An animal model study on the gene expression profile of meniscal degeneration

Meniscal degeneration, often seen in middle-aged and elderly individuals and associated with osteoarthritis (OA), leads to loss of meniscal function, early cartilage degeneration, and subchondral bone loss. While historically considered a hallmark of OA, the meniscus also protects articular cartilage, and its degeneration worsens joint health and quality of life. Direct human studies are limited by ethics and scarce normal meniscal tissue, necessitating animal models. Small-animal models (guinea pigs, rats, rabbits) have translational limitations due to joint size and loading differences. Mini-pig knees better approximate human weight-bearing. This study aimed to elucidate molecular mechanisms of meniscal degeneration using a Wuzhishan mini-pig model based on ACL and LCL resection (modified Pond-Nuki model), followed by gene expression profiling and validation to identify pathways and processes implicated in degeneration.

Prior work indicates the normal meniscus protects underlying cartilage, and meniscal degeneration contributes to OA progression. Animal models have demonstrated meniscal and osteochondral changes after destabilization, but small animals have limitations for human translation. The Pond-Nuki ACL resection model induces meniscal degeneration in large animals; mini-pigs offer knee loading comparable to humans. Previous studies reported histological and gene expression changes in degenerative meniscus after ACL injury and increased COL1A1 expression. Inflammation and nitric oxide (NO) signaling promote meniscal matrix catabolism and pro-inflammatory gene expression; NO suppresses autophagy via JNK inhibition, exacerbating degeneration. Interventions such as hyaluronic acid, selenium, and IL-10 can mitigate NO-driven degeneration. Sex differences in OA and meniscal pathology suggest roles for sex hormones and muscle function. Meniscal calcification is common; mechanical stress and inflammatory mediators upregulate ANKH and ENPP1, altering calcium signaling and impairing repair. Phosphocitrate reduces calcification and may protect meniscus. Iron overload has been linked to accelerated OA and meniscal degeneration. IGF signaling supports meniscal repair; metabolic dysfunction (e.g., type II diabetes mellitus) and inflammatory pathways (MAPK, MMPs, ADAMTS) contribute to joint degeneration.

Study design and animals: Seven male Wuzhishan mini-pigs (mean age 6.8±0.3 months; mean body weight 19.4±3.4 kg) were used. Only males were included to avoid sex hormone variability. Anesthesia: intramuscular xylazine hydrochloride 0.3 ml/kg and pentobarbital sodium 20 mg/kg. Surgical procedures: Right rear limbs (experimental, Ba group) underwent lateral parapatellar approach with medial patella luxation. ACL was clamped and transected by 1.0 cm at the distal end; LCL was exposed along the joint line and resected by 1.0 cm. Wounds were irrigated with 100 ml sterile saline and capsule closed with 3-0 silk. Left rear limbs (control, Aa group) had sham incision without manipulation of ligaments, menisci, cartilage, or bone. Sides were not randomized to facilitate identification. Postoperative care: Penicillin and tramadol administered for 7 days; animals monitored twice daily and allowed free movement after wound healing. Tissue collection: At 26 weeks, animals were euthanized by acute exsanguination. Medial menisci were harvested using a standardized technique without contacting articular surfaces. Samples from the red-white zone of the pars intermedia were cut into small pieces, placed into cryovials, immediately frozen, and stored in liquid nitrogen. Equipment was treated with hydrogen peroxide to remove RNase. RNA extraction and QC: Total RNA was extracted with E.Z.N.A Total RNA Kit II after DNase digestion. RNA quantity/quality were measured by NanoDrop ND-1000; integrity assessed by denaturing agarose gel electrophoresis. Microarray labeling and hybridization: Agilent One-Color protocol was used. Total RNA was linearly amplified and labeled with Cy3 (Agilent Quick Amp kit). Labeled cRNA was purified (RNeasy Mini), quantified for concentration and specific activity. One microgram cRNA was fragmented (10x blocking agent, 25x fragmentation buffer) at 60°C for 30 min, then diluted with 2× GE hybridization buffer. Hybridization (100 µl) was performed on Agilent Whole Pig Genome Microarray for 17 h at 65°C. Arrays were washed, fixed, and scanned on Agilent G2505C. Data processing and analysis: Feature Extraction software v11.0.1.1 obtained raw data; GeneSpring GX v12.1 performed quantile normalization. Genes detected in at least 7 of 14 samples were analyzed. Differentially expressed genes (DEGs) were identified using volcano plot filtering with fold-change >2.0 and P<0.05 (Student t-test in GeneSpring). Unsupervised hierarchical clustering was conducted in R. Functional enrichment analyses included Gene Ontology (biological process, cellular component, molecular function) using topGO and KEGG pathway analysis. Microarray data are MIAME-compliant and available in GEO (GSE145402). RT-PCR validation: Twelve DEGs were selected spanning key GO terms and pathways (e.g., PRL, ACP5, DMRT1, ACTA1, CYP17A1, SSTR2, EPHX1, ABCC8, ADIPOQ, SLC2A4, ADCY4, TRPA1). cDNA synthesis used GeneAmp PCR System 9700; qPCR used ViiA 7 Real-Time PCR System. Cycling: 95°C 10 min; 40 cycles of 95°C 10 s, 60°C 60 s. Each reaction was run in triplicate and experiments repeated twice. Expression was normalized to GAPDH using 2^-ΔΔCt. Statistics: Student t-test with P<0.05 (SPSS 19.0). Ethics: Approved by the Medical Ethics Committee of Hainan General Hospital (Med-Eth-Re [2020] 5); all procedures followed relevant guidelines and regulations.

