Microparticles for controlled growth differentiation factor 6 delivery to direct adipose stem cell‐based nucleus pulposus regeneration

Abstract

Currently, there is no effective long‐term treatment for intervertebral disc (IVD) degeneration, 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 IVD degeneration and promote regeneration. Growth differentiation factor 6 (GDF6) can induce ASC differentiation into anabolic nucleus pulposus (NP) cells and hence holds promise for IVD regeneration. Here, we optimised design of novel poly(DL‐lactic acid‐co‐glycolic acid) (PLGA)–polyethylene glycol–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 quantitative polymerase chain reaction, 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 with or exceeding media supplemented rhGDF6. Microparticle‐delivered rhGDF6 also up‐regulated sulphated glycosaminoglycan and aggrecan secretion in comparison with 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 an NP‐like matrix suggesting suitability of microparticles for controlled growth factor release in regenerative strategies for treatment of IVD degeneration.

Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation

“…rhGDF6 significantly increased the expression of NP-marker genes (SOX9, ACAN, COL2A1, KRT8, KRT18, KRT19, FOXF1, CA12 and T) in NP cells and sGAG production following 14 days in culture. These results are similar to those observed for rhGDF6 stimulation of human MSCs [ 16 , 37 ] and provide further evidence of an anabolic role for GDF6 in healthy IVD homeostasis. rhGDF6 stimulation had no effect on the expression of catabolic enzymes (MMPs, ADAMTSs) in comparison to unstimulated controls, although levels were not induced in the system (e.g., through stimulation with a catabolic cytokine, such as IL-1β), hence GDF6 may not be able to reduce expression below basal levels and further work is required to interrogate this aspect in more detail.…”

Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research

“…Firstly, they are inherently reliant upon the remaining degenerated disc cells to be healthy and sufficient in number to synthesize ECM and ultimately regenerate the disc tissue. Secondly, GF are often short-lived, limiting clinical use toward sustained regeneration [ 324 ]; hence, recent works were investigating the use of microparticles as a GF delivery vehicle [ 325 , 326 , 327 ]. Arguably, this is a likely contributing factor to the lack of efficacy from the GDF5 clinical trial.…”

“…Furthermore, controlled administration of biologicals is extremely valuable for IVD TE, where cell and/or growth factor therapeutics would rely on single surgical procedures rather than on repeated delivery. In vitro, delivery of GDF6 through MP was demonstrated to be more efficient than repeated exogenous delivery to differentiate adipose stem cells into NP cells [ 327 ], which provides a promising direction for single surgical procedure approaches. MP have also been used to release the anti-inflammatory factor cyclooxygenase-2 in a canine dog model of DD [ 438 ], leading to encouraging results of preventing disease progression and reducing the inflammatory signature prostaglandin E 2 , a crucial nociception mediator.…”

High BMPR2 expression leads to enhanced SMAD1/5/8 signalling and GDF6 responsiveness in human adipose-derived stem cells: implications for stem cell therapies for intervertebral disc degeneration

“…8,9 Work from our laboratory and others has demonstrated that stimulation of MSCs and ASCs with recombinant human (rh)GDF6 promotes their differentiation to an NP-like phenotype. 10-13 Furthermore, rhGDF6 induces greater increases in NP-marker genes and proteoglycan production than other candidate growth factors, namely rhGDF5 and rhTGFβ3, particularly in ASCs. 10…”