Introduction

We report a single-centre, prospective, randomised study for pedicle screw insertion, by using a Computer Assisted Surgery (CAS) technique with three dimension (3D) intra-operative images intensifier versus conventional surgical procedure.

In recent years internal fixation of the spine by using posterior approach with minimally invasive and percutaneous technique were increasingly used in trauma. The percutaneous surgery lose information and navigation is supposed to provide better data because the lost information is found again. We hypothesise that a percutaneous minimal invasive dorsal procedure by using 3D intra-operative imaging for vertebral fractures allows short operating times with correct screw positioning and does not increase radiation exposure.

59 patients were included in this prospective, monocentric and randomised study. 29 patients (108 implants) were operated on by using conventional surgical procedure (CP) and 30 patients (72 implants) were operated on by using a 3D fluoroscopy-based navigation system (3D fluo). In the two groups, a percutaneous approach was performed for transpedicular vertebroplasty or percutaneous pedicle screws insertion. In the two groups surgery was done from T4 level to L5 levels. Patients (54 years old on average) suffered trauma fractures, fragility fractures or degenerative instabilities. Evaluation of screw placement was done by using post-operative CT with two independent radiologists that used Youkilis criteria. Operative and radiation running time were also evaluated.

With percutaneous surgery, the 3D fluo technique was less accurate with 13.88% of misplaced pedicle screws (10/72) compared with 11.11% (12/108) observed with CP. The radiation running time for each vertebra level (two screws) reached on average 0.56 mSv with 3D fluo group compared to 1.57 mSv with the CP group. The time required for instrumentation (one vertebra, two screws) with 3D fluo was 19.75 minutes compared with CP group 9.19 minutes. The results were statistically significant in terms of radiation dose and operative running time (p < 0.05), but not in terms of accuracy (p= 0.24).

With percutaneous procedures, 3D fluoroscopy-based navigation (3D fluo) system has no superiority in terms of operative running time and to a lesser degree in terms of accuracy, as compared to 2D conventional procedure (CP), but the benefit in terms of radiation dose is important. Other advantages of the 3D fluo system are twofold: up-to-date image data of patient anatomy and immediate availability to assess the anatomical position of the implanted screws.

Direct arthroscopic cartilage assessment remains the gold standard. It is recommended by the International Cartilage Repair Society (ICRS) to systematically assess cartilage status during arthroscopy but this examination is highly subjective, poorly reproducible, time-consuming and lacks precision. US has shown good potential for cartilage evaluation but is limited in extra-articular conditions. It is also difficult to manually maintain a perfect perpendicularity between the ultrasound beam and the curved surface of the cartilage. Therefore, we have developed a navigated intra-articular US probe (NIAUS). The NIAUS probe could contribute to a more exhaustive and direct intra-articular evaluation of cartilage integrity. Navigation enables control of the US echo pulse perpendicularity and its localisation relative to the joint. Our objectives were (1) to evaluate automatic cartilage thickness measurement with the NIAUS probe in comparison to high definition MRI on cartilage samples, (2) to generate a real-time 3D map of the thickness parameter on samples, and (3) to demonstrate the feasibility of a full NIAUS probe cartilage scan on a specimen distal femur in arthroscopic conditions.

The NIAUS probe is a 4.5mm probe consisting of a 64 element linear array transducer with a central frequency of 13 MHz and a motorised head. The NIAUS probe is navigated. The rotating US head position is controlled by navigation in order to enable constant perpendicular acquisition of cartilage. The NIAUS probe thickness measurement (1) was evaluated on bone and cartilage samples of 9 tibial plateaus. The cartilage thickness was measured via automatic segmentation. Each sample was also scanned in a high resolution MRI (4,7 Tesla) and cartilage thickness was semi-automatically extracted for comparison. During NIAUS scan, (2) a visual 3D map was generated. Finally (3), we scanned two distal femurs with the NIAUS probe in arthroscopic navigated conditions on one specimen and a 3D map of the distal femur thickness was generated in real time.

