55 – CHARACTERIZATION OF NOVEL CULTURE CONDITIONS WHICH PREVENT ARTICULAR CHONDROCYTE DEDIFFERENTIATION

Abstract

Purpose: Traditionally, chondrocyte growth and characterization studies have been conducted using non-physiologic, normoxic, monolayer culture systems that have the major drawback of dedifferentiation. Recently, however, the use of novel 3D culture systems, cytokine supplementation or hypoxic culturing techniques have shown that chondrocyte dedifferentiation can be greatly reduced. Unfortunately, to date, no single culture technique has been identified that completely prevents the dedifferentiation-related changes in ECM gene expression. We hypothesized that combining a high density culture condition with an hypoxic environment would improve chondrocyte phenotype retention as determined by gene expression and protein production when compared to current standard culture conditions.

Method: Freshly isolated normal human articular chondrocytes were maintained in three culture conditions:

1. conventional monolayer culture,

2. high density monolayer culture (HDMC) and

3. embedded in alginate and compared to freshly isolated positive controls (FIC) and fibroblasts as negative controls.

The conventional monolayer cultures were harvested at confluence while HDMC and alginate-embedded chondrocytes (AEC) were maintained in culture for 8 weeks. Parallel experiments were conducted under normoxic (21% O2) and hypoxic (5% O2) conditions for all three experimental groups. Chondrocytes were harvested, RNA was extracted and quantitative RT-PCR was performed using primers for collagens (I, II, VI, IX and XI), aggrecan, SOX-9, HIF-1, 3 different integrins and GAPDH. In addition, collagen and GAG content was quantified when possible using Sircol and Blyscan assays respectively.

Results: HDMC cultures in hypoxic conditions showed a 2.5 fold increase in wet weight, a 6.9 fold increase in GAG content and a 1.3 fold increase in collagen content relative to normoxic HDMCs. With respect to gene expression levels, only the HDMCs in hypoxic culture conditions yielded mRNA expression levels of collagen II, IX, XI, aggrecan, HIF-1, SOX-9 and one Integrin that were consistent with the levels seen in freshly isolated chondrocytes (positive control). Importantly, HDMC culture in hypoxic conditions also yielded the lowest levels of collagen I of any experimental condition.

Conclusion: This research demonstrated that high density monolayer culture in hypoxic conditions prevented the severe loss of chondrocyte phenotype typically associated with conventional monolayer culture. Cells cultured in these conditions demonstrated gene expression levels similar to those seen in FICs, which are superior to those seen following conventional culture conditions such as the use of alginate beads. These culture conditions provide a novel opportunity to maintain chondrocyte phenotype over a prolonged period of time while generating extracellular matrix that may be beneficial for treatment of full thickness cartilage defects.