Serum‐free Cultures of Primary Equine Articular Chondrocytes Demonstrate Differences in Collagen Production And Degradation in Response to Extracellular Matrix Protection Factor (ECPF‐1), A Novel Osteoarthritis Therapeutic

Abstract

Reproducible, long term culture of primary, articular chondrocytes has been difficult regardless of mammalian species of origin because articular chondrocytes plated in monolayer with serum‐containing media will rapidly de‐differentiate. Several limitations of chondrocyte cell culture have been overcome with the development of a three‐dimensional system consisting of alginate beads that more closely resembles the architecture of cartilage in vivo. But most studies described in the literature are not completely serum‐free because they include low serum or serum equivalents in the media, especially in reports of equine primary articular chondrocyte cultures. In this report, we describe the development and utility of a serum, hormone and albumin free, three‐dimensional alginate culture system for primary, equine, aged, articular chondrocytes (EACs). The goals of this project were to measure equine articular chondrocyte cell yield and survival, production and degradation of collagen type II, a clinically relevant endpoint of osteoarthritis, and the effects of treatment with a novel OA therapeutic on collagen metabolism in these articular chondrocytes. Articular chondrocytes were isolated from the radiocarpal joint of aged, horses euthanized for reasons other than joint disease following the standards and protocols of University of Pennsylvania's New Bolton Center. Three horses ranging in age from 10 years to 21 years (two geldings and a mare) were included in this study. An average of ten million articular chondrocytes were isolated from the lateral or medial aspect of the distal radius. Articular chondrocytes were released from the tissue through trypsin and collagenase digestion, plated at 2.5×106 cells per milliliter alginate and fed serum‐free media (Control = Dulbecco's Modified Eagle's Medium with antibiotic/antimycotic, sodium pyruvate and glutamine) or serum‐free media plus 2500nM ECPF‐1 (Treated) and conditioned media was collected on days 2 and 5 in culture. At day 2 in culture, 2500nM ECPF‐1 increased intact collagen type II production two‐fold (401.24ng/35mm culture Control versus 985.74ng/35mm culture Treated) and maintained a 1.3 fold increase through day 5 in culture (1476.43ng/culture Control versus 1845.98ng/culture Treated). Total amount of degraded collagen type II present in day 5 conditioned media demonstrated a slight decrease following 2500nM ECPF‐1 treatment when compared to control. Thus we have shown that primary, aged, equine articular chondrocytes can be reared in serum and hormone free media and that ECPF‐1 treatment does reduce collagen type II degradation while increasing production of intact collagen type II in aged equine chondrocytes.Support or Funding InformationThis research was supported with funds from the Division of Research at PCOM.