Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

Cell-based strategies for IVD repair: clinical progress and translational obstacles.

Intervertebral disc (IVD) degeneration is a major cause of low back pain, a prevalent and chronic condition that has a striking effect on quality of life. Currently, no approved pharmacological interventions or therapies are available that prevent the progressive destruction of the IVD; however, regenerative strategies are emerging that aim to modify the disease. Progress has been made in defining promising new treatments for disc disease, but considerable challenges remain along the entire translational spectrum, from understanding disease mechanism to useful interpretation of clinical trials, which make it difficult to achieve a unified understanding. These challenges include: an incomplete appreciation of the mechanisms of disc degeneration; a lack of standardized approaches in preclinical testing; in the context of cell therapy, a distinct lack of cohesion regarding the cell types being tested, the tissue source, expansion conditions and dose; the absence of guidelines regarding disease classification and patient stratification for clinical trial inclusion; and an incomplete understanding of the mechanisms underpinning therapeutic responses to cell delivery. This Review discusses current approaches to disc regeneration, with a particular focus on cell-based therapeutic strategies, including ongoing challenges, and attempts to provide a framework to interpret current data and guide future investigational studies.

Site-Directed Differentiation of Human Adipose-Derived Mesenchymal Stem Cells to Nucleus Pulposus Cells Using an Injectable Hydroxyl-Functional Diblock Copolymer Worm Gel.

Adipose-derived mesenchymal stem cells (ASCs) have been identified for their promising therapeutic potential to regenerate and repopulate the degenerate intervertebral disk (IVD), which is a major cause of lower back pain. The optimal cell delivery system remains elusive but encapsulation of cells within scaffolds is likely to offer a decisive advantage over the delivery of cells in solution by ensuring successful retention within the tissue. Herein, we evaluate the use of a fully synthetic, thermoresponsive poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA) diblock copolymer worm gel that mimics the structure of hydrophilic glycosaminoglycans. The objective was to use this gel to direct differentiation of human ASCs toward a nucleus pulposus (NP) phenotype, with or without the addition of discogenic growth factors TGFß or GDF6. Accordingly, human ASCs were incorporated into a cold, free-flowing aqueous dispersion of the diblock copolymer, gelation induced by warming to 37 °C and cell culture was conducted for 14 days with or without such growth factors to assess the expression of characteristic NP markers compared to those produced when using collagen gels. In principle, the shear-thinning nature of the biocompatible worm gel enables encapsulated human ASCs to be injected into the IVD using a 21G needle. Moreover, we find significantly higher gene expression levels of ACAN, SOX-9, KRT8, and KR18 for ASCs encapsulated within worm gels compared to collagen scaffolds, regardless of the growth factors employed. In summary, such wholly synthetic worm gels offer considerable potential as an injectable cell delivery scaffold for the treatment of degenerate disk disease by promoting the transition of ASCs toward an NP-phenotype.

Exploiting the role of nanoparticle shape in enhancing hydrogel adhesive and mechanical properties.

The ability to control nanostructure shape and dimensions presents opportunities to design materials in which their macroscopic properties are dependent upon the nature of the nanoparticle. Although particle morphology has been recognized as a crucial parameter, the exploitation of the potential shape-dependent properties has, to date, been limited. Herein, we demonstrate that nanoparticle shape is a critical consideration in the determination of nanocomposite hydrogel properties. Using translationally relevant calcium-alginate hydrogels, we show that the use of poly(L-lactide)-based nanoparticles with platelet morphology as an adhesive results in a significant enhancement of adhesion over nanoparticle glues comprised of spherical or cylindrical micelles. Furthermore, gel nanocomposites containing platelets showed an enhanced resistance to breaking under strain compared to their spherical and cylindrical counterparts. This study opens the doors to a change in direction in the field of gel nanocomposites, where nanoparticle shape plays an important role in tuning mechanical properties.

Combinatorial conditioning of adipose derived-mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration.

