Defining the Human Nucleus Pulposus Microenvironment and Its Impact on Cell Matrix Synthesis and Metabolic Activity

Introduction Intervertebral disc (IVD) degeneration remains the leading cause of low back pain worldwide. Regenerative therapies focused on restoring extracellular matrix (ECM) composition and disc height often overlook the IVD microenvironment, which remains to be fully characterized. This study first aims to profile the microenvironment of human nucleus pulposus (NP) tissue across degeneration grades from discectomy procedures, quantifying glucose, oxygen, pH, lactate, osmolarity, and 13 cytokines (TNF, IL‐1β, IL‐6, MMP‐3, β‐NGF, BNDF, IL‐10, TIMP‐1, ‐2, ‐3, FGF, and ADAMTS4 and 5). Profiling within the same samples enabled correlation analysis between all parameters. Second, this study investigated how clinically relevant microenvironmental conditions influence NP cell matrix synthesis and metabolic activity. Materials and Methods Microenvironmental profiling: NP tissue was obtained via informed consent from patients undergoing discectomy. pH was measured using a fiber optic microsensor. Oxygen was quantified using Image‐iT green hypoxia reagent. Glucose and lactate were quantified via colorimetric assays, and osmolarity was measured using a vapor pressure osmometer. Cytokines were analyzed by multiplex and enzyme‐linked immunosorbent assay (ELISA). Cellular response: NP cells were formed into microtissues and primed with TGF‐β3 for 7 days, followed by 14 days of clinically relevant microenvironmental insult with different combinations of glucose and pH or cytokine exposure and pH. Cell viability, DNA, GAG, collagen, and metabolic rates were assessed. Results and Discussion Across a broad donor cohort, microenvironmental parameters, cytokine concentrations, and ECM were maintained with increasing degeneration grades, despite notable donor variability. NP microtissues demonstrated resilience across clinically relevant ranges of glucose, pH, and cytokine exposure. This study establishes experimentally defined microenvironmental ranges that are representative of the human NP microenvironment and supported by donor‐specific in silico modeling. It further demonstrates that human NP cells within a native matrix are not highly sensitive to clinically relevant changes in microenvironmental conditions, an important consideration for cell‐based regenerative strategies.