A novel modified culture medium for enhancing redifferentiation of chondrocytes for cartilage tissue engineering applications

Abstract

The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days.Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83-2.75, 5.00-7.50, and 1.00-1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.

Development of chondrocyte‐laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration

“…In this study, the viability of chondrocytes encapsulated in alginate microbeads was slightly reduced when the microbeads were incubated with the SCB treatment medium for 3 days. Our previous report demonstrated that the viability of chondrocyte pellet was negatively affected by SCB in a dose‐dependent manner 30 . However, all the tested SCB treatment medium exhibited no cytotoxicity (over 80% viability) due to the low SCB concentrations used in this study.…”

“…Our previous report demonstrated that the viability of chondrocyte pellet was negatively affected by SCB in a dose-dependent manner. 30 However, all the tested SCB treatment medium exhibited no cytotoxicity (over 80% viability) due to the low SCB concentrations used in this study. Members of the transforming growth factor-β (TGF-β) superfamily are well known for regulating chondrogenesis 34 and inducing cell proliferation.…”

“…59 Previous studies have reported that SCB had an adverse effect on cell viability due to the excessive concentration of ions in the culture medium hindering the growth rate. 30,47 In this study, the viability of chondrocytes encapsulated in alginate microbeads was slightly reduced when the microbeads were incubated with the SCB treatment medium for 3 days. Our previous report demonstrated that the viability of chondrocyte pellet was negatively affected by SCB in a dose-dependent manner.…”

“…To decrease the crosslinking density of alginate microbeads, a culture medium containing SCB was used for trapping Ca 2+ and replacing it with Na + in a crosslinked structure of calcium alginate hydrogels. The culture media containing various concentrations of SCB solution were prepared as described in our previous report 30 . In brief, the stock SCB solution was prepared by dissolving sodium citrate, sodium chloride, and EDTA in deionized water to a final concentration of 55, 150, and 30 mM, respectively, and adjusted pH to 6.8.…”

“…The SCB consists of sodium chloride and two calcium chelators, which are ethylenediaminetetraacetic acid (EDTA) and sodium citrate, that strongly trap calcium from the calcium‐crosslinked alginate structure and consequently solubilize the alginate hydrogels 21,29 . Very recently, we have successfully developed a novel and effective protocol to promote the redifferentiation of chondrocyte pellets using the modified culture media containing SCB at specific concentrations 30 . Thus, it is interesting to study a new approach of using the treatment medium supplemented with SCB to partially dissolve alginate microbeads in order to obtain the microbeads with specific properties and microenvironments favorable to the chondrogenesis process.…”