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 Ca. 2+. 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 Ca. 2+. 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 Ca. 2+. [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 Ca. 2+. 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). Ca. 2+. 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, Ca. 2+. and cincalcet did not significantly increase the expression of catabolic enzymes save ADAMTS5. Similar effects were observed in whole organ cultures, as Ca. 2+. and cinacalcet decreased proteoglycan and collagen content. Although both Ca. 2+. and cinacalcet increased the expression of alkaline phosphatase (ALP), only in Ca. 2+. -treated IVDs was there evidence of calcium deposits in NP and AF tissues as determined by von Kossa staining. Biomechanical studies on Ca. 2+. and cinacalcet-treated IVDs demonstrated decreases in complex modulus (p<0.01 and p<0.001, respectively; n=5), however, only Ca. 2+. -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. Ca. 2+. may be a contributing factor in IVD degeneration and calcification

Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc. Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10. ∧. 6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of mouse IVDs, suggesting the development of sensory neuron invasion of the disc. We provide evidence that prolonged tungsten exposure can induce disc fibrosis and increase the expression of markers associated with pain. Tungsten toxicity may play a role in disc degeneration disease

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

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

Aim. Cefuroxime is a time-dependent antibiotic widely used as intravenous perioperative prophylaxis in spine surgery. A previous study has indicated that a single dose of cefuroxime provided insufficient spine tissue concentrations for spine procedures lasting more than 2–3 hours. Due to the fact that postoperative pyogenic spondylodiscitis is associated with prolonged antimicrobial therapy and high relapse rates, we aimed to evaluate if a twofold increase of standard dosage of 1.5g cefuroxime given as one double dose or two single doses with 4-hours intervals will lead to sufficient cefuroxime spine tissue concentrations throughout the dosing interval. Method. This is preliminary data for 8 out of 16 female pigs. Data from all 16 pigs will be included for the conference. Eight pigs were randomized into two groups: Group A received one double dose of cefuroxime (3g) as a bolus, and Group B received two single doses of cefuroxime (2×1.5g) with 4-hours intervals. Measurements were obtained from plasma, subcutaneous tissue (SCT), vertebral cancellous bone and the intervertebral disc (IVD) for 8-hours thereafter. Microdialysis was applied for sampling in solid tissues. The cefuroxime concentrations were determined using ultra-high performance liquid chromatography. Results. The time with concentrations above the minimal inhibitory concentration (T>MIC) for the clinical breakpoint MIC for Staphylococcus aureus of 4 μg/ml, was superior in all compartments when administering cefuroxime as two single doses with 4-hours intervals. For the target MIC of 4 μg/ml, the mean T>MIC in all compartments ranged between 53–73% and 85–95% for Group A and B, respectively. For both groups the area under the concentration-curve (AUC) was higher for plasma compared to the remaining compartments, and the lowest AUCs were found in the vertebral cancellous bone and the IVD. There were no differences in AUC between the two groups. Furthermore, the maximal concentrations were lower for both vertebral cancellous bone and IVD compared to both SCT and plasma. When comparing the two groups, higher maximal concentrations were found in all compartments for Group A. Tissue penetration was incomplete and delayed for all compartments and comparable between the two groups. Conclusions. Despite comparable pharmacokinetic results between the two groups, Group B exhibited superior T>MIC in all compartments for the clinical breakpoint MIC for Staphylococcus aureus of 4 μg/ml. As such administration of cefuroxime as two single doses with 4-hours intervals provided sufficient cefuroxime spine tissue concentrations for a minimum of 85% of an 8-hour dosing interval, which may be acceptable for most spine procedures

