Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells

Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool.