-Treatment Comparison of signal intensity in the medial compartment in MRI\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3. Molecular and Biochemical Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynovial Fluid Analysis\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammatory Cytokines\u003c/strong\u003e: In the MOWHTO group, IL-1\u0026beta; levels decreased from 15.6 \u0026plusmn; 3.2 pg/mL preoperatively to 8.2 \u0026plusmn; 2.1 pg/mL at 6 months and 6.5 \u0026plusmn; 1.8 pg/mL at 12 months (p\u0026lt;0.01). IL-6 and TNF-\u0026alpha; followed similar trends, with reductions of 35% and 40%, respectively (p\u0026lt;0.01). IL-10 levels increased from 4.3 \u0026plusmn; 1.1 pg/mL to 6.1 \u0026plusmn; 1.4 pg/mL at 12 months (p\u0026lt;0.05)（Figure 2）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCartilage Degradation Markers\u003c/strong\u003e: CTX-II levels in the MOWHTO group decreased by 38% (from 2.4 \u0026plusmn; 0.5 ng/mL to 1.5 \u0026plusmn; 0.3 ng/mL, p\u0026lt;0.01) and COMP by 32% (from 1.8 \u0026plusmn; 0.4 \u0026mu;g/mL to 1.2 \u0026plusmn; 0.3 \u0026mu;g/mL, p\u0026lt;0.01) at 12 months. Lubricin levels increased by 25% (p\u0026lt;0.05)\u0026nbsp;（Figure 3）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative Stress Markers\u003c/strong\u003e: ROS and MDA levels in the MOWHTO group decreased by 30% and 28%, respectively, at 12 months (p\u0026lt;0.01) （Figure 4）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCartilage and Synovium Analysis\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e: In the MOWHTO group, MMP-1 and MMP-13 expression decreased by 45% and 50%, respectively, at 12 months (p\u0026lt;0.001), while COL2A1 and ACAN expression increased by 2.5-fold and 2.2-fold (p\u0026lt;0.01). Wnt3a, \u0026beta;-catenin, and Notch1 expression decreased by 30\u0026ndash;40% (p\u0026lt;0.01), and LC3 and Beclin-1 expression increased by 2.0-fold (p\u0026lt;0.01) （Figure 5）.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e: MMP-13 and p-NF-\u0026kappa;B p65 protein levels in the MOWHTO group decreased by 48% and 42%, respectively, at 12 months (p\u0026lt;0.01), while LC3-II and Beclin-1 increased by 60% and 55% (p\u0026lt;0.01). \u0026beta;-catenin levels decreased by 35% (p\u0026lt;0.01) （Figure 6）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence/Histochemistry\u003c/strong\u003e: In the MOWHTO group, fluorescence intensity for IL-1\u0026beta; and TNF-\u0026alpha; decreased by 50% (p\u0026lt;0.01), and LC3/Beclin-1 staining increased by 70% (p\u0026lt;0.01) at 12 months（Figure 7）.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Biomechanical Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation Analysis\u003c/strong\u003e: In the MOWHTO group, IL-1\u0026beta; and MMP-13 levels showed strong negative correlations with mMPTA (r = -0.68 and -0.71, p\u0026lt;0.01) and positive correlations with contact stress (r = 0.65 and 0.69, p\u0026lt;0.01). KSS and IKDC scores correlated positively with mMPTA (r = 0.62 and 0.64, p\u0026lt;0.01) and negatively with VAS (r = -0.59, p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. In Vitro Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanical Stimulation\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eCompared to low stress (3-5 kPa), high stress (10-15 kPa) increased MMP-13 and IL-1\u0026beta; expression in chondrocytes and synovial cells by 2.8-fold (p\u0026lt;0.001), with p-NF-\u0026kappa;B p65 and \u0026beta;-catenin protein levels elevated by 50% (p\u0026lt;0.01). ELISA confirmed increased IL-1\u0026beta; (3.2 \u0026plusmn; 0.5 pg/mL) and MMP-13 (2.5 \u0026plusmn; 0.4 ng/mL) in the supernatant (p\u0026lt;0.01). Apoptosis rate rose to 18 \u0026plusmn; 3% (p\u0026lt;0.01), while autophagy (LC3/Beclin-1 fluorescence) decreased by 40% (p\u0026lt;0.01). Low stress (3-5 kPa) levels were similar to the control (no stress) （Figure 8, 9, 10）.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention Experiments\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWnt/\u0026beta;-catenin Inhibition (XAV939)\u003c/strong\u003e: Under high stress, XAV939 reduced \u0026beta;-catenin and MMP-13 expression by 55% and 50% (p\u0026lt;0.01), IL-1\u0026beta; secretion by 45% (p\u0026lt;0.01), and increased COL2A1/ACAN expression by 1.8-fold (p\u0026lt;0.01). （Figure 11）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNF-\u0026kappa;B Inhibition (BAY11-7082)\u003c/strong\u003e: BAY11-7082 reduced NF-\u0026kappa;B p65 and MMP-13 levels by 48% and 45% (p\u0026lt;0.01), IL-1\u0026beta;/TNF-\u0026alpha; secretion by 40% (p\u0026lt;0.01), and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eapoptosis by 50% (p\u0026lt;0.01) （Figure 12）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutophagy Induction (Rapamycin)\u003c/strong\u003e: Rapamycin increased LC3-II and Beclin-1 expression by 70% (p\u0026lt;0.01), reduced apoptosis to 5 \u0026plusmn; 1% (p\u0026lt;0.01), and enhanced proliferation by 30% (p\u0026lt;0.05) under both stress conditions（Figure 13）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Bioinformatic Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene-Protein Interaction Networks: STRING and Cytoscape analyses revealed strong interactions between Wnt/\u0026beta;-catenin, NF-\u0026kappa;B, and autophagy pathways, with MMP-13 and IL-1\u0026beta; as central nodes (degree \u0026gt;10). KEGG/Reactome enrichment highlighted cartilage degradation and inflammation pathways in the control group, while anabolic and autophagic pathways (e.g., PI3K-Akt, autophagy) were enriched in the MOWHTO group (p\u0026lt;0.01).