Purpose of study and background. We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Methods and Results. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O. 2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as collagen II and aggrecan and NP phenotypic markers FOXF1 and PAX1, whereas Bgel induced expression of collagen I and osteopontin, indicative of osteogenic differentiation. Conclusion. NPgel and Bgel were able to differentiate patient derived MSCs from different sources into both NP and osteogenic lineages, which may give rise to novel treatment strategies for IVD degeneration and spinal fusion, enabling choice for cell source according to patients' circumstances and needs. C Le Maitre and C Sammon hold a patent for the hydrogel described. Funded by MRC and Versus Arthritis

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. Methods. Osteoblasts isolated from patients with AIS were selected for each functional subgroup and compared with osteoblasts obtained from healthy matched controls. We used the latest gene chip human genome array Affymetrix (HuU133 Plus 2.0 array) that allows for the analysis of the expression level of 38 000 well characterised human genes. Raw data were normalised with robust multiarray analysis method. Statistical analysis was done by the EB method with FlexArray software. Selection criteria for in-depth analysis include the magnitude of change in expression (at least □} 3-fold) and 5% false discovery rate as stringency selection. Validation of selected candidate genes was done by qPCR and at the protein level by Western blot and ELISA methods. Plasma OPN concentrations were measured by ELISA on a group of 683 consecutive patients with AIS and were compared with 262 healthy controls and 178 asymptomatic offspring, born from at least one scoliotic parent, and thus considered at risk of developing the disorder. The regulation of OPN signalling pathway in normal and AIS cells were validated in vitro by cellular dielectric spectroscopy (CDS). Results. Of 38 000 human genes tested, we have found eight genes specifically associated with the functional subgroup 1, 16 genes with the functional subgroup 2, and 11 genes with the functional subgroup 3. Interestingly, only 19 genes were shared and affected to the same extent in all AIS functional subgroups exhibiting a similar curve pattern (double major), suggesting their role in the formation of this curve pattern. Indeed, most of these genes encode for regulatory proteins such as transcription factors regulating axial skeleton, somite development, and extracellular matrix proteins. Mean plasma OPN concentrations were significantly increased in patients with AIS and correlated with disease severity. Increased plasma OPN concentrations were also detected in the asymptomatic at-risk group, suggesting that these changes precede scoliosis onset. CDS experiments clearly showed that OPN exposure triggers a Gi-coupled receptor signalling dysfunction, which is exacerbated by oestrogens. Conclusions. Our data further support our functional method of stratification of patients with AIS and allow the identification of genes triggering scoliosis onset versus those predisposing to the development of a specific curve pattern. Furthermore, our clinical and experimental data show that OPN is essential for scoliosis onset and curve progression, thus offering a first molecular concept to explain the pathomechanism leading to the asymmetrical growth of the spine in AIS. Acknowledgments. This research project was supported by grants from La Fondation Yves Cotrel de l'Institut de France, Canadian Institutes of Health Research, and Paradigm Spine LLC