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.

Purpose: Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological mechanism and full characterization of each individual stage through which the BM-MSC differentiate into adult chondrocytes is not yet understood. The physiological micro-environment of the chondrocytes is intensely hypoxic which triggers over-expression SLC2A proteins (GLUTs) in their membranes as a compensatory mechanism for energy production within the glycolytic cycle.

Method: We cultured and differentiated BM-MSC, and adult chondrocytes in hypoxic (5% O2 tension) and normoxic (20% O2) conditions. Within this cell populations we screened for the presence of the 12 GLUT genes as well as quantification of the variation of the 12 GLUTs gene translation by simple pcr and rt-pcr. The expression profile of the GLUT proteins was investigated using western blot analysis and immunohistochemistry. Functional characterization of the GLUTs expressed in the different cell populations was carried out by the means of radio-isotope labeled hexose fluxes done accordingly to the substrate specificity and kinetic properties particular to each SLC2A isoforms.

Results: Our data showed that the functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSC comprised an extensive expression of fructose-transporting GLUTs as opposed to the glucose-only transporting isoforms expression in normoxic BM-MSC. The flux data showed clear similarities in functional GLUT profiles between BM-MSC cultured in hypoxic conditions, adult chondrocytes. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-gluose, 3-orthomethyl-glucose and galactose) in these cellular populations under both hypoxic and normoxic conditions and in the presence and absence of Cytochalasin B (a GLUT1-specific inhibitor) showed that SLC2A class II transporters (GLUTs 5, 7, 9 and 11) play a more important role in the uptake of sugars by the normal hypoxic chondrocytes when compared to the ubiquitously-expressed GLUT1.

Conclusion: Use of this approach allows the correct culturing conditions to be identified that would select for those chondrocyte precursors from the total BM-MSC population that would have the best potential for producing viable articular cartilage. In addition, specific substrates for GLUTs isoforms could be used for physiologic, non-invasive and real time imaging of cartilage, BM-MSC and cartilage autograft by means of Positron Emission Tomography.

Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration.

Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes. Real time polymerase chain reaction was utilized to quantify and compare the essential markers, collagen II, collagen I and aggrecan, in the stem cell derived chondrocytes. The CD44 enriched and CD44 depleted populations were compared.

Results: The cells isolated possessed a cell morphology and surface antigen profile consistent with a MSC population. In addition, both experimental groups demonstrated multipotent ability. Real time PCR analysis of the chondrogenic cells demonstrated that the CD44 positive population expressed collagen II and aggrecan at a significantly higher level than the CD44 negative population.

Conclusion: To date no group has successfully identified a relationship between a MSC subpopulation and the multipotent progenitors responsible for generating cartilage. This work demonstrated that there are MSC sub-populations with different potential for chondrogenic expression and represents an important step towards identifying MSC subpopulations with enhanced cartilage formation potential.

Current research strategies for studying articular cartilage (AC) repair include the observation of chondrocyte behaviour in monolayer cultures, the use of artificial matrices and animal models. Since AC relies on the diffusion of joint synovial fluid for nourishment, we hypothesised that it should be possible to develop a research model in which full-depth AC explants are maintained under established tissue culture conditions. Successful maintenance of explants for prolonged periods of time would represent a novel approach and provide a very powerful research tool to address a wide range of chondrocyte biology and matrix synthesis questions. The objective of the project was to examine the cell viability within an AC explant model maintained in tissue media.

AC samples were obtained from the femoral condyles of total knee arthroplasty patients. Cylindrical dowels (10mm in diameter) were harvested from these samples. The dowels consisting of full-depth AC with several mm of subchondral bone attached were placed in tissue culture flask (T-25) containing 15mls of the respective culture media and maintained at 37oC in an incubator containing 5% CO2. Dowels were cultured in a variety of different media formulations (DMEM/F12, CGM (chondrocyte growth medium)) as well as PBS (phosphate buffered saline) which served as a negative control. AC chondrocyte viability was evaluated after five weeks. After having determined the best medium for cartilage maintenance, a second study with a broader range of end-points was undertaken. All dowels collected, rated 2/4 on the Outerbridge scale for osteoarthritis, and were then grown for zero, four, eight or twelve weeks in DMEM/F12 and CDM (chondrocyte differentiating medium). At each time interval, the dowels were evaluated for viability (live/dead stain), general morphology (trichrome stain), distribution of matrix proteins and proteoglycans (aggrecan, Types I and II collagen – immunofluorescence).

After five weeks in PBS, there were no viable cells in the explant. Viability in the explants maintained in DMEM/F12 was 71% compared to 59.6% in the CGM treatment. The viability of the cells in the second study was 90% with DMEM/F12. After twelve weeks, the explant models stained well for general morphology and the distribution of proteoglycans and collagen was well maintained.