Aim. Pyogenic spondylodiscitis is associated with prolonged antimicrobial therapy and high relapse rates. Nevertheless, tissue pharmacokinetic studies of relevant antimicrobials in both prophylactic and therapeutic situations are still sparse. Previous approaches based on bone biopsy and discectomy exhibit important methodological limitations. The objective of this study was therefore to assess the concentration of cefuroxime in intervertebral disc (IVD), vertebral body cancellous bone, subcutaneous adipose tissue (SCT) and plasma pharmacokinetics after single dose administration by use of microdialysis (MD) in a large animal model. Method. Ten female pigs were assigned to receive 1,500 mg of cefuroxime intravenously over 15 min. Measurements of cefuroxime were obtained from plasma, SCT, the vertebral cancellous bone and the IVD for 8 hours thereafter. MD was applied for sampling in solid tissues. The cefuroxime concentration in both the MD and plasma samples was determined using ultra-high performance liquid chromatography. Results. For both the IVD and the vertebral cancellous bone, the area under the concentration-curve from zero to the last measured value was significantly lower than that of free plasma. Tissue penetration of cefuroxime was incomplete for the IVD, whereas for vertebral cancellous bone and SCT it was not. Furthermore, the penetration of cefuroxime from plasma to IVD was delayed. Additionally, a noticeable prolonged elimination rate of cefuroxime in the IVD was found. The maximal concentration and the elimination of cefuroxime were reduced in IVD compared to both SCT and vertebral cancellous bone. Due to this delay in elimination of cefuroxime, the time with concentrations above the minimal inhibitory concentration (T>MIC) was significantly higher in IVD than in SCT, vertebral cancellous bone and free plasma for MICs up to 6 μg/ml. Conclusions. MD was successfully applied for serial assessment of the concentration of cefuroxime in the IVD and the vertebral cancellous bone. Penetration of cefuroxime from plasma to IVD was found to be incomplete and delayed, but due to a prolonged elimination, the best results regarding T>MIC was found in IVD

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to test intervertebral disc (IVD) regeneration, application becomes costly when used as a screening method. The bovine IVD organ culture system offers an inexpensive alternative, however, in the current paradigm, the bony vertebrae is removed to allow for nutrient diffusion to disc cells. This provides limitations on the conditions and strategies one can employ in investigating IVD regeneration and mechanisms in degenerative disc disease (i.e. complex loading). Although one method has been attempted to extend the survival of bovine vertebrae containing IVDs (vIVD) cell viability declined after two weeks in culture. Our goal was to develop and validate a long-term organ culture model with vertebral bone, which could be used subsequently for studying biological repair of disc degeneration and biomechanics. Preparation of vIVDs: Bovine IVDs from the tails of 22–28-month-old steers were prepared for organ culture by parallel cuts through the adjacent vertebral bodies at 1cm from the endplates using an IsoMet®1000 Buehler precision sectioning saw. vIVDs were split into two groups: IVDs treated with PrimeGrowth Media kit (developed by Intervertech and licensed to Wisent Bioproducts) and IVDs with DMEM. The PrimeGrowth group was incubated for 1h in PrimeGrowth Isolation Medium (Cat# 319–511-EL) and the DMEM group for 1h in DMEM. After isolation, IVDs were washed in PrimeGrowth Neutralisation Medium (Cat# 319–512-CL) while the other IVDs were washed in DMEM. The discs isolated with PrimeGrowth and DMEM were cultured for up to 5 months in sterile vented 60 ml Leakbuster™ Specimen Containers in PrimeGrowth Culture Medium (Cat# 319–510-CL) and DMEM with no mechanical load applied. Live/Dead Assay: vIVDs cultured for 1 or 5 months were dissected and cell viability was assessed in different regions by confocal microscopy using Live/Dead® (Invitrogen) fluorescence assay. Glucose Diffusion: After one month of culture, vIVDs were incubated for 72h in diffusion medium containing PBS (1x), CaCl2 (1mM), MgCl2 (0.5mM), KCl2 (5mM), 0.1% BSA and 150µM 2-NDBG, a D-glucose fluorescent analogue. Discs were dissected and IVD tissues were incubated in guanidinium chloride extraction buffer. Extracts were measured for fluorescence. After 5 months in culture, vIVDs prepared with PrimeGrowth kit demonstrated approximately 95% cell viability in all regions of the disc. However, dramatic reductions (∼90%) in vIVD viability were measured in DMEM group after 1 month. vIVD viability was related to the amount of 2-NDBG incorporated into the disc tissue. We have developed a novel method for isolating IVDs with vertebral bone capable of long-term viability. This method may not only help in the discovery of novel therapeutics in disc regeneration, but could also advance our understanding on complex loading paradigms in disc degeneration

