Potential Therapeutic Efficacy of a Novel Metalloproteinase Inhibitor, Extracellular Matrix Protection Factor 1, in Human Osteoarthritic Chondrocyte Primary Cultures

Date of Award

2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Marina D’Angelo, PhD

Second Advisor

Christopher Adams, PhD

Third Advisor

Mindy George-Weinstein, PhD

Fourth Advisor

Marcus Bell, PhD

Abstract

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix 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 of primary, human osteoarthritic articular chondrocytes (HOACs) for the purposes of studying chondrocyte behavior and testing potential disease modifying OA drugs. In this study, we tested the effects of a member of the novel class of MMP inhibitors, Extracellular Matrix Protection Factor 1 (ECPF-1), on collagen degradation by HOACs. Primary human osteoarthritic articular chondrocytes (HOACs) were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were separated and identified as the side of greatest or least pathology. The number of live cells were determined and plated at 2.5 x 106 live cells per milliliter of alginate. Cells were treated with ECPF-1 (250nM or 2500nM) at the time of plating and day 2 of culture. Control cultures received serum-free media only. Days 2 and 5 conditioned media (CM) and day 5 alginate-associated matrix (AAM) were isolated and assayed for the amount of total collagen types I and II, and the percentages of the total collagen that were intact and degraded. Media and AAM were also analyzed for the presence of Interleukin 1β (IL-1β), Transforming Growth Factor β2, and MMP-13. Treatment with both doses of ECPF-1 reduced the degradation of collagens type I and II in the media. Treatment with ECPF-1 did not affect the amounts of intact and degraded collagens within the 5-day AAM. ECPF-1 decreased the amount of the inflammatory cytokine IL-1β in day 2 and day 5 conditioned media, but not in the AAM. Treatment with ECPF-1 also decreased the amount of latent and activated TGF-β2, and MMP-13 in the day 5 CM and AAM. This study demonstrates that HOACs reared in serum-free medium in alginate beads are responsive to the novel therapeutic, ECPF-1. Our culture model is a reproducible and cost effective system in which to test potential disease modifying OA drugs.

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