Entwicklung von bioresponsiven photopolymerisierbaren Hydrogel-Scaffolds zur Chondrogenese von Affen-Stammzellen
Zusammenfassung der Projektergebnisse
The chondrogenesis of MSCs is limited since maturing chondrocytes can become hypertrophic, eventually leading to endochondral ossification. ACSC, which are isolated from the superficial layers of cartilage, are a new promising cell type for cartilage repair, because they do not readily differentiate down the endochondral pathway. The purpose of this research fellowship was to evaluate new protocols for the chondrogenesis of MSCs, and second to characterize ACSCs, a new type of stem cells for cartilage tissue engineering. We explored the use of cell-conditioned medium from cells that were cultured under normoxic or hypoxic conditions in order to decrease hypertrophy in MSCs. MSCs were cultured in a 3D pellet culture system with chondrogenic induction medium and received cell-conditioned medium from chondrocytes that were cultured in parallel under normoxic or hypoxic conditions. Cell pellets that received CCM from chondrocytes cultured under hypoxic conditions showed a decrease in the expression of hypertrophy associated genes, and an increase in matrix associated genes. In addition the results demonstrated that the use of hCCM can increase matrix production. Hypoxia can decrease hypertrophy in MSCs during pellet culture. We therefore evaluated the effects of hypoxia in cultures of ACSCs supplemented with chondrogenic induction medium. Hypoxia facilitated an increase in the gene expression for type II collagen, aggrecan and SOX9 as well as a decrease in collagen type X, I, and MMP13. These results indicate that hypoxia can further improve the chondrogenesis of ACSCs. In order to further characterize ACSCs we proposed to compare the RNA expression of ACSC clones with MSC clones as well as to identify surface markers that are unique to ACSCs. We are currently exploring methods to clone MSCs using hypoxic isolation and expansion, first described by Adesida et al. Once we successfully isolate and expand MSC clones we will then perform the RNA sequencing. The characterization of ACSC markers has begun with FACS analysis using an array of 242 antibodies. ACSCs were isolated and expanded using a fibronectin assay. The first successful run of ACSCs clones revealed three markers that were unique to ACSCs. These will be confirmed in other clones and with immunohistochemical analysis of sections from normal and osteoarthritic cartilage. Summarizing, this DFG research fellowship allowed me to work and learn from leading scientists in the field of cartilage tissue engineering. Our work will add to the finding that hypoxia is important for all in vitro work with chondrocytes, MSCs and ACSCs. We are currently working on the characterization and identification of ACSCs, a promising new cell type for cartilage repair in the future.
