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Institute of Medical Sciences

Bone and Cartilage Biology

Bone is a dynamic tissue, made and maintained by various cell types: osteoblasts that synthesize bone, osteocytes which act as mechanosensors, osteoclasts which resorb bone, cartilage cells lining the bones in joints, endothelials cells and bone marrow cells, including mesenchymal stem cells, which live in the central bone marrow region of the long bones. We study several aspects of the cell biology of most of the cell types mentioned above. Some of this work is covered under regenerative medicine

Our work on osteoclasts is extensive and covers the role of small GTPases in their survival and behaviour, vesicular transport, the study of their ultrastructural pathology in osteoclast diseases, the effects of cannabinoids and mechanisms of osteoclast apopotosis.

Figure 1: Osteoclast in a transmission electron microscopical image. We are studying the formation of the ruffled border and other membrane compartments within osteoclasts and the vesicular transport to such specialized membrane regions. We are also interested in understanding better the place of the osteoclasts in its cellular environment. As illustrated in this image, osteoclasts are in close contact with other bone cells (such as osteoblasts), but also with bone marrow cells and endothelium.

With programme grant funding from the Arthritis Research Campaign, we have begun to systemically study the expression of small GTPases, especially Rab GTPases, in osteoclasts and start to elucidate their specific roles in this cell type. This work uses biochemical and proteomic approaches to first enrich and identify Rabs at the molecular level and then employs molecular tools to modulate their expression and study their function. Rabs are crucially involved in vesicular transport in osteoclasts (Coxon and Rogers, 2003) and our studies are focusing increasingly on the identification and characterisation of the vesicular compartment within osteoclasts and the study of the role of vesicular transport in formation of the ruffled border and in bone resorption. A range of methodologies is developed to perform such studies including novel methods for transfection of postmitotic multinucleated cells (e.g., Taylor et al., 2007) and a range of imaging modalities, such as confocal microscopy, live cell imaging, micro computer tomography and ultrastructural microscopy (e.g., Coxon et al. 2008 and Figures 1-3)

Figure 2: Confocal microscopic image of a resorbing osteoclast on dentine. The dentine surface, labeled with a fluorescent bisphosphonate, is shown in purple. Polarisation of the cell is shown by staining for F-actin (green), which reveals the sealing zone ("actin ring"), while staining of cortical actin reveals the outline of the cell. Acidic vesicles are stained by Lysotracker (red), and the nuclei are stained using TO-PRO-3 (blue). The black area corresponds to the dentine removed by the resorptive activity of the osteoclast.

These studies link with our ongoing programme of work in osteopetrosis, a genetically and clinically heterogeneous group of diseases caused by osteoclast dysfunction. Mutations in the genes for carbonic anhydrase 2 CAII, the proton pump TCIRG1, the chloride channel CLC7, and OSTM1 are all known to cause osteoclast-rich recessive osteopetrosis in humans with lack of ruffled border formation. There are however additional genes, yet to be identified, that account for some additional forms of osteopetrosis. In collaboration with the group of Wim van Hul in Antwerp, we recently found mutations in the gene PLEKHM1 which give rise to an intermediate recessive form of osteopetrosis (Van Wesenbeeck et al., 2007 ) again with normal osteoclast formation, but lack of ruffled border formation. Interestingly, the Plekhm1 protein was found to colocalise with Rab7 in vesicles prompting further studies into the role of Plekhm1 in vesicular trafficking. In collaboration with the group of Anna Villa in Milan, we have studied osteoclast-poor osteopetrosis, where osteoclast formation is disrupted. We found that mutations in RANKL/TNFSF11 (Sobacchi et al., 2007) and mutations in RANK/TNFRSF11a (Guerrini et al., 2008) are responsible for some of these cases. These findings are important for the clinical management of the disease: while patients with mutations that lead to intrinsic osteoclast defects may benefit from bone marrow transplantation, other treatments are required for those with a RANKL defect. We are now performing detailed signalling studies trying to identify the exact functional defect in RANKL/RANK interactions in the various mutant proteins.