Aim. Pyogenic spondylodiscitis remains a therapeutic challenge, as demonstrated by divergent treatment guidelines. The combination of moxifloxacin and rifampicin may be an attractive treatment option for cases caused by staphylococci; however, previous studies have reported a reduction in plasma concentrations of moxifloxacin when co-administered with rifampicin. The magnitude of this reduction in spinal tissues is not known. We aimed to investigate the interaction of rifampicin on moxifloxacin tissue concentrations in vertebral cancellous bone, intervertebral disc and subcutaneous adipose tissue in steady-state conditions using microdialysis in a porcine model. Method. Twenty female pigs were randomized into two groups of ten pigs: Group A received moxifloxacin 400 mg orally once daily for three days preoperatively. Group B received moxifloxacin 400 mg orally for three days preoperatively combined with rifampicin 450 mg twice daily for seven days preoperatively. Measurements were obtained from plasma, vertebral cancellous bone, intervertebral disc and subcutaneous adipose tissue for 24 h. Microdialysis was applied for sampling in solid tissues. Results. Co-administration of moxifloxacin and rifampicin demonstrated a reduction of free moxifloxacin concentrations in spinal tissues. The peak drug concentration (C. max. ) and the area under the concentration-time curve (AUC. 0–24. ) in all tissue compartments decreased in the range of 66–79% and 65–76%, respectively. Conclusions. Using microdialysis, we demonstrated a significant reduction of moxifloxacin C. max. and AUC. 0–24. in the spinal tissues when co-administered with rifampicin. Further studies are warranted to understand the clinical implications of this finding for the treatment of pyogenic spondylodiscitis

Aims. Vancomycin may be an important drug for intravenous perioperative antimicrobial prophylaxis in spine surgery. We assessed single-dose vancomycin intervertebral disc, vertebral cancellous bone, and subcutaneous adipose tissue concentrations using microdialysis in a pig model. Methods. 8 female pigs received 1,000 mg of vancomycin intravenously as a single dose over 100 minutes. Microdialysis probes were placed in the C3-C4 intervertebral disc, C3 vertebral cancellous bone, and subcutaneous adipose tissue, and vancomycin concentrations were obtained over 8 hours. Venous blood samples were obtained as reference. Results. Ranging from 0.24 to 0.60, vancomycin tissue penetration, expressed as the ratio of tissue to plasma area under the concentration-time curve from 0 to the last measured value, was incomplete for all compartments. The lowest penetration was found in the intervertebral disc. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 4 μg/mL were 3, 17, 25, and 156 min for plasma, subcutaneous adipose tissue, vertebral cancellous bone and the intervertebral disc, respectively. In contrast to the other compartments, a mean MIC of 8 μg/mL was not reached in the intervertebral disc. An approximately 3-time longer elimination rate was observed in the intervertebral disc in comparison to all the other compartments (p < 0.001), and the time to peak drug concentration was higher for all tissues compared with plasma. Conclusions. Preoperative administration of 1,000 mg of vancomycin may provide adequate vancomycin tissue concentrations with a considerable delay, though tissue penetration was incomplete. However, in order also to achieve adequate intervertebral disc concentrations in all individuals and accommodating a potentially higher MIC target, supplemental application of vancomycin may be necessary

