Introduction: Fluoride ion (F-) is well known to be incorporated in hydroxyapatite (HA) and form fluoridated hydroxyapatite (FHA). Fluoride ion stimulates osteoblastic cells[1],[2] and modifies protein adsorption on the calcium phosphate. We previously reported that synthetic octacalcium phosphate (OCP), a precursor of HA, promotes osteoblastic cell differentiation and facilitates bone regeneration[3],[4]. Because of the structure similarity to HA, OCP prepared with F- possibly improves osteogenic capability of original OCP. The aim of this study is to investigate the structural characteristics, protein adsorption, and cellular response of fluoride-containing calcium phosphate (F-CaP) derived from OCP. F-CaPs were prepared two different methods, such as hydrolysis of OCP in F- solution and co-precipitation of OCP with F- .
Materials and Methods: F-CaP prepared by hydrolysis (HF-CaP) was synthesized by incubating OCP in 150 mM Tris buffer (pH = 7.1~9.9) containing 50 ppm F- at 37oC. F-CaP prepared by co-precipitation (CF-CaP) was synthesized by adding calcium solution into phosphate solution including 12 and 96 ppm F- at 70oC[5]. The chemical composition and crystal structure of the F-CaPs were examined using inductively coupled plasma (ICP), fluoride electrode and x-ray diffraction (XRD), and field emission scanning electron microscope (FE-SEM). The solubility of the products was evaluated by Ca2+ concentration released after immersing them at pH = 7.4 and 4.0. Protein adsorption on F-CaPs was analyzed using bovine serum albumin (BSA) and cytochrome c. For cell attachment and proliferation test, mouse bone marrow stromal ST-2 cells were seeded onto coatings of F-CaPs and OCP. The number of cells on the coatings or polystyrene culture plate (no coating) was determined using the WST-8 assay.
Results: Chemical composition and XRD analysis showed that HF-CaP tended to convert to FHA depending on the F- content of the solids. On the other hand, CF-CaP possessed not only the reflection of OCP, but also that of fluoridated hydroxyapatite (FHA). The solubility of HF-CaP was decreased depending on the fluoride content, whereas that of CF-CaP was comparable to OCP regardless of fluoride contens. HF-CaP adsorbed more cytochrome c than CF-CaP and OCP, whereas BSA adsorption was inhibited on HF-CaP. The difference of protein adsorption behaviors between two types of F-CaPs affected cell attachment and proliferation of ST-2 cells. CF-CaP with high F- content promoted cell attachment and proliferation more than OCP. In contrast, the cell numbers on the HF-CaP with middle F- content were lowest among all the groups.
Discussion and Conclusion: The present study confirmed that one of the CF-CaP with high F- content enhances the attachment and proliferation of mouse bone marrow stromal cells. The synthesis processes of incorporating F- to OCP had crucial effects on the crystal structure and morphology as well as the protein adsorption on F-CaP. CF-CaP preserved a small amount of OCP in FHA and maintained and Ca2+ concentration in the medium at a level similar to the original OCP. It seems likely that CF-CaP with high F- content may work as a better substrate for coating an implant surface or bone substitute material.
References:
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