NIAUS thickness measurement (1) absolute error compared to MRI for 9 plateaus ranged from 0.15mm to 0.32mm in median, p25=0.07 and 0.18, p75=0.28 and 0.5 respectively. 3D maps of the sample cartilage thickness (2) were generated in real time during the NIAUS scan. The cadaveric procedure (3) was conducted without incident via the two anterior portals and a 3D map of the distal femurs cartilage thickness was generated.

A precise US arthroscopic grading and scoring of cartilage during surgery could help for better standardisation, prediction of results and making “live” decisions. Our in vitro experiment shows good results compared to MRI for NAIUS cartilage thickness measurement, and our cadaveric study demonstrate the feasibility of a NIAUS scan in arthroscopic conditions. Our results are encouraging and a clinical trial is currently being designed for preliminary in vivo NIAUS evaluations of cartilage thickness compared to MRI.

Purpose

To investigate the prognostic effect of surgical margins in soft tissue sarcoma on Local Recurrence (LRFS), Metastasis (MFS) and Disease Free Survival (DFS).

Introduction

From the many human studies that attempt to identify genes for adolescent idiopathic scoliosis (AIS), the view emerging is that AIS is a complex genetic disorder with many predisposing genes exhibiting complex phenotypes through environmental interactions. Although advancements in genomic technology are transforming how we undertake genetic and genomic studies, only some success has been reached in deciphering complex diseases such as AIS. Moreover, the present challenge in AIS research is to understand the causative and correlative effects of discovered genetic perturbations. An important limitation to such investigations has been the absence of a method that can easily stratify patients with AIS.

To overcome these challenges, we have developed a functional test that allows us to stratify patients with AIS into three functional subgroups, representing specific endophenotypes. Interestingly, in families with multiple cases of AIS, a specific endophenotype is shared among the affected family members, indicating that such a transmission is inherited. Moreover, increased vulnerability to AIS could be attributable to sustained exposure to osteopontin (OPN), a multifunctional cytokine that appears to be at the origin of the Gi-coupled receptor signalling dysfunction discovered in AIS. We examined the molecular expression profiles of patients with AIS and their response to OPN.

Background: A navigated 8 in 1 femoral cutting guide for TKA that does not require primary fixation or intramedullary guides was developed. The hypothesis of our study were twofold: 1) the navigation system allows for precise alignment and adjustment of a new femoral 8 in 1 cutting guide with negligible variance in the initially planned vs. achieved implant position; 2) resulting femoral cuts are very accurate without relevant cutting errors.

Material and Methods: We demonstrate our approach with the Universal Knee Instrument (UKI, Precimed Inc. USA), a versatile 8 in 1 TKA guide designed to perform all femoral cuts with a single jig. We integrated an array of “adjustable constraints” into the UKI by machining four threaded holes directly through the template. Adaptation to a navigation system has been performed by integrating the adjustable constraints protocol on the open platform Surgetics Station (PRAXIM-medivision, France), which uses image-free BoneMorphing technology. Based on navigated bone morphing the required preadjustment of the guide was done mechanically, with depth control by mini screws. Testings on 10 cadavers compared the planned vs. achieved positions of the jig before, after fixation, final implant position and planned vs. achieved cutting procedures.

Results: Results revealed for valgus/varus deviations before fixation −0.1°±0.7°, after 0.0°±0.8° (p=0.51), final implant position 0.9°±1.7° (p=0.93). For flexion before fixation −0.3°±1.3° after −0.3°±1.8° (p=0.44), final position 2.9°±2.5° (p=0.65). Distal cut height before fixation 0.0°±0.4°, after 0.1°±0.3° (p=0.61), final position 0.3°±1.0° (p=0.1). Axial rotation before −0.3°±1.1°, after fixation 0.2°±1.4° (p=0.57), final implant position 0.8°±2.7° (p=0.89). Anterior-posterior positions before fixation 0.7°±1.4°, after 1.0°±1.6° (p=0.27), final position 3.4°±1.3° (p=0.13). Highest deviations in the planned vs. actual cut position was found for the posterior cut −3.1°±2.4° in sagittal and anterior cut 0.8°±1.9° in the coronal plane. The highest mean errors in the final implant position where on the order of 3 degrees/mm in flexion and anterior-posterior positioning.