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1-KO) exhibit progressive ectopic calcification of spinal tissues, including the annulus fibrosus (AF) of intervertebral discs (J Bone Miner Res 28:1135–49, 2013, Bone 90:37–49, 2016). Our purpose was twofold: (1) to compare ectopic calcifications in ENT1-KO mice with those in human DISH, and (2) to investigate the molecular pathways underlying pathological calcification in ENT1-KO mice. Studies were performed with age-matched wild-type (WT) and ENT1-KO mice, as well as human cadaveric vertebral columns meeting radiographic criteria for DISH. Mouse and human specimens were scanned using high-resolution, micro-computed tomography (micro-CT). As well, some samples were decalcified and processed for histological assessment. Calcified lesions in selected specimens were examined using energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). To investigate molecular changes associated with ectopic calcification, we isolated AF tissue from thoracic intervertebral discs of WT and ENT1-KO mice. Tissues were then subjected to transcriptomic and proteomic analyses. Micro-CT of ENT1-KO mice revealed ectopic calcification of spinal tissues, first appearing in the cervical-thoracic region and extending caudally with advancing age. Histological examination of calcified lesions in mice revealed accumulations of amorphous, eosinophilic, acellular material in paraspinal ligaments and entheses, intervertebral discs, mandibular symphysis, and sternocostal articulations. There was no evidence of inflammation associated with these lesions. EDX of calcified lesions revealed a high content of calcium and phosphorus in a molar ratio of ∼1.6, with hydroxyapatite detected by micro-XRD. Ten human cadaveric spines (three females and seven males, mean age 81 years) that met radiographic criteria for DISH were analysed in detail by micro-CT. Remarkable heterogeneity in the density and morphology of ectopic calcifications was observed. Analyses of calcifications by EDX and XRD again yielded a calcium/phosphorus ratio of ∼1.6 and a crystalline diffraction pattern matching hydroxyapatite. Histological examination of human lesions revealed regions of mature ossification and other areas of irregular amorphous calcification that resembled lesions in ENT1-KO mice. Microarray analysis of AF tissue from WT and ENT1-KO mice showed extensive dysregulation of transcription in affected tissues. Cell cycle-associated transcripts were the most affected, including the E2f family of transcription factors and proliferating cell nuclear antigen. In addition, expression of genes involved in the regulation of mineralization and bone development were dysregulated. Proteomic analyses confirmed transcriptomic changes and revealed alterations in known modulators of biomineralization such as matrix Gla-protein. Many of the characteristics of ectopic calcification in ENT1-KO mice resemble those of DISH in humans. Human lesions were found to be heterogeneous with regions of pathological ossification and amorphous calcification, the latter resembling lesions in the mouse model. Our studies of the molecular events associated with ectopic calcification in ENT1-KO mice may provide insights into the pathogenesis of DISH in humans. ENT1-KO mice may also be useful for evaluating therapeutics for the prevention of ectopic calcification in DISH and related disorders

Aim. Vertebral osteomyelitis (VO) is an infection of the spine mostly caused by bacterial pathogens. The pathogenesis leading to destruction of intervertebral discs (IVD) and adjacent vertebral bodies (VB) is poorly described. We aimed to investigate the connection between infection, bone- and disc-metabolism in VO patients. Method. Fourteen patients with VO (infection group) and 14 patients with incomplete burst fractures of the spine (fracture group as controls) were included prospectively. Demographic data, treatment details, laboratory infection markers, and patient-reported outcome were assessed. Tissue biopsies from affected IVDs and adjacent VBs were analyzed for mRNA-expression levels of 18 target genes including chemokines, adipokines and genes involved in bone-metabolism by RT-qPCR. Results. The Receptor activator of NF-κB/Osteoprotegerin (RANK/OPG) expression ratio was elevated in VB and IVD of the infection group (p<0.001 and p=0.028, respectively). The RANK-ligand (RANKL)/OPG expression ratio was elevated in VB of the infection group (p<0.01). Expressions of the chemokines IL8 and CCL20 were higher in VB samples of the infection group. The expression of leptin was higher in IVD tissue, the mRNA expression of omentin and resistin was lower in VBs of the infection group. OPG mRNA expression was lower in infected VB and in IVD tissue compared to the fracture group. Conclusions. We identified similar expression patterns of pro-inflammatory cytokines and the RANK/RANKL/OPG axis in VBs and IVDs of patients with VO. This finding suggests that common immuno-metabolic pathways are involved in mechanisms leading to tissue degradation in VBs and IVDs during VO

