Meniscal injuries affect over 1.5 million people across Europe and the USA annually. Injury greatly reduces knee joint mobility and quality of life and frequently leads to the development of osteoarthritis. Tissue engineered strategies have emerged in response to a lack of viable treatments for meniscal pathologies. However, to date, constructs mimicking the structural and functional organisation of native tissue, whilst promoting deposition of new extracellular matrix, remains a bottleneck in meniscal repair. 3D bioprinting allows for deposition and patterning of biological materials with high spatial resolution. This project aims to develop a biomimetic 3D bioprinted meniscal substitute.

Meniscal tissue was characterised to effectively inform the design of biomaterials for bioprinting constructs with appropriate structural and functional properties. Histology, gene expression and mass spectrometry were performed on native tissue to investigate tissue architecture, matrix components, cell populations and protein expression regionally across the meniscus. 3D laser scanning and magnetic resonance imaging were employed to acquire the external geometrical information prior to fabrication of a 3D printed meniscus. Bioink suitability was investigated through regional meniscal cell encapsulation in blended hydrogels, with the incorporation of growth factors and assessed for their suitability through rheology, scanning electron microscopy, histology and gene expression analysis.

Meniscal tissue characterisation revealed regional variations in matrix compositions, cellular populations and protein expression. The process of imaging through to 3D printing highlighted the capability of producing a construct that accurately replicated meniscal geometries. Regional meniscal cell encapsulation into hydrogels revealed a recovery in cell phenotype, with the incorporation of growth factors into the bioink's stimulating cellular re-differentiation and improved zonal functionality.

Meniscus biofabrication highlights the potential to print patient specific, customisable meniscal implants. Achieving zonally distinct variations in cell and matrix deposition highlights the ability to fabricate a highly complex tissue engineered construct.

Acknowledgements: This work was undertaken as part of the UK Research and Innovation (UKRI)-funded CDT in Advanced Biomedical Materials.

Background

Involving research users in setting priorities for research is essential to ensure research outcomes are patient-centred and to maximise research value and impact. The Musculoskeletal (MSK) Disorders Research Advisory Group Versus Arthritis led a research priority setting exercise across MSK disorders.

Background

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 in vivo administration.

Background

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.

Study purpose and background

Novel regenerative therapies have the potential to restore function and relieve pain in patients with low back pain (LBP) caused by intervertebral disc (IVD) degeneration. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation into nucleus pulposus (NP) cells of the IVD, which have potential for IVD regeneration. We have also shown that GDF6 stimulation activates the Smad1/5/8 and ERK1/2 signalling cascades. The aim of this study was to progress our understanding of the immediate/early response mechanisms in ASCs (N=3) which may direct GDF6-induced differentiation.

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6.

Clinical trials are underway to elucidate a successful MSC-based therapy for the repair and regeneration of intervertebral disc (IVD) tissue. Currently, there is a lack of knowledge surrounding the relationship between naïve MSCs and the inflammatory microenvironment of the degenerate disc. To inform a phase II clinical trial, this study tests the hypothesis that cytokines, IL-1ß and TNFα regulate the expression of neuropeptides and neurotrophic factors from MSCs, thus exacerbating pain in those patients that have the presence of sensory nerve fibres within the IVD. Patient-matched MSCs derived from bone marrow (BM) or adipose (AD) tissue were stimulated with IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours in either 21% or 5% O2. qRT-PCR was performed to assess expression of trophic factors involved in the survival or nerves (NGF & BDNF), blood vessels (VEGF) as well as pain related peptides (SP & CGRP) and inflammatory factors. Conditioned culture medium was analysed using ELISAs to identify secretion of soluble factors. IL-1β did not regulate neurotrophic factor expression from BM-MSCs under normoxic or hypoxic conditions. However, TNFα increased NGF, BDNF, SP and CGRP under normoxic conditions. In ADMSCs, VEGF was increased following IL-1β and TNFα stimulation; with TNFα also increasing NGF and CGRP under normoxic conditions. When exposed to hypoxia, the trophic effect of TNFα on human BM-MSCs was reduced. Overall this data suggests a role for priming or pre-stimulation of naïve MSCs prior to implantation to prevent exacerbation of pain from sensory nerve fibres.

Background

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.

Background

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.

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.

Background

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.

Background

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.

Introduction: Intervertebral disc (IVD) degeneration is a major underlying factor in the pathogenesis of chronic low back pain. The healthy IVD is both avascular and aneural, however during symptomatic degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves. This study aimed to identify whether class 3 semaphorins were expressed by cells of the IVD and addresses the hypothesis that they may play a role in repelling axons surrounding the healthy disc thus maintaining its aneural condition.

Methods: Forty human IVD samples were investigated using RT-PCR and immunohistochemistry to identify the expression of sema3A, 3F and their receptors; neuropilins (1 & 2) and plexins (A1-4). Serial sections were stained for PGP9.5 and CD31 to correlate semaphorin expression with nerve and blood vessel ingrowth respectively.

Results: Sema3A protein, localised primarily to the OAF, was expressed highly in the healthy disc. In degenerate samples sema3A expression decreased significantly in this region, although chondrocyte clusters within the degenerate NP exhibited strong immunopositivity. mRNA for sema3A receptors was also identified in healthy and degenerate tissues. CD31 and PGP9.5 were expressed most highly in degenerate tissues correlating with low expression of sema3A.

Conclusions: This study is the first to establish the expression of semaphorins and their receptors in the human IVD with a decrease seen in the degenerate symptomatic IVD. Sema3A may therefore, amongst other roles, act as a ‘barrier’ to neuronal ingrowth into the healthy disc.

Conflicts of Interest: None declared

Sources of Funding: Arthritis Research Campaign

Introduction: Intervertebral disc (IVD) degeneration involves loss of disc matrix leading to instability and pain. Autologous cells are the ideal choice for bioengineering a new IVD, but removal of cells from the IVD is problematic. Our aim was to direct mesenchymal stromal cells (MSCs) down a chondrocytic lineage to mimic disc chondrocyte phenotype.

Methods: MSCs were either maintained in monolayer, pelleted into micromass aggregates or transferred to alginate beads. Pellet cultures were used in immunohis-tochemistry for type II collagen and aggrecan and in situ hybridisation for SOX-9 mRNA. Monolayer and alginate cells were cultured in the presence or absence of chondrogenic medium for 4 and 11 days. Monolayer cultured MSCs were also transfected with a SOX-9 adenovirus and cultured in the presence or absence of TGF-_1. Realtime quantitative PCR was used to analyse expression of chondrocyte markers.

Results: IHC showed increased expression of type II collagen and aggrecan in pellet cultures, while ISH showed that SOX-9 was not expressed by monolayer MSCs, but increased after pelleting. Realtime PCR using alginate-cultured MSCs showed down regulation of type I collagen mRNA expression and up-regulation of SOX-9 that was increased by chondrocgenic medium. SOX-9 transduced monolayer MSCs showed increased type II collagen, aggrecan, SOX-6 and SOX-9 mRNA over controls, while type I collagen levels showed no significant change. Stimulation of transfected MSCs with TGF-_1 showed similar increases in chondrocyte genes.

Discussion & conclusions: Adult human MSCs were induced to differentiate along a chondrocytic phenotype, which was mediated by culture conditions. Alginate and pellet culture produce a cell that has more chondrogenic characteristics than monolayer cells. SOX-9 transduced monolayer MSCs appeared to produce a more chondrocytic phenotype which was modulated by TGF-_1. Results suggest SOX-9 transfected monolayer MSCs may be used as a source of chondrocytes for repair of degenerate IVD.