Bone is a dynamic and complex organ continuously engaged in a remodeling process of resorption and formation. The coupled regulation of osteoclast and osteoblast activity is dependent upon a variety of local and systemic factors. My research is aimed at identifying and characterizing specific molecules involved in bone remodeling and examining the mechanisms by which these factors may influence the structure and function of normal and pathological bone. This is achieved through the combined use of cellular and molecular in vitro techniques along with detailed histological and histomorphometric analysis of genetically manipulated mice and rodent models of tumor-induced bone disease and injury.

Bone Remodeling. The remodeling process of bone comprises the coupled activity of bone resorbing osteoclasts and bone forming osteoblasts. This system is tightly controlled by a number of soluble regulatory factors and through cellular interactions within the bone microenvironment.

BONE REMODELING AND THE OSTEOCLAST

Bone remodeling is a sensitive, dynamic process which begins with a period of osteoclast recruitment and bone resorption. The formation and activity of these large, multinucleated cells is mediated primarily via osteoclastogenic factors and cell-to-cell interactions with various constituents of the bone microenvironment. The inappropriate expression and altered levels of these factors results in the dysregulation of osteoclastogenesis and uncoupling of the bone remodeling process. This frequently leads to the debilitating bone loss associated with tumor-induced bone disease and disorders such as osteoporosis and rheumatoid arthritis.

Active osteoclasts. Large, multinucleated osteoclasts are seen resorbing trabecular bone in vivo (A, H&E orig. mag. x 400, B, TRAP orig. mag. x 100).

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approx. 2.5 million people in the US alone, and causes pain, swelling and damage to cartilage and bone in multiple joints. While the initial cause of RA remains unknown the development of this condition is through the dysregulated action of cells of the immune system and the untimely release of inflammatory factors, leading to synovial swelling and bone destruction.

Increased levels of inflammatory factors within the bone environment have been shown to induce osteoclast formation and consequently stimulate bone loss. Recently a newly identified member of the TNF superfamily, LIGHT, was shown to induce T-cell activation and proliferation. As T-cells and T-cell products have been implicated in osteoclast formation and function, the role of LIGHT in RA-associated bone loss was investigated.

These studies, performed at the University of Oxford alongside Dr. Afsie Sabokbar and Prof. Nick Athanasou clearly demonstrate an increase in TRAP+ multinucleated cells following LIGHT treatment. These cells were also capable of resorbing large areas of dentine. In addition, increased levels of soluble LIGHT protein were found in the serum of RA patients, suggesting that LIGHT may play a role in the unbalanced bone remodeling seen in RA-osteoarticular tissue.

Increasing Light Concentrations
LIGHT-induced osteoclast formation. LIGHT treated PBMCs form TRAP+ multinucleated osteoclasts (Edwards et al 2006).

Since there is no cure for RA, recent approaches to the treatment of the debilitating bone loss associated with this disease have focused on the neutralization of potent inflammatory factors such as TNFα. These biologic agents specifically target parts of the immune system that lead to inflammation as well as joint and tissue damage. Etanercept (Enbrel) for example, is a recombinant soluble TNFα receptor which can bind to TNFα and decreases its role in inflammatory diseases. Identification and characterization of factors such as LIGHT, will allow for a greater understanding of the processes involved in RA-associted osteoarticular disease and allow for the development of novel therapeutic targets to combat the effects of this destructive condition.

Dysregulated bone remodeling can also occur as a result of tumor metastases to bone. Osteoclasts are recruited by tumor-derived factors to degrade bone, leading to uncontrolled bone resorption and facilitation of tumor progression. The increase in osteoclast activity leads to the release of bone matrix proteins which can stimulate tumor cell proliferation and survival, ultimately resulting in a viscous cycle of osteoclast formation, bone degradation and tumor cell proliferation. This system represents an extreme example of how the bone remodeling process can be manipulated in pathological conditions, resulting in decreased bone volume and increased tumor burden.

