Introduction: The recent conflicts in Iraq and Afghanistan have led to unprecedented numbers of survivors of combat trauma with significant extremity injury[1]-[3]. These patients are so numerous and have such a high chance of developing heterotopic ossification (HO) that one author has described the current situation as a “military HO epidemic”[4]. The accepted methods of preventing this condition are not as effective or appropriate for combat injured patients as we could hope for. Once established, the only effective treatment for this debilitating condition is major surgery to excise this tissue. Our novel approach is to target the mineral component of HO directly, in order to prevent its formation and dissolve it before it is able to cause the wide range of debilitating symptoms seen in affected patients. We chose to investigate the potential of the family of molecules known as polyphosphates due to their demonstrated ability to dissolve hydroxyapaite[5].
Materials and Methods: We incubated monoliths of the calcium phosphate phases hydroxyapatite and brushite in a selection of polyphosphates in order to investigate their dissolving ability. This was quantified by measuring mass loss over time. Hydroxyapatite sol was also synthesised in the presence of these reagents in order to investigate their effect on hydroxyapatite crystal synthesis. The polyphosphate hexametaphosphate was incubated with hydroxyapatite sol in order to investigate the role of pH in controlling its dissolving ability. A simple analogue for endochondral ossification utilising a hydroxyapatite-gellan system was used to investigate the ability of hexametaphosphate to dissolve the mineral phase from within a scaffold. Finally, mechanical testing was undertaken on samples of ex-vivo murine bone to establish the effect of hexametaphosphate on biological hydroxyapatite in its native setting.
Results and Discussion: Hexametaphosphate was shown to be a potent hydroxyapatite and brushite dissolving agent with a relative mass loss of 3.12% and 11.6% per day respectively. This effect was found to be highly pH sensitive with higher dissolving ability at lower pH values for all concentrations of hexametaphosphate on test. Hexametaphosphate was shown to inhibit the synthesis of hydroxyapatite as demonstrated by an increased amount of unreacted precursor on X-ray diffraction of the product. In the simple qualitative endochondral ossification model, hexametaphosphate was shown to dissolve the mineral component selectively without affecting the gellan matrix. Finally, incubation of isolated murine tibiae in hexametaphosphate reduced their stiffness and maximum force to failure by 49% and 41% respectively without reducing their yield force. Taken as a whole, these results demonstrate that hexametaphosphate is able to dissolve hydroxyapatite in multiple forms and at physiological pH.
Conclusions: The results of this experimental series demonstrate proof of concept that targeting the mineral component of HO directly is feasible and may represent a novel treatment and preventative strategy in this disease. The key future challenges will be to demonstrate efficacy in an in vivo model of HO and to design a formulation that will allow the treatment to be deployed without affecting normal bone. Hexametaphosphate is a relatively benign and readily available chemical that may, in time, be formulated into a workable treatment that will improve quality of life not only for those currently affected by HO but also those who will, unfortunately, be injured in future conflicts.
The RAMC Charity Drummond Committee; Joint Forces Command
References:
[1] FORSBERG, J. A., PEPEK, J. M., WAGNER, S., WILSON, K., FLINT, J., ANDERSEN, R. C., TADAKI, D., GAGE, F. A., STOJADINOVIC, A. & ELSTER, E. A. 2009. Heterotopic ossification in high-energy wartime extremity injuries: prevalence and risk factors. The Journal of Bone & Joint Surgery, 91, 1084-1091.
[2] POTTER, B. K., BURNS, T. C., LACAP, A. P., GRANVILLE, R. R. & GAJEWSKI, D. 2006. Heterotopic ossification in the residual limbs of traumatic and combat-related amputees. J Am Acad Orthop Surg, 14, S191-7.
[3] POTTER, B. K., FORSBERG, J. A., DAVIS, T. A., EVANS, K. N., HAWKSWORTH, J. S., TADAKI, D., BROWN, T. S., CRANE, N. J., BURNS, T. C., O'BRIEN, F. P. & ELSTER, E. A. 2010. Heterotopic ossification following combat-related trauma. J Bone Joint Surg Am, 92 Suppl 2, 74-89
[4] ALFIERI, K. A., FORSBERG, J. A. & POTTER, B. K. 2012. Blast injuries and heterotopic ossification. Bone Joint Res, 1, 192-7.
[5] MCGAUGHEY, C. 1983Binding of polyphosphates and phosphonates to hydroxyapatite, subsequent hydrolysis, phosphate exchange and effects on demineralization, mineralization and microcrystal aggregation. Caries Res, 17, 229-241