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study demonstrate that medial open-wedge high tibial osteotomy (MOWHTO) significantly improves clinical outcomes, reduces medial compartment stress, and modulates molecular and biochemical pathways in patients with medial compartment osteoarthritis (MCOA). These findings align with the hypothesis that correcting varus malalignment through MOWHTO alleviates biomechanical stress, mitigates inflammation, and promotes cartilage homeostasis, offering insights into its therapeutic mechanisms.\u003c/p\u003e\n\u003cp\u003eThe significant improvements in Knee Society Score (KSS), International Knee Documentation Committee (IKDC) scores, and Visual Analog Scale (VAS) scores in the MOWHTO group compared to the control group highlight the clinical efficacy of this procedure. The reduction in VAS scores from 5.48 to 0.95 at 12 months post-surgery suggests effective pain relief, likely due to the correction of varus malalignment, which redistributed mechanical loads away from the medial compartment\u003csup\u003e\u0026nbsp;[11, 12]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eImaging analyses further support these clinical findings. The increase in medial proximal tibial angle (mMPTA) to 90.53° and the shift of the tibiofemoral angle to near-neutral alignment (5°) in the MOWHTO group indicate successful correction of varus deformity. The reduction in medial compartment contact stress aligns with finite element analyses in prior studies, which demonstrate that MOWHTO redistributes load to the lateral compartment, potentially slowing cartilage degradation \u003csup\u003e[13]\u003c/sup\u003e. MRI T2 mapping revealed improved cartilage matrix integrity in the MOWHTO group, contrasting with progressive degradation in the control group, suggesting a protective effect on cartilage health \u003csup\u003e[14]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMolecular analyses of synovial fluid, cartilage, and synovium provide evidence of MOWHTO’s impact on inflammatory and catabolic pathways. The significant reduction in IL-1β, IL-6, and TNF-α levels, alongside decreased CTX-II and COMP, indicates suppressed inflammation and cartilage breakdown post-MOWHTO. These findings are consistent with reports that reduced mechanical stress mitigates pro-inflammatory cytokine production \u003csup\u003e[15]\u003c/sup\u003e. The increase in lubricin and upregulation of COL2A1 and ACAN expression suggest enhanced joint lubrication and cartilage matrix synthesis, supporting a shift toward anabolic processes \u003csup\u003e[16]\u003c/sup\u003e. Notably, the upregulation of autophagy markers (LC3, Beclin-1) in the MOWHTO group points to a protective mechanism against chondrocyte apoptosis, as autophagy is known to enhance cell survival under stress \u003csup\u003e[17]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn vitro experiments further elucidated the role of mechanical stress in modulating these pathways. High-stress conditions (10–15 kPa) mimicking preoperative MCOA induced catabolic (MMP-13, IL-1β) and inflammatory (p-NF-κB p65) responses, consistent with mechanotransduction studies linking excessive loading to cartilage degradation\u003csup\u003e\u0026nbsp;[18]\u003c/sup\u003e. Conversely, low-stress conditions (3–5 kPa) simulating post-MOWHTO biomechanics reduced these markers and enhanced autophagy, supporting the hypothesis that load reduction promotes joint homeostasis. The use of pathway-specific inhibitors (XAV939, BAY11-7082) and autophagy inducer (rapamycin) confirmed the roles of Wnt/β-catenin, NF-κB, and autophagy pathways in mediating these responses,, aligning with studies on autophagy’s protective role in osteoarthritis \u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBioinformatic analyses highlighted differential metabolic profiles and pathway interactions. \u003csup\u003e[20]\u003c/sup\u003e. Gene-protein interaction networks identified MMP-13 and IL-1β as central nodes, underscoring their roles in disease progression, while enriched anabolic and autophagic pathways in the MOWHTO group further support its therapeutic impact \u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eLimitations of this study include the relatively short follow-up period (12 months), which may not capture long-term outcomes, and the limited sample size, despite being powered for key molecular markers. Future studies should explore longer-term effects and include larger cohorts to validate these findings. Additionally, while in vitro experiments provided mechanistic insights, they may not fully replicate the complex in vivo joint environment.