BONE REMODELING AND THE OSTEOBLAST

Osteoblast differentiation and bone formation can be controlled by a number of regulatory molecules. TGF-β for example, is abundant in the bone matrix and is liberated in large quantities following a period of bone resorption. Bone Morphogenetic Proteins (BMPs) are bone-derived molecules of the TGF-β superfamily, which play a clear role in the bone remodeling process and are critical to embryonic development. BMPs have been shown to stimulate osteoblast differentiation and bone formation along with influencing osteoclast formation and function, although the mechanisms which underlie this process are still emerging.

Our work has identified a number of regulatory mechanisms governing BMP expression and bone formation, and in collaboration with Prof. Steve Harris’ group at UTHSCSA, have characterized the skeletal phenotype of osteoblast specific BMP-2 and -4 KO mice. These mice were created by members of the Harris lab and have become a valuable tool in assessing the role of BMP and BMP-inductive molecules in skeletal physiology and bone development.

BMP-4cKO mouse and control littermate. BMP-4cKO animals are considerably smaller in size and weight than littermate controls. Also, these mice have decreased BMD and display a number of patterning defects such as a distorted sternum and various degrees of polydactyly.
Figure A	Figure B
Decalcified histological sections of control (A) and BMP-4cKO (B) L4 vertebrae. A considerable decrease in trabecular bone is seen in mice deficient in osteoblast-derived BMP-4 (H&E/Orange G, orig. mag. x40).
Undecalcified vertebrae from BMP-2cKO mice. Von Kossa/Van Gieson staining of L4 vertebrae demonstrating mineralized bone (black) and unmineralized osteoid (red) underlying active osteoblasts.

In addition to bone formation, the BMP family has also been shown to mediate osteoclast formation and activity. These dual effects emphasize the dynamic nature of this family of regulatory molecules and demonstrates the diverse role of BMP-family members in the delicate process of bone remodeling.

BONE REMODELING AND INJURY

Following the trauma of a bone fracture, the normal process of bone remodeling becomes drastically altered as the bone environment attempts to compensate for the loss of mechanical strength and to manage the repair of the injured site. Even though 7 million skeletal fractures occur in the U.S. each year, the treatment has remained essentially unchanged for centuries. Recent studies investigating the regulatory factors controlling bone remodeling indicate that a number of compounds may be used to enhance local growth factor production and stimulate bone formation and repair. Given the unique attribute of statins to regulate BMP-2 expression and stimulate bone growth, these compounds were tested in a pinned-rat fracture model for improved fracture healing and biomechanical parameters.

Day 0 2 Weeks 6 Weeks Vehicle-Treated Statin-Treated

Radiographic assessment of statin-treated bone fractures. Increased fracture healing is evident in groups treated with statins compared to vehicle-treated controls.

Radiological evaluation of statin treated bones showed enhanced fracture repair at 2 weeks with complete healing by week 6. Bone mineral density, breaking force and stiffness were also increased following statin treatment. In addition, detailed histological analysis illustrated a clear increase in the rate of bone remodeling and consequent healing process following statin administration. In treated samples, the fracture callus was seen to develop at a faster rate and contain more trabecular bone, with a greater number of osteoclasts and proliferating cells than vehicle treated controls.

Vehicle Treated
Statin Treated
Increased rates of repair are observed in statin-treated bone fractures. Histological analysis of fractured bones clearly illustrated a dramatic increase in the rate of remodeling and repair following statin administration.

This investigation clearly demonstrates how pharmacological manipulation of bone remodeling can lead to an improved response in the healing process of the skeleton. The rapid, initial production of temporary, woven bone followed by an increased and sustained osteoclast recruitment and bone remodeling period clearly demonstrate the potential of statins as a new and effective therapeutic modality to hasten fracture repair.

These investigations explore a range of factors influential in the bone remodeling process and continue to identify novel mediators of bone formation and resorption. Collectively, these studies examine and expand upon existing information relating to the molecules involved in the regulation of bone remodeling and offer novel therapeutic approaches to combating the devastating bone loss associated with the dysregulation of this sensitive and dynamic process.

