Intervertebral disc degeneration (IVDD) associated with low back pain is a major contributor to global disability. Current treatments are poorly efficient in the long-term resulting in medical complications. Therefore, minimally invasive injectable therapies are required to repopulate damaged tissues and aid regeneration. Among injectable biomaterials, self-assembling peptide hydrogels (SAPHs) represent potential candidates as 3D cell carriers. Moreover, the advent of graphene-related materials has opened the route for the fabrication of graphene-containing hydrogel nanocomposites to direct cellular fate. Here, we incorporated graphene oxide (GO) within a SAPH to develop a biocompatible and injectable hydrogel to be used as cell carrier to treat IVDD. Hydrogel morphology and mechanical properties have been investigated showing high mechanical properties (G'=12kPa) comparable with human native nucleus pulposus (NP) tissue (G'=10kPa), along with ease of handling and injectability in dry and body fluid conditions. Hydrogel nanocomposites resulted biocompatible for the encapsulation of bovine NP cells, showing higher viability (>80%) and metabolic activity in 3D cell culture over 7 days, compared to GO-free hydrogels. Moreover, GO has demonstrated to bind TGF-β3 biomolecules with high efficiency, suggesting the use of GO as local reservoir of growth factors within the injected hydrogel to promote extracellular matrix deposition and tissue repair.Introduction
Methods and results
Mesenchymal stem cells (MSCs) are undergoing evaluation as a potential new therapy for immune and inflammatory-mediated conditions such as IVD degeneration (IDD). Both adipose (ASCs) and bone-marrow (BMSCs) derived MSCs have been widely used in this regard. The optimal tissue source and expansion conditions required to exploit the regenerative capacity of these cells are not yet fully elucidated. In addition the phenotypic response of transplanted cells to the disease environment is not well understood. In this study, ASCs and BMSCs were exposed to a combination of hypoxic conditioning and selected inflammatory mediators, conditions that mimic the microenvironment of the degenerate IVD, in an effort to understand their therapeutic potency for Donor-matched ASCs and MSCs were pre-conditioned with either IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours under hypoxic conditions (5% O2). Conditioned media was collected and 45 different immunomodulatory proteins were analysed using human magnetic Luminex® assay. Secreted levels of several key cytokines and chemokines, both pro- and anti-inflammatory, were significantly upregulated in ASCs and BMSCs following the conditioning regime. Under all conditions tested, ASCs expressed significantly higher levels of IL-4, IL-6, IL-10, IL-12, TGF-α, and GCSF compared to BMSCs. Pre-conditioning with TNFα resulted in significantly higher levels of IL-10 while preconditioning with IL-1β resulted in higher levels of IL-6, IL-12 and GCSF.Background
Methods and Results
Degeneration of the intervertebral disc (IVD) is a leading cause of lower back pain, and a significant clinical problem. Inflammation mediated by IL-1β and TNF-α drives IVD degeneration through promoting a phenotypic switch in the resident nucleus pulposus (NP) cells towards a more catabolic state, resulting in extracellular matrix degradation. Bone marrow mesenchymal stem cells (MSCs) produce bioactive factors that modulate local tissue microenvironments and their anti-inflammatory potential has been shown in numerous disease models. Thus MSCs offer a potential therapy for IVD degeneration. In a clinical setting, adipose-derived stem cells (ASCs) might represent an alternative and perhaps more appealing cell source. However, their anti-inflammatory properties remain poorly understood. Here we assess the anti-inflammatory properties of donor-matched human ASCs and MSCs using qPCR and western blotting.Background
Methods
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. 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 (LCP) was calculated for intact molecules. The proportion of non-intact/fragmented to intact aggrecan and the molecular area of all monomers was determined.Introduction
Methods
While the human embryonic, foetal and juvenile intervertebral disc (IVD) is composed of large vacuolated notochordal cells, these morphologically distinct cells are lost with skeletal maturity being replaced by smaller nucleus pulpous cells. Notochordal cells are thought to be fundamental in maintaining IVD homeostasis and, hence, their loss in humans may be a key initiator of degeneration, leading ultimately to back pain. Therefore, it is essential to understand the human notochordal cell phenotype to enable the development of novel biological/regenerative therapies. CD24+ notochordal cells and CD24- sclerotomal cells were sorted from enzymatically-digested human foetal spines (7.5–14 WPC, n=5) using FACS. Sorting accuracy was validated using qPCR for known notochordal markers and Affymetrix cDNA microarrays performed. Differential gene expression was confirmed (qPCR) and Interactive Pathway Analysis (IPA) performed.Background
Methods
Currently, there is a focus on the development of cell based therapies to treat intervertebral disc (IVD) degeneration, particularly for regenerating/repairing the central region, the nucleus pulposus (NP). Recently, we demonstrated that GDF6 promotes NP-like differentiation in mesenchymal stem cells (MSCs). However, bone marrow- (BM-MSCs) and adipose- (Ad-MSCs) showed differential responses to GDF6, with Ad-MSCs adopting a more NP-like phenotype. Here, we investigated GDF6 signalling in BM-MSCs and Ad-MSCs, with the aim to improve future IVD stem cell therapies. GDF6 receptor expression in patient-matched BM-MSCs and Ad-MSCs (N=6) was profiled through western blot and immunocytochemistry (ICC). GDF6 signal transduction was investigated through stimulation with 100 ng ml−1 GDF6 for defined time periods. Subsequently smad1/5/9 phosphorylation and alternative non-smad pathway activation (phospho-p38; phospho-Erk1/2) was analysed (western blot, ELISA). Their role in inducing NP-like gene expression in Ad-MSCs was examined through pathway specific inhibitors.Background
Methods
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. 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.Background
Methods
Signalling by growth differentiation factor 6 (GDF6/BMP13) has been implicated in the development and maintenance of healthy NP cell phenotypes and GDF6 mutations are associated with defective vertebral segmentation in Klippel-Feil syndrome. GDF6 may thus represent a promising biologic for treatment of IVD degeneration. This study aimed to investigate the effect of GDF6 in human NP cells and critical signal transduction pathways involved. BMP receptor expression profile of non-degenerate and degenerate human NP cells was determined through western blot, immunofluorescence and qPCR. Phosphorylation statuses of Smad1/5/9 and non-canonical p38 MAPK and Erk1/2 were assessed in the presence/absence of pathway blockers. NP marker and matrix degrading enzyme gene expression was determined by qPCR following GDF6 stimulation. Glycosaminoglycan and collagen production were assessed through DMMB-assay and histochemical staining.Background
Methods
Intervertebral disc degeneration is implicated as a major cause of chronic lower back pain. Current therapies for lower back pain are aimed purely at relieving the symptoms rather than targeting the underlying aberrant cell biology. As such focus has shifted to development of cell based alternatives. Notochordal cells are progenitors to the adult nucleus pulposus that display therapeutic potential. However, notochordal cell phenotype and suitable culture conditions for research or therapeutic application are poorly described. This study aims to develop a suitable culture system to allow comprehensive study of the notochordal phenotype. Porcine notochordal cells were isolated from 6 week post natal discs using dissection and enzymatic digestion and cultured Background
Methods & Results
