Aims

This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

Aims. Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods. The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Results. Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Conclusion. Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis. Cite this article: Bone Joint Res 2023;12(11):677–690

Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model were constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. We demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α–impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α–induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Our results revealed that melatonin can reverse TNF-α–impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD

Cite this article: Bone Joint Res 2023;12(3):199–201.

Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the transcription factor Nrf2 was up- or downregulated when the melatonin receptor was activated or blocked by melatonin or luzindole, respectively. Conclusion. Melatonin protects NPCs against reactive oxygen species-induced pyroptosis by upregulating the transcription factor Nrf2 via melatonin receptors. Cite this article: Bone Joint Res 2023;12(3):202–211

Aims. This study examined whether systemic administration of melatonin would have different effects on osseointegration in ovariectomized (OVX) rats, depending on whether this was administered during the day or night. Methods. In this study, a titanium rod was implanted in the medullary cavity of one femoral metaphysis in OVX rats, and then the rats were randomly divided into four groups: Sham group (Sham, n = 10), OVX rat group (OVX, n = 10), melatonin day treatment group (OVX + MD, n = 10), and melatonin night treatment group (OVX + MN, n = 10). The OVX + MD and OVX + MN rats were treated with 30 mg/kg/day melatonin at 9 am and 9 pm, respectively, for 12 weeks. At the end of the research, the rats were killed to obtain bilateral femora and blood samples for evaluation. Results. Micro-CT and histological evaluation showed that the bone microscopic parameters of femoral metaphysis trabecular bone and bone tissue around the titanium rod in the OVX + MD group demonstrated higher bone mineral density, bone volume fraction, trabecular number, connective density, trabecular thickness, and lower trabecular speculation (p = 0.004) than the OVX + MN group. Moreover, the biomechanical parameters of the OVX + MD group showed higher pull-out test and three-point bending test values, including fixation strength, interface stiffness, energy to failure, energy at break, ultimate load, and elastic modulus (p = 0.012) than the OVX + MN group. In addition, the bone metabolism index and oxidative stress indicators of the OVX + MD group show lower values of Type I collagen cross-linked C-telopeptide, procollagen type 1 N propeptide, and malondialdehyde (p = 0.013), and higher values of TAC and SOD (p = 0.002) compared with the OVX + MN group. Conclusion. The results of our study suggest that systemic administration with melatonin at 9 am may improve the initial osseointegration of titanium rods under osteoporotic conditions more effectively than administration at 9 pm. Cite this article: Bone Joint Res 2022;11(11):751–762

Adolescent idiopathic scoliosis (AIS), defined by an age at presentation of 11 to 18 years, has a prevalence of 0.47% and accounts for approximately 90% of all cases of idiopathic scoliosis. Despite decades of research, the exact aetiology of AIS remains unknown. It is becoming evident that it is the result of a complex interplay of genetic, internal, and environmental factors. It has been hypothesized that genetic variants act as the initial trigger that allow epigenetic factors to propagate AIS, which could also explain the wide phenotypic variation in the presentation of the disorder. A better understanding of the underlying aetiological mechanisms could help to establish the diagnosis earlier and allow a more accurate prediction of deformity progression. This, in turn, would prompt imaging and therapeutic intervention at the appropriate time, thereby achieving the best clinical outcome for this group of patients.

Cite this article: Bone Joint J 2022;104-B(8):915–921.

Aims

The aim of this study was to determine whether there is an increased prevalence of scoliosis in patients who have suffered from a haematopoietic malignancy in childhood.

The October 2014 Research Roundup. 360 . looks at: unpicking syndesmotic injuries: CT scans evaluated; surgical scrub suits and sterility in theatre; continuous passive motion and knee injuries; whether pain at night is melatonin related;venous thromboembolic disease following spinal surgery; clots in lower limb plasters; immune-competent cells in Achilles tendinopathy; and infection in orthopaedics

