Advertisement for orthosearch.org.uk
Results 1 - 3 of 3
Results per page:
Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 73 - 73
1 Jul 2020
Albiero A Piombo V Diamanti L Birch M McCaskie A
Full Access

Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells.

DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software.

We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways.

In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_1 | Pages 114 - 114
1 Jan 2013
Rankin K Nisar S Morfitt H Biswas S Lunec J Birch M Gerrand C
Full Access

Background

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a role in the progression of several common solid cancers. Given that osteosarcoma features extensive local invasion and haematogenous metastases, we hypothesised that osteosarcoma cells utilise MT1-MMP to drive these processes. Moreover, since hypoxia regulates MT1-MMP expression in breast cancer we investigated the effects of hypoxia on MT1-MMP expression in osteosarcoma cells.

Aims

Examination of MT1-MMP expression in osteosarcoma biopsy tissue in relation to clinical outcome

Assessment of MT1-MMP, together with hypoxia inducible factors HIF-1α and HIF-2α expression in a panel of osteosarcoma cell lines under normoxia and hypoxia


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_II | Pages 88 - 88
1 Feb 2012
Shyamsundar S Morgan R Birch M Campbell P McCaskie A Fenwick S
Full Access

Clinical proteomics is an exciting new sub-discipline of proteomics that involves the application of proteomic technologies at the bedside to identify new biomarkers, associated with specific diseases. In this study to compare serum protein profiles between identical age-matched groups of fracture and non-fracture controls, we looked at the initial proteomic profile of 10 patients who had fractures and compared them to age-matched controls to see if there was any specific difference indicative of fracture.

Materials and Methods

10 patients with single fractures of the long bones, wrist or ankle gave a blood sample upon presentation at the fracture clinic. 10 healthy, age-matched, non-fracture volunteers also donated blood. Plasma was isolated and the albumin and IgG fractions removed before loading equal amounts of each sample onto 2 dimensional polyacrylamide gels for analysis by isoelectric point in the first dimension and molecular mass in the second dimension. Protein profiles between fracture patients and non-fracture controls were contrasted using Phoretix 2D analysis software.

Data analysis differentiated between the average gel of the patient group and the average gel of the control group. More than 300 protein spots were observed in both the control and patient group. Seven protein spots were identified which showed a statistically significant (p<0.05) difference between the control and patient samples. Of these, three spots (X, Y, Z) were clear, distinct and present in at least 80% of these gels. All the three spots were up regulated in the patient group as opposed to the control group. These proteins are currently being investigated further by MALDI-TOF TOF for specific protein identification.

Discussion

Proteomic analysis is already a powerful tool in the identification of disease markers. We aim to show here that there are differences seen in blood plasma profiles in fracture patients compared to non-fracture healthy controls. The differences seen may help us to understand the fracture repair process better.