Research Techniques:

• Handling of human and murine, general and orthopaedic tissue samples for histological analysis, encompassing specimen preparation and processing, microtomy of frozen and fixed tissue including calcified and decalcified, plastic and paraffin embedded bone, tinctorial and immunohistochemical staining and detailed bone histomorphometric analysis.
• Animal handling and dissection including new-born calvariae and mature bone marrow stroma isolation, calvariae injection, subcutaneous and tumor cell inoculation and bone fracture analysis.
• Cell culture techniques employing numerous cell lines and primary cells including the isolation and culture of human monocytic cells and murine bone marrow stromal cells, for use in such techniques as the assessment of osteoclast formation and bone resorption following controlled differentiation.
• Molecular techniques including chromatin immunoprecipitation assays, realtime PCR, in situ hybridisation, gene specific site-directed mutatagenesis and DNA construct preparation, bacterial transformation, culture, isolation, purification and transfection; previous procedures include promoter studies, design of dominant negative constructs and siRNA transfections.
• Biochemical techniques including flow cytometry, ELISA and western blotting.

Recent Publications:

Adamopoulos, PB Wordsworth, JR Edwards, DJ Ferguson, NA Athanasou. Osteoclast differentiation and bone resorption in multicentric reticulohistiocytosis. Hum Pathol. 2006 Sep;37(9):1176-85.

JR. Edwards, SG. Sun, CM. Shipman, I. Adamapoulos, NA Athanasou, A Sabokbar. LIGHT (TNFSF14), a novel mediator of bone resorption, is elevated in rheumatoid arthritis. Arthritis Rheum. 2006 May;54(5):1451-62.

JR. Edwards, M. Zhao, M. Qiao, MA. Harris, SE. Harris, GR. Mundy. The Activated Phospho-CREB Transcription Factor Stimulates BMP-2 Expression in Murine Osteoblast Cells. J Bone Miner Res. 2005  Sept;20 Supp 1:S1-S512 (abstract)

A Loughry, S Fairchild, N Athanasou, J Edwards, FC Hall. Inflammatory arthritis and dermatitis in thymectomized, CD25+ cell-depleted adult mice. Rheumatology (Oxford). 2005 Apr;44(4):569.

N. Dalbeth, J. Edwards, S. Fairchild, M. Callan, F. Hall. The non-thiol angiotensin-converting enzyme inhibitor Quinapril suppresses inflammatory arthritis. Rheumatology. 2005 Jan;44 (1):24-31.

JR Edwards, DG Jackson, P McNally, CLMH Gibbons, NA Athanasou. The Role of Lymphatics in the Growth and Spread of Primary and Secondary Bone Tumours.  Journal of Pathology, 2004; Sep, 204 Suppl:1-54 (abstract).

H. Xu, JR. Edwards, O. Espinosa, S. Banerji, DG. Jackson, N. Athanasou. Expression of a lymphatic endothelial cell marker in benign and malignant vascular tumours. Hum Pathol. 2004 Jul;35(7):857-61.

H. Xu, J. Edwards, S. Banerji, R. Prevo, DG. Jackson, N. Athanasou. Distribution of lymphatic vessels in normal and arthritic human synovial tissue. Annals of Rheumatic Disease. 2003; 62 (12):1227-9

L. Danks, SG. Sun, JR. Edwards, A. Sabokbar, N. Athanasou. RANKL and M-CSF are required for osteoclast differentiation by macrophages in breast cancer. Journal of Pathology, 2003; 201: 48 (abstract)

A. Sabokbar, HC. Blair, S. Sun, JR. Edwards, L. Danks, N. Athanasou. TRAIL is expressed in quantities similar to RANKL in human periprosthetic pseudomembrane, osteoarthritic stromal cells and osteoblasts, Calcified Tissue International, 2003; 72 (4): 331 (abstract)