Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Degenerative disc disease (DDD) is a common cause of lower back pain. Calcification of the intervertebral disc (IVD) has been correlated with DDD, and is especially prevalent in scoliotic discs. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. Our data indicate that Ca²⁺ and expression of the extracellular calcium-sensing receptor (CaSR) are significantly increased in mild to severely degenerative human IVDs. In this study, we evaluated the effects of Ca²⁺ and CaSR on the degeneration and calcification of IVDs.

Human donor lumbar spines of Thompson grade 2, 3 and 4 through organ donations within 24 hs after death. IVD cells, NP and AF, were isolated from tissue by sequential digestion with Pronase followed by Collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on IVD tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting and RT-qPCR were performed on cultured NP and AF cells to demonstrate expression of CaSR, matrix proteins aggrecan and collagen, catabolic enzymes and calcification markers. IVD cells were cultured in increasing concentrations of Ca²⁺ [1.0-5.0 mM], CaSR allosteric agonist (cincalcet, 1 uM), and IL-1b [5 ng/mL] for 7 days. Ex vivo IVD organ cultures were prepared using PrimeGrowth Disc Isolation System (Wisent Bioproducts, Montreal, Quebec). IVDs were cultured in 1.0, 2.5 mM Ca²⁺ or with cinacalcet for 21 days to determine effects on disc degeneration, calcification and biomechanics. Complex modulus and structural stiffness of disc tissues was determined using the MACH-1 mechanical testing system (Biomomentum, Laval, Quebec).

Ca²⁺ dose-dependently decreased matrix protein synthesis of proteoglycan and Col II in NP and AF cells, similar to treatment with IL-1b. (n = 4). Contrarily to IL-1b, Ca²⁺ and cincalcet did not significantly increase the expression of catabolic enzymes save ADAMTS5. Similar effects were observed in whole organ cultures, as Ca²⁺ and cinacalcet decreased proteoglycan and collagen content. Although both Ca²⁺ and cinacalcet increased the expression of alkaline phosphatase (ALP), only in Ca²⁺-treated IVDs was there evidence of calcium deposits in NP and AF tissues as determined by von Kossa staining. Biomechanical studies on Ca²⁺ and cinacalcet-treated IVDs demonstrated decreases in complex modulus (p<0.01 and p<0.001, respectively; n=5), however, only Ca²⁺-treated IVDs was there significant increases stiffness in NP and AF tissues (p<0.001 and p<0.05, respectively; n=3).

Our results suggest that changes in the local concentrations of calcium and activation of CaSR affects matrix protein synthesis, calcification and IVD biomechanics. Ca²⁺ may be a contributing factor in IVD degeneration and calcification.

Low back pain is more common in women than men, yet most studies of intervertebral disc (IVD) degeneration do not address sex differences. In humans, there are sex differences in spinal anatomy and degenerative changes in biomechanics, and animal models of chronic pain have demonstrated sex differences in pain transduction. However, there are few studies investigating sex differences in annular puncture IVD degeneration models. IVD puncture is known to result in progressive biomechanical alterations, but whether these IVD changes correlate with pain is unknown. This study used a rat IVD injury model to determine if sex differences exist in mechanical allodynia, biomechanics, and the relationship between them, six weeks after IVD injury. Procedures were IACUC approved. 24 male & 24 female four-month-old Sprague-Dawley rats underwent a sham or annular puncture injury surgery (n=12 male, 12 female). In injury groups, three lumbar IVDs were each punctured three times with a needle, and injected with tumor necrosis factor-alpha. Mechanical allodynia was tested biweekly using von Frey filaments. Six weeks after IVD injury, rats were euthanized and motion segments were dissected for non-destructive axial tension-compression and torsional rotation biomechanical testing. Two-way ANOVA with Bonferroni corrections identified statistically significant differences (p < 0 .05) and correlations used Pearson's coefficient. Annular puncture injury induced a significant increase in mechanical allodynia compared to sham in male but not female rats up to six weeks after injury. There was a significant sex effect on both torque range and torsional stiffness, with males exhibiting greater stiffness and torque range than females. Tensile stiffness, compressive stiffness, and axial range of motion showed no sex difference. Males and females showed similar patterns of correlation between variables when sham and injury groups were analyzed together, but correlations were stronger in males. Most correlations were clustered within testing approach: axial biomechanics negatively correlated, torsional biomechanics positively correlated, and von Frey thresholds positively correlated. Surprisingly, mechanical allodynia did not correlate with any biomechanics after injury, and the axial and torsional biomechanics showed little correlation. This study demonstrates that males and females respond to IVD injury differently. Given the absence of correlation between pain and biomechanics, pain cannot be attributed completely to biomechanical changes. This may explain why spinal fusion surgery, an intervention limited to the spine, has produced inconsistent results and is controversial for patients with low back pain. Thus, in addressing low back pain, we must consider both spinal tissues and the nervous system. Further, the limited correlation between axial and torsional biomechanics indicates that IVD injury may have distinct effects on nucleus pulposus and annulus fibrosus. Biomechanics did not differ between sham and injury at week six, suggesting healing after injury. It remains possible that acute biomechanical changes may initiate chronic pain pathogenesis. We conclude that the observed sex differences demonstrate the need for inclusion of both males and females in IVD injury and pain studies, and suggest that males and females may require different treatments for conditions that appear similar