As the intervertebral disc is largely avascular, needle injection is the most practical method for delivery of therapeutic agents used in treatments for degenerative disc disease. Intradiscal pressure increases during injection, and insufficient recovery time prior to needle retraction may result in injectate leakage. In order to determine the maximum pressure and post-injection recovery time for a given injection volume and rate, an analytical model of intradiscal injection was developed and calibrated experimentally. A governing equation was derived defining intradiscal pressure as a function of effective permeability, initial elastic stiffness, nonlinear stiffness term, and injection rate. The equation was solved using a fourth order Runge-Kutta routine with a 0.05s time step and a ramp-dwell injection. The model was calibrated by performing controlled intradiscal injections on five bovine caudal intervertebral discs. Three had adjacent vertebrae intact, while two were separated from vertebrae and constrained between porous stainless steel platens. A syringe driven by a linear actuator was used to inject phosphate buffered saline through a 21g hypodermic needle inserted radially into the disc to a depth of one half of the disc diameter. Injection was performed at a rate of 75μL/s to a volume of 250μL followed by a 240s dwell. Fluid pressure was recorded during both the injection phase and subsequent recovery phase. For each experimental pressure vs time trace, model parameters were varied in order to obtain an optimal fit. The model was run with the average parameter values across a grid of possible injection protocols, with injection volume ranging from 30 to 300μL and injection time ranging from 0.1 to 5s. For each case, peak pressure and time required to reach a 1kPa threshold were recorded. Experimentally measured peak pressure ranged from 68 to 88kPa. Pressure at the end of the 240s dwell ranged from 49 to 69kPa. There was no apparent difference between discs with and without endplates. Leakage of fluid following needle retraction was observed in all specimens. Experimental data were well fit by the analytical model, which predicted higher peak pressure and longer recovery time with increasing volume, from approximately 1500s at 30μL to nearly 3000s at 300μL. The model was nearly insensitive to injection rate. The experimental data confirm pressurization of the disc during injection and injectate leakage resulting from insufficient recovery time. The model predicts that the time required to recover to below threshold leakage pressure is impractically long for both laboratory and clinical injection protocols. Similar behavior with and without endplates confirms that fluid flow is limited by permeability of the tissue itself, not the boundary conditions. Slow recovery is likely attributable to the fact that peak injection pressures were lower than the hydraulic swelling pressure of the nucleus pulposus, which has been reported to be approximately 140kPa. Due to the high swelling pressure of the nucleus pulposus, it is unlikely that intradiscal injection procedures can be performed without substantial injectate leakage following needle retraction

Tenomodulin (Tnmd) is the best known mature tendon factor for tendon and ligament tissues with reported important regulatory roles1. In addition, Tnmd C-terminal cysteine-rich domain has been descibed to exert anti-angiogenic functions in in vitro angiogenic assays as well as in vivo models of tendon injury and age-associated cardiac valve diseases1, 2. Interestingly, Tnmd expresson in the intervertebral disc (IVD), which is normally avascular tissue, has been also suggested3. Hence, the purpose of this study was first, to map the exact expression pattern of Tnmd during IVD development and aging and second, by implementing Tnmd-knockout mouse model, to examine if Tnmd plays a role in IVD homeostasis. Histological analyses (hematoxylin/eosin, Safranin O, CD31 for endothelium, TUNEL for apoptosis and type X collagen and Runx2 for hypetrophy) were performed on Tnmd −/−, Tnmd −/− and chondromodulin I Chmd 1 −/− (Tnmd only homolog) double knockout and wild type mice WT (n = three to five) to examine IVD degeneration. Real time PCR was implemented to explore gene expression chnages in annulus fibrous (AF) between Tnmd −/− and WT mice. In addition, outer AF (OAF) cells were isolated from both genotypes to further determine cellular phenotype and assess effects on co-culture with human umbical vein endothelial cells (HUVECs). Statistical differences between two groups were determined with t-test. In multiple comparisons, one-way ANOVA was followed by Bonferroni post-hoc correction. Tnmd was expressed in a temporal manner in OAF and to very low extent in NP. Tnmd −/− mice exhibited more rapid progression of age-related IVD degeneration. These signs included smaller collagen fibril diameter, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis and inflamatory cell infiltration in OAF as well as more hypertrophic-like chondrocytes in the NP. In addition, Tnmd−/− Chm1 −/− mice displayes not only accelerated IVD phenotpye, but also ectopic bone formation in the IVD. Lastly, the abscence of Tnmd in OAF-derived cells significantly promoted HUVECs migratory capacity. These findings provide clear evidence that Tnmd plays a critical role in IVD homeostasis

