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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I6 - Development of a Blood-Joint Transwell System to Investigate the Effect of High Blood Glucose Exposure in a Diabetic Osteoarthritis Model

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kedar Krishnan

Undergraduate Student
Johns Hopkins University
Horsham, Pennsylvania, United States

Co-Author(s)

Neeraj Sakhrani, MS (he/him/his)

PhD Student
Columbia University
Flushing, New York, United States

Primary Investigator(s)

CH

Clark Hung

Primary Investigator
Columbia University, United States

Introduction: Osteoarthritis (OA) is a degenerative joint disease marked by cartilage deterioration and synovial inflammation. Previous studies have shown that type 2 diabetes mellitus (DM) and its associated hyperglycemia may increase the progression and incidence of OA. The connection between both diseases has historically been attributed to increasing age and obesity induced joint loading. Furthermore, the complex interactions of the blood-joint barrier across endothelial cells, synovium, and articular cartilage necessitates the development of in vitro models that accurately recapitulate physiological conditions of the joint space. In this study, we develop a blood-joint transwell system combining human umbilical vein endothelial cells (hUVECs), fibroblast-like synoviocytes (FLS), and articular chondrocytes (ACs) (Fig. 1). The transwell system allows for cross-talk between the cell types either across the porous membrane or via paracrine factors released into shared media. As such, the system can recapitulate cellular signaling in the joint and model underlying pathophysiological mechanisms implicated in DM and OA.

Materials and

Methods: Cell Isolation: Healthy human synovium and cartilage grafts were provided by MTF Biologics. Tissue samples were digested to isolate primary FLS and ACs. Single-donor hUVECs were sourced from Angio-Proteomie. Red Blood Cell (RBC) Isolation: Human blood samples were obtained from New York Blood Center, and RBCs were extracted via Ficoll-Paque technique. Transwell Culture: Cells were seeded on 24 mm transwells with 8 μm membrane inserts. ACs were expanded on the plate bottom (1x10^6 cells; DMEM + 10% FBS, 1% antibiotic/antimycotic, 1ng/mL TGF-β1, 5ng/mL FGF-2). FLS were seeded onto inverted transwell inserts (5x10^5 cells; αMEM + 10% FBS, 1% antibiotic/antimycotic, 5ng/mL FGF-2). Following overnight attachment and expansion, basal media was replaced with low-glucose DMEM, and transwell inserts were flipped. hUVECs were seeded onto the apical side (5x10^5 cells; EGM-2). Blood Treatment: After 24h, apical media was replaced with euglycemic (EG; 5 mM D-glucose) or hyperglycemic (HG; 100 mM D-glucose) treated RBCs (+BLD; 40% v/v in DMEM), with parallel controls in low-glucose DMEM (CTL). Media in the basal compartment was replaced with 50% v/v synovial fluid in DMEM (n=4).

Study 1: Following treatment, cell viability was assessed via Calcein and Ethidium Homodimer stains. Study 2: Basal media was analyzed for nitric oxide (NO) and lactate dehydrogenase (LDH). Study 3: Gene expression was performed via qPCR for makers of matrix degradation, matrix synthesis, inflammatory cytokines, and glucose uptake. Statistics: Genes were normalized to GAPDH and day 0 controls. Media comparisons were assessed using one-way ANOVA with Tukey HSD post-hoc tests at α=0.05.

Results, Conclusions, and Discussions:

Results: Cell viability remained consistent for hUVEC and AC across all treatment conditions (Fig. 2). Slight cell death was observed in FLS with 5mM and 100mM blood glucose exposure compared to DMEM controls (Fig. 2). NO and LDH release into the basal media were significantly elevated with blood exposure compared to non-blood treated controls (Fig. 3; p< 0.001). NO levels were further elevated under HG culture conditions (Fig. 3A; p=0.031). Gene expression analysis revealed significant differences across all cell types and treatment groups. Markers for ECM degradation (MMP1, MMP2, MMP3, MMP13), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), oxidative stress (AGER, ROMO1), and glucose transport (GLUT1) were upregulated with blood exposure compared to controls, with higher expression observed under HG treatment (Fig. 4). Conversely, matrix synthesis (ACAN, COL1A1, COL2A1, FN1) and insulin receptor (INSR) genes exhibited decreased expression under both blood and HG exposure (Fig. 4).

Discussions: FLS viability was decreased in groups with blood exposure, suggesting HG conditions may promote cell death. Media analysis demonstrated increased inflammation (NO) and cytotoxicity (LDH) with hyperglycemic conditions as well as blood exposure, reflecting features of both OA and DM disease states. Gene expression across all cell types confirmed that elevated blood glucose promotes ECM degradation, joint inflammation, and oxidative stress characteristic of OA. Elevated expression of glucose transport proteins (GLUT1) and downregulation of insulin receptor activity (INSR) in HG blood groups may be indicative of increased glucose transport and insulin resistance, resembling the DM diseased state.

Conclusions: The blood-joint transwell recapitulates cellular interactions of the joint space. This in vitro system provides a physiologic model to assess the effects of the vascularized synovial membrane on the underlying avascular articular cartilage surface. This study demonstrated that DM-induced high blood glucose levels can contribute to synovial inflammation and cartilage breakdown, both of which are characteristic features of the OA disease state.

Acknowledgements (Optional): Amazon Summer Undergraduate Research Experience (SURE), NSF GRFP, Musculoskeletal Transplant Foundation (MTF)