Myung Chul Chang

FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde

FT-IR analysis was performed for the hydroxyapatite (HAp)/collagen (COL) nanocomposite cross-linked by glutaraldehyde (GA). The amide bands I, II and III from COL matrix, and phosphate and carbonate bands from HAp were identified. The amide B band arising from C-H stretching mode showed a sensitive conformation by the degree of cross-linking. The amide I band showed a complicate conformational change by the degree of cross-linking. The characteristic amide I band at 1685 cm(-1), which is known as an aging parameter in the biological bone, did not show a monotonous tendency by the degree of cross-linking. The relative contents of the organics in the cross-linked HAp/COL nanocomposite were evaluated as an integration ratio between the amide I band at 1600-1700 cm(-1) and PO(4)(3-) band at 900-1200 cm(-1). The increase of the organics content by the cross-linking is enabled by the further organization of Ca(2+) ions of HAp crystals in HAp/COL nanocomposite. The complicate conformational behavior in the amide I, II and III bands seems to be affected by the cross-linking induced directional arrangement of HAp/COL nanocomposite fibrils.

Preparation of a porous hydroxyapatite/collagen nanocomposite using glutaraldehyde as a crosslinkage agent

Hydroxyapatite (HAp) has been intensively investigated as one of suitable bone substitutes in order to give the biocompatible, bioactive, biodegradable and osteoconductive properties of the natural bone [1, 2]. HAp has been frequently modified by the organics of PMMA, polylactide, chondroitin sulfates, chitosan and collagen (COL) [3–11]. It is known that the human bone is an extracellular matrix mainly composed of HAp nanocrystals and COL fibers. HAp nanocrystals are aligned their c-axes along COL fibers [6]. This HAp embedded collagen nanostructure could have been reproduced using a biomimetic coprecipitation method [6]. In this study we tried to prepare a porous structure of HAp/COL nanocomposite using glutaraldehyde (GA) as a cross-linkage agent. The pure Ca(OH)2 was obtained through the hydration of CaO calcined at 1150 ◦C for 3 h [8]. The slurry of HAp/COL nanocomposites was prepared by the simultaneous titration method using the pH controller as described in detail by Kikuchi et al. [6]. Homogeneous suspension including 0.1994 mol of Ca(OH)2 dispersed in 2 1 of H2O and 59.7 mM of H3PO4 aqueous solution with 5 g of COL was gradually added into a reaction vessel through tube pumps. The weight ratio of HAp/Col was fixed at 80/20. The temperature and pH of reaction solution in vessel was set as 38 ◦C and 8.4, respectively. After the coprecipitation process, the slurry obtained was aged at 38 ◦C for 12 h and pH has gradually lowered to 7.0. Then an aqueous solution of glutaraldehyde (0.2%) was slowly dropped into the slurry solution 38 ◦C; The total amount of GA was regulated to be 30, 90, 300 and 600 molecules per collagen molecule. The samples obtained were hereafter denoted by HCG30, HCG90, HCG300 and HCG600, respectively; the pure collagen and the HAP/collagen composite without cross-linkage were abbreviated as COL and HAP/COL, respectively. Crosslinkage of COL with GA involves the reaction of the free amine groups of lysine or hydroxlysine amino acid residues of the polypeptide chains with the GA aldehyde groups [14]. The HAp/COL slurry was filtered using glass filter and gently washed five times with ion-exchanged water. The precipitates obtained were dried in a freeze dryer (Advantage, Vir Tis, USA) at −30 ◦C under vacuum, or were naturally dried in the air at 25 ◦C. The samples were dipped in ion-exchanged water or in simulated body-fluid in order to check dissolution and water absorption. The precursors used were CaCO3 (alkaline analysis grade, Wako, Japan), H3PO4 (AP grade, Wako, Japan), collagen (MW 300,000, Nitta Gelatin, Japan) and GA (25% aqueous solution, MW 100, AP grade, Wako, Japan). Collagen was extracted from porcine dermis and telopeptide was removed by treating with an enzyme. A length of COL fiber is 300 nm, which is the length of α chain of COL. The collagen was diluted in a phosphoric water solvent by the supplier (Nitta Gelatin) and the typical property was pH 2.23, conc. 10 mg/l. and 20 mM (phosphoric acid). The microstructures of the composites were observed by scanning electron microscopy (SEM, TOPCON, Japan). A chemical reaction between HAP nanocrystals and functional groups of collagen was evaluated using a diffuse reflection FT-IR (Spectrum 2000, Perkin-Elmer, UK). Three-point bending strength and Young’s modulus were measured by a universal testing machine (AGS-H, Shimadzu, Japan) at a cross-head speed of 500 μm/min with a span of 15 mm and the typical samples size was 5 × 3 × 20 mm3. Fig. 1 shows the SEM photographs of the samples prepared. Peculiar pore structures could be observed for the cross-linked samples (HCG30, HCG90, HCG300 and HCG600). Figs 1B and D show the microstructure of naturally dried HCG30, and Figs 1A and E those of a freeze-dried HCG30 sample and a naturally dried HCG300 sample, respectively. One of HCG30 samples (Fig. 1C) was semi-dried at room temperature in air and then completely dried in a freeze dryer. From Fig. 1C we can confirm the strong development of COL bundles. The fractured COL bundles (a white arrow) due to the sectioning process show an evidence for the selfalignment of COL bundles. The COL bundles are well aligned and shows woven networks (a black arrow). From the freeze dried sample (A) we can also observe the randomly distributed pore channels. From the naturally dried samples (B, E) we can observe the columnar open pore channels in matrices and many small pores in the channels. Fig. 1D shows a microstructure for a perpendicular section for Fig. 1C and we can observe many open pores (white arrows). Therefore the open channels probably formed a 3-dimensional network. When glutaraldehyde was added into the composite slurry during the preparation process, the size of the

