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:

conventional monolayer culture,

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.

Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time.

Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with real time PCR, probing specifically for the genes responsible for the production of collagen I, collagen II, aggrecan, and GAPDH. This was done in duplicate.

Results: Collagen II gene expression was maintained over seven weeks in cells left at confluence but was decreased in passaged cells. Collagen I gene expression decreased over seven weeks in cells left at confluence, but remained the same in passaged cells. Aggrecan gene expression remained the same in both groups.

Conclusion: Current culture and expansion techniques that employ passaging (as used in clinical scenarios) result in significant alterations in gene expression that are inconsistent with the current definition of a “chondrocyte”. Culturing chondrocytes at confluence can produce gene expression more similar to native chondrocytes but even these cells have expression of collagen type I that should not be present in chondrocytes. The results of this study suggest that further investigation is required to develop chondrocyte culture and expansion techniques that minimize the de-differentiation of chondrocytes by maintaining collagen II gene expression and eliminating/preventing collagen I gene expression.