Extracellular matrix stability of primary mammalian chondrocytes and intervertebral disc cells cultured in alginate-based microbead hydrogels

Abstract

Three-dimensional alginate constructs are widely used as carrier systems for transplantable cells. In the present study, we evaluated the chondrogenic matrix stability of primary rat chondrocytes and intervertebral disc (IVD) cells cultured in three different alginate-based microbead matrices to determine the influence of microenvironment on the cellular and metabolic behaviours of chondrogenic cells confined in alginate microbeads. Cells entrapped in calcium-, strontium- or barium- ion gelled microbeads were monitored with the live/dead dual fluorescent cell viability assay kit and the 1, 9-dimethylmethylene blue (DMB) assay designed to evaluate sulfated glycosaminoglycan (s-GAG) production. Expression of chondrogenic extracellular matrix (ECM) synthesis was further evaluated by semi-quantitative RT-PCR of sox9, type II collagen and aggrecan mRNAs. Results indicate that Ca- and Sr-alginate maintained significantly higher population of living cells compared to Ba-alginate (p<0.05). Production of s-GAG was similarly higher in Ca- and Sralginate microbead cultures compared to Ba-alginate microbeads. Although there was no significant difference between strontium and calcium on day 14 of culture, Sr-alginate showed remarkably improved cellular and metabolic activities on long-term cultures, with chondrocytes expressing as much as 31% and 44% greater s-GAG compared to calcium and barium constructs respectively while IVD cells expresses 63% and 74% greater s-GAG compared to calcium and barium constructs respectively on day 28. These findings indicate that Sr-alginate represent a significant improvement over Ca- and Ba-alginate microbeads for the maintenance of chondrogenic phenotype of primary chondrocytes and IVD cells.