The striped dolphin (Stenella coeruleoalba) is a small pelagic dolphin. The melon, present in most odontocetes, is a rounded structure located in the center of the forehead, between the blowhole and the tip of the head....The striped dolphin (Stenella coeruleoalba) is a small pelagic dolphin. The melon, present in most odontocetes, is a rounded structure located in the center of the forehead, between the blowhole and the tip of the head. The dolphin's melon, as well as the blubber, consists of a lipid component similar to that of adipose tissue, with large, rounded cells closely related to each other and a muscular component. The melon primarily plays a role in echolocation, but this organ can also be involved in immune function. Numerous studies have demonstrated the accumulation of contaminants, such as heavy metals, in the fatty tissues of cetaceans (melon and blubber), which alter the immune system and stimulate an inflammatory response. This study aims to describe the involvement of Stenella coeruleoalba melon in immune function. Histological samples of the melon of Stenella coeruleoalba, were used in this study. To describe the morphology of this organ, the sections of the tissue were stained with Giemsa, Masson, and Mallory staining techniques. Immunoperoxidase and immunofluorescence techniques using specific antibodies such as MHC II, Langerin/CD 207, TLR2, and CD14 were used to characterize melon immune cells, providing novel insights into their immunological features. The results showed scattered immune cells among melon adipocytes immunoreactive to the tested antibodies; macrophages are the principal immune cells infiltrating adipose tissue, and their function is to secrete proinflammatory mediators. Furthermore, the adipocytes themselves appear to be labeled with the antibodies used. This study demonstrates the involvement of the dolphin melon in immune function, helping us better understand the immune system of cetaceans, which is still little understood. Furthermore, it could also demonstrate that analyses of blubber collected from deceased individuals of highly protected species can provide valuable information on their health status and exposure to contaminants.
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) resistance to PARP inhibitors (PARPi) remains poorly understood, the mechanism of resistance remain poorly understood. MATERIAL AND METHODS: We integrated multi-omics d...BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) resistance to PARP inhibitors (PARPi) remains poorly understood, the mechanism of resistance remain poorly understood. MATERIAL AND METHODS: We integrated multi-omics data from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA-PAAD) datasets, Cancer Cell Line Encyclopedia (CCLE), and the Genomics of Drug Sensitivity in Cancer (GDSC) performed weighted gene co-expression network analysis (WGCNA) to identify hub genes linked to PARPi resistance. GSE86394 from the Gene Expression Omnibus (GEO) database was utilized to differential expressed genes (DEGs) and performed functional enrichment analyses. CCK8, flow cytometry, Transwell assay were conducted to detected cell viability, apoptosis, and migration. Xenograft models was conducted to investigate the effect of YARS2 on PARPi-resistant in vivo. Immunohistochemistry (IHC) was used to detect the expression of various proteins. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was utilized to assess cells apoptosis. Gene and protein expression were detected by quantitative reverse transcription PCR (qRT-PCR) and western blotting (WB), respectively. RESULTS: YARS2 was identified as a PARPi resistance hub gene, whose high expression correlated with poor prognosis in PDAC. YARS2 was significantly upregulated in PARPi-resistant PDAC cells, and its knockdown reversed PARPi resistance in vitro and in vivo. Mechanistically, YARS2 knockdown suppressed the expression of the c-MYC, E2F1 and elevated levels of phosphorylated histone H2AX (γ-H2AX). However, the overexpression of c-MYC was reversed the inhibitory role of YARS2 knockdown in PARPi-resistant PDAC cells. CONCLUSION: Our findings reveal that YARS2 mediates PARPi resistance via the MYC/E2F1 pathway in PDAC, highlighting YARS2 may serve as a potential target for therapy to counteract PARPi resistance.
