Effects of Heat Treatment of Calcium Hydroxyapatite Particles on the Protein Adsorption Behavior

Abstract

The effects of heat treatment of calcium hydroxyapatite (Hap) on the protein adsorption behavior were examined using typical proteins of bovine serum albumin (BSA: isoelectric point (iep) = 4.7, molecular mass (Ms) = 67,200 Da, acidic protein), myoglobin (MGB: iep = 7.0, Ms = 17,800 Da, neutral protein), and lysozyme (LSZ: iep = 11.1, Ms = 14,600 Da, basic protein). The TEM, XRD, and gas adsorption measurements ascertained that all of the Hap particles examined were highly crystallized and nonporous. The Hap single phase was continued up to the heat treatment temperature of 600 degrees C. However, after treatment above 800 degrees C in air, the beta-Ca3(PO4)2 (beta-TCP) phase slightly appeared. TG and ICP-AES measurements suggested that all of the Hap particles are Ca2+-deficient. Also, it was indicated from FTIR and XPS measurements that a partially dehydrated oxyhydroxyapatite (pd-OHap) was formed after treatment at high temperature. The saturated amounts of adsorbed BSA (nsBSA) did not vary on the Hap particles after heat treatment at 200 and 400 degrees C. However, nsBSA values were increased by raising the heat treatment temperature above 600 degrees C. The adsorption coverage of BSA was increased up to ca. 1.4. This adsorption coverage of BSA (thetaBSA) over unity suggests that the BSA molecules densely adsorbed and a part of BSA molecules adsorbed as end-on type on the Hap particle surface or BSA molecules became contracted. Similar adsorption behavior was observed on the LSZ system, but the adsorption coverage of LSZ (thetaLSZ) values are much less than thetaBSA. On the other hand, no effect of the heat treatment of Hap particles was observed on the adsorption of MGB. The increases of nsBSA and nsLSZ were explained by the increase of calcium and phosphate ions in the solutions dissolved from beta-TCP formed after heat treatment of Hap, especially treated at high temperature. The dissolved Ca2+ and PO(4)3 - ions may act as binders between proteins and Hap surfaces; the adsorption of Ca2+ ions on the Hap surface offers an adsorption site for BSA owing to its positive charge. In the case of adsorption of positively charged LSZ molecules, PO43- ions act as a binder in an opposite way. Since the MGB molecules are neutral, no binding effect of either ion was observed.