TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds
Introduction
Articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) are common cell sources for articular cartilage engineering; however there are complications associated with the use of each cell type. While ACs are isolated, expanded, and clinically implanted in autologous chondrocyte transplantation [1], [2], obtaining sufficient numbers of ACs presents a challenge, as their isolation can cause significant donor site morbidity [2]. Furthermore, in vitro expansion of ACs can lead to rapid dedifferentiation and a fibroblastic phenotype [2], resulting in an inferior tissue-engineered construct. Conversely, bone marrow-derived MSCs are readily available, easily expandable, and capable of chondrogenic differentiation [3]. While achieving MSC chondrogenesis has been demonstrated, it is not without challenges. After chondrogenic induction, MSCs often exhibit a hypertrophic phenotype [4], [5] followed by calcification of the extracellular matrix (ECM) [5], [6]. Additionally, compared to ACs the amount of cartilage-like ECM produced by differentiating MSCs is minimal [7], [8], [9] and leads to constructs with inferior mechanical properties [10]. More recently, co-cultures of ACs and MSCs are being investigated as a potential cell population for articular cartilage engineering [11], [12]. Such a cell population has several advantages, including the ability to reduce the required number of ACs, while achieving equal or greater levels of chondrogenesis compared to ACs alone [13]. Furthermore, the co-culture scheme has been shown to reduce the mineralization and hypertrophy that is often seen in chondrogenic MSCs [4], [14].
Studies investigating the mechanism of enhanced chondrogenesis in AC/MSC cultures have primarily concluded that the trophic effects of MSCs enhance AC chondrogenesis [8], [13], [15]. Most commonly, co-cultures have been investigated using standard serum-free culture medium with 10 ng/ml of TGF-β3 [4], [13], [14], [15], [16] or TGF-β1 [8], [11], [17] as a chondrogenic inducer in vitro; however, reduced concentrations of TGF-β3 in AC/MSC co-cultures have not been investigated. Due to the ability of TGF-β3 to enhance chondrogenesis of both ACs and MSCs and thus possibly enhancing the co-culture effects, the present study hypothesized that co-cultures may have enhanced sensitivity to chondrogenic stimuli, and thus may be capable of chondrogenic induction with a reduced concentration of TGF-β3, compared to AC or MSC cultures alone. Furthermore, due to the continued presence of MSCs and thus continued chondrogenic stimulation, it was hypothesized that the co-cultured cell populations would have a more stable phenotype upon the removal of TGF-β3.
Section snippets
Experimental design
Bovine ACs and rabbit bone marrow-derived MSCs were seeded in monoculture or in co-culture (1:3 ratio of ACs:MSCs) on electrospun poly(ɛ-caprolactone) (PCL) scaffolds and cultured for two weeks in a chemically defined, serum-free culture medium, supplemented with one of four concentrations of TGF-β3: 0 ng/ml, 1 ng/ml, 3 ng/ml or 10 ng/ml. After the initial chondrogenic stimulation period, all groups were cultured for an additional two weeks to evaluate the stability of the construct phenotypes
Two week biochemical assays
The quantity of DNA was measured in all cell populations at the start of the culture to determine the baseline value and measured again after two weeks of culture (Fig. 1A). At that time, ACs and co-cultures had proliferated, resulting in an increase in DNA content from the initial levels; however, the MSCs did not exhibit any change in DNA content from day 0. In general, the levels of DNA were independent of TGF-β3 concentration with the exception of non-induced co-cultures having lower DNA
Discussion
Co-cultures of ACs and MSCs are a promising cell source for cartilage engineering with the potential to overcome several challenges associated with the individual use of corresponding monocultures. The enhanced proliferation, matrix production, and chondrogenic gene expression of co-cultures has generally been attributed to the ACs, whose chondrogenic activity is upregulated by the presence of MSCs [8], [13], [15]. In the present study, we hypothesized that co-cultures of ACs and MSCs would
Conclusions
The current work evaluated the sensitivity of co-cultures of ACs and MSCs to TGF-β3 by quantifying the synthetic capacity and chondrogenic gene expression of the cultures after exposure to a range of TGF-β3 concentrations over two weeks of culture. The results demonstrated that while TGF-β3 was able to enhance the chondrogenic phenotype of ACs and MSCs, AC/MSC co-cultures required a reduced concentration of TGF-β3 in order to achieve enhanced chondrogenesis compared to either cell type alone.
Acknowledgments
This work was supported by the National Institutes of Health grant R01 AR057083.
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