Casein kinase 1 alpha (CK1α), a conserved serine/threonine kinase, regulates diverse cellular processes including signal transduction, cell cycle progression, and DNA replication. To define its spatiotemporal dynamics du...Casein kinase 1 alpha (CK1α), a conserved serine/threonine kinase, regulates diverse cellular processes including signal transduction, cell cycle progression, and DNA replication. To define its spatiotemporal dynamics during mammalian ovarian development, we systematically analyzed CK1α protein expression from embryonic day (E) 14.5 to postnatal day (P) 56 in the mouse ovary using Western blotting, immunofluorescence (IF) on tissue sections, and IF on isolated primary granulosa cells and oocytes. Reanalysis of published single-cell RNA sequencing data revealed progressive upregulation of Csnk1a1 transcripts from E9.5 through early postnatal stages. At the protein level, CK1α expression significantly increased from E14.5 to P56, with stabilization after P28. Immunofluorescence showed CK1α localized predominantly to oocytes within primordial follicles from E14.5 to P7. Starting at P7, CK1α expression expanded to include granulosa cells of developing primary and secondary follicles. Quantitative fluorescence analysis confirmed that CK1α signal in granulosa cells increased significantly from P7 onward, while the signal in the oocytes gradually decreased throughout development process. In vitro validation confirmed robust CK1α localization in both isolated granulosa cells and oocytes, co-localizing with FSHR and DDX4, respectively. This study provides the first comprehensive spatiotemporal atlas of CK1α expression in the wild-type developing mouse ovary, revealing stage-specific localization patterns that suggest distinct roles in oocyte maintenance and granulosa cell differentiation during folliculogenesis.
Several acrylate-based materials are commercially available as embedding resins for the histological analysis of non-decalcified hard tissues, including implants. However, commercial embedding resins are imported, expens...Several acrylate-based materials are commercially available as embedding resins for the histological analysis of non-decalcified hard tissues, including implants. However, commercial embedding resins are imported, expensive, and take a long time to buy. We conducted several tests to apply a mixture of methyl methacrylate and oleyl alcohol (oleyl Alc) as an embedding resin on a non-decalcified bone specimen. Further, we observed differences in the compressive strength of the block resin and the fused bone sample, changes in crystallinity, and a hydrophilic change in the block. Hematoxylin and eosin (H&E) and Masson's Trichrome (MT) staining were performed for the histological analysis of dog bones to compare staining characteristics. The block to which the resin with the optimal composition ratio was applied for embedding was confirmed experimentally to achieve optimal performance.
Impaired angiogenesis is a critical factor contributing to delayed wound healing in diabetes patients. This study aimed to investigate the role and underlying mechanism of semaphorin 3B (SEMA3B) and its upstream regulato...Impaired angiogenesis is a critical factor contributing to delayed wound healing in diabetes patients. This study aimed to investigate the role and underlying mechanism of semaphorin 3B (SEMA3B) and its upstream regulator Yes-associated protein (YAP) in diabetic wound healing. Human umbilical vein endothelial cells (HUVECs) were treated with advanced glycation end products (AGEs) to simulate a diabetic environment in vitro. AGEs significantly suppressed the viability, proliferation, and tube formation capacity of HUVECs, which was accompanied by downregulated expression of both SEMA3B and YAP. Consistent with these results, decreased expression of SEMA3B and YAP was detected in the skin and wound tissues of diabetic mice. Overexpression of SEMA3B significantly reversed AGE-induced endothelial dysfunction. Mechanistically, YAP was identified as a positive regulator of SEMA3B transcription. Overexpression of YAP restored endothelial function through the upregulation of SEMA3B, whereas YAP inhibition exacerbated functional impairment. In vivo experiments demonstrated that topical application of a YAP inhibitor delayed wound healing in diabetic mice concomitant with reduced SEMA3B expression. Furthermore, topical application of recombinant semaphorin 3B protein (rSEMA3B) significantly promoted wound healing in diabetic mice, confirming its therapeutic effect. This study reveals the crucial regulatory role of the YAP/SEMA3B axis in diabetic wound healing, demonstrating that YAP improves endothelial function and promotes angiogenesis by upregulating SEMA3B, thereby accelerating wound closure. These findings suggest a novel potential therapeutic target for the treatment of diabetic wounds.