Conclusion: A novel ‘CAS-enabled 8 in 1 jig’ has been developed and validated. The system allows for direct execution of a complex, multi-planar CAS plan with single navigated device. The instrumentation is considerably simplified and eliminates the problems associated with sequential jigs.

Planning resection margins for soft tissue sarcomas is a compromise between functional sacrifice and therapeutic safety. In practice, the histological analysis of the resection margins often shows that the preoperative objective has not been achieved. We studied the prevalence and factors of risk of this surgical outcome.

This was a prospective monocentric study of 133 patients. The resection objectives, pathological results and operative reports were examined. Margins were classified according to the UICC (R0, R1, R2). Data were included in a grid which also included patient related and tumour related preoperative information. Inadequate resection was noted as planned R0 with R1 or R2 outcome. Statistical analysis was performed with Statview 5.0.

The prevalence of inadequate resection was 25.2%. Among the factors analysed, the aspect of tumor limits (badely or well defined) was significantly related to poor surgical results (odds ration 2.85 [1.47–5.52], p < 0.005). No other significant risk factor could be identified. Margins greater than two mm were associated with adequate surgery in every case.

No preoperative risk factor predictive of inadequate resection margins was clearly identified in this study. Postoperatively, the microscopic aspect of the proliferation limits at the final pathology examination is for us significantly associated with inadequate resection. However the current classification for resection margins lacks precision, especially regarding R0 and R1 when margins are small, in defining the risk of inadequate resection. This appears to be the source of the difficulties encountered in interpreting pathology samples and therefore in choosing the right treatment. Further follow-up is needed to clarify such questions.

We conclude that where resection margins are thin (less than two mm), the definition of R0 or R1 resections should be clarified to optimize patient care. To achieve this, potential risk factors for inadequate resection such as tumor limits should be taken into account and further studied.

Purpose of the study: Planning resection margins for soft tissue sarcomas is a compromise between functional sacrifice and therapeutic safety. In practice, the histological analysis of the resection margins often shows that the preoperative objective has not been achieved. We defined this as anatomo-surgical discordance and studied its prevalence and factors of risk.

Materials and Methods: This was a prospective mono-centric study of 133 patients. The resection objectives, pathological results and operative reports were examined. Margins were classified according to the UICC (R0, R1, R2). Data were included in a grid which also included patient related and tumour related preoperative information. Discordance was noted as planned R0 with R1 or R2 outcome. Statistical analysis was performed with Statview 5.0.

Results: The prevalence of anatomo-surgical discordance was 25.2%. Among the factors analysed, the aspect of the margins was significantly related to poor surgical results (odds ration 2.85 [1.47–5.52], p=0.0031). No other significant risk factor could be identified. Margins greater than 2mm were associated with adequate surgery in every case.

Discussion: No preoperative risk factor predictive of inadequate resection margins was clearly identified in this study. Postoperatively, the microscopic aspect of the proliferation margins at the final pathology examination is for us significantly associated with inadequate resection. But the current classification of resection margins (R0 and R1), especially for poorly delimited tumours lacks precision. This appears to be the source of the difficulties encountered in interpreting pathology samples and therefore in choosing the right treatment. Further follow-up is needed to clarify such questions.

Conclusion: We were unable to identify any preoperative factor predictive of inadequate resection. A poorly-defined microscopic aspect of the tumour is significantly associated with inadequate resection but the current classification system raises certain interpretation problems for resections with margins less than 2 mm. Concerning these cases, the definition of margins must be clarified to optimize patient care.

Introduction: Over the last three years, we have demonstrated the complex role of melatonin, a hormone produces mainly in the brain, in the development of scoliosis and in particular by reporting for the first time that cells from AIS patients cannot respond to melatonin, which contrasted with similar cells isolated from healthy subjects. We have determined that this phenomenon is caused by chemical modifications affecting the activity of Gi proteins, a group of small proteins normally associated with both melatonin receptors. Interestingly, previous studies showed that melatonin deficiency could also induce a scoliosis suggesting that the asymmetrical growth of the spine in humans and in melatonin deficient animals could be caused by a common downstream effector regulated by melatonin. This study was then designed to determine and characterise the early biochemical, cellular and molecular changes underlying the formation of spinal deformities in growing pinealectomized chicken and in bipedal C57Bl/6 mice, a naturally melatonin deficient strain of mice.

