Synovial fibroblasts support vascular function in both health and inflammatory disease

Introduction: The synovium is a vascularized, joint-lining tissue present in the knee, as well as in other articulating joints. After an acute trauma, the synovium undergoes marked changes in cellular composition [1, 2]. While advances in single cell sequencing have elucidated these cellular changes, the distinct roles of such cells have yet to be identified in both health and disease (inflammation). In this study, we aimed to develop a microphysiologic model of the human synovium to determine the specific role of synovial fibroblasts in maintaining vascular health. Endothelialized vessels were engineered within collagen gels (one of the primary matrix components of the synovium), exposed to IL1B (inflammation/injury) [3], and functional outcomes (permeability, collagen remodeling, synoviocyte-vessel colocalization) were assessed over 8 days in culture. Using this 3D model system, we can interrogate the unique role of individual cell populations through the intentional design of controls (±synoviocytes), (±endothelial cells), (±IL1B)—which would be impossible in any complementary animal model. Overall, we hypothesized that healthy human synoviocytes would enhance the functional properties of our vascularized lumens, including the generation of a basement membrane-like barrier. In the presence of IL1B, we posited that the synoviocytes would have a diminished, yet still beneficial effect.

Materials and

Methods: Device fabrication. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated using custom 3D printed molds. PDMS rods were extruded through 23-gauge needles, creating ~340µm diameter lumens. The top and bottom PDMS layers were aligned, and a PDMS rod was inserted through the inner channel of each device. Device endothelialization and culture. Collagen (3 mg/mL) was injected into the hexagonal chamber of the devices (± human synoviocytes; Cell Applications) and polymerized. After removing the PDMS rods, human umbilical vein endothelial cells (HUVECs) (20,000/µL) were injected into each hollow channel. The devices were rotated, and cultured in endothelial growth media (EGM). After 1 day of culture, the devices (n = 8-10/group) were cultured in EGM (-IL1B) or EGM + 100 pg/mL IL1B (+IL1B) for 7 additional days with media changed once daily. Permeability. Vessel permeability was quantified, as in [4], by injecting 10-kDa FITC-dextran into a channel, and serially imaging the device midplane. Image series were uploaded into a MATLAB script [4] to quantify Pd. Staining. VE-cadherin staining: Devices were fixed, permeabilized, blocked, and incubated in VE-cadherin primary antibody (1:300). Subsequently, devices were rinsed and stained Alexa Fluor 647 donkey anti-rabbit IgG (1:500). Live/dead: Devices were rinsed and stained with ethidium homodimer-1 (dead), and calcein-AM (live). Collagen Remodeling. 8% FITC-labeled collagen was mixed into the bulk collagen solution prior to polymerization. Synoviocytes were stained with DiD prior to encapsulation, and RFP-HUVECs enabled separate tracking of endothelial cells. All analyses were performed in FIJI. Statistics. 2-way ANOVAs, and an unpaired t-test (GraphPad Prism) were performed.

Results, Conclusions, and Discussions: Results and Discussions: Microfluidic devices were fabricated in a high-throughput manner (Fig. 1A), enabling investigation into the impact of synovial fibroblasts on vascular function in both health and disease (inflammation). As shown, the seeded HUVECs formed confluent lumens (Fig. 1B). Additionally, within the collagen gels, human donor synoviocytes maintained their viability (Fig. 1C). All assessments were performed on days 4 or 8 post-fabrication (Fig. 1D). We first measured how synovial fibroblasts influence vascular permeability over time in culture (Fig. 1E), and discovered that the vessels matured rapidly with the addition of synoviocytes (Fig. 1F). Next, we introduced the pro-inflammatory cytokine IL1B daily to the cell culture media, and reassessed permeability at Day 8 (Fig. 1G). Interestingly, the lumens without synoviocytes demonstrated a strong increase in permeability in response to IL1B treatment. However, the lumens surrounded by synoviocytes maintained their permeability. To probe the mechanisms behind this finding, we fabricated vessels using FITC-labeled collagen, enabling tracking of matrix remodeling. On Day 8, we imaged the bottom half of the vessels (Fig. 2A, B), and binarized the images to quantify collagen accumulation around the vessel periphery. These images highlight how endothelial cells alone remodel their environment (Fig. 2C); however, the synoviocytes elevate this endothelial cell-mediated response. In comparison to the -IL1B baseline, +IL1B appears to diminish the layer of collagen around the vessels. As mentioned, we believe that synoviocytes instruct the endothelial cells to remodel their surrounding environment, creating a ‘basement membrane,’ as we see no remodeling in the absence of endothelial cells (data not shown). Lastly, we observed significantly more synovial fibroblasts surrounding our healthy vessels, in comparison to the +IL1B group (Fig. 2D). Future work will identify cell-secreted factors from both synoviocytes and endothelial cells that contribute to matrix remodeling.

Conclusions: This model is the first-of-its-kind to investigate the role of synovial fibroblasts in health and inflammatory disease, focusing on synovial-vascular interactions. To further enhance the physiologic relevance of our model, ongoing studies are integrating fluid flow into our microphysiological system.

Acknowledgements (Optional): The authors would like to thank the Schmidt Science Fellowship in partnership with the Rhodes Trust, as well as the Center for Engineering Mechanobiology (CEMB).

References:
[1] Knights, A. J., et al., Annals of the Rheumatic Diseases., 82:272-282, 2023. [2] Knights, A. J., et al., bioRxiv, 2023. [3] Sward, P., et al., Osteoarthritis and Cart., 20:1302-1308, 2012. [4] Polacheck, W. J., et al., Nature Protocols, 14:1425-1454, 2019.