Introduction. Melatonin-deficient rats are known to develop scoliosis when rendered bipedal. In a previous study we have shown that melatonin-deficient bipedal mice with scoliosis had lower bone density than did mice without scoliosis. Published work suggests that children with AIS have lower bone density than do healthy children. The aim of this study is to establish whether osteoporosis causes scoliosis. We hypothesised that bipedal rats with lower bone density would have increased spinal malalignment compared with the control group. Methods. 50 female Sprague-Dawley rats were rendered bipedal at 3 weeks of age by amputation of the forelimbs and tails. Two groups were formed: control group (n=25), in which rats received no drug; and the experiment group (n=25), in which rats received daily subcutaneous 1 U/g heparin injections. Animals were kept in standard cages, and food and water was provided at the top of the cages to encourage more time standing erect. DEXA scans were done on week 4 to assess bone density. Radiographs were taken on week 40 to assess spinal alignment in both control and experiment groups. Results. 19 rats in the heparin group and 23 rats in the control group were available for evaluation at the end of the study. At week 4, DEXA scans showed significant difference between the bone densities of the control and heparin groups (p<0·05), with the heparin group having lower bone density. The incidence of curves between the heparin and control groups were not statistically significant (p>0·01) (table). The magnitude of curves in scoliotic rats for the heparin group was 11·8° (SD 3·75) and for the control group 10° (4·3). The difference between the groups was not significant (p>0·05). Conclusions. This study involved rats with normal melatonin levels and both groups showed a high frequency of scoliosis incidence. Although no significant differences were recorded between groups, the results suggest that bipedality is a cause for scoliosis, and low bone mineral density may further increase this tendency

The aim of school screening is to identify most or all the individuals with unrecognized idiopathic scoliosis (IS) at an early stage when a less invasive treatment is more effective. The present study summarises the contribution of school screening in research of IS epidemiology, natural history and aetiology. In addition, school screening is a unique tool for research of IS in humans, as in most published articles, all aetiopathogenetic factors are studied in animals and not in humans. Such contribution is beyond the original aim of school screening but is very important to expand our knowledge and adequately understand the pathogenesis of IS. The role of biological factors such as the menarche, the lateralization of the brain, the handedness, the thoracic cage, the intervertebral disc, the melatonin secretion, as well as the role of environmental factors such as the light and the impact of the geographical latitude in IS prevalence were studied in children referred from school screening. The present study provides evidence to support that school screening programs should be continued not only for early detection of IS but also as a basis for epidemiological surveys until we learn much more about the aetiology of IS

Melatonin’s concentration is high in early childhood and declines gradually thereafter. In the elderly serum melatonin levels are very low. Melatonin, the “light of night”, among other functions is involved in human sexual maturation and in osteogenesis. Hormesis is the response of cells or organisms to an exogenous (eg drug or toxin) or intrinsic factors (eg hormone), where the factor induces stimulatory or beneficial effects at low doses and inhibitory or adverse effects at high doses [bimodal dose-response] or vice versa. At the age around 10 years, when idiopathic scoliosis may appear, the circulating melatonin level is about 120 pg/ml – positive hormesis for menses – and menarche appears. Melatonin deficiency may result in a delay of the age at menarche and consequently the girl is susceptible to scoliosis. In these terms melatonin could be certainly involved in the scoliosis pathogenesis. Around the age of 45 years when the circulating melatonin levels are about 20 pg/ml – negative hormesis for menses, menopause starts and the woman has an increased risk for osteoporosis and fractures. It is documented the bone-protecting effect of melatonin in ovariectomized rats which can depend in part on the free radical scavenging properties of melatonin. Additionally, melatonin may impair development of osteopenia associated with senescence by improving non-rapid eye movement sleep and restoring GH secretion. Whether modulation of melatonin blood levels can be used as a novel mode of therapy for scoliosis and augmenting bone mass in diseases deserves to be studied

