Differentiation of embryonic stem cells through controlled release of growth factors from microspheres

Abstract

The development of microspheres for the sustained delivery of protein and small drug delivery has been utilised in tissue engineering and drug delivery applications. However problems exist in obtaining a controlled and predictable release pattern of the encapsulated molecules from these materials. In this study, microspheres with a zero order release kinetic profile and no lag phase were developed from a novel PLGA based polymer blend. The novel PLGA based polymer blend was made from blending PLGA with varying compositions of the triblock co-polymer PLGA-PEG-PLGA. These blends were subsequently used in the fabrication of lysozyme and dexamethasone loaded microspheres. Blending of the triblock copolymer with PLGA resulted in a reduction of the glass transition temperature (36.1ºC against 59.7ºC) and an increased mechanical strength (25.25 ± 1.26MPa against 0.26 ± 0.05MPa) for PLGA and 30% triblock w/w microspheres respectively. An incremental increase in the triblock composition within the Triblock/PLGA blends resulted in a corresponding reduction in glass transition temperature of the microspheres. Varying the triblock composition within the microspheres showed no significant effect on entrapment efficiency (EE) of lysozyme (protein) and dexamethasone (drug) within fabricated microspheres (EE ~ 60% for and 75% for loading weight 5% w/w for lysozyme and dexamethasone microspheres respectively). Controlled release experiments showed incorporation of the triblock increased the burst release of the protein and drug molecules from the microspheres and improved their release kinetics, with zero-order release profile (post burst phase) observed at a triblock composition of 30% w/w. A positive correlation between the amount of triblock within the triblock / PLGA blend and the rate of protein and drug release was also observed. The induction of osteogenesis and chondrogenesis within stem cells seeded on dexamethasone and ascorbate phosphate, and TGF-β3 loaded scaffolds was successfully demonstrated. Zonal release of TGF-β3 and BMP4 proteins from a bilayered scaffold was also demonstrated. However experiments conducted to demonstrate the tissue zonation within a bone cartilage bilayered construct developed from embryonic stem cell seeded TGF-β3 and BMP4 loaded bilayered scaffolds yielded inconclusive data. These results suggests that protein and drug loaded injectable microspheres for tissue engineering applications can be formed from triblock/PLGA blends, and that by varying the triblock composition, the temperature at which the microspheres form scaffolds, the release kinetics and the mechanical strength of the resulting scaffolds can be controlled