BACKGROUND: Mesenchymal stem cells (MSCs) are becoming an increasingly attractive option for regenerative therapies due to their availability, self-renewal capacity, multilineage potential, and anti-inflammatory properties. Clinical trials are underway to test the efficacy of stem cell-based therapies for the repair and regeneration of the degenerate intervertebral disc (IVD), a major cause of back pain. Recently, both bone marrow-derived MSCs and adipose-derived stem cells (ASCs) have been assessed for IVD therapy but there is a lack of knowledge surrounding the optimal cell source and the response of transplanted cells to the low oxygen, pro-inflammatory niche of the degenerate disc. Here, we investigated several neurovascular factors from donor-matched MSCs and ASCs that may potentiate the survival and persistence of sensory nerve fibers and blood vessels present within painful degenerate discs and their regulation by oxygen tensions and inflammatory cytokines. METHODS: Donor-matched ASCs and MSCs were conditioned with either IL-1ß or TNFα under normoxic (21% O2) or hypoxic (5% O2) conditions. Expression and secretion of several potent neurovascular factors were assessed using qRT-PCR and human magnetic Luminex assay. RESULTS: ASCs and MSCs expressed constitutive levels of key neurotrophic factors; and stimulation of ASCs with hypoxia triggered increased secretion of both angiogenic factors (Ang-2 and VEGF-A) and neurotrophic (NGF and NT-3) compared to MSCs. We also report increased transcriptional regulation of pain-associated neuropeptides in hypoxia stimulated ASCs compared to those in normoxic conditions. We demonstrate transcriptional and translational upregulation of NGF, NT-3, Ang-1, and FGF-2 in response to cytokines in ASCs in 21% and 5% O2. CONCLUSIONS: This work highlights fundamental differences between the neurovascular secretome of donor-matched ASCs and MSCs, demonstrating the importance of cell-selection for tissue specific regeneration to reduce ectopic sensory nerve and blood vessel survival and improve patient outcomes.

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two -omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three -omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

Nucleus pulposus phenotypic markers to determine stem cell differentiation: fact or fiction?

Progress in mesenchymal stem cell (MSC) based therapies for nucleus pulposus (NP) regeneration are hampered by a lack of understanding and consensus of the normal NP cell phenotype. Despite the recent consensus paper on NP markers, there is still a need to further validate proposed markers. This study aimed to determine whether an NP phenotypic profile could be identified within a large population of mature NP samples.qRT-PCR was conducted to assess mRNA expression of 13 genes within human non-degenerate articular chondrocytes (AC) (n=10) and NP cells extracted from patients across a spectrum of histological degeneration grades (n=71). qRT-PCR results were used to select NP marker candidates for protein expression analysis.Differential expression at mRNA between AC and non-degenerate NP cells was only observed for Paired Box Protein 1 (PAX1) and Forkhead box F1 (FOXF1). In contrast no other previously suggested markers displayed differential expression between non-degenerate NP and AC at mRNA level. PAX1 and FOXF1 protein expression was significantly higher in the NP compared to annulus fibrosus (AF), cartilaginous endplate (CEP) and AC. In contrast Laminin-5 (LAM-332), Keratin-19 (KRT-19) and Hypoxia Inducible Factor 1 alpha (HIF1α) showed no differential expression in NP cells compared with AC cells.A marker which exclusively differentiates NP cells from AF and AC cells remains to be identified, raising the question: is the NP a heterogeneous population of cells? Or does the natural biological variation during IVD development, degeneration state and even the life cycle of cells make finding one definitive marker impossible?

Syndecan-4 in intervertebral disc and cartilage: Saint or synner?