Methods: For this study, 145 newly hatched chickens (Mountain Hubbard) were purchased at a local hatchery and divided into three distinct groups. First group, pinealectomized (n=100), underwent complete removal of the pineal gland. The second group, sham (n=20), underwent superficial cranial incision without the ablation of the pineal gland. The third group, control (n=25), the chickens did not undergo any surgical procedure. All surgeries were performed by the same surgeon between day three and five after hatching. At days 14, 21 and 28 chicken underwent radiographic examination with a DEXA bone densitometer (PIXImus II, Lunar Corp., Madison, WI). Each digital image was evaluated for the presence of scoliosis and the degree of curvature was measured. Cobb angle threshold value of 10° and higher was retained as a significant scoliotic condition. Blood samples (1 to 2 ml) were taken from a peripheral wing vein of each chicken (from 6 am to 9 am) at the age of 14, 21 and 28 days. Sera were collected by centrifugation and immediately stored at −80°C until assayed. Serum melatonin concentrations were determined using an ELISA method (IBL, Hamburg, Germany). At day 28, chicken were euthanised and tissues were collected to extract mRNA for expression analysis or proteins for subsequent detection. C57Bl/6 mice (n=50) were purchased from Charles-Rivers and bipedal mice were generated by removing the forelimbs and tail after weaning (three weeks old) according to a protocol approved by our institutional animal health care committee. Sera of AIS patients and matched healthy controls were also analysed to determine the levels of circulating P factor using an ELISA assay.

Results: Our results demonstrated a more dynamic variation of circulating melatonin level only in pinealectomised chicken developing a scoliosis, which allowed us to separate scoliotic chicken in two distinct groups. In the first group, the animals showed a biphasic response with a strong decrease of melatonin level between days 14 to 21, followed by a rapid recovery to almost reach the normal values at day 28. In the second group, pinealectomised chickens showed a linear decrease of circulating melatonin over the three-week period while, non-scoliotic pinealectomised chicken showed non-significant variations in melatonin concentration with values close to those obtained with the shams. At the molecular level, expression analysis demonstrated higher expression of a gene encoding a protein that has been termed P factor only in paraspinal muscles of pinealectomized chicken developing a scoliosis. Accumulation of P factor was also confirmed at the protein level by Western blot analysis. Bipedal C57Bl/6 mice, which are naturally melatonin deficient, developed also scoliotic deformities in a proportion of 45% over a two-month period. Interestingly, we observed that genetically modified mice devoid of P factor (n=60) or one of its receptor (n=40) in the same genomic background (C57Bl/6) cannot develop a scoliosis in the same conditions. Moreover, P factor circulating levels in scoliotic patients showed a 2–4 fold increase when compared to healthy matched individuals.

Conclusions: These results showed for the first time a more dynamic variation in circulating melatonin levels among pinealectomised chicken, which was unsuspected by previous studies. Interestingly, a transient decrease of circulating melatonin level was sufficient to induce scoliotic deformities during the first two weeks even if melatonin concentration was subsequently recovered a week later. This may explain why melatonin injection in pinealectomised chicken is not always efficient in preventing scoliosis. Taken together, these observations further suggest that a melatonin decrease below a certain threshold during a specific postnatal window may be sufficient to trigger a scoliosis and reconcile the data concerning AIS patients showing in most of the studies no significant variation when analysed at late stages. The study of early molecular changes in animal models also led us to identify a novel factor, which appears essential to initiate scoliosis through a specific signalling action. The clinical relevance of the P factor in AIS and related spinal syndromes is further strengthened by the detection of high levels of P factor only in scoliotic patients and could pave the way for the development of innovative diagnosis tools as well as the first pharmacological treatments to prevent scoliosis deformities in children.