BACKGROUND: The inference of molecular information from hematoxylin-eosin (HE) specimens may reduce the ancillary testing burden in digital pathology. OBJECTIVE: To assess whether deep convolutional networks predict, at...BACKGROUND: The inference of molecular information from hematoxylin-eosin (HE) specimens may reduce the ancillary testing burden in digital pathology. OBJECTIVE: To assess whether deep convolutional networks predict, at the per-nucleuslevel, continuous DAB optical density (OD) and binary positivity for a biologically coherent panel of DNA replication-related proteins (CDC6, CDT1, MCM7, ORC1, CDC7, and Geminin) plus Ki-67 directly from HE nuclear images. METHODS: We constructed a same-specimen paired HE/IHC dataset from 21 endometrioid carcinoma cases (7 per grade). After color unmixing via NMF-derived stain vectors and HoVer-Net-based nuclear segmentation, 100 × 100 nucleus-centered HE crops were paired one-to-one with IHC-derived per-nucleus OD. ImageNet-initialized backbones (ResNet-50 baseline vs. EfficientNet-B0 and MobileNetV3-Small) were trained with regression (OD) and classification (positivity) heads, and multi-task learning across markers was also evaluated. Case-wise splits ensured no patient overlap across training/validation/test. RESULTS: Across markers, the per-nucleus prediction of protein expression from HE nuclear morphology was feasible with moderate discriminative performance, with the strongest signal being observed for MCM7 (AUC-ROC ≈ 0.71; F1 ≈ 0.72). Performance was dependent on the markers used: MCM7 and Ki-67 consistently showed stronger discrimination (AUC-ROC ≈ 0.70-0.72), Geminin and CDC6 were moderately predictable in some settings, and CDT1 remained near the chance level (AUC-ROC ≈ 0.50). Among the architectures evaluated, ResNet-50 demonstrated the most stable generalization, and multi-task training yielded modest average gains, but was not consistently beneficial. Regarding the Ki-67 labeling index, the nuclear-crop approach showed moderate agreement with WSI-based digital IHC (ROI level r ≈ 0.54; MAE ≈ 15.2 pp). The major limitations of this study were the small cohort size (21 cases) and lack of external validation. CONCLUSIONS: Per-nucleus protein expression-both binary positivity and continuous OD-was shown to be inferable from HE nuclear morphology alone. These results suggest clinical utility and will motivate future studies using larger external cohorts and self-supervised pretraining.
The current study provides the first histological whole-brain atlas of the lizard, Hemidactylus flaviviridis. Macroscopic analysis of the lizard brain demonstrates its gross morphology, including various fissures and maj...The current study provides the first histological whole-brain atlas of the lizard, Hemidactylus flaviviridis. Macroscopic analysis of the lizard brain demonstrates its gross morphology, including various fissures and major divisions, forebrain, midbrain, and hindbrain. Serial coronal sections stained with cresyl violet provided a detailed insight into the architecture of the lizard brain with a well-defined ventricular system featuring lateral ventricles in the telencephalon, third ventricle in the diencephalon, and fourth ventricle in the rhombencephalon. The forebrain comprised telencephalon and diencephalon, wherein telencephalon was further subdivided into pallium and subpallium. The dorsal pallium consisted of cortical region divided into medial, dorsal, dorsomedial, and lateral cortex, while ventral pallium contained anterior dorsal ventricular and posterior dorsal ventricular ridge. At the posterior ends of telencephalon, anterior commissure, optic tract, diagonal band of Broca, and preoptic area were observed. The diencephalon included epithalamus, thalamus, and hypothalamus. On either side of the third ventricle various hypothalamic nuclei were present. The midbrain housed optic tectum, torus semicircularis, and substantia nigra, while the hindbrain included cerebellum as well as different nuclei (vestibular, anterior raphe, reticular and lateral reticular). The study demonstrates that the major subdivisions as well as anatomical details of the wall lizard brain follows general vertebrate brain plan and provide an essential reference for neurobiological studies in reptiles.
Hypoxia is a characteristic, although heterogeneous, of solid tumors, and experimental hypoxia is still far too frequently addressed as a single replaceable phenomenon. Yashiro et al. demonstrate in their recent article...Hypoxia is a characteristic, although heterogeneous, of solid tumors, and experimental hypoxia is still far too frequently addressed as a single replaceable phenomenon. Yashiro et al. demonstrate in their recent article in Acta Histochemica that the signaling of lysophosphatidic acid receptor in gastric cancer cells differs significantly in cobalt chloride-induced but not physiological hypoxia with resulting differences in motility and cisplatin response. It is not an observation merely at the receptor level but it reveals a deeper reproducibility and translational issue in tumor biology. Pathway attribution, interpretation of drug-response and prioritization of targets can be misleading when chemical hypoxia is to be used as a surrogate to oxygen deprivation without explicit validation. We believe that this paper must motivate the field-wide to abandon convenience-driven hypoxia modelling in favour of decision-grade hypoxia phenotyping. There should be future work to incorporate physiologically accurate oxygen control, orthogonal confirmation of hypoxic conditions, spatially resolved histochemical reporters and standard reporting of receptor-context interactions. This framework would enhance mechanistic accuracy, enhance cross-study comparability, and expedite translation of hypoxia-informed therapeutic interventions in gastric cancer and other diseases.