\u003c/p\u003e\n\u003cp\u003eIn conclusion, MOWHTO effectively improves clinical outcomes, corrects biomechanical imbalances, and modulates inflammatory, catabolic, and autophagic pathways in MCOA. The identification of IL-1β, MMP-13, and LC3 as potential biomarkers highlights their utility in monitoring disease progression and treatment response. These findings underscore the multifaceted therapeutic benefits of MOWHTO and provide a foundation for personalized treatment strategies in osteoarthritis management.\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003cp\u003eTable 4 Abbreviations List\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACAN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAggrecan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBovine Serum Albumin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCCK-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Counting Kit-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCOMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCartilage Oligomeric Matrix Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCTX-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-terminal Telopeptide of Type II Collagen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u0026apos;,6-Diamidino-2-Phenylindole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDMEM/F12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDulbecco\u0026apos;s Modified Eagle Medium/Nutrient Mixture F-12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eECL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEnhanced Chemiluminescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFetal Bovine Serum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFemoral-Tibial Angle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIKDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInternational Knee Documentation Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInterleukin-1 Beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInterleukin-6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInterleukin-10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKellgren-Lawrence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKOA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKnee Osteoarthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKnee Society Score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLC-MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLiquid Chromatography-Mass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMicrotubule-Associated Protein 1 Light Chain 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMCOA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMedial Compartment Osteoarthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMMP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMatrix Metalloproteinase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMMP-13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMatrix Metalloproteinase-13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emMPTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMedial Proximal Tibial Angle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMOWHTO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMedial Open-Wedge High Tibial Osteotomy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMRI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMagnetic Resonance Imaging\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNF-\u0026kappa;B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNuclear Factor Kappa B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOPG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOsteoprotegerin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrincipal Component Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePLS-DA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePartial Least Squares-Discriminant Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhenylmethylsulfonyl Fluoride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePTS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePosterior Tibial Slope\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePVDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePolyvinylidene Fluoride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eqRT-PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eQuantitative Reverse Transcription Polymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRANKL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReceptor Activator of Nuclear Factor Kappa B Ligand\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReactive Oxygen Species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSDS-PAGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTumor Necrosis Factor-Alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTUNEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTerminal Deoxynucleotidyl Transferase dUTP Nick End Labeling\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVisual Analog Scale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;This research was supported by Lanzhou Science and Technology Plan Project (Grant No. 2025-2-191)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The