Figure 3: microCT reconstruction of trabecular bone from the proximal tibia of a mouse

Work on the pathogenetic mechanisms in late onset Paget's disease of bone focus on the nature of the inclusion bodies in osteoclasts and on the cellular role of Sequestosome-1 in bone cells (Helfrich and Hocking, 2008). Given the recent realisation that Sequestosome-1 is a critical protein in autophagy, our work is beginning to explore the role of this process in osteoclasts. In a related project we are studying how mutations in the signal peptide region of in RANK/TNFRSF11a in patients with rare, early-onset, forms of Pagetic bone disease (such as Familial Expansile Osteolysis) lead to osteoclast hyperactivity.

In collaboration with the cannabinoid group in Aberdeen, we found that cannabinoid receptors are critically involved in bone physiology and we now study in more detail how the cannabinoidsregulate aspects of osteoclast biology.

We have recently found that matricellular proteins (CCN proteins), especially the protein CYR61 are important regulators of osteoclast function (Crockett et al., 2007). Dr. Julie Crockett has received a career establishment award from the European Calcified Tissue Society to explore this area further.

Studies on mechanisms of osteoclast apoptosis are building on earlier work defining the role of caspases in osteoclastic cell death (Benford et al., 2001). More recently, we have identified Mcl-1 as an important player in osteoclast apoptosis and we are identifying further pathways important for osteoclast survival.

In studies in other bone cell lineages, we culture explants of cartilage to investigate how the chondrocytes respond to mechanical load and damage caused by excessive load (Burgin and Aspden, 2008, Jeffrey and Aspden, 2007, Plumb and Aspden, 2005). Recent work has started to explore the role of Wnt signaling and FGF18 in mechanically loaded human cartilage. We also expose osteocytes to various types of mechanical load, including fluid shear, to investigate which genes are regulated by strain and are studying the expression of a range of cell-cell and cell-matrix adhesion molecules in this process. Several years ago we found that nitric oxide (NO) is an important regulator of bone turnover and current studies are trying to elucidate the mechanism by which NO acts in bone and which NO synthase isoforms are involved. Recent work has started to study the differentiation of osteogenic cells from mesenchymal stem cells in the bone marrow focussing especially on differences seen in this process between patients with osteoarthritis and patients with osteoporosis.

Research in these areas is funded by the Arthritis Research Campaign, the Wellcome Trust, the Chief Scientist Office, Action Medical Research, Medical Research Scotland, The National Association for the Relief of Paget's Disease, Procter and Gamble, Novartis and the European Calcified Tissue Society.

Personnel include Prof Richard Aspden, Dr Fraser Coxon, Dr. Julie Crockett, Dr Miep Helfrich and Prof Mike Rogers.

Relevant recent publications:

  1. Guerrini, MM, Sobacchi, C, Cassani, B, Abinun, M, Kilic, SS, Pangrazio, A, Moratto, D, Mazzolari, E, Clayton-Smith, J, Orchard, P, Coxon FP, Mellis, D, Helfrich M, Crockett J, Vellodi, A, Ilhan Tezcan, I Notarangelo LD, Rogers MJ, Vezzoni P, Frattini A, Villa A. 2008 Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to RANK TNFRSF11A mutations. Am. J. Human. Genet. In press.
  2. Helfrich, MH and Hocking, LJ. Genetics and aetiology of Pagetic disorders of bone. Arch Biochem Biophys. 473:172-82, 2008.
  3. Coxon, FP, Thompson, K, Roelofs, AJ, Ebetino, FH and Rogers, MJ. Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells. Bone. 42:848-60, 2008.
  4. L.V. Burgin and R.M. Aspden. Impact testing to determine the mechanical properties of articular cartilage in isolation and on bone. Journal of Materials Science: Materials in Medicine 19: 703-711, 2008.
  5. Burgin, LV and Aspden, RM. A drop tower for controlled impact testing of biological tissues. Med Eng Phys. 29:525-30, 2007.
  6. Crockett, JC, Schütze,N, Tosh, D, Jatzke,S, Duthie, A, Jakob, F, Rogers, MJ. The matricellular protein CYR61 inhibits osteoclastogenesis by a mechanism independent of alphavbeta3 and alphavbeta5. Endocrinology. 148:5761-8, 2007.
  7. Helfrich, MH, Crockett, JC, Hocking, LJ and Coxon, FP The pathogenesis of osteoclast diseases: some knowns but still many unknowns. BoneKEy-Osteovision. 4:61-77, 2007. DOI 10:1138/20060249.
  8. Jeffrey, JE and Aspden RM. Cyclooxygenase inhibition lowers prostaglandin E2 release from articular cartilage and reduces apoptosis but not proteoglycan degradation following an impact load in vitro. Arthritis Res Ther. 9:R129, 2007.
  9. Ralston, S.H., Afzal, M.A., Helfrich, M.H., Fraser, W.D., Gallagher, J.A., Mee, A. & Rima, B. Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone. J Bone Miner Res 22, 569-577, 2007.
  10. Sobacchi, C, Frattini, A, Guerrini, M, Abinum, M, Pangrazio, A, Susani, L, Bredius, R, Mancini, G, Cant, A, Bishop, N, Grabowski, P, Del Fattore, A, Messina, C, Errigo, G, Coxon, FP, Scott, DI, Teti, A, Rogers, MJ, Vezzoni, P, Villa, A and Helfrich MH. Osteoclast-poor osteopetrosis due to mutations in RANKL. Nat. Gen., 39: 980-962, 2007.
  11. Taylor, A., Rogers, M.J., Tosh, D and Coxon, F.P. A novel method for efficient generation of transfected human osteoclasts. Calcif Tiss Int 80, 132-136, 2007.
  12. Van Wesenbeeck, L., Odgren, P.R., Coxon. F.P., Frattini, A., Moens, P., Perdu, B., MacKay, C.A., Van Hul,. E., Timmermans, J-P., Vanhoenacker, F., Jacobs, R., Peruzzi, B., Teti, A., Helfrich, M.H., Rogers, M.J., Villa, A. and Van Hul, W. Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J. Clin Invest117, 919-930, 2007.
  13. Dunford, JE, Rogers, MJ, Ebetino, FH, Phipps, RJ, and Coxon, FP. Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42 and Rho GTPases. J Bone Miner Res21, 684-694, 2006.
  14. Jeffrey, JE and Aspden RM. The biophysical effects of a single impact load on human and bovine articular cartilage. Proc Inst Mech Eng [H]. 220:677-86, 2006.
  15. Plumb, MS, Treon, K and Aspden RM. Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro. Biochim Biophys Acta. 1760:762-7, 2006.
  16. Yao, L, Bestwick, CS, Bestwick, LA, Maffulli, N, and RM. Aspden. Phenotypic drift in human tenocyte culture. Tissue Engineering 12, 1843-9, 2006.
  17. Plumb, MS and Aspden RM. The response of elderly human articular cartilage to mechanical stimuli in vitro. Osteoarthritis Cartilage. 13:1084-91, 2005.
  18. Van 't Hof, RJ, MacPhee, J, Libouban, H., Helfrich, MH and Ralston, SH. Decreased levels of bone turnover in mice deficient in nNOS; Possible indications for a central mechanism of regulation for bone turnover. Endocrinology, 145:5068-74, 2004.
  19. Coxon, FP and Rogers, MJ. The role of prenylated small GTP-binding proteins in the regulation of osteoclast function. Calcif Tissue Int. 72:80-4. 2003.
  20. Benford, HL, McGowan, NWA, Helfrich, MH, Nuttall, ME, and Rogers, MJ. Visualisation of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone, 28:465-473, 2001.