The ECM of the intervertebral disc and articular cartilage contains a highly organised network of collagens and proteoglycans which resist compressive forces applied to these tissues. A pathological hallmark of the intervertebral disc is the imbalance between production of anabolic and catabolic factors by the resident cells. This process is thought to be mediated by pro-inflammatory cytokines, predominantly TNF-α and IL-1ß, which upregulate expression of matrix degrading enzymes such as MMPs and ADAMTSs. This imbalance ultimately results in tissue degeneration causing failure of the biomechanical function of the tissues. A similar cascade of events is thought to occur in articular cartilage during development of osteoarthritis. Within these skeletal tissues a small, cell surface heparan sulphate proteoglycan; syndecan-4 (SDC4) has been implicated in maintaining physiological functions. However in the degenerating niche of the intervertebral disc and cartilage, dysregulated activities of this molecule may exacerbate pathological changes. Studies in recent years have elucidated a role for SDC4 in mediating matrix degradation in both intervertebral discs and cartilage by controlling ADAMTS-5 function and MMP3 expression. Discourse presented in this review highlights the potential of SDC4 as a possible therapeutic target in slowing the progression of ECM degradation in both degenerative disc disease and osteoarthritis.

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc.

BACKGROUND: Chronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs. METHODS: Immunohistochemistry was performed with 61 human IVD samples, to identify and localise nerves (neurofilament 200 [NF200]/protein gene product 9.5) and blood vessels (CD31) within different regions of the IVD. RESULTS: Immunopositivity for NF200 was identified within all regions of the IVD within post-mortem tissues. Nerves were seen to protrude across lamellar ridges and through matrix towards NP cells. Nerves were identified deep within the NP and were in many cases, but not always, seen in close proximity to fissures or in areas where decreased matrix was seen. Fifteen percent of samples were degenerate and negative for nerves and blood vessels, whilst 16 % of all samples were degenerate with nerves and blood vessels. We identified 52% of samples that were degenerate with nerves but no blood vessels. Interestingly, only 4% of all samples were degenerate with no nerves but positive for blood vessels. Of the 85 samples investigated, only 6 % of samples were non-degenerate without nerves and blood vessels and 7% had nerves but no blood vessels. CONCLUSIONS: This study addresses the controversial topic of nerve and blood vessel ingrowth into the IVD in a large number of human samples. Our findings demonstrate that nerves are present within a large proportion of NP samples from degenerate IVDs. This study shows a possible link between nerve ingrowth and degeneration of the IVD and suggests that nerves can migrate in the absence of blood vessels.

Class 3 semaphorins expression and association with innervation and angiogenesis within the degenerate human intervertebral disc.

Nerve and blood vessel ingrowth during intervertebral disc degeneration, is thought to be a major cause of low back pain, however the regulation of this process is poorly understood. Here, we investigated the expression and regulation of a subclass of axonal guidance molecules known as the class 3 semaphorins, and their receptors; plexins and neuropilins within human NP tissue and their regulation by pro-inflammatory cytokines. Importantly this determined whether semaphorin expression was associated with the presence of nerves and blood vessels in tissues from human intervertebral discs. The study demonstrated that semaphorin3A, 3C, 3D, 3E and 3F and their receptors were expressed by native NP cells and further demonstrated their expression was regulated by IL-1ß but to a lesser extent by IL-6 and TNFα. This is the first study to identify sema3C, sema3D and their receptors within the nucleus pulposus of intervertebral discs. Immunopositivity shows significant increases in semaphorin3C, 3D and their receptor neuropilin-2 in degenerate samples which were shown to contain nerves and blood vessels, compared to non-degenerate samples without nerves and blood vessels. Therefore data presented here suggests that semaphorin3C may have a role in promoting innervation and vascularisation during degeneration, which may go on to cause low back pain.

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration.

INTRODUCTION: The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells. METHODS: Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1ß) stimulated NP cells. RESULTS: Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1ß induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1ß. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1ß and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1ß. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines. CONCLUSIONS: The release of cytokines, in particular IL-1ß during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1ß is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

Inflammatory cytokines induce NOTCH signaling in nucleus pulposus cells: implications in intervertebral disc degeneration.

The objective of the study was to investigate how inflammatory cytokines, IL-1ß, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKß significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/ß2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1ß as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1ß and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.