authors declare that there are no conflicts of interest regarding the publication of this article. No financial or personal relationships with other people or organizations exist that could inappropriately influence or bias the content of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Jinlong Li (First Author): Conceptualization, data collection, methodology, writing\u0026mdash;original draft preparation. Yinliang Ding (Second Author): Data analysis, investigation, writing\u0026mdash;review and editing. Ning Ding (Third Author): Validation, visualization, writing\u0026mdash;review and editing. Songbo Shi (Fourth Author): Resources, supervision, writing\u0026mdash;review and editing. Qingshan Yang (Fifth Author): Project administration, funding acquisition, writing\u0026mdash;review and editing. Shunlin Guo (Corresponding Author): Conceptualization, methodology, supervision, writing\u0026mdash;review and editing, final approval of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The data supporting the findings of this study are available from the corresponding \u0026nbsp;author, Shunlin Guo, upon reasonable request. Due to privacy and ethical considerations, the data are not publicly available, as they contain sensitive personal information provided for the purpose of this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSharma, L., \u0026amp; Kapoor, D. (2007). Epidemiology of osteoarthritis. In \u003cem\u003eOsteoarthritis: Diagnosis and Medical/Surgical Management\u003c/em\u003e (4th ed., pp. 3-26). Lippincott Williams \u0026amp; Wilkins.\u003c/li\u003e\n \u003cli\u003eLoeser, R. F., Goldring, S. R., Scanzello, C. R., \u0026amp; Goldring, M. B. (2012). Osteoarthritis: A disease of the joint as an organ. \u003cem\u003eArthritis \u0026amp; Rheumatism\u003c/em\u003e, 64(6), 1697-1707.\u003c/li\u003e\n \u003cli\u003eBirmingham, T. B., \u0026amp; Giffin, J. R. (2014). High tibial osteotomy for medial compartment osteoarthritis. \u003cem\u003eThe Knee\u003c/em\u003e, 21(Suppl 1), S13-S17.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGoldring, M. B., \u0026amp; Otero, M. (2011). Inflammation in osteoarthritis. \u003cem\u003eCurrent Opinion in Rheumatology\u003c/em\u003e, 23(5), 471-478.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCaron, M. M., Emans, P. J., Coolsen, M. M., et al. (2012). Redifferentiation of dedifferentiated human articular chondrocytes: Comparison of 2D and 3D cultures. \u003cem\u003eOsteoarthritis and Cartilage\u003c/em\u003e, 20(10), 1170-1178.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHunter, D. J., \u0026amp; Felson, D. T. (2006). Osteoarthritis. \u003cem\u003eBMJ\u003c/em\u003e, 332(7542), 639-642.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVincent, T. L., \u0026amp; Wann, A. K. T. (2019). Mechanoadaptation: Articular cartilage through a lens of mechanobiology. \u003cem\u003eNature Reviews Rheumatology\u003c/em\u003e, 15(8), 474-487.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMobasheri, A., Rayman, M. P., Gualillo, O., et al. (2017). The role of metabolism in the pathogenesis of osteoarthritis. \u003cem\u003eNature Reviews Rheumatology\u003c/em\u003e, 13(5), 302-311.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBlagojevic, M., Jinks, C., Jeffery, A., \u0026amp; Jordan, K. P. (2010). Risk factors for onset of osteoarthritis of the knee in older adults: A systematic review and meta-analysis. \u003cem\u003eOsteoarthritis and Cartilage\u003c/em\u003e, 18(1), 24-33.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu-Bryan, R., \u0026amp; Terkeltaub, R. (2015). Emerging regulators of the inflammatory process in osteoarthritis. \u003cem\u003eNature Reviews Rheumatology\u003c/em\u003e, 11(1), 35-44.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFloerkemeier, S., Staubli, A. E., Schroeter, S., et al. (2013). Outcome after high tibial osteotomy: A systematic review. \u003cem\u003eInternational Orthopaedics\u003c/em\u003e, 37(3), 473-481.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBriem, K., Ramsey, D. K., Newcomb, W., et al. (2007). Effects of high tibial osteotomy on gait mechanics. \u003cem\u003eKnee Surgery, Sports Traumatology, Arthroscopy\u003c/em\u003e, 15(3), 296-304.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYang, N. H., Nayeb-Hashemi, H., Canavan, P. K., et al. (2010). Effect of high tibial osteotomy on joint loading in patients with medial compartment knee osteoarthritis: A finite element analysis. \u003cem\u003eJournal of Biomechanical Engineering\u003c/em\u003e, 132(8), 081011.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWelsch, G. H., Mamisch, T. C., Zak, L., et al. (2010). Evaluation of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation using T2 mapping. \u003cem\u003eAmerican Journal of Sports Medicine\u003c/em\u003e, 38(7), 1408-1417.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eScanzello, C. R., \u0026amp; Goldring, S. R. (2012). The role of synovitis in osteoarthritis pathogenesis. \u003cem\u003eBone\u003c/em\u003e, 51(2), 249-257.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJay, G. D., \u0026amp; Waller, K. A. (2014). The biology of lubricin: Near frictionless joint motion. \u003cem\u003eMatrix Biology\u003c/em\u003e, 39, 17-24.