Intervertebral disc and endplate cells response to IL-1ß inflammatory cell priming and identification of molecular targets of tissue degeneration.

Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1ß treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression.

OBJECTIVE: To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). DESIGN: An in vitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. RESULTS: All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) tested and at the median frequency (1 Hz) and time interval (12 h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. CONCLUSIONS: We have described for the first time the activation of MAPKs in human AF cells in response to CTS and showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiology and possibly to the design of novel therapeutic interventions.

Revision surgery after PSO failure with rod breakage: a comparison of different techniques.

STUDY DESIGN: Author experience and literature review. OBJECTIVES: To compare different revision techniques in the treatment of implant failure after pedicle subtraction osteotomy (PSO). The complication rate of pedicle subtraction osteotomy is substantially higher than other corrective procedures available for the treatment of spinal sagittal imbalance: in particular, hardware failures and mechanical complications affect this technique and their biomechanical explanation is still purely speculative. METHODS: The author's experience and the literature regarding the revision techniques for PSO failures are discussed. RESULTS: In this paper, eight consecutive revision cases due to rod breakage after PSO surgery are reported. In our experience, the main goals are to restore the spinal balance, through a posterior approach (correction and hardware revision and implementation) and to get a solid anterior fusion (both through a traditional anterior approach or minimally invasive transpsoas approach). CONCLUSION: The efficacy of PSO should be balanced with the high risk of the procedure reported in the literature. Management of revision surgery after PSO may require the addition of anterior column support to maintain correction and reduce complications.

Molecular MR imaging for the evaluation of the effect of dynamic stabilization on lumbar intervertebral discs.

The dynamic stabilization of lumbar spine is a non-fusion stabilization system that unloads the disc without the complete loss of motion at the treated motion segment. Clinical outcomes are promising but still not definitive, and the long-term effect on instrumented and adjacent levels is still a matter of discussion. Several experiments have been devised in order to gain a better understanding of the effect of the device on the intervertebral disc. One of the hypotheses was that while instrumented levels are partially relieved from loading, adjacent levels suffer from the increased stress. But this has not been proved yet. The aim of this study was to investigate the long-term effect of dynamic stabilization in vivo, through the quantification of glycosaminoglycans (GAG) concentration within instrumented and adjacent levels by means of the delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) protocol. Ten patients with low back pain, unresponsive to conservative treatment and scheduled for Dynesys implantation at one to three lumbar spine levels, underwent the dGEMRIC protocol to quantify GAG concentration before and 6 months after surgery. Each patient was also evaluated with visual analog scale (VAS), Oswestry, Prolo, Modic and Pfirrmann scales, both at pre-surgery and at follow-up. Six months after implantation, VAS, Prolo and Oswestry scales had improved in all patients. Pfirrmann scale could not detect any change, while dGEMRIC data already showed a general improvement in the instrumented levels: GAG was increased in 61% of the instrumented levels, while 68% of the non-instrumented levels showed a decrease in GAG, mainly in the posterior disc portion. In particular, seriously GAG-depleted discs seemed to have the greatest benefit from the Dynesys implantation, whereas less degenerated discs underwent a GAG depletion. dGEMRIC was able to visualize changes in both instrumented and non-instrumented levels. Our results suggest that the dynamic stabilization of lumbar spine is able to stop and partially reverse the disc degeneration, especially in seriously degenerated discs, while incrementing the stress on the adjacent levels, where it induces a matrix suffering and an early degeneration.

Parametric FE mesh generation: application to the cervical spine.

A voxel-based reconstruction algorithm, targeted at the generation of finite element (FE) meshes of structures with schematic geometry, is presented. The algorithm is able to generate three dimensional fully hexahedral FE meshes of structures composed of volumes with a schematic geometry. In order to be meshed, each volume must be described in terms of a set of surfaces which enclose the volume. Due to its schematic nature, the method allows the generation of fully parameterized FE models, thus it facilitates the investigation of the mechanical relevance of geometrical parameters by speeding up the mesh generation process. The algorithm was employed in the automatic construction of an FE model of the C3-C7 spinal segment with schematic geometry, made up exclusively of hexahedral elements. Non-linear simulations were carried out in different loading conditions: flexion- extension, lateral bending and axial rotation. The results were compared to data retrieved from the literature in order to ensure the validity of the model. Moment-rotation curves for each loading condition were determined. The range of motion was obtained for each spinal unit and loading condition. Both principal and coupled rotations were determined in lateral bending and axial rotation, for each spinal unit. The intradiscal pressure was also computed in the nucleus pulposus, for all the intervertebral levels. Geometrical parameterization of the models offers potential for the biomechanical investigation of pathologic conditions and surgical procedures, such as spinal fusion and disc arthroplasty, even on a patient-specific basis.

