Despite developing refinements of chemotherapy regimens for osteosarcoma, multi-drug resistant cases are frequently seen and patients with metastatic or recurrent disease continue to have a very poor prognosis. Recently, the expression of the longevity gene Sirt1 was found to be relatively higher expressed in tumors compared with the normal tissues. Association of high level of Sirt1 expression with the development of multi-drug resistance in tumor cells has also been indicated. Thus, it is interesting to study the therapeutic potential of regulating Sirt1 activity for the treatment of osteosarcoma.

In the present study, we evaluated the effects of two Sirt1 activators, resveratrol and isonicotinamide, on growth and apoptosis in four human osteosarcoma cell lines, HOS, Saos-2, U-2 OS and MG-63. We found that Sirt1 protein was expressed in all osteosarcoma cell lines. Instead of promoting cell survival, both resveratrol and isonicotinamide decreased cell growth and induced cell apoptosis in a dose-dependent fashion. Furthermore, the pro-apoptotic effect of resveratrol could be enhanced by L-asparaginase-induced nutrition restriction of cultured osteosarcoma cells.

Our results demonstrated that Sirt1 activators elicited pro-apoptotic effects in osteosarcomas. Thus, Sirt1 could be a potential target in the treatment of osteosarcoma. However, due to the non-specificity of the Sirt1 activators used further studies, such as knock-down of Sirt1 by siRNA, are needed to confirm the effect of Sirt1 activation on malignant cells.

Introduction: Alcohol can induce adipogenesis by bone marrow stromal cells and may cause osteonecrosis of the femoral heads. Currently, there are no medications available to prevent alcohol-induced osteonecrosis. The purpose of this study was to evaluate the effects of puerarin on adipocytic differentiation of bone marrow stromal cells and on the prevention of alcohol-induced osteonecrosis.

Materials and Methods: In the in vitro study, bone marrow stromal cells were treated with ethanol as model groups, with ethanol and puerarin as experimental groups, and without ethanol or puerarin to serve as controls. In the in vivo study, model group mice received ethanol intragastrically and normal saline by intramuscular injection. The experimental group received the same dose of alcohol intragastrically and puerarin by intramuscular injection, and the control group received water intragastrically and normal saline by intramuscular injection daily, for 4, 6, 8, and 10 months, respectively.

Results: It was found that in the in vitro experimental group, the number of adipocytes, contents of triglycerides and levels of PPARγ mRNA expression were significantly decreased, and alkaline phosphatase activity, contents of osteocalcin and levels of osteocalcin mRNA expression were significantly increased compared with cells in model groups. In the in vivo experimental group, cholesterol, and triglyceride in serum were significantly decreased, and alkaline phosphatase activity was significantly higher, compared with the model group. Fat cell hypertrophy and proliferation, thinner and sparse trabeculae, diminished hematopoiesis, and increased empty osteocyte lacunae in the subchondral region of the femoral head were observed in the model groups. However, no significant changes were seen in femoral heads of the experimental and the control group.

Discussion: The results showed that puerarin can inhibit adipogenic differentiation by bone marrow stromal cells both in in vitro in cell culture and in vivo animal experiments. These findings indicate that puerarin can prevent alcohol-induced adipogenesis and osteonecrosis.

Purpose: To determine if some subsets of healthy subjects displayed other than a typical gait pattern and to identify which subsets have similar kinematic pattern to patients with knee osteoarthritis.

Methods: The healthy subject dataset consisted of 106 asymptomatic volunteers. These subjects were over 17 years of age, pain-free, had no record of surgery to the lower limb and no evidence or history of arthritic disease at the time of testing. The patient population consisted of 12 patients diagnosed with knee OA, evaluated within 6 months prior to the tests. The 3D movements of right knee joint were recorded using a functional knee analyzer with magnetic sensors while subjects walked on a treadmill at their own preferred speed. The magnetic sensors are non-invasive electromagnetic devices, which track the 3D positions and orientations of sensors relative to a source. The system has been shown to be accurate, especially in the frontal and transversal planes. K-means clustering analysis was chosen to identify the gait patterns among healthy subjects based on three components of the knee joint angles, and analyses of variance were performed to determine which parameters were different between subsets.

Results: Three gait groups or patterns were identified in the healthy subjects. The first group (G1) was characterized by a kinematic profile similar to the OA group. The second group (G2) had the highest external rotation angle, which was significantly different from OA group. The abduction angles were always greater in the G2 and G3 than in the OA group. This might be attributed to a valgus static alignment in G2 and G3 comparing to a varus alignment in the patient with OA.

Conclusions: The newly developed functional knee analyzer provided a non-invasive way to accurately measure 3D kinematic data which enabled cluster analysis to distinguish three gait patterns from 106 healthy subjects. The results suggested a strong correlation between static alignment and dynamic ad-abduction angles during the gait, which need to be investigated. Funding: Other Education Grant Funding Parties: NSERC, CIHR and FCAR

Introduction: Intervertebral disc (IVD) degeneration involves loss of disc matrix leading to instability and pain. Autologous cells are the ideal choice for bioengineering a new IVD, but removal of cells from the IVD is problematic. Our aim was to direct mesenchymal stromal cells (MSCs) down a chondrocytic lineage to mimic disc chondrocyte phenotype.

Methods: MSCs were either maintained in monolayer, pelleted into micromass aggregates or transferred to alginate beads. Pellet cultures were used in immunohis-tochemistry for type II collagen and aggrecan and in situ hybridisation for SOX-9 mRNA. Monolayer and alginate cells were cultured in the presence or absence of chondrogenic medium for 4 and 11 days. Monolayer cultured MSCs were also transfected with a SOX-9 adenovirus and cultured in the presence or absence of TGF-_1. Realtime quantitative PCR was used to analyse expression of chondrocyte markers.

Results: IHC showed increased expression of type II collagen and aggrecan in pellet cultures, while ISH showed that SOX-9 was not expressed by monolayer MSCs, but increased after pelleting. Realtime PCR using alginate-cultured MSCs showed down regulation of type I collagen mRNA expression and up-regulation of SOX-9 that was increased by chondrocgenic medium. SOX-9 transduced monolayer MSCs showed increased type II collagen, aggrecan, SOX-6 and SOX-9 mRNA over controls, while type I collagen levels showed no significant change. Stimulation of transfected MSCs with TGF-_1 showed similar increases in chondrocyte genes.

Discussion & conclusions: Adult human MSCs were induced to differentiate along a chondrocytic phenotype, which was mediated by culture conditions. Alginate and pellet culture produce a cell that has more chondrogenic characteristics than monolayer cells. SOX-9 transduced monolayer MSCs appeared to produce a more chondrocytic phenotype which was modulated by TGF-_1. Results suggest SOX-9 transfected monolayer MSCs may be used as a source of chondrocytes for repair of degenerate IVD.