Physicochemical properties and release characteristics of growth factor‐modified calcium phosphate bone cement

The addition of growth factors, such as recombinant human transforming growth factor‐β1 (rhTGF‐β1) to calcium phosphate cements (CPCs) may improve bone regeneration. Previously we have shown that the differentiation of pre‐osteoblastic cells from adult rat long bones was stimulated by rhTGF‐β1 in CPC. CPC that was intermixed with rhTGF‐β1 and then applied in rat calvarial defects enhanced bone growth around the cement and increased the degradation of the cement. It is still unknown however whether the addition of rhTGF‐β1 changes the material properties of the CPC, and what the release characteristics are of rhTGF‐β1 from the CPC. We therefore determined here the release of rhTGF‐β1 in vitro from the cement pellets as implanted in the rat calvariae. The possible intervening effects of rhTGF‐β1‐intermixing on clinical compliance of CPC were studied by assessing its compressive strength and setting time, as well as crystallinity, calcium to phosphorus ratio, porosity and microscopic structure. CPC was prepared by mixing calcium phosphate powder (58% α‐tricalcium‐phosphate, 25% dicalcium‐phosphate anhydrous, 8.5% calcium‐carbonate and 8.5% hydroxyapatite), with liquid (3 g/ml). The liquid for standard CPC consisted of water with 4% sodium hydrogen phosphate, while the liquid for modified CPC, was mixed with an equal amount of 4 mM hydrochloride with 0.2% bovine serum albumin. The hydrochloride liquid contained the rhTGF‐β1 in different concentrations for the release experiments. Most of the incorporated rhTGF‐β1 in the cement pellets was released within the first 48 hr. Approximately 0.5% rhTGF‐β1 (intermixed at 100 ng to 2.5 mg/g CPC) was released within the first 4 hr increasing to 1% after 48 hr. rhTGF‐β1 release continued at 0.1% up to at least 8 weeks. Modification of CPC slightly increased the initial setting time at 20°C from 2.6 to 5 min, but did not affect the final setting time of the CPC at 20°C, nor the initial and final setting time at 37°C. The compressive strength was increased from 18 MPa (standard CPC) to 28 MPa (modified CPC) only at 4 hr after mixing. The compressive strength diminished in the modified CPC between 24 hr and 8 weeks from 55 to 25 MPa. X‐ray diffraction revealed that both standard and modified CPC changed similarly from the basic components, alpha‐tri‐calcium phosphate and dicalcium phosphate anhydrous, into an apatite cement. The calcium to phosphorus ratio as determined by an electron microprobe did not differ for standard CPC and modified CPC. Standard and modified CPC became a dense and homogeneous structure after 24 hr, but the modified CPC contained more crystal plaques compared to the standard CPC, as observed by scanning electron microscopy (SEM). SEM and back scattered electron images revealed that after 8 weeks both cements showed an equally and uniform dense structure with microscopic pores (less than 1 μm). Both CPCs showed fewer crystal plaques at 8 weeks than at 24 hr. This study shows that the calcium phosphate cement was not severely changed by modification of the CPC for rhTGF‐β1. Clinical handling may be affected by the prolonged setting time of modified cement, but it is still within preferable limits. Compressive strength was for both standard and modified cements within the range of thin trabecular bone, and therefore both CPCs can withstand equal mechanical loading. The faster diminishing compressive strength of modified cement (from 24 hr to 8 weeks) likely results in early breakdown, and therefore might be favourable for bone regeneration. Together with our previous studies showing the beneficial effects of adding rhTGF‐β1 to CPC on bone regeneration, we conclude that the envisaged applications for CPC in bone defects are upgraded by intermixing of rhTGF‐β1. Therefore the combination of CPC with rhTGF‐β1 forms a promising synthetic bone graft.