Evaluation of systemic metal diffusion after spinal pedicular fixation with titanium alloy and stainless steel system: a 36-month experimental study in sheep.

It is known that titanium alloys cause more extensive local metallosis due to fretting corrosion than stainless steel implants. The aim of the present study was to investigate possible systemic metal releases (Ti, Al, V, Cr, Ni) in sheep where L4-L5 were implanted with titanium alloy (Ti6Al4V, ASTM F 136) and stainless steel (AISI 316 L). 16 sheep were used: 8 were implanted with Ti6Al4V and 8 with stainless steel. At 6, 12, 24 and 36 months, the following examinations were performed: histology, atomic absorption spectrophotometry (AAS) and scanning electron microscopy (SEM), on liver, lung, kidney, brain, spleen and lumbo-aortic lymph nodes. Hair, urine and arteria blood samples were also analysed by AAS before implantation and at sacrifices. A histologic and ultrastructural study was performed on peri-implant tissues, too. Particular attention was paid to avoid contamination from dissection instruments or use of containers. In basal and in samples at 6 and 12 months, no metals were found in blood, urine, hair or other target tissues of the animals implanted with either Ti6Al4V or stainless steel. Regarding Al, V, Co and Ni, negative results in all tissues and body fluids were obtained also at 24 and 36 months. On the contrary, Ti traces were found in lumbo-aortic lymph nodes and lungs of one sheep only (10 and 30 ng/g, respectively) at 24 months. At 36 months, a systemic diffusion of Ti was observed in all tissues of both sheep instrumented with Ti6Al4V (2-16.5 ng/g), except for body fluids and hair. Metal research in target tissues by light and SEM micro-probe analysis provided negative results. Current data suggest that the amount of Ti found in organs after stable pedicular fixation is extremely low and not biologically available. This observation would lead us to exclude the hypothesis of any toxic reaction and such a release seems to be due to the passive diffusion through lymphatic fluids. Additional studies are needed to confirm if this long-term release of Ti particles might cause tissue damage.

Atomic absorption spectrophotometry (AAS) for the evaluation of metallosis in prostheses and artificial organs: a new approach.

To study the presence of metals in body fluids and tissues after implantation of metallic biomaterials and possible related diseases, a new approach in Atomic Absorption Spectrophotometry (AAS) was developed. This technique was compared to three traditional methods: mineralisation with acid digestion (method A) also known as "wet method", dry ashing (with or without oxygen) (method B); classic Kjeldaal (method C). The new approach (method D) modifies the mineralisation phase and the instrument operating instructions. Al, Na, Cr, K, Ni, Co, Ti, Fe, Hg, Pb, V, Sb and Cu levels were tested with the four methods on bone, muscle, cartilage, skin, brain, lymph nodes, blood, urine, and hair. Test results were checked by the addition method. Results demonstrated a significantly higher percentage of Al, Cr, Ni, Ti and Hg recovery with the new approach. The advantages of method D are no residue, no redox reaction, insignificant loss of analytes and enhanced sensitivity (at ppb level vs ppm of the other methods). This approach should be considered especially when testing heavy metals and complex matrices. Its disadvantages are that it is more time consuming and requires the presence of an operator.

[Neurological manifestations in Marfan's syndrome. A critical review and presentation of a case]. / Manifestazioni neurologiche nella sindrome di Marfan. Revisione critica e presentazione di un caso.

Marfan's syndrome is a heritable disorders of the connective tissue known mainly for its skeletal, cardio-vascular and ocular changes. A recent case of intracranial hematoma in a girl with Marfan's syndrome, hyperkyphosis and scoliosis led the Authors to investigate neurological problems in this syndrome, finding in the literature a greater number of reports than expected.

