Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p<0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics. Support or Funding Information Support was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix (ECM) in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human OA chondrocytes (HOACs). However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of live cells were determined and plated at 1.8 × 106 cells/0.5ml alginate per well of a 12‐well plate. Cells were cultured in complete serum‐free media for 11 days with RNA and DNA collected at days 2, 5, 8 and 11. Total RNA and DNA were isolated via Trizol reagent protocol. Quantitation of nucleic acids was accomplished with the NanoDrop system. Over the eleven days in culture there was no statistically significant difference in the total amount of RNA isolated either in the least pathology samples (day 2=4.3ugRNA/culture versus day 11=2.8ugRNA/culture) or the greatest pathology samples (day 2=5.3ugRNA/culture versus day 11=6.2ugRNA/culture). The same was true for the total DNA isolated from the least pathology samples (day 2=1.9ugDNA/culture versus day 11=1.6ugDNA/culture) or the greatest pathology samples (day 2=8ugDNA/culture versus day 11=1.7ugDNA/culture). Future studies include quantitative, real‐time PCR to measure expression of metabolic agents including MMPs and collagens present in OA chondrocytes. This study is the first of its kind to isolate RNA and DNA from completely serum‐free, three‐dimensional cultures of primary human OA chondrocytes. Support or Funding Information This study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research

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.