(M-501) Utilizing tissue chip to elucidate the crosstalk between cartilage and synovial tissue in pain activation

Introduction::
INTRODUCTION: Osteoarthritis (OA) is a painful and debilitating disease and is the 11^th global contributor to disability. To date there are no disease modifying OA drugs that have reached FDA approval, leaving pain management to non-steroidal anti-inflammatory drugs or invasive surgeries such as total joint arthroplasty (TJA). Synovitis, the inflamed state of the synovium, is known to contribute mediators that potentially lead the generation of pain and also further progress cartilage degeneration. Similarly, cartilage degradation products have been shown to cause synovial inflammation. The dynamic relationship between the cartilage and synovium during OA works as a negative feedback loop producing inflammatory cytokines, chemokines, and damaging-associated molecular patterns (DAMPs). As cartilage is aneural and avascular in nature, the synovium is currently considered as the primary contributor to subsequent nociceptive firing during early OA progression. The objective of this study is to use a recently established cartilage-synovial tissue chip to investigate the interaction of cartilage degradation products and an innervated synovial-like tissue to understand pain activation in OA.




Materials and Methods:: Engineering Tissue Constructs and assembling tissue chip:
Human bone marrow-derived mesenchymal stem cells (hBMSCs, P5) were differentiated into fibroblasts and human blood derived monocytes were polarized into M0 macrophages. At the time of complete differentiation, the two cell types were encapsulated in 10% methacrylated gelatin (GelMA) to form a synovial-like tissue. Human primary chondrocytes were encapsulated in 15% GelMA to form the cartilage construct. A two-chamber microfluidic chip containing the synovium and two-dimensionally seeded human dorsal root ganglion (DRG) neurites allowed for the innervation of the synovium
(Fig.1). Medium from the cartilage was provided to the synovial-like construct to allow for tissue-crosstalk. Assessment of these tissues were accomplished using histology, qPCR, and ELISAs.
2)Characterization of Synovium following inflamed cartilage treatment:

To create an OA model and study the effect of cartilage degradation products on the synovial tissue, the cartilage was treated with interleukin (IL) 1β (10 ng/mL) for three days prior to connection with the innervated synovial chips. Synovial constructs were treated with conditioned medium from the inflamed cartilage for two days to demonstrate the effect of soluble cartilage factors. Synovial tissue samples and their conditioned medium were collected for qPCR and Luminex assay respectively to assess the production of inflammatory mediators (Fig. 2).
3)Assessment of Neuronal Activation:
DRG neurites were assessed using live imaging to investigate their response to the conditioned medium in the synovial-like chamber. Prior to seeding, neurons were transfected with GCaMP, which allowed for the assessment of calcium signaling and pain activity
(Fig.3).


Results, Conclusions, and Discussions::
Cartilage tissue constructs treated with IL-1 β showed robust inflammation and degradation compared to the negative control. Specifically, results form the Luminex assay showed a significant increase of matrix metalloproteinases (MMPs) and proinflammatory cytokines in the condition medium (Fig.2A). Treatment of synovial-like tissue with cartilage conditioned medium demonstrated a M1 polarizing effect on the tissue compared to the healthy control. For example, Luminex assay results showed an increase in CCL-2, PLGF, G-CSF, and CXCL-1 in the synovial constructs following treatment with the inflamed cartilage conditioned medium (Fig.2B). Interestingly, fluorescence imaging of rodent and human dorsal root ganglion (DRG) neurites showed activation when they were treated with conditioned medium from the OA-induced cartilage constructs (Fig.3), suggesting the generation of pain activity.

Understanding the mechanism of inflammation caused by cartilage degradation particles on the synovium is essential for understanding nociceptive activation in OA pain. Here we aim to engineer human based tissues to elucidate those mechanisms and study the mechanisms of pain activation during OA. Currently we are working on establishing a micro-electrode array to study neuronal firing in response to the synovial-like tissue’s treatment with medium from the OA induced cartilage. We are also testing the re-circulation of the medium to examine if inflamed synovial tissues will further accelerate the degradation of cartilage. The final objective of this study is to use our system serve as a platform for personalized pain medicine for the assessment and treatment of OA patients. OA related pain is not well understood and complex. Through investigating the mechanisms behind cartilage degradation products and subsequent synovial inflammation, we can begin to understand the crosstalk of cartilage and the synovium in pain activation, providing a novel platform to study OA pain in human and screen new therapeutics.






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