BCP CRYSTALS IN OSTEOARTHRITIS; UNRAVELLING THE CHANGING CHONDROCYTE SECRETOME

Purpose: Calcium containing crystals were long considered innocent bystanders, however recent evidence shows otherwise. Injection of BCP crystals in the murine knee leads to the development of synovial inflammation and increased cartilage degradation. In vitro, these findings are further supported by increased IL-6 and matrix metalloproteinases (MMPs). Despite the progress in understanding the response of various cell types to BCP crystals, most studies focus on a single aspect of this response. In the present study, we aimed to map the changes in the articular chondrocyte’s secretome, in response to BCP crystals, in an unbiased manner.

Methods: BCP crystals were synthesized as previously described and validated with FTIR. Human knee OA articular chondrocytes (OA HACs) were cultured in DMEM/F12 supplemented with 10% FCS, 1% P/S and 1% NEAA. Stimulations were performed with 50 μg/ml BCP crystals. Gene expression analysis was performed by RT-qPCR and normalized to PPIA. Secretion of IL-6 was determined by ELISA. Label-free LC-MS/MS quantitative proteomics was performed on 48 hour-conditioned medium from control and BCP-stimulated HACs of 14 individual donors (passage 2, METC permit 2017-0183). Donors comprised six females and eight males, with an average age of 71.6 years and a BMI of 30.8 kg/m².

Results: To verify the biological activity of the synthetic BCP crystals, we determined the IL-6 secretion of HACs upon stimulation. In a pool of 12 OA HAC donors, IL-6 secretion rose continuously over a period of 48 hours. A significant increase was observed from 4 hours onwards (Fig 1A). Since IL-6 can result from de novo synthesis or release from intracellular storage, we evaluated gene expression levels. Twenty-four and forty-eight hours post BCP stimulation, IL-6 expression was significantly increased (Fig 1B), hinting towards de novo synthesis. To determine if BCP crystal exposure potentially altered matrix composition, the expression levels of extracellular matrix (ECM) components COL1A1, COL2A1 and ACAN were determined. We observed a significant decrease in all measured ECM components at 24 and 48 hours (Fig. 1C). This decreased expression was accompanied by an increase in MMP-1, while we observed no change in MMP-13 (Fig. 1C). To map the changes in the chondrocyte secretome upon BCP crystal exposure, we performed label-free LC-MS/MS quantitative proteomics. We identified 158 proteins in the HAC secretome, of which 4 were downregulated (Fig. 1D) and 16 were upregulated when comparing control to the BCP condition (Fig. 1E). The downregulated proteins are involved in autophagy (prelamin A/C, p=0.046) and cell-cell interactions (desmoplakin, p=0.013). The most upregulated factor in response to BCP crystal stimulation compared to control was MMP-1, which increased 7.1-fold. Upregulated factors function in various pathways such as extracellular matrix reorganization, either by an increased expression of a collagen subtypes (COL6A3, p=0.015) or activation of matrix degradative components (MMP-1, p<0.0001). Other proteins found to be upregulated in response to BCP crystals function in fibrinolysis, such as SERPINE 2 and annexin A2. In addition, proteins involved in apoptosis, cell-cycle regulation and immune system activation were altered upon BCP crystal stimulation.

Conclusions: Due to the high prevalence of BCP crystals in OA there is a need for better understanding the chondrocyte response to BCP crystals. In this study, we aimed to map the changes in the chondrocyte's secretome in an unbiased manner. Our results show that BCP crystal exposure results in a complex and diverse response, including matrix reorganization, apoptosis, fibrinolysis and cell-cycle regulation. BCP crystals therefore seem a viable target for development of an OA disease-modifying intervention, either by directly inhibiting crystal growth or targeting of the BCP-driven secreted factors.