This study aimed to produce a high-quality mesenchymal stem cell-derived secretome (MSC-sec) using a three-dimensional culture system to enhance its therapeutic potential for skin wounds. Human umbilical cord-derived MSC...This study aimed to produce a high-quality mesenchymal stem cell-derived secretome (MSC-sec) using a three-dimensional culture system to enhance its therapeutic potential for skin wounds. Human umbilical cord-derived MSCs were cultured on optimally modified gelatin sponge (GS) scaffolds (ethanol-plus-polylysine), which maintained high cell viability. The secretome from these GS-cultured MSCs (GS-MSC-sec) contained significantly higher concentrations of key growth factors (KGF, VEGF, PDGF) compared to conventional 2D culture. In vitro, GS-MSC-sec enhanced fibroblast proliferation, migration, and angiogenesis. In a rat full-thickness skin wound model, concentrated GS-MSC-Section (5 ×) not only accelerated wound healing but also promoted wound-induced hair neogenesis. This enhanced regeneration was associated with upregulation of Wnt/β-catenin and TLR3/STAT3 signaling and downregulation of BMP/Smad signaling. These findings demonstrate that GS-MSC-sec possesses significant therapeutic potential for promoting skin wound healing and regeneration, with implications for treating alopecia.
We investigated whether the effect of treatment with quercetin-loaded microcapsules 10 mg/kg promotes anti-inflammatory action and prevents damage to the intestinal wall in the jejunum of rats with adjuvant-induced arthr...We investigated whether the effect of treatment with quercetin-loaded microcapsules 10 mg/kg promotes anti-inflammatory action and prevents damage to the intestinal wall in the jejunum of rats with adjuvant-induced arthritis. Thirty Holtzman rats were distributed into 5 groups: C (control), control administered with quercetin-loaded microcapsules 10 mg/kg (CQ), adjuvant-induced arthritis (AIA), adjuvant-induced arthritis administered with ibuprofen 17.5 mg/kg (AI) and adjuvant-induced arthritis administered with quercetin-loaded microcapsules 10 mg/kg (AQ). After 60 days, the jejunums were collected for histological and immunohistochemical techniques. CD20 , CD45, CD68, CD206, 5-HT cells, mast cells, intraepithelial lymphocytes, goblet cells, intestinal wall morphometry and cell proliferation were evaluated. AIA triggered intestinal inflammation assessed by the increase in immune cells. AIA also caused hypertrophy of the intestinal wall, evidenced by the increase in the height and width of the villi, crypts depth, mucosa height, total intestinal wall and increased cell proliferation in the crypts. Quercetin-loaded microcapsules in rats with AIA (AQ) showed anti-inflammatory action and prevented depletion of goblet cells, the widening of the villi, crypts depth, increased 5-HT cells and increased cell proliferation. Ibuprofen (AI group) showed anti-inflammatory action, prevented damage to the intestinal wall, goblet cells and 5-HT cells. In CQ, increased macrophages, intraepithelial lymphocytes, cell proliferation, and intestinal wall thickness. Chronic AIA caused inflammation, intestinal wall alteration and increased 5-HT expression in the mucosa. Quercetin-loaded microcapsules showed beneficial effects on the alterations in the intestinal mucosa architecture caused by AIA, being a promising antioxidant and anti-inflammatory compound for the treatment and prevention of arthritis progression.
Leber's Hereditary Optic Neuropathy (LHON) is the most prevalent mitochondrial inherited disorder, primarily caused by primary mitochondrial mutations. Clinically, LHON is characterized by degeneration of optic nerves th...Leber's Hereditary Optic Neuropathy (LHON) is the most prevalent mitochondrial inherited disorder, primarily caused by primary mitochondrial mutations. Clinically, LHON is characterized by degeneration of optic nerves that leads to acute or subacute sudden or painless central vision loss. Currently no effective treatment has been established for LHON. Recent studies have highlighted the significance of intercellular mitochondrial transfer, which facilitates communication between cells and presents a novel therapeutic avenue. In this study, we investigated the formation of tunnelling nanotubes (TNTs) and the subsequent mitochondrial transfer between Bone Marrow Mesenchymal Stem Cells (BM-MSCs) and LHON ND4 mutant cells within the coculture system. Our findings demonstrated that mitochondrial transfer from BM-MSCs to LHON mutant cells via TNTs effectively rescued the mutant LHON cells by reducing apoptosis, restoring mitochondrial membrane potential and reducing reactive oxygen species (ROS) generation. These results provide compelling evidence of cell-cell communication between mesenchymal stem cells and LHON mutant cells, indicating a potential regenerative capacity through the reduction in mitochondrial mutation load. This study would help to implement further research in this area for the protective effect of mitochondria transfer and future cell-based treatment approaches for LHON.