To our surprise, the DMEM/F12 medium actually demonstrated the highest cell viability. Typically, AC requires joint motion to pressurise the synovial fluid into the matrix, which augments the transport of nutrients to the cells. Given that this study did not include any form fluid pressurization, it is surprising that such high cell viability was observed. This suggests that passive diffusion alone may provide adequate nourishment in this model system. In conclusion, the explant model for studying AC damage and repair examined in this research appears to be quite promising. This novel approach may serve as the foundation for subsequent research into new treatment strategies for AC injury.

Articular cartilage (AC) has a poor innate healing capacity following significant injury. Autologous chondrocyte implantation is a repair technique which utilises in vitro-expanded chondrocytes combined with a periosteal patch. The chondrocytes are enzymatically digested from arthroscopically harvested tissue at an initial surgery and expanded in monolayer culture prior to implantation at a second procedure. Unfortunately, in vitro expanded chondrocytes appear unable to retain their fundamental phenotype resulting in dedifferentiated cells which produce a matrix of inferior quality. This study compares the matrix-component gene expression profiles of chondrocytes in their native chondrons and through multiple divisions in monolayer culture. We hypothesised that there would be a rapid decline of matrix-component gene expression within a few cell replications in monolayer culture. The goal is to understand more fully the process of chondrocyte dedifferentiation and to compare matrix-component gene expression during cellular expansion in vitro.

Human AC was obtained from tissue donors and operative patients. A portion of the AC was stored at −80°C for use as a control while the remainder was homogenised and enzymatically digested with collagenase. The released cells were plated in monolayer culture and passaged (2:1) when they approached confluence. RNA was extracted from the frozen cartilage control and the passaged chondrogenic cell lines from which cDNA was generated. Real time PCR was performed with primers specific for collagen I, collagen II, aggrecan, and GAPDH. Gene expression was quantified and profiles from the cells in their native chondron and passaged cells (p0-p9) were compared.

Cells, when removed from the extra-cellular matrix and plated in monolayer, experienced an immediate upregulation of collagen I which persisted throughout all passages. In contrast, there was a stepwise decrease in collagen II with each successive passage until p8-p9 when the expression became undetectable. Aggrecan expression only decreased minimally as the cells were passaged.

Rapid dedifferentiation of monolayer cultured chondrocytes is a persistent barrier to AC tissue engineering including ACI. This study quantified the expression of relevant genes relating to AC generation and is an important first step to understanding cellular events, as alternative expansion techniques and cellular alternatives are sought.

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

Background: The neurocentral junction often has been identified as a potential cause of adolescent idiopathic scoliosis (AIS). Disparate growth at this site has been thought to lead to pedicle asymmetry, which then causes vertebral rotation in the transverse plane and ultimately, the development of scoliotic curves.

Objectives:

To develop a model that incorporates pedicle growth and growth modulation into an existing finite element model of the thoracic and lumbar spine already integrating vertebral growth and growth modulation

Methods: The model was personalised to the geometry of a non-pathological subject and used as the reference spinal configuration. Left/right asymmetry of pedicle geometry (i.e. initial length) and left/right asymmetry of the pedicle growth rate alone or in combination with other AIS potential pathogenesis (anterior, lateral, or rotational displacement of apical vertebra) were simulated over a period of 24 months. The Cobb angle and local scoliotic descriptors (wedging angle, axial rotation) were assessed at each monthly growth cycle.

Results: Simulations with asymmetrical pedicle geometry did not produce significant scoliosis, vertebral rotation or wedging. Simulations with asymmetry of pedicle growth rate did not cause scoliosis independently and did not amplify the scoliotic deformity caused by other initial deformations tested by Villemure (2004).

Discussion and Conclusion: The results of this biomechanical model do not support the hypothesis that asymmetrical neurocentral junction growth is a cause of AIS. This concurs with recent animal experiments in which neurocentral junction growth was unilaterally restricted and no scoliosis, vertebral wedging or rotation was noted. With regards to addressing the aetiology of scoliotic curve development, biomechanical modelling represents a powerful tool to investigate cause and affect relationships since AIS patients typically present to the scoliosis clinic well after curves have manifested.

Contact person and Presenter: Carl-Éric Aubin, Ph.D., Canada Research Chair “CAD Innovations in Orthopedic Engineering”, Department of Mechanical Engineering, Ecole Polytechnique, Montreal, Canada, Tel: (514) 340-4711, ext. 4437; Fax: (514) 340-5867; E-mail: carl-eric.aubin@polymtl.ca

Vertebral growth remains a mystery, especially with regards to the contribution of different growth plates and the mechanisms of growth after closure of these plates. As an example of vertebral growth in general, the growth of the vertebral canal was assessed in a rat model using fluorochromes. Although 80–90% of vertebral canal growth was due to growth plates, the remaining canal growth occurred via periosteal absorption and deposition. This is contrary to the traditional idea that periosteal mechanisms do not change the shape or dimensions of bone and suggests that the vertebrae exhibit a different model of growth than typical bones.

Vertebral growth remains largely a mystery. The contributions of different growth plates and the mechanisms of growth after closure of these plates requires further exploration. As an example of vertebral growth, vertebral canal growth was assessed in a living rat model using fluorochromes.

