Microparticles for controlled GDF6 delivery to direct ASC-based nucleus pulposus regeneration

Currently, there is no effective long-term treatment for intervertebral disc degeneration (IVDD), making it an attractive candidate for regenerative therapies. Hydrogel delivery of adipose stem cells (ASCs) in combination with controlled release of bioactive molecules is a promising approach to halt IVDD and promote regeneration. Growth differentiation factor (GDF)6 can induce ASC differentiation into anabolic nucleus pulposus (NP) cells and hence holds promise for IVD regeneration. Here, we optimised design of novel PLGA-PEG-PLGA microparticles to control GDF6 delivery and investigated effect of released GDF6 on human ASCs differentiation to NP cells. Recombinant human (rh)GDF6 was loaded into microparticles and total protein and rhGDF6 release assessed. The effect of microparticle loading density on distribution and gel formation was investigated through scanning electron microscopy. ASC differentiation to NP cells was examined after 14 days in hydrogel culture by qPCR, histological and immunohistochemical staining in normoxic and IVD-like hypoxic conditions. RhGDF6-microparticles were distributed throughout gels without disrupting gelation and controlled rhGDF6 release over 14 days. Released GDF6 significantly induced NP differentiation of ASCs, with expression comparable to or exceeding media supplemented rhGDF6. Microparticle-delivered rhGDF6 also up-regulated sGAG and aggrecan secretion in comparison to controls. In hypoxia, microparticle-delivered rhGDF6 continued to effectively induce NP gene expression and aggrecan production. This study demonstrates the effective encapsulation and controlled delivery of rhGDF6, which maintained its activity and induced ASC differentiation to NP cells and synthesis of a NP-like matrix suggesting suitability of microparticles for controlled growth factor release in regenerative strategies for treatment of IVDD.