Investigating Diabetic Osteoarthritis with a 3D Blood-Joint Spheroid Model

Introduction: Osteoarthritis (OA) is a degenerative joint disease characterized by synovial inflammation and cartilage damage. The prevalence of OA has been associated with type 2 diabetes mellitus (DM), a chronic disorder resulting in high blood glucose levels. The connection between both diseases has historically been attributed to similar risk factors, including increasing age and joint loading due to obesity. However, pathophysiological mechanisms underlying both DM and OA have not been thoroughly explored due to associated complications involved with treating this patient population. Therefore, the complex interactions between blood vessels, synovium, and cartilage highlights the need for advanced in vitro models that can accurately recapitulate native properties of the joint. In this study, we develop a novel blood-joint spheroid featuring a tri-layer cellular model consisting of human umbilical vein endothelial cells (hUVECs), fibroblast-like synoviocytes (FLS), and articular chondrocytes (ACs). This model aims to replicate cellular crosstalk within the joint and characterize the effects of DM-induced hyperglycemia in the bloodstream on OA-associated degradation of cartilage. Study 1 characterized viability across all three cell layers following blood exposure at varying glucose levels representative of healthy and DM diseased conditions. In Study 2, spheroids were dissociated followed by cell sorting and gene expression for markers of extracellular matrix synthesis, degradation, proinflammatory cytokines, oxidative stress, and glucose regulation. Study 3 characterized the release of nitric oxide (NO), glycosaminoglycans (GAG), and glucose uptake in media following blood and high glucose exposure to the spheroids.

Materials and

Methods: Cell Isolation: Healthy human synovium and cartilage were obtained from MTF Biologics and digested to isolate FLS and ACs. Primary hUVECs were obtained from Angio-Proteomie. Red Blood Cell (RBC) Extraction: Human blood samples were sourced from NYBC, and RBCs were isolated via Ficoll-Paque technique. Spheroid Culture: Spheroids were formed with a chondrocyte core and layered with FLS and hUVECs. Using round bottom plates, ACs were seeded at 8,000 cells, centrifuged to form the spheroid core, and incubated overnight to allow the cells to coalesce (Fig. 1A). The same protocol was repeated for the FLS layer on day 2 (Fig. 1B) followed by the outer hUVEC layer on day 3 (Fig. 1C). Spheroids were separated into euglycemic (EG; 5 mM D-glucose) or hyperglycemic (HG; 100 mM D-glucose) treated RBCs (+BLD; 40% v/v in DMEM), with parallel no blood controls for 2 days (N=5). Study 1: Cell viability staining was performed using Calcein AM and Ethidium Homodimer. Study 2: Following blood glucose treatment, spheroids were labeled with FITC-CD31, PE-CD90, and APC-CD151 to separate hUVECs, FLS, and ACs respectively, using a MA900 Cell Sorter (Sony). Gene expression was analyzed using qPCR for degradation (MMP1, MMP2, MMP3, MMP13), synthesis (ACAN, COL1A1, COL2A1, FN1), inflammation (IL-1β, IL-6, IL-8, TNF- α), oxidative stress (AGER, ROMO1), and glucose regulation (GLUT1, INSR). Study 3: Media was assayed for NO, GAG, and glucose uptake. Statistics: For qPCR, genes were normalized to GAPDH and day 0 controls. Media comparisons were made using one-way ANOVA with Tukey HSD post-hoc tests (α=0.05).

Results, Conclusions, and Discussions:

Results: Cell viability decreased through the depth of the spheroid model under blood and HG culture conditions (Fig. 1J-L), while no significant cell death was observed in DMEM controls (Fig. 1D-F). Following spheroid dissociation and cell sorting (Fig. 2A-C), gene expression analysis yielded significant differences across all cell types and treatment conditions. Markers of matrix degradation, inflammatory cytokines, oxidative stress, and glucose transport were generally upregulated in blood groups compared to controls, with significantly higher expression with HG treatment (Fig. 2D). Matrix synthesis genes and insulin receptor activity showed an opposite trend with increased expression in DMEM controls and downregulation under blood and HG exposure. Media analysis confirmed glucose uptake in spheroids treated with HG blood (Fig. 2E). GAG and NO release into the media were significantly elevated with blood exposure compared to DMEM controls (Fig. 2F-G). NO levels were also higher with HG culture conditions compared to non-blood treated spheroids (Fig. 2F).

Discussions: Spheroids exposed to HG and blood media conditions had decreased viability toward the inner AC core compared to DMEM controls, suggesting hyperglycemic episodes can induce cell death to joint tissues. Elevated expression of glucose transporters (GLUT1) and decreased insulin receptor activity (INSR) was observed in HG+BLD spheroids, potentially indicative of DM-induced insulin resistance. Gene expression across all three cell types demonstrated that hyperglycemic environments promote matrix degradation, inflammation, and oxidative stress characteristic of OA. Media analysis confirmed increased inflammation (NO) and glucose uptake with HG culture conditions, mimicking characteristics of the DM and OA disease states.

Conclusions: The blood-joint spheroid offers a promising platform to investigate the effects of hyperglycemic environments on endothelial dysfunction, synovial inflammation, and cartilage breakdown. This 3D model recapitulates the cellular interactions of the joint space, allowing for future studies that explore therapies to mitigate the pathogenesis of DM and OA.

Acknowledgements (Optional): NSF Graduate Research Fellowship Program (GRFP), NIH P41EB027062 (TERC), MTF Biologics, Columbia Stem Cell Initiative (CSCI) Flow Cytometry Core