Introduction: Reactive oxygen species (ROS) have important roles in the pathogenesis of ischemia reperfusion injury (I/R) of skeletal muscles Melatonin was proved to be an antioxidant agent and many experimental models showed that it reduces I/R injury in many tissues. The objective of present study was to detect protective antioxidant effect of melatonin on I/R injury of skeletal muscles. Material and Methods: Albimino wistar rats were randomly allocated into 3 groups. There were 8, 10, 10 rats in sham, I/R and I/R + melatonin (Mel) groups respectively. Right hind limb ischemia was achieved by clamping femoral arteries in all groups except for control group. Melatonin (10 mg/kg) was administered intraperitoneally in I/R + Mel group 48, 24, 1 hour before reperfusion. After a period of 2 hour ischemia followed by 1.5 hour reperfusion, muscles and venous blood samples were collected for biochemical analysis and histopathological examination. Plasma antioksidant enzyme activities of süperoxide dismutase (SOD), glutathion peroxidase (GSH-Px), and levels of MDA and NO. were investigated. Enzyme activities of catalase (CAT), protein carbonyl (PC), SOD, GSH-Px and levels of MDA and NO. were analysed in muscle tissues. Results: Antioxidant enzyme activities and levels of MDA and NO. in plasma were significantly higher in I/R group compared to control group (p< 0,001). Muscle tissues of I/R groups revealed significant higher antioxidant enzyme activity and MDA, NO. levels with respect to control group (p< 0,001). Levels of these parameters in muscle and plasma revealed significant reduction in I/R + Mel group with respect to I/R group (p< 0.001). Histopathological examination of ischemic muscles in I/R group showed significant degeneration and inflammation compared to control group whereas melatonin administered ischemic muscles showed significant reduction of degeneration and inflammation with respect to I/R group (p< 0.001). Conclusions: Levels of NO. and MDA and antioxidant enzyme activity were significantly higher and also revealed significant degeneration and inflammation in I/R group. These results support the opinion that ROS is an important factor in the pathogenesis of I/R injury in skeletal muscles. We attribute the increasing enzyme activities in I/R group to a compensatory mechanism against ROS. Levels of NO. and MDA and antioxidant enzyme activity in tissue and plasma of I/R + Mel group were significantly lower and additionally revealed significant improvement in inflammation and degenaration. This proves the potential ROS scavenging effect of melatonin in reduction of I/R injury. In conclusion we suggest that melatonin may be used in the treatment of I/R injury due extremity injuries with vascular compromise, extremity surgery with prolonged tourniquet time and compartment syndrome

AIS has different image than paralytic scoliosis or scoliosis accompanying some diseases of the spinal cord in electromyographical and electroneurographical examinations (EMG and ENG). These differences are concerned to different progression, characteristic properties in skeletal system pathology or curves angles at the thoracic and lumbosacral spine. There are always two sites in patients with AIS where changes in transmission from the motor cortex to the motoneuronal centres in lumbosacral region appear. These phenomena were shown in motor evoked potentials studies which were induced with the magnetic field (MEP) in areas of motor cortex and recorded from centres of cervical and lumbosacral spinal cord as well as from muscles of upper and lower extremities. Changes in efferent transmission are greater twice in recordings from muscles of lower extremities and in oververtebral recordings at L5-S1 regions what suggests, that secondary slowing down takes place at the level of the apical thoracic vertebrae of primary curve (mostly at Th7–8), predominantly on the concave than convex side of scoliosis. MEP study confirmed a previous finding with somatosensory evoked potentials (SEPs) similarly about two focuses of disturbances in of afferent transmission on the spinal centres-supraspinal centres pathway. MEP showed changes in the efferent transmission on the supraspinal centres-spinal motor generator pathway. Such changes are not observed in scolioses other than idiopathic. Results of the complex neurophysiological studies suggest that the primary origin of AIS is the brain stem area at the level of thalamus where changes of afferent and efferent transmission are detected. There is a close relationship of this structure with the pineal gland and secretion of neurotransmitters at this level in correlation to disturbances in melatonin secretion and other neurohormones. Disorders in melatonin secretion and other neurohormones may induce a scoliosis what was shown in previous genetic and experimental neurophysiological studies on animals, together with cutting of the pineal stalk. Some aspects of this problem were also mentioned in our previous clinical neurophysiological studies [1–3]. Results of studies suggest that in patients with AIS, there are structural and functional changes in the area of thalamus, which cause disturbances in afferent and efferent transmission at this level. Pathology in the pineal secretion of neurohormones can be one of the factors influencing the formation and progression of AIS, as a disease of probably secondary origin to the functional changes in brain. Results of MEP studies discussed in this report confirm that the primary origin of AIS takes place at the level of the brain stem but not in the spinal cord