Research project supported by La Fondation Yves Cotrel de l’Institut de France

Introduction: Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, which appears to be caused by a melatonin signalling dysfunction proved recently in osteoblasts. This pathology occurs and progresses during the time of pre-puberty and puberty growth. This period is known to be under the hormonal control and coincides with many biological changes related to the secretion of estrogens, of which estradiol (E2) is the most active. The female prevalence of AIS disease is clearly evident. Indeed, in Quebec the spine deformities considered clinically significant (at least 11° of deformity) are found in a girl:boy ratio of approximately 2:1 for reduced scoliosis, and this ratio increases to 10:1 for scoliosis of more than 30o of deformation. However, the reason for this female prevalence as well as the role of estrogens and estrogen receptors in AIS is not clear despite the fact that these hormones are known for their impact on bone and bone growth, including the spine.

The purpose of the present study was to investigate the role of E2 on the responsiveness of the AIS cells to the melatonin, to determine the expression of estrogens receptors (ERα and ERβ) in AIS tissues and to clarify the impact of estrogen receptor gene polymorphisms in the pathogenesis of AIS.

Methodology: The effects of oestrogen on the AIS osteoblasts (n=10) response to the melatonin was determined by measuring the reduction of forskolin-induced cAMP accumulation. The forskolin treated osteoblasts were incubated in the presence of increasing amounts of melatonin (10–11 to 10-5 M) with or without physiological concentrations (10-10 M) of 17-β-estradiol for 16 hours, and the intracellular cAMP measured by radio-immunoassay using Biotrak Kit. Using RT-PCR, we determined ERα and ERβ mRNA expression in osteoblasts from AIS patients (n=14). Polymorphisms of the first intron of the ERα gene, which contains the XbaI and PvuII polymorphisms, were investigated by PCR following digestion with restriction enzyme and using the genomic DNA from lymphocytes isolated from scoliotic patients (n=33). Using the restriction enzymes XbaI and PvuII, the allelic variants XX, Xx, xx, PP, Pp, and pp were identified in 33 AIS patients (uppercase letters represent absence, and lowercase letters represent presence of restriction sites).

Results: The intracellular level of cAMP was significantly increased (p< 0.01) in the presence of a physiological concentration of 17-β-estradiol (10-10 M) when compared to the level observed in the presence of melatonin alone (10-9 M) (melatonin + estradiol: 109.46 ± 20.07; melatonin 76.09 ± 12.32 (mean ± SD)). As previously described by Dr Moreau’s team, the same pattern (three type of response to melatonin) takes place in the presence of 17-β-estradiol. We observed the loss of ERβ gene expression in 8/ 14 AIS patients contrasting with ERα gene expression that was found in all AIS patients. The XbaI and PvuII polymorphisms were found in 70% (23/33) and 80% (26/33) of the cases respectively. Of the 33 cases, 21 presented both digestion sites, 24 presented PvuII digestion site (6 homozygote, 18 heterozygote) and 23 (8 homozygote, 15 heterozygote) presented XbaI digestion site. The allelic variants were found as follows: XX: n=8, Xx: n=15, xx: n=8, PP: n=6, Pp: n=18 and pp: n=6. Classified by their location in the spine, seven right thoracic, one left thoracic, one right thoracolumbar, three left thoracolumbar and nine right thoracic-left lumbar were found among the patients presenting PvuII positive polymorphism. Among the patients with XbaI positive polymorphism, six right thoracic, one left thoracic, one right thoracolumbar, three left thoracolumbar and eight right thoracic left lumbar were found.

Conclusion: These results show the antagonistic effects of the 17-β-estradiol on AIS osteoblasts response to the melatonin. Thus estrogens interference with melatonin signalling activity would act as a triggering or aggravating factor in the pathogenesis of AIS. At the molecular level, it is possible that estrogens attenuate the response of AIS cells to melatonin through the desensitization of melatonin receptors. The loss of ERβ expression in a significant number of AIS patients appears to be important for the change of the ERα/ERβ receptors ratio that consequently may perhaps alter estrogens signalling pathways. The XbaI and PvuII polymorphisms are present in a significant number of AIS patients but this was not dependant of the curve pattern. These results clearly support the interplays and crosstalk between estrogens and melatonin signalling pathways in AIS aetiopathogenesis.

Supported by the Fondation Yves Cotrel, Institut de France

Introduction: Experimental pinealectomy in chickens shortly after hatch produces scoliosis with morphological characteristics similar to that of human idiopathic scoliosis (Coillard et al., 1996). The objective of this study was to develop a finite element model (FEM) incorporating vertebral growth to analyse how bone growth modulation by mechanical loading affects development of scoliosis in chicken.

Materials and Methods: We have adapted the experimental set-up of Bagnall et al. (1999) to study spine growth of pinealectomised chickens. Three groups were followed for a period of six weeks:

wild-type (controls) (n=25);

The experimental data was used to adapt a FEM previously developed to simulate the scoliosis deformation process in human (Villemure et al. 2002). The FEM consists of 7 thoracic vertebrae and the first lumbar, the intervertebral discs and the zygapophyseal joints. The geometry was measured on specimens using a calliper. The material properties of human spines were used as initial approximation. The growth process included a baseline growth (0.130 mm/day) and a growth modulation behaviour proportional to the stress and to a sensitivity factor. It was implemented through an iterative process (from the 14th to the 28th day). Asymmetric loads (2–14 Nmm) were applied to represent different paravertebral muscle abnormalities influenced by the induced melatonin defect.

Results: Within the pinealectomised group, 55% of the animals (n = 42) developed a scoliosis. In the FEM model, by varying the value of the applied moment, different scoliosis configurations were simulated. The resulting Cobb angle varied between 6° and 37°, while the maximal vertebral wedging appeared at T4 or T5 (range between 5° to 28°). A descriptive comparison of the simulation results with the experimental deformation patterns (n = 41; mean Cobb angle: 26°) was made as a preliminary validation. In 2 typical cases, the scoliotic shapes were quite similar to that seen in the scoliotic chickens.

Discussion and Conclusion: The basic mechanisms by which the metabolism of the growing spine is affected by mechanical factors remain not well known, and especially the role of tissue remodelling and growth adaptation in scoliosis. The agreement between the experimental study and preliminary simulation results shows the feasibility of the model to simulate the scoliotic deformation process in pinealectomised chickens. When completely developed and validated this modelling approach could help investigating the pathomechanisms involved in the scoliotic deformation process. Especially, computer simulations could be used to complement bio-molecular and mechanobiological studies concerning the neuroendocrinal hypothesis implicating melatonin signalling dysfunction, which could trigger a complex cascade of molecules and mechanoreceptors leading to an accumulation of specific factors in specialised tissues (Moreau et al. 2004), directly or indirectly implicated in proprioception, and which can be implicated in the pathomechanisms of scoliotic deformities.

Introduction: Spinal deformities and scoliosis in particular, represent the most prevalent type of orthopaedic deformities in children and adolescents. At present, the most significant problem for clinicians is that there is no proven method or test available to identify children or adolescents at risk of developing AIS or to identify which of the affected individuals are at risk of progression. As a consequence, the application of current treatments, such as bracing or surgical correction, has to be delayed until a significant deformity is detected or until a significant progression is clearly demonstrated, resulting in a delayed and less optimal treatment. Among patients with AIS needing treatment, 80% to 90% will be treated by brace and 10% will need surgery to correct the deformity by spinal instrumentation and fusion of the thoracic and/or lumbar spine. About 15000 such surgeries are done every year in North America, resulting in significant psychological and physical morbidity. Moreover, there is no pharmacotherapy available to either prevent or reduce spinal deformities due mainly to our limited knowledge of AIS aetiopathogenesis. We have recently reconciled the role of melatonin in AIS aetiopathogenesis by demonstrating a melatonin signalling dysfunction occurring in a cell autonomous manner in cells derived from AIS patients exhibiting severe scoliotic deformities. This defect could potentially explain the majority of abnormalities reported in AIS since melatonin receptors and signalling activities are normally found in all tissues and systems affected in AIS, thus offering a very innovative and unifying concept to explain the aetiology of AIS. Moreover, several lines of evidence suggested that inactivation of Gi proteins by an increased phosphorylation of serine residues could be at the source of this signalling defect in AIS. The goals of that study were to assess the possibility to establish a molecular classification of AIS patients and to demonstrate the feasibility to correct this melatonin signalling defect in cells of AIS patients using therapeutic compounds.