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions. OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN. Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β. Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s)

Cervical spinal arthrodesis is the standard of care for the treatment of spinal diseases induced neck pain. However, adjacent segment disease (ASD) is the primary postoperative complication, which draws great concerns. At present, controversy still exists for the etiology of ASD. Knowledge of cervical spinal loading pattern after cervical spinal arthrodesis is proposed to be the key to answer these questions. Musculoskeletal (MSK) multi-body dynamics (MBD) models have an opportunity to obtain spinal loading that is very difficult to directly measure in vivo. In present study, a previously validated cervical spine MSK MBD model was developed for simulating cervical spine after single-level anterior arthrodesis at C5-C6 disc level. In this cervical spine model, postoperative sagittal alignment and spine rhythms of each disc level, different from normal healthy subject, were both taken into account. Moreover, the biomechanical properties of facet joints of adjacent levels after anterior arthrodesis were modified according to the experimental results. Dynamic full range of motion (ROM) flexion/extension simulation was performed, where the motion data after arthrodesis was derived from published in-vivo kinematic observations. Meanwhile, the full ROM flexion/extension of normal subject was also simulated by the generic cervical spine model for comparative purpose. The intervertebral compressive and shear forces and loading-sharing distribution (the proportions of intervertebral compressive and shear force and facet joint force) at adjacent levels (C3-C4, C4-C5 and C6-C7 disc levels) were then predicted. By comparison, arthrodesis led to a significant increase of adjacent intervertebral compressive force during the head extension movement. Postoperative intervertebral compressive forces at adjacent levels increased by approximate 20% at the later stage of the head extension movement. However, there was no obvious alteration in adjacent intervertebral compressive force, during the head flexion movement. For the intervertebral shear forces in the anterior-posterior direction, no significant differences were found between the arthrodesis subject and normal subject, during the head flexion/extension movement. Meanwhile, cervical spinal loading-sharing distribution after anterior arthrodesis was altered compared with the normal subject's distribution, during the head extension movement. In the postoperative loading-sharing distribution, the percentage of intervertebral disc forces was further increased as the motion angle increased, compared with normal subject. In conclusion, cervical spinal loading after anterior arthrodesis was significantly increased at adjacent levels, during the head extension movement. Cervical spine musculoskeletal MBD model provides an attempt to comprehend postoperative ASD after anterior arthrodesis from a biomechanical perspective