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

Objective: To clarify whether serum melatonin levels in adolescent idiopathic scoliosis correlate with curve progression, and whether the exogenous melatonin treatment is effective in patients with decreased levels of endogenous melatonin in adolescent idiopathic scoliosis. Method: A total of 63 adolescents were studied; 38 with adolescent idiopathic scoliosis and 25 age matched control subjects. We divided the patients into stable (28 patients) and progressive (10 patients) groups based on the scoliotic curve measured radiographically at three to six month intervals. The level of melatonin was considered low if it fell below the mean – 2.0 standard deviation established in normal adolescents throughout the 24 hour period or nocturnal (0:00 – 6:00 hour) integrated concentration. Oral melatonin replacement (3mg / before bedding) was administered in patients with decreased endogenous melatonin. The patients with low melatonin were treated with a brace, melatonin or both combined. During melatonin treatment, the level of melatonin was measured yearly for a period ranging from three to six years. Results: In all subjects the melatonin levels showed diurnal variations; low during the day and high at night. Of 38 patients with adolescent idiopathic scoliosis, 22 patients had normal melatonin and 16 had low melatonin. Of 22 patients with a normal melatonin, 10 of 15 treated with brace and 6 of 7 untreated patients had stable scoliosis, and the remaining six had a progressive scoliosis. Of 16 patients with low melatonin, eight of nine treated only with melatonin, and four of seven treated with melatonin and brace had stable scoliosis. The remaining four had a progressive course. Of the 10 patients who had progressive scoliosis in normal and low levels of melatonin, nine had greater than 40 degrees of curve at the initial examination. Conclusion: These findings suggest that transient melatonin deficiency may be associated with deterioration of scoliosis and that melatonin level may serve as a useful predictor for progression of spine curvature in patients with idiopathic scoliosis. Also, the results of this study suggest a possible role of melatonin supplement in the prevention of progressive scoliosis especially in mild cases showing less than a 40° curve. Supported by the Fondation Yves Cotrel, 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

It is customary to analyse scoliosis as a mechanical failure: first there is a straight spine (=normal), then an habitual and collapsing posture (=disease) and finally, structural remodelling (Hueter-Volkmann effect = scoliosis). This hypothesis makes two practical predictions:. There is a disease process causing the pathological posture. The purpose of gatherings such as this is to identify this pathology, thus far without success. Early diagnosis will permit early non-operative treatment which will halt or reverse the remodelling and reduce the occurrence of severe deformity and the need for corrective spinal surgery. The failure of school scoliosis screening to achieve this end is well documented, but the consequence for the underlying hypothesis has not been analysed. Screening failed, not because it was unable to detect scoliosis, but because scoliosis did not behave as the hypothesis predicted. Disease process: All theories presume some form of neurological or muscular deficit as the final pathway but while the variety is wide, e.g. (historically) anterior poliomyelitis; more recently proprioceptive defect, melatonin or calmodulin disorder, there is no clear evidence for such a deficit in adolescent idiopathic scoliosis (AIS). Of 1342 screening referrals to this centre, 10 had a neurological diagnosis (most of which were already known to the patients) and 598 had radiologically confirmed AIS. In contrast, 1707 referrals to the general clinics included 410 syndromic cases and 420 AIS. Patients with a neurological problem, by and large, find their own way to medical attention. The hypothesis does not explain the natural history or the aetiology, and awkward observations, such as the association with growth (. Goldberg et al . Spine. 18. (5):. 529. –535.1993. , . Eur Spine J. 2. :. 29. –36.1993. and, most recently, . Ylikoski M. . Journal of Pediatric Orthopaedics B. 14. :. 320. –324, . 2005. ) or the higher incidence in ballet dancers (. Warren et al. . New England Journal of Medicine. 314. (21):. 1348. –1353. 1986. ) and rhythmic gymnasts (. Tanchev et al. . Spine. 25. (11):. 1367. –1372. 2000. ) are ignored. Screening: Screening programmes (e.g. . Goldberg et al., . Spine. 20. (12):. 1368. –1374, . 1995. ) showed that there was no precise demarcation between “scoliosis” and “normal,” and that there was no benefit in terms of the need for surgical correction from screening or bracing, (. Goldberg et al. . Spine. 26. (1):. 42. –47, . 2001. ). Discussion: his information has been in the public domain for some years and, in the meanwhile, there have been huge advances in biology and medicine which must have relevance. When the predictions of a hypothesis are not confirmed, that hypothesis must at least be re-examined, and it is not necessary to wait until a replacement can be suggested. The undisputed aspects of scoliosis, such as association with growth rate and maturation, lateralisation, gender predominance, normal distribution of Cobb angle and asymmetry over the wider population, essential health and normality of those with even severe deformity, increased incidence in other conditions, all suggest a different model. This is an opportune time to pause and reconsider the underlying model of scoliosis in the light of what we have learned about scoliosis and what is now known in other disciplines about how morphology is determined and evolved

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);. shams (surgical controls) (n=20);. pinealectomised (n=76). 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