The pathogenesis of falling bone mineral density (BMD) as a universal feature of advancing age is poorly understood. 1. Frequently culminating in the development of osteoporosis, the process is attributable to more than 500,000 fragility fractures occurring every year in the UK Such injuries are associated with great levels of morbidity, mortality and a £3.5 billion cost to the healthcare economy. 2. . With age, humans are known to accumulate somatic mitochondrial DNA (mtDNA) mutations in mitotic and post mitotic tissue, and stem cell precursors. 3. Compelling evidence in recent years, particularly that provided by animal models suggests that these mutations are intrinsic to the ageing process. 4–6. We provide evidence for the first time that mitochondrial dysfunction contributes significantly to the failure of bone homeostasis and falling BMD. We have utilised a mouse model that accumulates mtDNA mutations at 3–5 times the rate of normal mice, consequently ageing and developing osteoporosis prematurely. 7. , to clearly demonstrate that osteoblasts are vulnerable to mtDNA mutations. We have developed a new quadruple immunofluorescent assay to show that mitochondrial respiratory chain dysfunction occurs in osteoblasts as a consequence (p < 0.0001). We show that this mitochondrial dysfunction is associated with reduced BMD in female and male mice by 7 (p = 0.003) and 11 (p = 0.0003) months of age respectively. Using osteoblasts derived from mesenchymal stem cells extracted from male and female mice with mitochondrial dysfunction aged 4, 7 and 11 months, we demonstrate a vastly reduced capacity to produce new mineralised bone in vitro when compared to wild type cell lines (p < 0.0001). Exercise was found to have no beneficial effect on osteoblast and whole bone phenotype in this mouse model. It is likely that mtDNA mutations accumulating over a longer time period in human ageing have significantly detrimental effects on bone biology and diminishing BMD

Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA

Mitochondrial dysfunction has been demonstrated in aging and osteoarthritic tissues. We investigated knee joints of prematurely aging mitochondrial DNA mutator mice (PolgD275A) to evaluate a relationship between mitochondrial dysfunction and osteoarthritis. Cartilage damage was evaluated using OARSI histopathology grading and osteoclast numbers were quantified by tartrate-resistant acid phosphatase staining in wild type, heterozygous and homozygous PolgD275A mice. Subchondral cortical plate and epiphyseal trabecular bone structures were determined by micro-computed tomography. Apoptosis in cartilage and subchondral bone tissues was studied using an indirect TUNEL method. Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type and heterozygous mutants. Subchondral osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). Wild type mice as well as hetero- and homozygous mutants displayed low-grade cartilage degeneration due to loss of cartilage proteoglycans. In contrast, chondrocyte hypertrophy was more abundant in the homozygous mice. There were no differences in chondrocyte apoptosis rates between groups. Prematurely ageing mtDNA mutator mice with or without further mechanic or metabolic stimuli might serve as a valuable model for further experimental studies on aging-induced osteoporotic OA phenotype

The hypoxic nucleus pulposus cells were thought to contain few, functionally redundant mitochondria. However in contrast to this widely held notion, new evidence shows presence of functional mitochondrial networks in disc cells. The lecture will discuss this evidence and provide insights into how microenvironmental cues govern mitochondrial function. The lecture will also discuss emerging evidence on how mitochondrial dysfunction of nucleus pulposus cells results in metabolic dysregulation and acquisition of a state that promotes inflammation and degeneration

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity. Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining. HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts. Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity

When transferring tissue regenerative strategies involving skeletal stem cells to human application, consideration needs to be given to factors that may affect the function of the cells that are transferred. Local anaesthetics are frequently used during surgical procedures, either administered directly into the operative site or infiltrated subcutaneously around the wound. The aim of this study was to investigate the effects of commonly used local anaesthetics on the morphology, function and survival of human adult skeletal stem cells.

Cells from three patients who were undergoing elective hip replacement were harvested and incubated for two hours with 1% lidocaine, 0.5% levobupivacaine or 0.5% bupivacaine hydrochloride solutions. Viability was quantified using WST-1 and DNA assays. Viability and morphology were further characterised using CellTracker Green/Ethidium Homodimer-1 immunocytochemistry and function was assessed by an alkaline phosphatase assay. An additional group was cultured for a further seven days to allow potential recovery of the cells after removal of the local anaesthetic.

A statistically significant and dose dependent reduction in cell viability and number was observed in the cell cultures exposed to all three local anaesthetics at concentrations of 25% and 50%, and this was maintained even following culture for a further seven days.

This study indicates that certain local anaesthetic agents in widespread clinical use are deleterious to skeletal progenitor cells when studied in vitro; this might have relevance in clinical applications.