【正文】 nanospheres regulate apatite crystal morphology during enamel formation.6Thus, ECM plays critical roles not only by providinga space to deposit mineral crystals but also by conducting nucleation, crystal morphology, and orientation of the biominerals with regard to hard tissuegrowth.7–9Several works have used calcification systems in various hydrogels and attempted to understand the role of the matrix protein in biomineralization.10,11For example, Boskey et al. established diffusion systems using gelatin and agarose gels anddemonstrated the promoting/inhibiting action of thenoncollagenous protein on HAp formation.12–14Hunter et al. used an acrylamide gel to investigatethe mineralization process in vitro.15,16Thus, formation of minerals in organic matrices is regarded asone of the mineralization methods that mimic hardtissue growth, and these organic/inorganic posite materials fabricated by the biomimetic processmight be useful in bone tissue engineering. However, the inorganic phase fabricated in the abovementioned gel systems mostly prised low crystalline apatite. It is desirable to control the propertiesof the inorganic phase in the posite materials foruse in bone tissue engineering, because the materialsrequire distinctive properties (., bioabsorbability)for each therapeutic application. To control this inorganic property, we attempted to fabricate the posite materials in a variety of conditions.Fluoride enhances the stabilization of the HApcrystals by substituting the hydroxyl ions in its crystalline lattice with fluoride ions. The fluoride ionCorrespondence to: T. Matsumoto。 contract grant number:18680039Contract grant sponsor: COE Frontier BiodentistryProgramC2112020 Wiley Periodicals, Inc.concentration in serum is 0– ppm17。 hence, weselected fibrin as the organic ponent for thisstudy. In this study, we performed in vitro mineralization using the fibrin gel under a variety of conditions and investigated the properties of the mineralsgenerated in the gel to obtain organic/inorganicposite materials with inorganic ponentsdiverse characteristics.MATERIALS AND METHODSIn vitro mineralization in the fibrin gelThe gel diffusion system was prepared as follows: Fibrinogen (4 mg/mL, Sigma) and thrombin solutions (units/mL, Sigma) were mixed in a ratio of 1:1. This solution (240 lL) was poured into a polypropylene tube (u 58 mm) and maintained in the incubator at 378C for 30 minto facilitate gelation. The Ca [100 mM (CH3COO)2CaC1H2O]and PO4(60 mM NH4H2PO4) solutions were poured intoopposite sides of the formed gel to facilitate mineralizationin the gel [Fig. 1(A)], and the entire system was maintained in the incubator at 378C for 3 days. The initial pHof the Ca and PO4solutions were and , respectively(noncontrolled). To investigate the influence of the solutionpH on mineralization, the pH values of both the solutionswere adjusted to and , respectively, by addition ofHCl or NaOH. The pH of fibrin gel was measured at eachmineralization condition to understand the pH changebefore and after mineralization. To investigate the effect offluoride on mineralization, 2–2020 ppm HF was added tothe PO4solutions. With regard to the control, mineralization in the solution system was performed by directlypouring the 25 mL Ca solution into the 25 mL PO4solution at 378C.Evaluation of the mineral productAfter mineralization, the gel was airdried for 24 h atroom temperature, and subsequently, the obtained products were identified using powder Xray diffraction (XRD)analysis (Ultima254。 40 kV and 30 mA, CuKa).Halfvalue breadths of the (002) peak were measured asthe index of crystallinity of obtained minerals.Mineral precipitation and crystal morphology wereobserved using scanning electron microscopy (SEM, JSM6390, JEOL). For the SEM observation, mineralized fibringels were fixed with 4% paraformaldehyde, rinsed sixtimes with phosphatebuffered saline (PBS), postfixed with1% OsO4, rinsed six times with PBS, and dehydrated in series of ethanol concentrations up to 100%. The gels werecriticalpointdried using CO2for 45 min, mounted, andsputtercoated with gold.Solubility experimentTo estimate the biodegradability of the obtained samples, in vitro solubility experiment was carried out. Theobtained samples were immersed in 10 mL of acetatebuffer solution mol/L at pH . After incubation at378C for 5 days, the remaining samples were pletelydissolved in 10 mL of HCl solution. The Ca concentrationin each solution was determined by an atomic absorptionspectrophotometer (AA6400F, Shimadzu) using a previously described method.27The solubility of the minerals inthe gel was defined as the ratio of the eluted Ca ions tothe initial Ca content in the posite gel.RESULTS