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. 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

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

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

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

The pineal hormone melatonin was recently shown to have a free radical scavenging ability which reduces lipid peroxidation and has also been shown to scavenge the hydroxyl radical and other reactive oxygen species. Melatonin stimulates several antioxidative enzymes, which increases its efficiency as an antioxidant. Also, melatonin stimulates osteoblast differentiation and mineralization of matrix and it may regulate osteoclastic activity via superoxide dismutase in vitro. Therefore, the effect of melatonin in fracture healing depends in part on the free radical scavenging and osteoblastic-osteoclastic regulatory properties of melatonin. In this study the effect of melatonin (MEL) on fracture healing in rat tibia model was studied by using biochemical, radiological and histopathologic methods. Male Sprague-Dawley rats (n=80) were randomized into control and melatonin groups with 8 rats per group according to the day of sacrifice (Days 1, 3, 7, 14, 28). Group B (melatonin group) received 30 mg/kg melatonin intraperitoneally (i.p.) for the duration of the experiment. Malondialdehyde (MDA) levels in MEL group decreased at days 3, 7, 14, and 28 compared to control values (p< 0.05). Superoxide dismutase (SOD) activity in MEL group decreased at days 3, 7, and 14, and returned to the first day value after 28 days. Myeloperoxidase (MPO) values in MEL group decreased at days 1, 3, and 7 (p< 0.001). Both radiologically and histopathologically fracture healing was significantly more advanced in the MEL group (p< 0.05, p< 0.05 respectively). We conclude that exogenously administering a pharmacological dose of melatonin has a positive effect on fracture healing and may be beneficial as a supportive agent in fracture cases

Scoliosis seen in the chicken after pinealectomy resembles adolescent idiopathic scoliosis in man. It has been suggested that in both species, deficiency of the pineal hormone, melatonin, is responsible for this phenomenon. In nine patients with adolescent idiopathic scoliosis and in ten age- and gender-matched controls, the circadian levels of serum melatonin and the excretion of urinary 6-hydroxy-melatonin-sulphate, the principal metabolite of melatonin, were determined. There were no statistically significant differences in the secretion of serum melatonin or the excretion of urinary 6-hydroxy-melatonin-sulphate between the patients and the control group. The hypothesis of melatonin deficiency as a causative factor in the aetiology of adolescent idiopathic scoliosis cannot be supported by our data

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: The development of scoliosis in pinealectomised chickens was first observed by Machida. 1. and since reported by others. That melatonin deficiency following pinealectomy may be a factor in causing scoliosis has been postulated. The relationship between pinealectomy, scoliosis and serum melatonin levels has been subject to experimental investigations. This study reports the incidence and type of scoliosis in pinealectomised, sham operated and unoperated chickens, and related serum melatonin levels. Methods: Serum melatonin levels were obtained at sacrifice up to six weeks postoperatively. Radiological and histological examination of the spine was performed. Results: The vertebral motion segment comprises a synovial joint lacking any discs. 19% of the un-operated group had a sharp angular deformity in contrast to the smooth curve seen in adolescent idiopathic scoliosis (AIS). There was a 38% incidence of scoliosis after sham operation (mostly of the angular variety) and a 75% incidence in the pinealectomy group (of which half were smooth curves similar to those in human AIS. Melatonin was not abolished by pinealectomy or sham operation but was at significantly lower levels than in the unoperated group. There was no difference in Melatonin levels between birds with the two types of curves. Discussion: The avian spine has fundamental structural differences with the human. There is a natural incidence of short angular scoliosis that increases with posterior fossa surgery in the chicken. We confirm that scoliosis similar to AIS forms after pinealectomy but it is not directly related to diminished melatonin levels

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: 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

We studied the possible role of melatonin deficiency in experimentally-induced scoliosis. A total of 90 chickens underwent pinealectomy on the third day after hatching: 30 were treated with serotonin, 30 with melatonin and 30 received no therapy (control group). Scoliosis developed in all the control group, in 22 of the serotonin group, and in only 6 of the melatonin group. The six melatonin-treated chickens with scoliosis had less severe spinal deformities than those in the serotonin-treated group. There were lower blood melatonin concentrations in chickens with scoliosis than in those without. Our findings suggest that melatonin deficiency contributes to the aetiology of this experimental scoliosis, probably by interfering with the normally symmetrical growth of the proprioceptive system involving the paraspinal muscles and the spine

Introduction: No appropriate animal model for studying adolescent idiopathic scoliosis (AIS) exists and this hampers research. In recent years, we have been examining a model in which scoliosis consistently develops in young chickens following pinealectomy and which has been shown to have many characteristics similar to those seen in AIS. Not all of the pinealectomised chickens develop scoliosis following the pinealectomy and so we have the opportunity to examine differences between the two groups. The obvious candidate for study of the mechanism underlying this phenomenon is melatonin which is the principal product of the pineal gland. In this study we have measured the serum melatonin levels of pinealectomised chickens that have developed scoliosis and compared these with similar measurements taken from chickens that have developed scoliosis. Methods and results: Newly-hatched chickens were obtained from a local hatchery and kept in a single pen with standard heating and lighting. A 12:12 light dark cycle was introduced immediately and the two-thirds of the chickens were pinealectomised three days later. The remainder acted as controls. At weekly intervals following surgery, the chickens were radiographed in a supine position while anaesthetised and the presence of scoliosis was determined from the radiographs. Three weeks after surgery the chickens were euthanised and blood samples were collected and analysed using radioimmunological techniques to determine levels of serum melatonin. The samples were collected in the presence of red light in the middle of the dark cycle when melatonin levels have been shown to be at their highest. Approximately 55% of the pinealectomised chickens developed scoliosis within the three weeks following surgery whereas none of the control chickens developed scoliosis. The results showed that the serum melatonin levels of pinealectomised chickens were significantly lower than the normal controls and were in fact all close to zero. However, there was no significant difference in serum melatonin levels between those chickens that developed scoliosis and those that did not. Conclusion: The results of this study have shown that pinealectomy significantly reduces serum melatonin levels close to zero in all chickens. The results also show that there is no significant difference in serum melatonin levels between those pinealectomised chickens that develop scoliosis and those that do not. Unless there is a subtle threshold level that is unable to be detected using our methodology or that melatonin levels in the days immediately after surgery are of critical importance, these results suggest that other causes for this phenomenon need to be examined. An understanding of the underlying cause would be of great importance and might represent a significant breakthrough in the study of AIS

The scoliosis observed in chickens after pinealectomy resembles that seen in humans with an adolescent idiopathic scoliosis, suggesting that melatonin deficiency may be responsible. However, to date there have been no studies of pineal gland glucose metabolism in patients with adolescent idiopathic scoliosis that might support this hypothesis. We examined the excretion of urinary 6-sulfatoxyl-melatonin as well as the glucose metabolism of the pineal gland in 14 patients with an adolescent idiopathic scoliosis and compared them with those of 13 gender-matched healthy controls using F-18 fluorodeoxyglucose brain positron emission tomography. There was no significant difference in the level of urinary 6-sulfatoxyl-melatonin or pineal gland metabolism between the study and the control group. We conclude that permanent melatonin deficiency is not a causative factor in the aetiology of adolescent idiopathic scoliosis

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: 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

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

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

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

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.