Spinal stenosis is a condition resulting in the compression of the neural elements due to narrowing of the spinal canal. Anatomical factors including enlargement of the facet joints, thickening of the ligaments, and bulging or collapse of the intervertebral discs contribute to the compression. Decompression surgery alleviates spinal stenosis through a laminectomy involving the resection of bone and ligament. Spinal decompression surgery requires appropriate planning and variable strategies depending on the specific situation. Given the potential for neural complications, there exist significant barriers to residents and fellows obtaining adequate experience performing spinal decompression in the operating room. Virtual teaching tools exist for learning instrumentation which can enhance the quality of orthopaedic training, building competency and procedural understanding. However, virtual simulation tools are lacking for decompression surgery. The aim of this work was to develop an open-source 3D virtual simulator as a teaching tool to improve orthopaedic training in spinal decompression. A custom step-wise spinal decompression simulator workflow was built using 3D Slicer, an open-source software development platform for medical image visualization and processing. The procedural steps include multimodal patient-specific loading and fusion of Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) data, bone threshold-based segmentation, soft tissue segmentation, surgical planning, and a laminectomy and spinal decompression simulation. Fusion of CT and MRI elements was achieved using Fiducial-Based Registration which aligned the scans based on manually placed points allowing for the identification of the relative position of soft and hard tissues. Soft tissue segmentation of the spinal cord, the cerebrospinal fluid, the cauda equina, and the ligamentum flavum was performed using Simple Region Growing Segmentation (with manual adjustment allowed) involving the selection of structures on T1 and/or T2-weighted scans. A high-fidelity 3D model of the bony and soft tissue anatomy was generated with the resulting surgical exposure defined by labeled vertebrae simulating the central surgical incision. Bone and soft tissue resecting tools were developed by customizing manual 3D segmentation tools. Simulating a laminectomy was enabled through bone and ligamentum flavum resection at the site of compression. Elimination of the stenosis enabled decompression of the neural elements simulated by interpolation of the undeformed anatomy above and below the site of compression using Fill Between Slices to reestablish pre-compression neural tissue anatomy. The completed workflow allows patient specific simulation of decompression procedures by staff surgeons, fellows and residents. Qualitatively, good visualization was achieved of merged soft tissue and bony anatomy. Procedural accuracy, the design of resecting tools, and modeling of the impact of bone and ligament removal was found to adequately encompass important challenges in decompression surgery. This software development project has resulted in a well-characterized freely accessible tool for simulating spinal decompression surgery. Future work will integrate and evaluate the simulator within existing orthopaedic resident competency-based curriculum and fellowship training instruction. Best practices for effectively teaching decompression in tight areas of spinal stenosis using virtual simulation will also be investigated in future work

The detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. Twenty one fully grown 12-week-old male Sprague-Dawley rats were used in this study. The rats were assigned to one of three groups randomly. One group was selected to be the baseline control group with intact intervertebral discs (n=7). In the other two groups, the vertebrae were immobilized with an external fixation device that fixed four caudal vertebrae (C7-C10) for 4 and 8 weeks, respectively. Four K-wires were fixed in parallel using two aluminum alloy cuboids which do not compress or stretch the target discs. The immobilized discs were harvested and then stained with hematoxylin/eosin, scanned using atomic force microscopy to obtain the modulus at both nano and micro scales, and analyzed the gene expression with real-time quantitative polymerase chain reaction. Significance of differences between the study groups was obtained using a two-way analysis of variance (ANOVA) with Fisher's Partial Least-Squares Difference (PLSD) to analyze the combined influence of immobilization time and scanning region. Statistical significance was set at P≤0.05. Compared to the control group, the inner layer of annulus fibrosus presented significant disorder and hyperplasia after immobilization for 8 weeks, but not in the 4 week group. The fibrils in inner layer showed an alteration in elastic modulus from 91.38±20.19MPa in the intact annulus fibrosus to 110.64±15.58MPa (P<0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33±0.04MPa to 0.47±0.04MPa (P<0.001). The upregulation of collagen II from 1±0.03 in control to 1.22±0.03 in 8w group (P = 0.003) was induced after immobilization, while other genes expression showed no significant alteration after immobilization for both 4 and 8 weeks compared to the control group (P>0.05). The biomechanical properties at both nano and micro scales altered in different degrees between inner and outer layers in annulus fibrosus after immobilization for different times. Meanwhile, the fibril arrangement disorder and the upregulation of collagen II in annulus fibrosus were observed using hematoxylin/eosin staining and real-time RT-PCR, respectively. These results indicate that immobilization not only influenced the individual collagen fibril at the nano scale, but also suggested alterations of micro-biomechanics and cell response. This work provides a better understanding of IVD degeneration after immobilization and benefits to the clinical treatment related to disc immobilization

Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly