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 a common chronic condition which involves the loss of articular cartilage, the cushion between joints that provides a smooth surface for the bones to glide. Three compounds that make up most of the articular cartilage are type II collagen, proteoglycans, and water. In OA there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage. The composition of the extracellular matrix changes in OA as evidenced by 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 alginate culture but measurable extracellular matrix endpoints of the OA profile are dampened when chondrocytes isolated from sides of greater and least pathology in the joint are pooled together in culture. In this study, we obtained HOACs from patients undergoing total knee arthroplasty. Femoral condyles and the tibial plateau were labeled greater or least pathology, determined by gross anatomical observation, the cells were isolated and plated separately in three‐dimensional, twelve‐well, alginate cultures at a density of 1.8 × 106 cells per 0.5 mL. Samples were treated for 48 hours from day of plating with either 250nM Extracellular Matrix Protective Factor‐1 (ECPF‐1) or hyaluronic acid conjugated ECPF‐1 (HA‐ECPF‐1). Collagen I degradation and sulfated proteoglycan synthesis were measured in alginate‐associated matrix of the treated cultures. HOACs isolated from the side of greatest pathology responded to treatment by reducing collagen type I degradation (8.42ng/culture Control, 7.25ng/culture ECPF‐1 and 4.25ng/culture HA‐ECPF‐1) and increasing retention of sulfated proteoglycans in the extracellular matrix (0.36ug/culture Control versus 0.56ug/culture ECPF‐1 treated). These data confirm that 48 hour treatment in serum free HOAC culture is sufficient to measure the therapeutic efficacy of an OA drug intervention and that both formulations of ECPF‐1 can slow the progression of OA by altering production of sulfated proteoglycan and collagen type I.Support or Funding InformationThis work was supported in part by a grant from the New Jersey Health Foundation and Philadelphia College of Osteopathic Medicine's Center for Chronic Disorders of Aging and the Division of Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Collagen, produced by fibroblasts, serves an integral role in the structural integrity of the extracellular matrix of the human gingiva, but during periodontal disease, matrix metalloproteases (MMPs) are upregulated and contribute significantly to the degradation of collagen. In this study, we test the effects of the novel MMP inhibitor, Extracellular Matrix Protection Factor‐2 (ECPF‐2) on total protein production by human gingival fibroblasts (HGVFs) reared in serum‐free media. Cells are enzymatically released from gingival tissue isolated during oral surgery and cultures produced from patient samples: normal, non‐inflamed (NTN); inflamed non‐smoker (IFN); inflamed previous smoker (IFP); inflamed current smoker (IFC). Subconfluent cultures were switched to 0.1% fetal bovine serum containing DMEM media overnight and then treated for 24 hours with complete serum‐free DMEM (Control); 5ug ECPF‐2; or 50ug ECPF‐2. Conditioned media was collected and concentrated and the cell layer was extracted with 0.5% CHAPS buffer. Total protein of the samples was quantified using the Pierce Modified Lowry Protein Assay. Regardless of treatment or patient pathology, all cultures tested produced approximately 1.3–1.5 ug/ml/culture of total protein. There was no statistically significant differences between patient samples or within treatment conditions per patient. These data suggest that our culture system allows for viable metabolism in serum‐free medium and treatment with the novel MMP inhibitor, ECPF‐2, does not affect the metabolic activity of HGVF cultures. Therefore, this culture system can be used to measure the therapeutic effects of ECPF‐2 on collagen metabolism associated with periodontal disease. Support or Funding Information Support from the PCOM Division of Research

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