Intervertebral disc (IVD) degeneration plays a major role in low back pain which is the leading cause of disability. Current treatments in severe cases require surgical intervention often leading to adjacent segment degeneration. Injectable hydrogels have received much attention in recent years as scaffolds for seeding cells to replenish disc cellularity and restore disc properties and function. However, they generally present poor mechanical properties. In this study, we investigated several novel thermosensitive chitosan hydrogels for their ability to mimic the mechanical properties of the nucleus pulposus (NP) while being able to sustain the viability of NP cells, and retain proteoglycans. CH hydrogels were prepared by mixing the acidic chitosan solution (2% w/v) with various combinations of three gelling agents: sodium hydrogen carbonate (SHC) and/or beta-glycerophosphate (BGP) and/or phosphate buffer (PB) (either BGP0.4M, SHC0.075M-BGP0.1M, SHC0.075M-PB0.02M or SHC0.075M-PB0.04M). The gelation speed was assessed by following rheological properties within 1h at 37°C (strain 5% and 1Hz). The mechanical properties were characterized and compared with that of human NP tissues. Elastic properties of the hydrogels were studied by evaluating the secant modulus in unconfined compression. Equilibrium modulus was also measured, using an incremental stress-relaxation test 24h after gelation in unconfined compression (5% strain at 5%/s followed by 5min relaxation, five steps). Cells from bovine IVD were encapsulated in CH-based gels and maintained in culture for 14 days. Cytocompatibility was assessed by measuring cell viability, metabolism and DNA content. Glycosaminoglycan (GAG) synthesis (retained in the gel and released) was determined using DMMB assay. Finally injectability was tested using human cadaveric discs. Unconfined compression confirmed drastically enhanced mechanical properties compared to conventional CH-BGP hydrogels (secant Young modulus of 105 kPa for SHC0.075PB0.02 versus 3–6 kPa for BGP0.04). More importantly, SHC0.075PB0.02 and SHC0.075BGP0.1 hydrogels exhibited mechanical properties very similar to NP tissue. For instance, equilibrium modulus was 5.2±0.6 KPa for SHC0.075PB0.02 and 8±0.8 KPa for SHC0.075BGP0.1 compared to 6.1±1.7 KPa for human NP tissue. Rheological properties and gelation time (G′=G″ after less than 15 s at 37°C, and rapid increase of G') of these hydrogels also appear to be adapted to this application. Cell survival was greater than 80% in SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels. Cells encapsulated in the new formulations also showed significantly higher metabolic activity and DNA content after 14 days of incubation compared to cells encapsulated in BGP0.4 hydrogel. Cells encapsulated in SHC0.075BGP0.1 and SHC0.075PB0.02 produced significantly higher amounts of glycosaminoglycans (GAG) compared to cells encapsulated in SHC0.075PB0.04 and BGP0.4 hydrogels. The total amount of GAG was higher in SHC0.075BGP0.1 hydrogel compared to SHC0.075PB0.02. Interestingly, both the SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels retained similar amounts of GAG. Injectability through a 25G syringe, filling of nuclear clefts and good retention in human degenerated discs was demonstrated for SHC0.075PB0.02 hydrogel. SHC0.075BGP0.1 appears to be a particularly promising injectable scaffold for IVD repair by providing suitable structural environment for cell survival, ECM production and mechanical properties very similar to that of NP tissue

Purpose. Nucleus pulposus (NP) replacements represent a less invasive alternative for treatment of early stage degenerative disc disease (DDD). Hydrogel based NP replacements are of particular interest as they can be injected/implanted using minimally invasive surgical (MIS) techniques to re-establish mechanical integrity and as a scaffold for regeneration. A thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) hydrogel crosslinked using polyethylene diacrylate has shown promise as a potential NP replacement for DDD in vitro. This study aims to assess the mechanical properties of this hydrogel when injected into an induced early stage DDD porcine model and to determine the optimal injection method for delivery. It is hypothesized that minimally invasive injection of the TMHA/EP material can restore mechanical behaviour of spinal motion segments in early stage DDD. Method. Intervertebral disc (IVD) degeneration was enzymatically induced in L2-L3 and L4-L5 lumbar levels in 10 Yorkshire boars using chondroitinase ABC (n=20 discs). An additional three animals served as healthy controls (n=6 discs). Following a four-week degradation period, the TMHA/EP solution (250microL in a 3:1 weight ratio) was injected into the degenerate NP of 16 discs by one of two MIS techniques: A direct 18G needle injection or a modified kyphoplasty technique (MKT) in which a balloon angiocatheter was inserted through an 11G trocar into the IVD and inflated to create a cavitary defect that was then filled with the hydrogel. Excised motion segments were tested in axial compression under a load of 400N and in axial rotation (AR), lateral bending (LB) and flexion/extension (FE) at 5Nm. Range of motion (ROM), neutral zone (NZ) length, NZ stiffness (NZStiff) and axial compressive stiffness (ACStiff) were quantified. Results. The degenerate control motion segments were, in general, found to be significantly less stiff and more flexible than the healthy controls. In comparison to the degenerate controls, direct injection of TMHA/EP demonstrated increased ACStiff and AR NZStiff (23%, 77%; p<0.05) and the MKT yielded a significant increase in AR NZStiff (88%) with a trend towards increased FE NZStiff (253%, p=0.089). Following TMHA/EP augmentation, direct injection and MKT treated IVDs demonstrated similar stiffness to healthy intact controls (p=0.519–1.000). Both ROM and NZ length in AR significantly increased following degeneration of the IVDs as compared to healthy controls (49%, 63%) In comparison to degenerate controls, both MIS techniques showed similar significant decreases in AR ROM (32%, 33%) and AR NZ length (35%, 32%). Both injection methods worked to restore motion to levels similar to healthy controls (p=0.173–1.00). Differences were not detected between the two treatment groups for all outcome variables (p=0.115–0.916). Conclusion. This study demonstrated the ability of the TMHA/EP composite to restore initial biomechanical function in early stage DDD independent of the MIS technique