Osteoarthritis (OA), a progressive and degenerative disease, affects millions of aging adults and results in the loss of cartilage. In healthy joints, articular cartilage provides a smooth and cushioned surface that allows bones to glide over each other with little friction. However, when OA begins to develop, degradation of the cartilage extracellular matrix begins to outweigh its synthesis. Production of growth factors, cytokines, and matrix‐degrading enzymes increases. Development of OA therapeutics is the gold standard in orthopedic medicine, but the complexity of the joint makes it a difficult system to manipulate. In this study, we optimize our novel, serum‐free, three‐dimensional culture system for human osteoarthritic articular chondrocytes (HOACs) obtained from patients with end‐stage osteoarthritis who have undergone a total knee arthroplasty. We altered the parameters of culture size, tissue harvest sites and the number of cultures pooled to determine whether clinically‐relevant endpoints are within measurable ranges. In previous studies, sides of greatest and least pathology, as determined from gross examination of the femoral condyles and tibial plateau, were separated and cells were isolated and plated within serum‐free, three‐dimensional alginate cultures on 35 mm plates at a plating density of 2.5×106 cells/milliliter. In this study, we combine both sides of the joint tissue (greatest and least pathology) together to increase the number of cells and cultures obtained from each patient. In addition, we scaled down the size of the cultures to 22 mm plates at a plating density of 1.8×106 cells/milliliter. The scaled‐down, serum‐free, three‐dimensional alginate cultures were maintained for five days, media fractions collected on the second and fifth day of the culture and the alginate‐associated matrix collected at termination of the cultures on day five. Intact and degraded collagens were measured by immunoassay. Both collagens type I and type II, intact and degraded, were detectable within the scaled‐down culture system. Pooling three cultures together provided adequate measurable material of the clinically‐relevant endpoints. Collagen I intact and degraded values were lower than collagen II intact and degraded regardless of the number of cultures pooled (n= 2, 3 or 4). In summary, our optimization techniques have provided more cultures per patient thus enabling measurement of clinically‐relevant endpoints of OA and a screening system to test potential OA therapeutics.Support or Funding InformationNew Jersey Health Foundation Research Award, Center for Chronic Disorders of Aging Small Grant, and PCOM's Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Osteoarthritis (OA) is characterized by an imbalance of cartilage extracellular matrix (ECM) production and degradation, due, in part, to increased cytokine production. It has been demonstrated that the cytokine, Interleukin‐1 beta (IL‐1β), is produced by OA articular chondrocytes and plays a prominent role in altering ECM metabolism by targeting the matrix metalloproteases (MMPs) that degrade the ECM. We are developing a novel, OA therapeutic, Extracellular Matrix Protection Factor‐1 (ECPF‐1) that targets MMP interaction with its ECM substrate. We have demonstrated a reduction in ECM degradation when primary, human OA chondrocyte (HOAC) cultures are treated with ECPF‐1. In this study, HOACs isolated from total knee arthroplasty were reared in three‐dimensional, serum‐free, alginate cultures. The sides of least and greatest pathology of the femoral condyles and tibial plateaus were determined by gross inspection and cells isolated were cultured separately (LP and GP). Chondrocytes were plated at 2.5×106 cells per milliliter of alginate and incubated in serum‐free medium for five days. Cells were released from the alginate and the RNA extracted from the pellet, reverse‐transcribed into cDNA and quantitative, real time PCR (qRT‐PCR) was performed with TaqMan Gene Expression assays for IL‐1β, collagen type I, collagen type II and 18s rRNA. In a previous study, IL‐1β production was detected in the conditioned media of HOAC cultures with the highest concentration of this cytokine present in the cultures established from the region of greatest pathology (GP). In this study, IL‐1β expression was detectable in mRNA produced by LP and GP HOAC cultures. The expression was reduced 2‐fold in LP cultures treated 24 hours with 2.5 uM ECPF‐1 and 1.4 fold in GP cultures. These results demonstrate the ability of ECPF‐1 to alter expression of a molecule involved in the feedback loop of ECM destruction. ECPF‐1 provides a tool with which to further define the cellular mechanisms responsible for OA chondrocyte pathology.Support or Funding InformationNew Jersey Health Foundation, Center for Chronic Disorders of Aging Small Grant, PCOM's Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.