Transforming growth factor-beta 1 incorporation in a calcium phosphate bone cement: Material properties and release characteristics

The bone regenerative properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human tran sforming growth factor-beta1 (rhTGF-beta1). Previously, we showed that rhTG F-beta1 in CPC stimulated the differentiation of preosteoblastic cells from adult rat long bones. The intermixing of rhTGF-beta1 in CPC, which was sub sequently applied to rat calvarial defects, enhanced bone growth around the cement and increased the degradation of the cement. However, it is unknown whether the addition of rhTGF-beta1 changes the material properties of CPC and what the characteristics of the release of rhTGF-beta1 from CPC are. T herefore, we determined in this study the release of rhTGF-beta1, in vitro, from the cement pellets as implanted in the rat calvariae, The possible in tervening effects of rhTGF-beta1 intermixing on the clinical compliance of CPC were studied through an assessment of its compressive strength and sett ing time, as well as its crystallinity, calcium-to-phosphorus ratio, porosi ty, and microscopic structure. We prepared CPC by mixing calcium phosphate powder (58% alpha -tricalcium phosphate, 25% anhydrous dicalcium phosphate, 8.5% calcium carbonate, and 8.5% hydroxyapatite) with a liquid (3 g/mL). T he liquid for standard CPC consisted of water with 4% disodium hydrogen pho sphate, whereas the liquid for modified CPC was mixed with an equal amount of 4 mM hydrochloride with 0.2% bovine serum albumin. The hydrochloride liq uid contained rhTGF-beta1 in different concentrations for the release exper iments. Most of the rhTGF-beta1 incorporated in the cement pellets was rele ased within the first 48 h. For all concentrations of intermixed rhTGF-beta 1 (100 ng to 2.5 mg/g of CPC, approximately 0.5% was released in the first 4 h, increasing to 1.0% after 48 h. Further release was only about 0.1% fro m 2 days to 8 weeks. CPC modification slightly increased the initial settin g time at 20 degreesC from 2.6 to 5 min but had no effect on the final sett ing time of CPC at 20 degreesC or the initial and final setting times at 37 degreesC. The compressive strength was increased from 18 MPa in the standa rd CPC to 28 MPa in the modified CPC only 4 h after mixing. The compressive strength diminished in the modified CPC between 24 h and 8 weeks from 55 t o 25 MPa. No other significant change was found with the CPC modification f or rhTGF-beta1. X-ray diffraction revealed that standard and modified CPCs changed similarly from the original components, alpha -tricalcium phosphate and anhydrous, dicalcium phosphate, into an apatite cement. The calcium-to -phosphorus ratio, as determined with an electron-microprobe, did not diffe r for standard CPC and modified CPC. Standard and modified CPCs became dens e and homogeneous structures after 24 h, but the modified CPC contained mor e crystal plaques than the standard CPC, as observed with scanning electron microscopy (SEM). SEM and backscattered electron images revealed that afte r 8 weeks the cements showed equally and uniformly dense structures with mi croscopic pores (<1 mum). Both CPCs showed fewer crystal plaques at 8 weeks than at 24 h. This study shows that CPC is not severely changed by its mod ification for rhTGF-beta1. The prolonged setting time of modified cement ma y affect the clinical handling but is still within acceptable limits. The c ompressive strength for both standard and modified cements was within the r ange of thin trabecular bone; therefore, both CPCs can withstand equal mech anical loading. The faster diminishing compressive strength of modified cem ent from 24 h to 8 weeks likely results in early breakdown and so might be favorable for bone regeneration. Together with the beneficial effects on bone regeneration from the addition of rhTGF-beta1 to CPC, as shown in our previous studies, we conclude that the envisaged applications for CPC in bone defects are upgraded by the inte rmixing of rhTGF-beta1. Therefore, the combination of CPC and rhTGF-beta1 f orms a promising synthetic bone graft. (C) 2001 John Wiley & Sons, Inc.