The development and progression of glioblastoma, the most aggressive malignant intracranial tumor with a poor prognosis, are influenced by mutations, oncogene overexpression, and epigenetic factors, particularly those re...The development and progression of glioblastoma, the most aggressive malignant intracranial tumor with a poor prognosis, are influenced by mutations, oncogene overexpression, and epigenetic factors, particularly those relating to DNA methylation status and histone post-translational modifications. Valproic acid (VPA), a well-known histone deacetylase (HDAC) inhibitor, has shown promise both alone and in combination with other agents against various solid tumors, including gliomas. Given VPA's reported effects on chromatin supramolecular organization and expression activity across different cell types, we studied textural features indicative of chromatin structural changes in U-251MG glioblastoma cells cultured in the presence of VPA, utilizing image cytometry and immunofluorescence techniques. For comparison, cells treated with 5-aza-CdR served as a positive control for DNA demethylation. Chromatin remodeling was observed in VPA-treated cells, which exhibited decreased HDAC activity and increased histone H3 acetylation, whereas no such changes were detected in 5-aza-CdR-treated cells. These findings suggest that, despite alterations in DNA methylation within glioblastoma cells and a possible effect of VPA on DNA demethylation, the chromatin remodeling phenomenon observed through image cytometry in VPA-treated U-251MG cells is more closely associated with induced changes involving histone modifications rather than with DNA demethylation. Although these findings are based on in vitro conditions, present findings of basic character may acquire pre-clinical perspective implications if corroborated by further results obtained from various glioblastoma cell cultures and animal models treated with drugs that may influence epigenetic markers, while bypassing the question of blood-to-brain drug flux.
Tenocytes migrating from cultured rat tendon explants have been shown to aggregate and gradually align in parallel rows with the tendon's long axis as the explant culture period extends to 12 days, leading to structure r...Tenocytes migrating from cultured rat tendon explants have been shown to aggregate and gradually align in parallel rows with the tendon's long axis as the explant culture period extends to 12 days, leading to structure remodeling of collagen bundles. It is well established that tendon architecture and mechanical load influence fibroblast phenotypes. In this study, we examined cells derived from tenocytes that migrated from tendon explants cultured for 8 and 12 days to determine whether their chromatin supraorganization differed based on their varying organization. Image analysis and immunofluorescence were used to assess features indicative of chromatin transcriptional expression or repression activities. While no differences occurred in Feulgen-DNA amounts, DNA methylation markers, or distribution of acetylated H3K9 when comparing cells derived from 8- and 12-day tendon cultures, we observed differences in light absorbance, fractal dimension, and concentration of stained chromatin at the image periphery (margination). Increased levels of the trimethylated H3K27 and decreased levels of 5-hydroxymethylcytosine (both repressive markers) in cells derived from the 12-day tendon explants provide evidence of chromatin condensation at that time. Elevated levels of the translational activating markers H3K4me2 and H3K4me3 in cells derived from tenocytes that extensively aligned in parallel rows in the explants during the extended tendon culture period, may suggest acquired or enhanced expression of specific gene sets, despite the concurrent indication of greater overall chromatin compaction. Further assays are needed to explore whether these findings correlate with a differential potential of tenocyte-derived cells for extracellular matrix synthesis and their implication in practical bioengineering applications.
Shaaban D, Seerley A, Crew L
… +5 more, Kaylor C, McElroy S, Guter E, Pounder J, Panter AG
Acta Histochem
· 2026 Jun · PMID 41619339
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There are significant risks in clinical, diagnostic, and research settings to those who investigate prion diseases, due to the difficult nature of inactivating prion proteins with standard decontamination methods. Formic...There are significant risks in clinical, diagnostic, and research settings to those who investigate prion diseases, due to the difficult nature of inactivating prion proteins with standard decontamination methods. Formic acid treatment has been shown to be effective for decontaminating infectious prions and commonly used in biosafety practice to prevent occupational exposure. However, the impact of formic acid protocols on the morphology of tissue samples has not been adequately documented. The goal of this study is to examine morphologic effects of formic acid treatment on central nervous system tissue, using mouse model brain hemisphere tissues that exhibit varying degrees of neurodegeneration as a model. This study included normal, non-diseased wild-type tissues and a 5xFAD model, which recapitulates aspects of Alzheimer's Disease (AD). A model exhibiting Chronic Wasting Disease (CWD), a prion disease of deer and elk, was also used to analyze the effects of formic acid on tissues with spongiform changes. Tissues from both formic acid and untreated control treatment groups were embedded in paraffin, sectioned, stained, and imaged microscopically. Anatomical regions were analyzed and evaluated quantitatively to determine the width, area, and structural integrity of the tissue between treatment groups. Our findings demonstrated that while formic acid has been previously reported to effectively inactivate prions, it compromised the morphology of mouse brain tissues. Furthermore, the effects of formic acid were not distributed equally between regions of the brain. Age did not play a role in the morphologic changes seen in the formic acid treatment group. Interestingly, the presence of neurodegeneration in the tissues did not appear to exacerbate the effects of morphological changes post-formic acid treatment. These results emphasize the need to explore alternative prion inactivation methods that ensure the safety and reliability of handling prion-infected tissues without compromising the integrity of tissues.
Lysophosphatidic acid (LPA) receptor-mediated signaling contributes to the pathogenesis of cancer. The tumor microenvironment (TME), composed of cancer cells and surrounding stromal cells, plays a key role in promoting m...Lysophosphatidic acid (LPA) receptor-mediated signaling contributes to the pathogenesis of cancer. The tumor microenvironment (TME), composed of cancer cells and surrounding stromal cells, plays a key role in promoting malignant traits, including resistance to anticancer drugs. In this study, we investigated the roles of LPA receptor-1 (LPA) and LPA in colon cancer DLD-1 cell functions modulated by 3T3 fibroblasts chronically exposed to chemotherapy. 3T3 cells were treated long-term with fluorouracil (5-FU), irinotecan (CPT-11), or cisplatin (CDDP) to generate 3T3-5FU, 3T3-CPT11, and 3T3-CDDP cells, respectively. Co-culture with anticancer drug-treated 3T3 cells altered LPA receptor expression in DLD-1 cells, characterized by reduced LPAR1 and elevated LPAR2 levels, compared with co-culture with untreated fibroblasts (n = 3 independent experiments). Under these conditions, LPA stimulation enhanced DLD-1 cell proliferation and motility. Pharmacological modulation of LPA signaling further supported receptor-specific effects: the LPA antagonist AM966 and the LPA agonist GRI-977143 promoted DLD-1 cell growth, while AM966 suppressed and GRI-977143 enhanced cell motility in co-culture with drug-treated fibroblasts. These findings suggest that chemotherapy-conditioned fibroblasts reshape LPA receptor-dependent signaling in colon cancer cells, suggesting potential therapeutic relevance of distinct roles for LPA and LPA within the drug-modified TME.
Osteoarthritis (OA) is a degenerative joint disease characterized by an increasing prevalence and complex pathogenesis. Despite significant research efforts, understanding its mechanisms and developing effective treatmen...Osteoarthritis (OA) is a degenerative joint disease characterized by an increasing prevalence and complex pathogenesis. Despite significant research efforts, understanding its mechanisms and developing effective treatments remain challenging. The development of in vitro models that accurately mimic the native articular microenvironment while preserving chondrocyte phenotype is crucial for advancing OA research. In this study, we constructed an Alginate-Hyaluronic Acid-Type I Collagen (Alg-HA-Col) composite hydrogel through physical mixing and chemical cross-linking. This composite matrix exhibited exceptional stability over 28 days under simulated physiological conditions. Neonatal murine chondrocytes encapsulated within Alg-HA-Col hydrogels for 28 days maintained their phenotype better than those cultured in monolayer, as evidenced by higher expression levels of SOX9, type II collagen, and aggrecan. Furthermore, by adjusting the component ratios, the stiffness of Alg-HA-Col hydrogels could mimic the stiffness of the pericellular matrix (PCM) in both physiological and pathological states, referred to as Firm and Soft. Chondrocytes cultured in Alg-HA-Col hydrogels with varying stiffness showed decreased expression of marker proteins, increased cellular mortality, and elevated inflammatory factor expression as stiffness decreased. Therefore, Alg-HA-Col hydrogels with tunable stiffness effectively simulated the physiological and pathological states of cartilage, providing an ideal three-dimensional (3D) matrix for chondrocyte culture. Additionally, this model's long-term culture stability offers potential applications in osteoarthritis drug therapy and cartilage tissue engineering.