Imparting bone mineral affinity to osteogenic proteins through heparin–bisphosphonate conjugates

Although numerous growth factors can promote the regeneration of bone upon parenteral administration, all exhibit undesirable side-effects that prevent their clinical utility. These side-effects arise due to the growth factors' inherent lack of bone affinity. The goal of this study was to develop a means to enhance the bone mineral affinity of osteogenic growth factors so as to minimize their extra-skeletal distribution. Heparin, a glycosaminoglycan that exhibits a high affinity to numerous growth factors, was modified with bisphosphonates to enhance its affinity to bone mineral (i.e. hydroxyapatite, HA). To this end, conjugation of 1-amino-1,1-diphosphonate methane (aminoBP) onto periodate-oxidized heparin using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide was attempted. Using this chemistry, the number of aminoBPs conjugated onto heparin was modulated by varying the reacting reagent concentrations (to a maximum of 7 aminoBPs per heparin). Increasing the number of aminoBPs resulted in a more than twofold increase in heparin's affinity for HA in vitro. Subsequently, the ability of aminoBP–heparin conjugates to enhance the mineral affinity of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) was explored. The results revealed that the complexation between the conjugates and growth factors had occurred and that the conjugates increased the bone mineral affinity of bFGF and BMP-2 in an aminoBP-dependent manner. In conclusion, the conjugation of aminoBP onto heparin is a feasible approach to enhance the affinity of heparin-binding, osteogenic growth factors to HA.