[Superior mesenteric artery syndrome after surgical correction of severe dorso-lumbar deformities. Authors' experience]. / La sindrome da compasso aorto-mesenterico dopo correzione chirurgica delle gravi deformità del rachide dorso-lombare. Nostra esperienza.

The authors report two cases of superior mesenteric artery syndrome observed in 22 operations for severe spinal deformities. Medical, dietary and postural treatment enabled the situation to be resolved in one case; whereas an intestinal derotation according to Strong-Valdoni was successfully performed in the other. The various surgical alternatives are discussed.

Electrical conductivity measurement: a new technique to detect iatrogenic initial pedicle perforation.

Pedicle screw fixation has achieved significant popularity amongst spinal surgeons for both single and multi-level spinal fusion. Misplacement and pedicle cortical violation occurs in over 20% of screw placement and can result in potential complications such as dysthesia, paraparesis or paraplegia. There have been many advances in techniques available for navigating through the pedicle; however, these techniques are not without drawbacks. A new electrical conductivity-measuring device, previously evaluated on the porcine model to detect the pedicle violation, was evaluated amongst nine European Hospitals to be used in conjunction with the methods currently used in that centre. This new device is based on two original principles; the device is integrated in the drilling or screwing tool. The technology allows real-time detection of perforation through two independent parameters, impedance variation and evoked muscle contractions. Data was collected twofold. Initially, the surgeon was given the device and a comparison was made between the devices ability to detect a breech and the surgeon's ability to detect one using his traditional methods of pedicle preparation. In the second module of the study, the surgeon was limited to using the electrical conductivity detection device as their sole guide to detect pedicle breaches. A comparison was made between the detection ability of the device and the other detection possibilities. Post-operative fine cut CT scanning was used to detect the pedicle breaches. Overall, the 11 trial surgeons performed a total of 521 pedicle drillings on 97 patients. Initially there were 147 drillings with 23 breaches detected. The detection rate of these breaches were 22/23 for the device compared to 10/23 by the surgeon. Over both parts of the study 64 breaches (12.3%) were confirmed on post-operative CT imaging. The electrical conductivity detection device detected 63 of the 64 breaches (98.4%). There was one false negative and four false positives. This gives the device an overall sensitivity of 98% and specificity of 99% for detecting a pedicle breach. The negative predictive value was 99.8%, with a positive predictive value of 94%. No adverse event was noted with the use of the electrical conductivity device. Electrical conductivity monitoring may provide a simple, safe and sensitive method of detecting pedicle breech during routine perforation of the pedicle, in the course of pedicle screw placement.

Posterior endoscopic discectomy (and other procedures).

Percutaneous approaches to lumbar discectomy were somewhat controversial, because of their limited indications. They have not proven to be as effective as standard open lumbar disc surgery, because of longer operating times and some technical problems in addressing all the different aspects of lumbar disc herniations. New percutaneous posterior procedures for lumbar disc disease have been described in the last few years, but the MicroEndoscopic Discectomy (MED) introduced by Foley and Smith in 1997 seems to be the most promising one. MED, and METR'x which evolved from it,. allow the surgeon to address not only contained lumbar disc herniations, but also free-fragment disc pathology and symptomatic lateral recess stenosis secondary to bony hypertrophy. The surgical technique is summarized and some preliminary clinical results of a prospective multicenter study with 13 months' mean follow-up are presented.

Ultrastructure of neuromelanin granules in dog substantia nigra at different ages.

The Authors study through ultrastructural observations the sequence of processes concerning the appearance and the depositing of the neuromelanic pigment in the neurons of dog mesencephalic substantia nigra nucleus at different ages. The results are as follows: in the one year old dog, in a low percentage of lysosome-like oval organelles, few masses, strongly electron-dense, appear on the matrix. In the five year old dog, these masses are deposited and increased in quantity inside many more organelles. In the specimens extracted from older dogs, these organelles, characterized moreover by the presence of lipidic globules, merging in groups, became larger and more irregular. The Authors discuss the results obtained with the ultrastructural observation, compared with those obtained with the optical methods.