Established studies proved that hydrostatic pressure had multiple effects on the biological behavior of the intervertebral disc (IVD). However, the conclusions of the previous studies were inconsistent, due to the difference in hydrostatic loading devices and observing methods used in these studies. The current study is aimed at investigating the role of dynamic hydrostatic pressure in regulating biological behavior of the notochordal nucleus pulposus (NP) and fibrocartilaginous inner annulus fibrosus (AF) and its possible mechanism using our novel self-developed hydrostatic pressure bioreactor. The differences in the biological behavior of the rabbit IVD tissues under different degree of hydrostatic pressure were evaluated via histological analysis. Results revealed that low-loading dynamic hydrostatic pressure was beneficial for cell survival and extracellular matrix (ECM) homeostasis in notochordal NP and fibrocartilaginous inner AF via upregulating N-cadherin (N-CDH) and integrin β1. In comparison, high-magnitude dynamic hydrostatic pressure aggravated the breakdown of ECM homeostasis in NP and inner AF via enhancing the Hippo-YAP/TAZ pathway-mediated cell apoptosis. Moreover, inner AF exhibited greater tolerance to physiological medium-loading degree of hydrostatic pressure than notochordal NP. The potential mechanism was related to the differential expression of mechanosensing factors in notochordal NP and fibrocartilaginous inner AF, which affects the fate of the cells under hydrostatic pressure. Our findings may provide a better understanding of the regulatory role of hydrostatic pressure on the cellular fate commitment and matrix metabolism of the IVD and more substantial evidence for using hydrostatic pressure bioreactor in exploring the IVD degeneration mechanism as well as regeneration strategies.

Established studies proved that mechanical compression loading had multiple effects on the biological behavior of the intervertebral disc (IVD). However, the regulating mechanism involved in this process remains unclear. The current study is aimed at exploring the potential bioregulators and signaling pathways involved in the compression‐associated biological changes of nucleus pulposus (NP) cells. Tandem mass tag‐ (TMT‐) based quantitative proteomics was exerted to analyze the differentially expressed proteins (DEPs) and signal pathways among the different groups of NP cells cultured under noncompression, low‐compression (LC), and high‐compression (HC) loading. Eight potential protective bioregulators for the NP cell survival under different compression loading were predicted by the proteomics, among which macrophage migration inhibitory factor (MIF) and oxidative stress‐related pathways were selected for further evaluation, due to its similar function in regulating the fate of the cartilage endplate‐ (CEP‐) derived cells. We found that deficiency of MIF accentuates the accumulation of ROS, mitochondrial dysfunction, and senescence of NP cells under overloaded mechanical compression. The potential molecular mechanism involved in this process is related to the mitophagy regulating role of MIF. Our findings provide a better understanding of the regulatory role of mechanical compression on the cellular fate commitment and matrix metabolism of NP, and the potential strategies for treating disc degenerative diseases via using MIF‐regulating agents.

Background: Nucleus pulposus (NP) degeneration is the core pathological change of intervertebral disc (IVD) degenerative diseases, but currently, no effective therapy is available. With the rapid development of biomaterials and tissue engineering in recent years, biomaterial-assisted cell transplantation becomes a promising therapy for IVD degeneration. However, the application is severely limited by the weak biological characteristics of NP cells (NPCs), such as a moderate proliferation ability, weak self-renewal capacity, and minimal extracellular matrix (ECM) synthesis capacity, caused by the current inappropriate cell seeding or grafting methods.

Thyroid cancer (THCA) is increasingly prevalent worldwide, particularly among women, highlighting the need for novel molecular biomarkers to improve early detection and prognostic assessment. The role of the cAMP response element modulator (CREM) gene in THCA remains poorly understood. This study aimed to investigate CREM’s expression, clinical significance, and association with the tumor immune microenvironment. Transcriptomic and clinical data from 507 THCA cases and 59 normal controls in The Cancer Genome Atlas (TCGA) were analyzed. CREM expression was compared across clinical subgroups using non-parametric tests. Survival outcomes were assessed via Kaplan-Meier curves and Cox regression. Receiver operating characteristic (ROC) analysis evaluated diagnostic value. Immune cell infiltration was quantified cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). Functional enrichment Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) and least absolute shrinkage and selection operator (LASSO)-Cox modeling identified CREM-associated genes, with protein-protein interaction (PPI) networks constructed via the STRING database. CREM expression was significantly downregulated in THCA compared to normal tissues (P<0.001), with diagnostic potential [area under the curve (AUC) =0.751]. Low CREM expression was associated with advanced tumor stage and poorer prognosis. High CREM levels predicted longer progression-free survival [hazard ratio (HR) =0.53, P=0.02]. CREM expression negatively correlated with infiltration by regulatory T cells (Tregs) and the expression of Treg-specific chemokines. CREM expression also negatively correlated with several immune checkpoints. IL37 and PSG6 were inversely correlated with CREM and associated with poor outcomes, while RCAN1 showed a positive correlation and favorable prognosis. CREM may function as a tumor suppressor and immune regulator in THCA. Its expression correlates with reduced immune infiltration and improved clinical outcomes, suggesting its potential as a prognostic biomarker and therapeutic target. Further experimental validation is warranted.

