Resveratrol counteracts IL-1ß-mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs.

When aiming at cell-based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL-1ß need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti-inflammatory properties. However, long-term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long-term model cultures for cell-based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL-1ß (1-10 ng/ml) or RSV (50 µM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL-1ß (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL-1ß-treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL-1ß treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL-1ß-mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long-term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.

Gap junctional intercellular communication in adipose-derived stromal/stem cells is cell density-dependent and positively impacts adipogenic differentiation.

Adipose-derived stromal/stem cells (ASCs) represent a widely used cell source with multi-lineage differentiation capacity in approaches for tissue engineering and regenerative medicine. Despite the multitude of literature on their differentiation capacity, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in human ASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in ASCs was demonstrated histologically and on the gene and protein expression level, and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of ASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid (AGA) significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, this study demonstrates the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of ASCs, and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.

Engineering vascularized adipose tissue using the stromal-vascular fraction and fibrin hydrogels.

The development of vascularized and functional adipose tissue substitutes is required to improve soft tissue augmentation. In this study, vascularized adipose tissue constructs were generated using uncultured cells from the stromal-vascular fraction (SVF) of adipose tissue as an alternative cell source to adipose-derived stem cells. SVF cell behavior and tissue formation were compared in a stable fibrin formulation developed by our group and a commercial fibrin sealant (TissuCol; Baxter) upon direct subcutaneous implantation in a nude mouse model. Further, the effect of in vitro adipogenic induction on SVF cell development was investigated by implanting stable fibrin constructs after 1 week of precultivation (adipogenic vs. noninduced control). Constructs were thoroughly analyzed before implantation regarding adipogenic differentiation status, cell viability, and distribution as well as the presence of endothelial cells. Before implantation, in vitro precultivation strongly promoted adipogenesis (under adipogenic conditions) and the formation of CD31(+) prevascular structures by SVF cells (under nonadipogenic conditions). Tissue development in vivo was determined after 4 weeks by histology (hematoxylin and eosin, human vimentin) and quantified histomorphometrically. In stable fibrin gels, adipogenic precultivation was superior to noninduced conditions, resulting in mature adipocytes and the formation of distinct vascular structures of human origin in vivo. Strong neovascularization by the implanted cells predominated in noninduced constructs. Without pretreatment, the SVF in stable fibrin gels displayed only a weak differentiation capability. In contrast, TissuCol gels strongly supported the formation of coherent and well-vascularized adipose tissue of human origin, displaying large unilocular adipocytes. The developed native-like tissue architecture was highlighted by a whole mount staining technique. Taken together, SVF cells from human adipose tissue were shown to successfully lead to adipose tissue formation in fibrin hydrogels in vivo. The results render the SVF a promising cell source for subsequent studies both in vitro and in vivo with the aim of engineering clinically applicable soft tissue substitutes.