A cytochrome C (cyt C) and apatite composite layer was formed on a NaOH- and heat-treated titanium substrate (Ti substrate) by immersing the Ti substrate for one day at 25 °C in supersaturated calcium phosphate solutions obtained by mixing infusion fluids. When the initial supersaturation level of the calcium phosphate solution was increased, the total amount of cyt C and apatite deposited on the Ti substrate increased. On the other hand, the cyt C content in the composite layer decreased with an increase in initial supersaturation level. The morphology of the composite layer markedly changed depending on the initial supersaturation level. Therefore, the initial supersaturation level affected the formation of the cyt C and apatite composite layer. It is expected that fibroblast growth factor-2 can be immobilized on NaOH- and heat-treated titanium substrates using the same method.
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