A method for characterising human intervertebral disc glycosaminoglycan disaccharides using liquid chromatography-mass spectrometry with multiple reaction monitoring.

Intervertebral disc (IVD) degeneration results in the depletion of proteoglycans and glycosaminoglycans (GAGs), which can lead to structural and mechanical loss of IVD function, ingrowth of nociceptive nerve fibres and eventually discogenic pain. Specific GAG types as well as their disaccharide patterns can be predictive of disease and degeneration in several tissues but have not been comprehensively studied within the IVD. A highly sensitive mass spectrometry based technique with multiple reaction monitoring (MRM) was used to provide characterisation of chondroitin sulphate (CS), hyaluronic acid (HA), heparan sulphate (HS) and their disaccharide sulphation patterns across different anatomical regions of human IVDs. Principal component analysis further distinguished important regional variations and proposed potential ageing variations in GAG profiles. CS was the GAG in greatest abundance in the IVD followed by HA and HS. Principal component analysis identified clear separation of GAG profiles between nucleus pulposus and annulus fibrosus in young and old specimens. Distinct patterns of predominantly expressed disaccharides of CS and HS between young and old IVD samples, provided preliminary evidence that important alterations in disaccharides occur within IVDs during ageing. This technique offered a novel approach to identify and quantify specific GAG disaccharides in human IVDs and the data presented were the first to offer insight into the spatial distribution as well as association with ageing of GAGs and GAG disaccharide sulphation patterns across the human IVD.

Marrow adipogenesis and bone loss that parallels estrogen deficiency is slowed by low-intensity mechanical signals.

UNLABELLED: Ovariectomized mice were used to assess the ability of low-intensity vibrations to protect bone microarchitecture and marrow composition. Results indicate that low-intensity vibrations (LIV), introduced 2 weeks postsurgery, slows marrow adipogenesis in OVX mice but does not restore the bone within the period studied. However, immediate application of LIV partially protects quality. INTRODUCTION: The aim of this study was to evaluate consequences of estrogen depletion on bone marrow (BM) phenotype and bone microarchitecture, and effects of mechanical signals delivered as LIV on modulating these changes. METHODS: LIV (0.3 g, 90 Hz) was applied to C57BL/6 mice immediately following ovariectomy or 2 weeks postestrogen withdrawal for 2 (ST-LIV) or 6 weeks (LT-LIV), respectively. Sham-operated age-matched controls (ST-AC, LT-AC) and ovariectomized controls (ST-OVX, LT-OVX) received sham LIV treatment. Bone microstructure was evaluated through µCT and BM adipogenesis through histomorphometry, serum markers, and genes expression analysis. RESULTS: LT-OVX increased BM adipogenesis relative to LT-AC (+136 %, p ≤ 0.05), while LT-LIV introduced for 6w suppressed this adipose encroachment (-55 %, p ≤ 0.05). In parallel with the fatty marrow, LT-OVX showed a marked loss of trabecular bone, -40 % (p ≤ 0.05) in the first 2 weeks following ovariectomy compared to LT-AC. Application of LT-LIV for 6w following this initial 2w bone loss failed to restore the lost trabeculae but did initiate an anabolic response as indicated by increased serum alkaline phosphatase (+26 %, p ≤ 0.05). In contrast, application of LIV immediately following ovariectomy was more efficacious in the protection of trabecular bone, with a +29 % (p > 0.05) greater BV/TV compared to ST-OVX at the 2w time period. CONCLUSIONS: LIV can mitigate adipocyte accumulation in OVX marrow and protect it by favoring osteoblastogenesis over adipogenesis. These data also emphasize the rapidity of bone loss with OVX and provide perspective in the timing of treatments for postmenopausal osteoporosis where sooner is better than later.