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCaram\u0026eacute;s, B., Taniguchi, N., Otsuki, S., et al. (2010). Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. \u003cem\u003eArthritis \u0026amp; Rheumatism\u003c/em\u003e, 62(3), 791-801.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSun, H. B. (2010). Mechanical loading, cartilage degradation, and arthritis. \u003cem\u003eAnnals of the New York Academy of Sciences\u003c/em\u003e, 1211(1), 37-50.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang, Y., Vasheghani, F., Li, Y. H., et al. (2015). Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis. \u003cem\u003eAnnals of the Rheumatic Diseases\u003c/em\u003e, 74(7), 1432-1440.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhai, G., \u0026amp; Aref-Eshghi, E. (2017). Metabolomics in osteoarthritis: A systematic review. \u003cem\u003eOsteoarthritis and Cartilage\u003c/em\u003e, 25(Supplement 1), S16-S17.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKanehisa, M., Furumichi, M., Tanabe, M., et al. (2017). KEGG: New perspectives on genomes, pathways, diseases and drugs. \u003cem\u003eNucleic Acids Research\u003c/em\u003e, 45(D1), D353-D361. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Medial open-wedge high tibial osteotomy, Medial compartment osteoarthritis, Inflammation, Cartilage degradation, Autophagy, Biomechanical stress, Molecular pathways, Wnt/β-catenin, NF-κB, Biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-7918672/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7918672/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eMedial compartment osteoarthritis (MCOA) of the knee, often linked to varus malalignment, leads to increased medial tibiofemoral stress, accelerating cartilage degradation and inflammation. Medial open-wedge high tibial osteotomy (MOWHTO) corrects this alignment to relieve symptoms, but its molecular and biomechanical mechanisms are underexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHypothesis/Purpose: \u003c/strong\u003eWe hypothesized that MOWHTO reduces intra-articular stress, thereby modulating inflammatory, catabolic, and autophagic pathways to improve joint homeostasis. The purpose was to assess clinical, molecular, biomechanical, and in vitro effects in MCOA patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design: \u003c/strong\u003eProspective clinical cohort study (n=63) combined with in vitro biomechanical simulations\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003ePatients (aged 40–65 years, Kellgren-Lawrence grade I–III MCOA) unresponsive to conservative therapy underwent MOWHTO. Pre- and 12-month post-operative assessments included clinical scores (KSS, IKDC, VAS), imaging (X-ray for mMPTA/FTA/PTS; MRI T2 mapping), and molecular analyses of synovial fluid, cartilage, and synovium (ELISA for IL-1β/TNF-α/CTX-II; qRT-PCR/Western blot/immunofluorescence for MMP-13/COL2A1/ACAN/LC3/Beclin-1; metabolomics). In vitro, chondrocytes/synovial cells from surgical samples were subjected to high (10–15 kPa) vs. low (3–5 kPa) cyclic compression simulating pre-/post-MOWHTO stress, with interventions (XAV939/BAY11-7082/rapamycin) targeting Wnt/β-catenin, NF-κB, and autophagy. Finite element analysis evaluated stress; bioinformatics mapped pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eMOWHTO improved KSS (63.79→90.32), IKDC (55.68→83.19), and VAS (5.48→0.95; all p\u0026lt;0.001), corrected alignment (mMPTA 84.02°→90.53°; p\u0026lt;0.01), and enhanced cartilage integrity (T2 55.3→48.7 ms; p\u0026lt;0.01). Molecularly, IL-1β/TNF-α/MMP-13/CTX-II decreased (35–50%; p\u0026lt;0.01), while COL2A1/ACAN/LC3/Beclin-1 increased (2–2.5-fold; p\u0026lt;0.01). In vitro, low stress reduced catabolism/inflammation and boosted autophagy; inhibitors/inducers confirmed pathway mediation. Bioinformatics showed downregulated inflammatory lipids and upregulated anabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eMOWHTO alleviates biomechanical stress, suppresses inflammation/cartilage degradation, and enhances autophagy, with IL-1β, MMP-13, and LC3 as biomarkers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance: \u003c/strong\u003eThese findings validate MOWHTO's efficacy for early MCOA, guiding patient selection and monitoring via biomarkers to optimize outcomes and delay arthroplasty.\u003c/p\u003e","manuscriptTitle":"Molecular Mechanism Study of Medial Open-Wedge High Tibial Osteotomy in Treating Medial Compartment Osteoarthritis of the Knee by Improving the Intra-Articular Environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 07:57:52","doi":"10.21203/rs.3.rs-7918672/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d621785d-fccd-4b45-a857-cef2de91d8a5","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T09:13:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 07:57:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7918672","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7918672","identity":"rs-7918672","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}