Methods: Primary cell cultures were prepared from musculoskeletal tissues of AIS patients (n=150) and age- and gender-matched controls (n=35) obtained intra-operatively. An informed consent was obtained for each subject as approved by our Institutional Ethical Committee. The osteoblasts, the bone-forming cells, were selected to assess whether or not an alteration of melatonin signalling pathway occurs in AIS and accordingly to identify which component of the melatonin transduction machinery could be involved. Co-immunoprecipitation experiments with membrane extracts were performed to identify interacting molecules with key components of melatonin signal transduction machinery. The functionality of melatonin signalling was assessed by investigating the ability of Gi proteins to inhibit stimulated adenyl cyclase activity in osteoblast cultures. Inhibition curves of cAMP production were generated by adding melatonin to the forskolin-containing samples in concentrations ranging from 10-11M to 10-5M in a final volume of 1 ml of _-MEM media containing 0.2% bovine serum albumin (BSA) alone or in presence of 2.5 _M of therapeutic compound A or therapeutic compound B (the nature of both compounds tested cannot be disclosed at this stage). The cAMP content was determined using an enzyme immunoassay kit (Amersham-Pharmacia Biosciences). All assays were performed in duplicate. A non-parametric test, the Wilcoxon matched pairs test was performed to verify the significance between 2 means. Significance was defined as P< 0.05.

Results: Osteoblasts from patients with AIS showed a lack or a markedly reduced inhibition of forskolin-stimulated adenyl cyclase activity by melatonin generating three distinct response-curves corresponding to three functional groups. In order to identify candidate genes involved in AIS aetiopathogenesis, we focused our attention on known kinases and phosphatases modulating Gi protein functions and characterised their interacting partners. Interestingly, PKC_ was initially targeted owing to its property to phosphorylate Gi proteins in vitro. Indeed, in normal osteoblast interactions occurring between MT2 melatonin receptor and RACK1 (a cytosolic protein that bind to and stabilises the actives form of PKC and permits its translocation to different sites within the cells) and PKC_ were detected although those interactions among different AIS patients were altered. Interestingly, treatment with compound A or B rescued melatonin signal defect in cells derived from 36% and 47% of AIS patients respectively. Overall, melatonin signal transduction was restored in cells of 64% of AIS patients (23/36) when treated by one of these therapeutic compounds.

Conclusions: The functional classification of AIS patients is correlated at the molecular level by distinct interactions between key molecules normally involved in melatonin signal transduction in spite that these patients exhibited the same curve type (right thoracic, Lenke type 1). Collectively, these data strongly argue that traditional curve pattern classification is not a relevant stratification of AIS patients to identify its genetic causes. Moreover, using that molecular system we have demonstrated also the possibility to identify therapeutic compounds to rescue the melatonin signalling defect observed in AIS without any prior knowledge of mutations in any defective genes causing AIS because we are measuring a function.

Research project supported by La Fondation Yves Cotrel de l’Institut de France

Purpose: Osteosarcoma (OS) is the most common type of cancer in children. OS demonstrates aggressive growth with a high risk of early, pulmonary metastasis. Here we investigated expression of malignancy-related factors in six osteosarcoma cell lines (SaOS, MNNG/ HOS, MG63, SW1353, SKES, SJSA), ten biopsies of primary OS and OS derived cells.

Methods: For this project, we are using RT-PCR and immunohistochemistry to detect PARP, ET-1, ETA and ETB receptor. We also, examined the expression of osteocrin by in situ hybridization. Zymography and Northern Blot were use to observe the presence of gela-tinases (MMP-2 and MMP-9) and finally, we showed the presence of ET-1(1–31) by Elisa-immunoassay.

Results: In OS tissues and cells, we observed ET-1, MMP-2, and ETA receptor overexpression, in contrast to under-expression of MMP-9 and ETB receptor. Additionally, in high malignant OS cells, MMP-2, MMP-3, MMP-13 and PARP were overexpressed and TIMP-1, TIMP-2 and TIMP-4 expressed at low levels. Using a new specific immunoassay for ET-1 (1–31) we showed presence of this alternative form of ET-1 in OS-derived cells. We also showed that, in cells exposed to ET-1, the inhibition of NF-kB pathway (NF-kB is an ubiquitous transcription factor playing a central role in differentiation, proliferation and malign transformation) drastically blocked MMP-2 production and activity, and that ET-1 induces NF-kB p65 unit translocation to the nucleus.

