Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion.

OBJECTIVE: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. DESIGN: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. RESULTS: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. CONCLUSIONS: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).

Microbial activity of biofilm during start-up period of anaerobic hybrid reactor at low and high upflow feeding velocity.

With an aim to shorten start-up time of an Anaerobic Hybrid Reactor (AHR), initial biofilm development was studied, particularly at different upflow feeding velocities. At a low (0.01 m x h(-1)) upflow velocity, initial biofilm was found to develop via the attachment of suspended biomass in the packed zone, while microbial growth on the film was insignificant. Contrarily, with higher (1.0 m x h(-1)) upflow velocity, initial biofilm development was from both microbial attachment and growth on supporting media. Biofilm thickness was determined using confocal laser scanning microscopy (CLSM), which indicated that the biofilm developed faster with the higher velocity, due to the contribution of the microbial growth on supporting media. When operated beyond the initial biofilm development with the lower velocity, both the activity of acetogens and the methanogens increased, although there was a lower amount of attached biomass on the supporting media. Whereas, both groups were found to decrease with higher upflow velocity, but acidogenic activity increased. It can be concluded that higher upflow velocity positively affected the initial stage of biofilm development and has the potential to accelerate attached biomass on supporting media during the initial phase. Subsequently, the upflow velocity should be reduced to the normal rate to enhance the methanogenic activity.