Title

Rescue of Osteoarthritic Chondrocyte Phenotype Through Long Term, Three-dimensional, Serum-free Culture

Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Marina D'Angelo, PhD

Second Advisor

Kathryn Behling, MD, PhD

Third Advisor

Jocelyn Lippman-Bell, PhD

Abstract

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 and functionally relevant components of the extracellular matrix (ECM) can be measured. However, when attempting to maximize samples per patient by pooling the greater and less damaged sides of the patient's knee, extracellular response of the HOACs was dampened when compared to cultures of lesser pathology HOACs alone. 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 x 106 cells per 0.5 mL. Samples were allowed to culture for up to 11 days. Collagen I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate-associated matrix of the cultures. Our data shows a reduction in collagen degradation and in loss of sulfated proteoglycans. Over 11 days in culture, collagen type I and II degradation was reduced while proteoglycan synthesis was increased in HOAC cultures. 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. ECPFs are a class of novel therapeutics, inhibiting MMPs. ECPF-1 specifically inhibits MMP- 13, the collagenase responsible for degrading collagen in the ECM. We treated HOACs cultured in alginate at time of plating with either 250nM Extracellular Matrix Protection Factor 1 (ECPF-1), or hyaluronic acid conjugated ECPF-1 (HA-ECPF-1) for 48 hours. HOA Cs from least pathology sides responded to treatment by reducing collagen I and II degradation. These data confirm that 48 hour treated 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 collagen I and II degradation products.

This document is currently not available here.

Share

COinS