Comparison of cartilage specific markers in articular and differentiated chondrocytes in pellet system

Abstract

Autologous cartilage replacement has the inherent advantage that the transplanted tissue is immunogenically neutral. However, chondrocyte isolation, proliferation, and dedifferentiation limitations resulted in the search for a cell type that would overcome the aforementioned limitations. Here we investigated if adipose-derived stem cells (ADSCs), which are easy to isolate in large quantities, in combination with a three dimensional culture system and growth factor would be a suitable alternative for autologous cartilage replacement. In this study ADSCs and human articular chondrocytes were allowed to differentiate in pellet culture in the presence or absence of transforming growth factor-beta 3 (TGF-beta 3) for 14 days and their chondrogenic potential was evaluated. Pellets were microscopically and histologically evaluated for structure and morphology. Matrix production was assayed using immunohistochemistry for collagen types I and II, and aggrecan. Images were evaluated via custom-made routines programmed in MATLAB. Oval to round cell pellets were formed with an overall homogenous dense central structure that consisted of isogenic groups with walled-off chondrocytes in their lacunae and encircled by basophilic extracellular matrix (ECM). Our results show that in the presence of TGF-beta 3, collagen type I expression was 2-fold and aggrecan expression was 3-fold higher in differentiated ADSCs compared with articular chondrocytes, whereas collagen type II was reduced by a factor of 1.58. Furthermore, the absence of TGF-beta 3 nearly completely suppressed collagen type I in both cell types, whereas aggrecan expression remained unaffected in ADSCs and was reduced 1.87-fold in articular chondrocytes. Pellet culture of ADSCs-derived chondrocytes represents a viable alternative for autologous cartilage replacement. The high cell density and more natural three-dimensional environment of pellet culture attenuated dedifferentiation of cells. Although we obtained a mixed chondrogenic phenotype with increased collagen type I expression, the extent of this increase was lower compared to reports in the literature. Furthermore, ADSCs-derived chondrocytes expressed large amounts of aggrecan, which is an essential component of the ECM in articular cartilage. Finally, TGF-beta 3 might be used to direct chondrogenic differentiation and reduce collagen type I expression by controlling concentration and treatment duration with TGF-beta 3.