Background. Stem cell therapy has been suggested as a potential regenerative strategy to treat IVD degeneration and GDF6 has been shown to differentiate adipose-derived stem cells (ASCs) into an NP-like phenotype. However, for clinical translation, a delivery system is required to ensure controlled and sustained GDF6 release. This study aimed to investigate the encapsulation of GDF6 inside novel microparticles (MPs) to control delivery and assess the effect of the released GDF6 on NP-like differentiation of human ASCs. Methods. GDF6 release from PLGA-PEG-PLGA MPs over 14 days was determined using BCA and ELISA. The effect of MP loading density on collagen gel formation was assessed through SEM and histological staining. ASCs were cultured in collagen hydrogels for 14 days with GDF6 delivered exogenously or via microspheres. ASC differentiation was assessed by qPCR for NP markers, glycosaminoglycan production (DMMB) and immunohistochemistry. Results. GDF6 release from MPs was controlled over 14 days equivalently to exogenous addition. SEM and histology confirmed that MPs were distributed throughout gels and that gel formation was not disrupted. In 3D cultures, GDF6 release from microspheres elicited equivalent ASC differentiation and NP-like matrix formation compared to exogenous delivery in media, indicating activity was not affected by MP encapsulation. Conclusions. This study demonstrates the effective encapsulation and controlled delivery of GDF6, which was able to maintain its activity and induce ASC differentiation into an NP-like phenotype and production of an NP-like ECM. Delivery of GDF6 microspheres in combination with ASCs is a promising strategy for IVD regeneration and treatment of back pain. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

Introduction. Given the predominant functional role which aggrecan has in the intervertebral disc, particularly within the nucleus pulposus, it is necessary to evaluate the quality of aggrecan produced by cells within tissue engineered disc constructs. The aim here was to characterise the nanostructure of aggrecan synthesised by nucleus pulposus cells treated with growth differentiation factor [GDF]-6) seeded in hydrogels in comparison to aggrecan isolated from healthy disc. Methods. Aggrecan was isolated from bovine nucleus pulposus (NP) tissue (n=3 [<18 months old]) and primary bovine NP cells cultured with (+GDF6) or without GDF6 (−GDF6) for 28 days (n=2) in type I collagen hydrogels. Isolated aggrecan monomers were visualised by atomic force microscopy and categorised as either intact (globular domains visible at both the N and C termini) or non-intact. Core protein contour length (L. CP. ) was calculated for intact molecules. The proportion of non-intact/fragmented to intact aggrecan and the molecular area of all monomers was determined. Results. Very few aggrecan molecules were intact (1.3% in NP compared to 4.3% +GDF6 and 0% -GDF6). There was no significant difference in the mean L. CP. between NP (389 ± 37 nm) compared to +GDF6 (379.2 ± 26 nm) or the molecular area between NP (3560 ± 2179 nm. 2. ) and –GDF6 (3586 ± 2071 nm. 2. ). However, the molecular area in both cases was significantly lower than +GDF6 (4774 ± 3715 nm. 2. ) p≤0.0001. Discussion & conclusions. Aggrecan structure can be altered by culture conditions. GDF6 treatment promoted the synthesis of more intact monomers, with greater over all molecular area. Conflicts of interest: None. Funding: Impact Research Scholarship and the Presidents Doctoral Scholarship, provided by the University of Manchester