Vertebral canal growth and presumably vertebral growth in general occurred by different mechanisms at different phases of development. Growth plates accounted for the majority of growth although periosteal mechanisms also resulted in changes in the size and shape of the vertebrae. This is contrary to the traditional concept of periosteal growth and suggests that vertebrae may exhibit a different model of growth than typical bones.

The growth of the vertebrae in a particular dimension and during a particular phase of development is dependent on different mechanisms of growth, which may play a role in interpreting vertebral growth anomalies.

The interspinous junction closed by the end of the first week, whereas the neurocentral junction closed between weeks three and four. By four weeks, the vertebral canal had achieved 80–90% of its growth in area and diameter. After growth plate closure, the canal continued to grow by periosteal mechanisms and was displaced posteriorly.

Thirty-six Sprague-Dawley rats (age one week-seven weeks) were injected with tetracycline and alizarin using a dosing interval of four days. Thoracic vertebrae were sectioned using a cryostat and examined under a fluorescence microscope. In addition to noting fluoro-chrome deposition, the dimensions of the growth plates and canal were noted.

Funding: Edmonton Orthopaedic Research Association and University of Alberta Department of Radiology and Diagnostic Imaging

This study was designed to examine the components of the MR image of the neurocentral junction (NCJ) and to explore the discrepancy between the age of closure of the NCJ as determined by anatomic and imaging studies. MR images of one hundred and fourteen porcine NCJs were correlated with anatomic and histologic sections. Whereas gross anatomic visualization did not reveal the NCJ site, MRI was sensitive for cartilage detection and accurately determined the age of NCJ closure although it overestimated the extent of closure. Based on this study, MRI characterization of the NCJ appears reliable and the NCJ cartilage does not close until adolescence.

This study examined the composition of the MR image of the neurocentral junction (NCJ) and the discrepancy between the age of closure of the NCJ as determined by anatomic and imaging studies.

MRI was sensitive for cartilage detection and accurately determined the age of NCJ closure (i.e. absence of cartilage on histologic examination).

Disparate NCJ development has been implicated as a potential cause of adolescent idiopathic scoliosis. Whereas autopsy studies have refuted this theory by suggesting that the NCJ closes before adolescence, MRI studies have resurrected this idea by suggesting later closure. MRI-histologic correlation suggests that the NCJ cartilage remains present until adolescence and therefore further exploration of the disparate growth hypothesis is required.

Gross anatomic visualization did not reveal the NCJ site, even after removal of the periosteum. In contrast, the presence or absence of an NCJ image correlated with the presence or absence of cartilage although MRI overestimated the extent of this cartilage.

Vertebrae were grossly examined for any evidence of the NCJ site. Sagittal and transverse MR images of one hundred and fourteen porcine NCJs in various stages of development (thirty-eight open, sixty-four closing, twelve closed) were correlated with anatomic and histologic sections acquired at the same position.

Funding: Edmonton Orthopaedic Research Association and University of Alberta Department of Radiology and Diagnostic Imaging

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.

Introduction: Although there are several known causes of scoliosis, most are of unknown cause and develop during adolescence, making adolescent idiopathic scoliosis (AIS) the most common form. It has long been hypothesised that unilateral closure of the neurocentral junction accompanied by continued growth on the opposite side could lead to vertebral rotation and subsequent lateral curvature. However, autopsy studies of neurocentral junction closure in children has revealed that these joints close at approximately six years of age consequently excluding this hypothesis as a cause of AIS. In contrast, a recent MRI study has suggested that in some children at least, the NCJ does not close until much later in development around the time of puberty thereby resurrecting this hypothesis as a potential cause of AIS. This study was designed to investigate closure time and pattern of closure of the NCJ in normal patients to determine whether further examination of this hypothesis might be warranted.

Methods and results: The morphology of the NCJs in 20 patients between the ages of 3 and 15 were observed in MR images taken for purposes other than spinal anomaly. The structure of individual NCJs were observed and reconstructed in 3-dimensions. The age at which NCJs became closed was determined and pattern of closure of a typical NCJ was created using the reconstructed images. The pattern of closure of the NCJs along the vertebral column was also determined and any differences between right and left sides at the same level was also noted.

The results showed that there was a sequence of closure along the vertebral column for the NCJs with those in the cervical and lumbar regions being the first to close and those at the approximate level of T8 being the last to close. While the NCJs in the cervical and lumbar regions close at 5–6 years of age, those in the thoracic region, that are the last to close, do so at approximately 12 years of age. No significant difference between the stage of closure of the left and right sides was seen at any level.

Conclusion: The results of this study have shown that the closure of the NCJs in those vertebrae that form at approximately the most common level for the apical vertebra associated with AIS (midthoracic) does not occur until the time of puberty. This contrasts sharply with previously held views on the age of closure. Although no significant difference in closure between left and right sides was seen among these particular patients it does not exclude unilateral closure as a cause of AIS at least in some patients. These results suggest that examination of this hypothesis should be resurrected and that further study is well warranted. MR examination of young patients with small, initial curves could be well worthwhile.