Metabolic dysfunction-associated steatohepatitis (MASH) progresses through macrophage activation and hepatic stellate cell (HSC)-driven fibrogenesis. As cell-level single-cell discoveries often suffer from pseudoreplication and fail to replicate across cohorts, in this study we critically re-evaluated human liver single-nucleus RNA-sequencing data using a rigorous per-donor statistical framework to dissociate invariant biological signals from technical artifacts. Macrophage and HSC compartments from dataset GSE202379 were profiled across clinical stages (healthy, metabolic dysfunction-associated steatotic liver disease, MASH). Cell-level statistics were aggregated into per-donor metrics, and continuous phenotypic shifts evaluated via the Jonckheere–Terpstra (JT) trend test. Key findings were tested for cross-cohort reproducibility in two independent cohorts (GSE136103 and GSE244832). A 249-gene scar-associated macrophage (SAMac) program showed reproducible, stage-dependent expansion in the discovery cohort (per-donor Jonckheere–Terpstra trend across the disease-only spectrum, MASLD to advanced MASH, z = 2.23, p = 0.026 [primary]; z = 2.74, p = 0.006 across all four stages including the healthy baseline) and directional replication in external data. Cell–cell communication analysis replicated the receptor-side topology of a conserved macrophage-to-HSC PDGF axis in both external cohorts and the ligand-driven PDGFC→PDGFRB edge in the external snRNA-seq cohort (GSE244832), with PDGFRB being a high-degree hub within the HSC fibrogenic interaction network. Importantly, a previously published global HSC activation module failed to validate (JT p = 0.77); however, its core extracellular matrix and RUNX1/2 sub-axis remained robust, whereas the widely claimed AP-1/JUNB regulatory driver was not supported at the transcript level in the single-nucleus RNA-seq cohort data. These findings are consistent with an isolation-associated immediate-early gene response in enzymatically dissociated scRNA-seq data, given the flat-to-downward transcript-level trend in cryopreserved-tissue cohorts; transcript-level data cannot, however, exclude protein- or phosphorylation-level AP-1 activity. Targetable downstream components within the validated core architecture were annotated. By implementing per-donor verification, we successfully separated robust pathological features of MASH from platform-dependent artifacts. The validated SAMac program and the macrophage–HSC PDGF axis, together with an associated RUNX subprogram, represent high-confidence, reproducibility-filtered candidate therapeutic nodes that require functional validation. These findings establish a reproducibility-first standard for single-cell translational research, and the validated programs are consistent with emerging models of inter-organ inflammatory circuitry in MASH.

Degenerative disc disease (DDD), characterized by the pathological deterioration of nucleus pulposus (NP) tissue, affects millions globally. Tissue engineering strategies offer potential to create tissue-engineered NP (TE-NP) analogs to address DDD. However, traditional "top-down' approaches face challenges in achieving uniform cell distribution and replicating the intradiscal extracellular matrix (ECM) environment. In contrast, a "bottom-up' strategy utilizing microscale seed units represents a promising alternative. This study introduces an innovative "bottom-up' approach for constructing TE-NP, leveraging bioreactor-cultivated NP microtissues (NP-MTs) as seed units and a novel methacrylate-modified decellularized nucleus pulposus matrix (DNPM-MA) hydrogel as a supporting biomaterial. NP-MTs cultivated under low-magnitude hydrostatic pressure exhibit nascent ECM surroundings adapting well to the intradiscal microenvironment. The DNPM-MA hydrogel, with its compositional and mechanical attributes, supports the growth, migration, proliferation, and ECM synthesis of NP-MTs, making it an ideal biomaterial for long-term cultivation. The combination of NP-MTs and the DNPM-MA hydrogel yielded superior tissue regeneration outcomes both in vitro and in vivo. Transcriptome and molecular assessments revealed a correlation between the biological properties of the DNPM-MA hydrogel and the attenuation of inflammaging within encapsulated NP-MTs. Overall, this innovative "bottom-up' constructed TE-NP exhibits superior regenerative potential and is a promising tissue engineering strategy for treating DDD.