Conclusions: Thus, we concluded that expression of malignancy-related factors in parallel to the histological evaluation of these tumors, could be beneficial for malignancy understanding. Tumor cells invasion and aggressiveness of OS cells are the results, in part, of the tumorigenic potential of alternative forms of ET-1 generated by MMPs and enhanced in malignant microenvironment. These factors could be predictive for tumoral progression. Funding: Educational Grant from the Canadian Orthopaedic Foundation. Funding Parties: Canadian Orthopaedic Foundation (Carrol A. Laurin Award) and MENTOR program of CIHR

Purpose: Recently, we highlighted a dysfunction in the melatonin signalling pathway in the osteoblasts from adolescent idiopathic scoliosis patients (AIS). The objective of this project is to verify if in the cells coming from the SIA patients, estrogens interfere with melatonin signalling pathways and to identify mechanisms through which these effects are carried out.

Methods: The effects of estrogens on the melatonin signalling pathway, in osteoblasts from AIS patients (n=7), were determined by measuring the capacity of the Gi proteins to inhibit the accumulation of cAMP. The osteoblasts were incubated in the presence of increasing amounts of melatonin (10–11 to 10–5 M) with or without 17-& #946;-estradiol in physiological concentrations (10–10 M) (n=7). Moreover, coimmunoprecipitations using anti-phosphoserine antibodies were carried out and then followed with a Western blot in order to detect melatonin receptors (MT1 and MT2).

Results: The intracellular level of cAMP is higher in the presence of a physiological concentration of 17-& #946;-estradiol among scoliotic patients compared to the level observed in the presence of melatonin alone. Moreover, the preliminary results of the coimmunoprecipitations seem to show an increase in the phosphorylation of proteins interacting with MT1 and MT2 receptors. The precise nature of these proteins remains to be identified.

Conclusions: These results seem to show the antagonistic effects of the 17-& #946;-estradiol on the melatonin signalling pathway in the osteoblasts from AIS patients. However, more cAMP dosages in the presence and absence of 17-& #946;-estradiol are underway so as to increase the number of patients. The results of this study could contribute to the development of the first molecular screening tests as well as the development of new therapeutic approaches.

Purpose: Osteosarcoma is a rare disease after the age of 40 years. Most malignant bone tumours at this age are secondary lesions and the primary tumours observed are generally chondrosarcomas. We report a small retrospective series to study the characteristic features and point out diagnostic difficultis as well as the prognosis in this age group.

Material and methods: We included in this series histologically proven osteosarcomas observed in patients over the age of 40 years who had been treated between 1990 and September 2002, excluding tumours occurring in patients with predisposing conditions (radiation, paget). The series included six women and nine men, mean age 54 years (41–79). Only three patients were over 60.

Results: During this same period, 59 osteosarcomas were treated. Diagnosis before 40 years corresponded to 25% of the cases reported at our interdisciplinary reunion. Three patients were referred after inadequate treatment, i.e. curettage-filling without diagnosis. Two others had undergone surgery for symptoms related to the osteosarcoma. Finally three patients had an erroneous or doubtful pathological diagnosis. There was no histological specificity or tumour localisation. The lower femur predominated. Ten patients had undergone neo-adjuvant chemotherapy with only two responders. Marginal surgery was achieved in three cases, intralesional surgery with immediate revision in one and large resection in ten. Six patients died from their disease (within two years of diagnosis) and one developed postoperative pulmonary embolism (46%). Two patients were living with active disease. Only six patients were living disease-free (40%). These included one patient with resected pulmonary metastasis and one patient who had experienced two episodes of locoregional recurrence which were operated (follow-up 12 and 8 months from last event). Four patients (26%) were in complete remission without any event since treatment (including two low-grade tumours).

Conclusion: Osteosarcoma after 40 years is, in our experience, more frequent than reported in the literature. In at least six of our patients (40%) this tumour was mis-diagnosed leading to non-optimal treatment. The prognosis is very poor with only 26% of the patients in complete remission without an event since treatment.