Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.

Injectable hydrogel induces regeneration of naturally degenerate human intervertebral discs in a loaded organ culture model.

Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc (IVD). This study investigates the ability of an injectable hydrogel (NPgel), to inhibit catabolic protein expression and promote matrix expression in human nucleus pulposus (NP) cells within a tissue explant culture model isolated from degenerate discs. Furthermore, the injection capacity of NPgel into naturally degenerate whole human discs, effects on mechanical function, and resistance to extrusion during loading were investigated. Finally, the induction of potential regenerative effects in a physiologically loaded human organ culture system was investigated following injection of NPgel with or without bone marrow progenitor cells. Injection of NPgel into naturally degenerate human IVDs increased disc height and Young's modulus, and was retained during extrusion testing. Injection into cadaveric discs followed by culture under physiological loading increased MRI signal intensity, restored natural biomechanical properties and showed evidence of increased anabolism and decreased catabolism with tissue integration observed. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. STATEMENT OF SIGNIFICANCE: Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc. This study investigated the potential regenerative properties of an injectable hydrogel system (NPgel) within human tissue samples. To mimic the human in vivo conditions and the unique IVD niche, a dynamically loaded intact human disc culture system was utilised. NPgel improved the biomechanical properties, increased MRI intensity and decreased degree of degeneration. Furthermore, NPgel induced matrix production and decreased catabolic factors by the native cells of the disc. This manuscript provides evidence for the potential use of NPgel as a regenerative biomaterial for intervertebral disc degeneration.

Senolytic Combination Treatment Is More Potent Than Single Drugs in Reducing Inflammatory and Senescence Burden in Cells from Painful Degenerating IVDs.

BACKGROUND: Low back pain is a global health problem directly related to intervertebral disc (IVD) degeneration. Senolytic drugs (RG-7112 and o-Vanillin) target and remove senescent cells from IVDs in vitro, improving tissue homeostasis. One drawback of using a single senolytic agent is the failure to target multiple senescent antiapoptotic pathways. This study aimed to determine if combining the two senolytic drugs, o-Vanillin and RG-7112, could more efficiently remove senescent cells and reduce the release of inflammatory factors and pain mediators in cells from degenerating human IVDs than either drug alone. METHODS: Preliminary data evaluating multiple concentrations of o-Vanillin and RG-7112 led to the selection of four treatment groups. Monolayer and pellet cultures of cells from painful degenerate IVDs were exposed to TLR-2/6 agonist. They were then treated with the senolytics o-Vanillin and RG7112 alone or combined. p16ink4a, Ki-67, caspase-3, inflammatory mediators, and neuronal sprouting were assessed. RESULTS: Compared to the single treatments, the combination of o-Vanillin and RG-7112 significantly reduced the amount of senescent IVD cells, proinflammatory cytokines, and neurotrophic factors. Moreover, both single and combination treatments significantly reduced neuronal sprouting in rat adrenal pheochromocytoma (PC-12 cells). CONCLUSIONS: Combining o-Vanillin and RG-7112 greatly enhanced the effect of either senolytic alone. Together, these results support the potential of senolytics as a promising treatment for IVD-related low back pain.

o-Vanillin Modulates Cell Phenotype and Extracellular Vesicles of Human Mesenchymal Stem Cells and Intervertebral Disc Cells.

Human mesenchymal stem cell (hMSC) and extracellular vesicle (EV) therapy is a promising treatment for discogenic low back pain (LBP). Although promising, major obstacles remain to be overcome. Cellular senescence reduces self-renewal and multipotent potentials, and the senescence-associated secretory phenotype creates an inflammatory environment negatively affecting tissue homeostasis. Reducing senescence could therefore improve regenerative approaches. Ortho-Vanillin (o-Vanillin) has senolytic activity and anti-inflammatory properties and could be a valuable supplement to MSC and EV therapy. Here, we used direct co-culture experiments to evaluate proteoglycan synthesis, inflammatory mediators, and senescent cells in the presence or absence of o-Vanillin. EV release and transfer between hMSCs and intervertebral disc cells (DCs) was examined, and the effect on hMSC differentiation and DC phenotype was evaluated in the presence and absence of o-Vanillin. This study demonstrates that o-Vanillin affects cell communication, enhances hMSC differentiation and improves DC phenotype. Co-cultures of DCs and hMSCs resulted in increased proteoglycan synthesis, a decreased number of senescent cells and decreased release of the cytokines IL6 and 8. Effects that were further enhanced by o-Vanillin. o-Vanillin profoundly increased EV release and/or uptake by hMSCs and DCs. DC markers were significantly upregulated in both cell types in response to conditioned media of o-Vanillin treated donor cells. Collectively, this study demonstrates that o-Vanillin affects hMSC and DC crosstalk and suggests that combining hMSCs and senolytic compounds may improve the outcome of cell supplementation and EV therapy for LBP.

Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration.

In this study, we used single-cell transcriptomic analysis to identify new specific biomarkers for nucleus pulposus (NP) and inner annulus fibrosis (iAF) cells, and to define cell populations within non-degenerating (nD) and degenerating (D) human intervertebral discs (IVD) of the same individual. Cluster analysis based on differential gene expression delineated 14 cell clusters. Gene expression profiles at single-cell resolution revealed the potential functional differences linked to degeneration, and among NP and iAF subpopulations. GO and KEGG analyses discovered molecular functions, biological processes, and transcription factors linked to cell type and degeneration state. We propose two lists of biomarkers, one as specific cell type, including C2orf40, MGP, MSMP, CD44, EIF1, LGALS1, RGCC, EPYC, HILPDA, ACAN, MT1F, CHI3L1, ID1, ID3 and TMED2. The second list proposes predictive IVD degeneration genes, including MT1G, SPP1, HMGA1, FN1, FBXO2, SPARC, VIM, CTGF, MGST1, TAF1D, CAPS, SPTSSB, S100A1, CHI3L2, PLA2G2A, TNRSF11B, FGFBP2, MGP, SLPI, DCN, MT-ND2, MTCYB, ADIRF, FRZB, CLEC3A, UPP1, S100A2, PRG4, COL2A1, SOD2 and MT2A. Protein and mRNA expression of MGST1, vimentin, SOD2 and SYF2 (p29) genes validated our scRNA-seq findings. Our data provide new insights into disc cells phenotypes and biomarkers of IVD degeneration that could improve diagnostic and therapeutic options.

Participation of L-Lactate and Its Receptor HCAR1/GPR81 in Neurovisual Development.

During the development of the retina and the nervous system, high levels of energy are required by the axons of retinal ganglion cells (RGCs) to grow towards their brain targets. This energy demand leads to an increase of glycolysis and L-lactate concentrations in the retina. L-lactate is known to be the endogenous ligand of the GPR81 receptor. However, the role of L-lactate and its receptor in the development of the nervous system has not been studied in depth. In the present study, we used immunohistochemistry to show that GPR81 is localized in different retinal layers during development, but is predominantly expressed in the RGC of the adult rodent. Treatment of retinal explants with L-lactate or the exogenous GPR81 agonist 3,5-DHBA altered RGC growth cone (GC) morphology (increasing in size and number of filopodia) and promoted RGC axon growth. These GPR81-mediated modifications of GC morphology and axon growth were mediated by protein kinases A and C, but were absent in explants from gpr81-/- transgenic mice. Living gpr81-/- mice showed a decrease in ipsilateral projections of RGCs to the dorsal lateral geniculate nucleus (dLGN). In conclusion, present results suggest that L-lactate and its receptor GPR81 play an important role in the development of the visual nervous system.

Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin.

BACKGROUND: There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. METHODS: Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). RESULTS: An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. CONCLUSIONS: Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

Senotherapeutic drugs for human intervertebral disc degeneration and low back pain.

Cellular senescence is a contributor to intervertebral disc (IVD) degeneration and low back pain. Here, we found that RG-7112, a potent mouse double-minute two protein inhibitor, selectively kills senescent IVD cells through apoptosis. Gene expression pathway analysis was used to compare the functional networks of genes affected by RG-7112, a pure synthetic senolytic with o-Vanillin a natural and anti-inflammatory senolytic. Both affected a functional gene network related to cell death and survival. O-Vanillin also affected networks related to cell cycle progression as well as connective tissue development and function. Both senolytics effectively decreased the senescence-associated secretory phenotype (SASP) of IVD cells. Furthermore, bioavailability and efficacy were verified ex vivo in the physiological environment of degenerating intact human discs where a single dose improved disc matrix homeostasis. Matrix improvement correlated with a reduction in senescent cells and SASP, supporting a translational potential of targeting senescent cells as a therapeutic intervention.

Pain in the lower back affects about four in five people during their lifetime. Over time, the discs that provide cushioning between the vertebrae of the spine can degenerate, which can be one of the major causes of lower back pain. It has been shown that when the cells of these discs are exposed to different stress factors, they stop growing and become irreversibly dormant. Such 'senescent' cells release a range of proteins and small molecules that lead to painful inflammation and further degeneration of the discs. Moreover, it is thought that a high number of senescent cells may be linked to other degenerative diseases such as arthritis. Current treatments can only reduce the severity of the symptoms, but they cannot prevent the degeneration from progressing. Now, Cherif et al. set out to test the effects of two different compounds on human disc cells grown in the laboratory. One of the molecules studied, RG-7112, is a synthetic drug that has been approved for safety by the US Food and Drug Administration and has been shown to remove senescent cells. The other, o-Vanillin, is a natural compound that has anti-inflammatory and anti-senescence properties. The results showed that both compounds were able to trigger changes to that helped new, healthy cells to grow and at the same time kill senescent cells. They also reduced the production of molecules linked to inflammation and pain. Further analyses revealed that the compounds were able to strengthen the fibrous matrix that surrounds and supports the discs. Cherif et al. hope that this could form the basis for a new family of drugs for back pain to slow the degeneration of the discs and reduce pain. This may also have benefits for other similar degenerative diseases caused by cell senescence, such as arthritis.

Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro.

Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.

Receptors of intermediates of carbohydrate metabolism, GPR91 and GPR99, mediate axon growth.

During the development of the visual system, high levels of energy are expended propelling axons from the retina to the brain. However, the role of intermediates of carbohydrate metabolism in the development of the visual system has been overlooked. Here, we report that the carbohydrate metabolites succinate and α-ketoglutarate (α-KG) and their respective receptor-GPR91 and GPR99-are involved in modulating retinal ganglion cell (RGC) projections toward the thalamus during visual system development. Using ex vivo and in vivo approaches, combined with pharmacological and genetic analyses, we revealed that GPR91 and GPR99 are expressed on axons of developing RGCs and have complementary roles during RGC axon growth in an extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent manner. However, they have no effects on axon guidance. These findings suggest an important role for these receptors during the establishment of the visual system and provide a foundational link between carbohydrate metabolism and axon growth.

Role of GPR55 during Axon Growth and Target Innervation.

Guidance molecules regulate the navigation of retinal ganglion cell (RGC) projections toward targets in the visual thalamus. In this study, we demonstrate that the G-protein-coupled receptor 55 (GPR55) is expressed in the retina during development, and regulates growth cone (GC) morphology and axon growth. In vitro, neurons obtained from gpr55 knock-out (gpr55(-/-) ) mouse embryos have smaller GCs, less GC filopodia, and have a decreased outgrowth compared with gpr55(+/+) neurons. When gpr55(+/+) neurons were treated with GPR55 agonists, lysophosphatidylinositol (LPI) and O-1602, we observed a chemo-attractive effect and an increase in GC size and filopodia number. In contrast, cannabidiol (CBD) decreased the GC size and filopodia number inducing chemo-repulsion. In absence of the receptor (gpr55(-/-) ), no pharmacologic effects of the GPR55 ligands were observed. In vivo, compared to their wild-type (WT) littermates, gpr55(-/-) mice revealed a decreased branching in the dorsal terminal nucleus (DTN) and a lower level of eye-specific segregation of retinal projections in the superior colliculus (SC) and in the dorsal lateral geniculate nucleus (dLGN). Moreover, a single intraocular injection of LPI increased branching in the DTN, whereas treatment with CBD, an antagonist of GPR55, decreased it. These results indicate that GPR55 modulates the growth rate and the targets innervation of retinal projections and highlight, for the first time, an important role of GPR55 in axon refinement during development.

Cannabinoid receptor CB2 modulates axon guidance.

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB2R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB2R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 (-/-)). The CB2R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB2R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 (-/-) mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB2R's implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB1R, but also involves CB2R.

Concerted action of CB1 cannabinoid receptor and deleted in colorectal cancer in axon guidance.

Endocannabinoids (eCBs) are retrograde neurotransmitters that modulate the function of many types of synapses. The presence of eCBs, their CB1 receptor (CB1R), and metabolizing enzymes at embryonic and early postnatal periods have been linked to developmental processes such as neuronal proliferation, differentiation, and migration, axon guidance, and synaptogenesis. Here, we demonstrate the presence of a functional eCB system in the developing visual system and the role of CB1R during axon growth and retinothalamic development. Pharmacological treatment of retinal explants and primary cortical neuron cultures with ACEA, a selective CB1R agonist, induced a collapse of the growth cone (GC). Furthermore the application of AM251, a CB1R inverse agonist, to the neuronal cultures increased the surface area of GC. In vivo, intraocular injection of ACEA diminished retinal projection growth, while AM251 promoted growth and caused aberrant projections. In addition, compared with their wild-type littermates, CB1R-deficient adult mice revealed a lower level of eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus. Finally, we found that pharmacological modulation of CB1R affected the trafficking of Deleted in colorectal cancer (DCC) receptor to the plasma membrane in a PKA-dependent manner. Moreover, pharmacological inhibition or genetic inactivation of DCC abolished the CB1R-induced reorganization of the GC. Overall, these findings establish a mechanism by which the CB1R influences GC behavior and nervous system development in concerted action with DCC.