- Differential expression: 893 DEGs identified between experimental (ACL+LCL resection) and sham groups (fold-change >2, P<0.05), including 537 upregulated and 356 downregulated genes. Unsupervised hierarchical clustering accurately separated experimental and control samples, indicating robust group-specific expression patterns. - Top DEGs: Upregulated (examples): TFAP2D, HOXD13, CES1, PAK5, C7H6orf15, GCNT7, VIL1, COL7A1, CD81, SLC7A8. Downregulated (examples): SPMI, INA, CPS1, PGA5, LYRM4, TMCO5A, LOC100520832, CFAP58, ESRP1, VMA21. - GO biological processes: Upregulated enrichment included cellular/response to hormone stimulus, C21-steroid hormone metabolic process, neuropeptide signaling, negative regulation of reactive oxygen species metabolism, regulation of nitric oxide biosynthesis, nitric oxide biosynthetic and metabolic processes. Downregulated enrichment included sex determination, male gonad development, male sex differentiation, development of primary male sexual characteristics, muscle fiber/cell development, mesenchyme morphogenesis, reproductive structure development. - GO cellular components: Upregulated enrichment included transcription factor complex, intrinsic component of membrane, cell-substrate adherens junction/junction. Downregulated enrichment included sarcomere, myofibril/contractile fiber, integral component of plasma membrane, endoplasmic reticulum membrane and subcompartments, plasma membrane part. - GO molecular functions: Upregulated enrichment included transcription regulatory region sequence-specific DNA binding, sequence-specific double-stranded DNA binding, transition metal ion binding, iron ion binding, regulatory region nucleic acid binding. Downregulated functions included transcription factor activity (RNA Pol II distal enhancer sequence-specific binding), enhancer binding, transcription regulator activity, neuropeptide binding, hormone activity. - Pathways: 36 KEGG pathways were differentially regulated. Prominent upregulated pathways included type II diabetes mellitus, taste transduction, prolactin signaling, longevity regulating pathway, ovarian steroidogenesis, neuroactive ligand-receptor interaction, inflammatory mediator regulation of TRP channels, pantothenate and CoA biosynthesis, AMPK signaling, and bile secretion. Downregulated pathways included thyroid hormone synthesis, cocaine/amphetamine addiction, metabolism of xenobiotics by cytochrome P450, legionellosis, glycerolipid metabolism, chemical carcinogenesis, acute myeloid leukemia, adherens junction, Salmonella infection, protein digestion and absorption, and hematopoietic cell lineage. - RT-PCR validation: Twelve DEGs representing key processes/pathways were validated, generally confirming microarray trends. Examples (RT-PCR fold-change in experimental vs control): ABCC8 ~3.37 (up, P<0.01), TRPA1 ~2.89 (up, P<0.01), PRL ~2.78 (up, P<0.01), ADIPOQ ~2.96 (up, P<0.01), SLC2A4 ~3.09 (up, P<0.01), CYP17A1 ~2.96 (up, P<0.01), ADCY4 ~2.89 (up, P<0.01), ACP5 ~2.82 (up), EPHX1 ~-1.49 (down, P<0.05). Overall, RT-PCR indicated the microarray data accurately reflected gene expression patterns.

The study addressed the molecular underpinnings of meniscal degeneration in a large animal model mimicking human knee loading by resecting ACL and LCL, which increases meniscal stress. The transcriptomic profile indicates activation of inflammatory and metabolic pathways (e.g., nitric oxide metabolism, inflammatory mediator regulation of TRP channels, AMPK and type II diabetes-related signaling) that are consistent with known catabolic responses in joint tissues. Upregulation of nitric oxide-related processes supports NO’s role in promoting matrix catabolism and inflammation during meniscal degeneration. Downregulation of male sex differentiation and related reproductive processes suggests altered androgen-related signaling, potentially affecting muscle function and dynamic knee stabilization, indirectly increasing meniscal loading and degeneration risk. Enrichment of transition metal ion and iron ion binding implicates mineral and iron homeostasis; relationships to calcification and iron overload-associated joint degeneration align with prior reports. The pathway findings highlight systemic metabolic influences (insulin/AMPK axis) and neuroendocrine signaling (prolactin, steroidogenesis) in meniscal pathology. The clustering and validation results strengthen the reliability of the identified DEGs and pathways. Overall, the data illuminate interconnected inflammatory, hormonal, metabolic, and biomechanical mechanisms contributing to meniscal degeneration, offering targets for therapeutic intervention and biomarkers for early detection.

Wuzhishan mini-pigs subjected to ACL and LCL resection provide a suitable model to study meniscal degeneration. Genome-wide expression profiling revealed numerous DEGs and enriched GO terms and pathways related to nitric oxide/inflammation, hormonal regulation, ion binding, and metabolic signaling (e.g., AMPK, type II diabetes pathways). RT-PCR validation confirmed key changes. These insights improve understanding of meniscal degeneration mechanisms and suggest potential molecular targets for treatment and early diagnosis. Future work should refine controls, dissect cell-type-specific contributions (e.g., single-cell analyses), and translate findings to human tissues.

- The contralateral limb served as the control; altered biomechanics due to unilateral surgery may influence gene expression in the control limb, making it an imperfect control. - Meniscal tissue contains multiple cell types; bulk RNA profiling reflects composite responses, limiting attribution to specific cell populations. Approaches such as cell isolation or single-cell sequencing are needed. - Findings from an animal model may not fully translate to humans and indicate possible mechanisms rather than definitive human pathology.