XPS study for the microstructure development of hydroxyapatite–collagen nanocomposites cross-linked using glutaraldehyde

X-ray photoelectron spectroscopy (XPS) was measured for the dry body of hydroxyapatite (HAP)-collagen (COL) nanocomposites cross-linked using glutaraldehyde (GA). Survey scan XPS showed the elemental spectra of N, C, O, Ca and P, which came from HAP and COL. The covalent bond formation between Ca2+ of HAP and RCOO- of COL molecule was confirmed by XPS. The bridge formation between COL fibers could be assessed from C1s and N1s band spectrum. The dehydration of swelling water during drying led to the reduction of linking distance for pendant GA between COL fibrils and contributed to further cross-linkage reaction. Cross-linkage induced the enlargement of length scale unit of COL. The modification of length scale was doing the key role of structure manipulation in the cross-linked HAP-COL nanocomposites.

The cross-linkage effect of hydroxyapatite/collagen nanocomposites on a self-organization phenomenon

Hydroxyapatite(HAp)/collagen nanocomposites were prepared by a coprecipitation method controlling the degree of cross-linkage between collagen molecules using glutaraldehyde. The precipitates filtered were dried in a freeze drier or naturally dried in the air at 25 °C. The naturally dried cakes had open channels of 5–15 μm in diameters, which were three-dimensionally and regularly developed over the whole samples, and showed a pretty good mechanical strength. The channels that were formed at spaces among the HAp/collagen particles, cross-linked one another, which had been filled up with water before its evaporation. The ordering state of the open channels depended on the degree of cross-linkage with glutaraldehyde; the optimal self-organized state was found when 30 molecules of glutaraldehyde were added per collagen molecule, though an excess amount of glutaraldehyde suppressed the appearance of the ordered state. From SEM and FT-IR measurements, it was indicated that the self-organization in the HAp/collagen nanocompsites continuously occurred during the drying process together with the removal of water and the increase of the density.

Modification of Hydroxyapatite/gelatin Nanocomposite with the Addition of Chondroitin Sulfate

In the preparation of hydroxyapatite (HAp)/gelatin (GEL) nanocomposite, GEL matrix was modified by the introduction of chondroitin sulfate (ChS) to obtain a strongly organized composite body. The formation reaction of the HAp/GEL-ChS nanocomposite was then investigated via XRD, DT/TGA, FT-IR, TEM and SEM. The organic-inorganic interaction between HAp nanocrystallites and GEL molecules was confirmed from DT/TGA and FT-IR. According to the DT/TGA results, the exothermal temperature zone between 300 and 550℃ showed an additional peak temperature that indicated the decomposition of the combined organics of the GEL and ChS. From the FT-IR analysis, calcium phosphate (Ca-P) was covalently bound with the GEL macromolecules modified by ChS. From TEM and ED, the matrix of the GEL-ChS molecules was mineralized by HAp nanocrystallites and the dense dried nanocomposite body was confirmed from SEM micrographs.

Osteoblastic response to the hydroxyapatite/gelatin nanocomposite and bio-calcium phosphate cement

The main purpose of the developingnew CPCs by modulating their components or ratios are to develop more proper biomaterials which can adapt to the body. In the present study, the properties of newly developed HAp/Gel Nanocomposites were tested using in-vitro experiments (The toxicity test, chromosomal aberration test, cytokinesis-block micronucleus assay, RT-PCR analysis). The data from the WST-1 experiments using HAp/Gel and human osteoblastic cells showed that this material does not induces cellular death. And this material did not induced any significant changes in the structures of chromosomes. The expression pattern of integrins after treatment with CPC or HAp/Gel powder represents that the cells in CPC forms focal adhesion as the cells on adhesive culture dish, but the cells in HAp/Gel do not. By performing several set of experiments, HAp/Gel is not toxic to hFOB-1.19 human osteoblast cell and does not cause any genetic problems. In contrast to CPC, HAp/Gel promotes the final differentiation of osteoblast into osteocyte. These results represent the improved bio-mimetic properties of HAp/Gel.

Modification of hydroxyapatite/gelatin nanocomposite using polyacrylamide

A hydroxyapatite (HAp)/gelatin (GEL) nanocomposite was mixed with mineralized polyacrylamide (PAM) to produce a macrocomposite. The mineralization of PAM was carried out by solution-precipitation using Ca(OH)2 and H3PO4. The crystal growth of HAp in PAM was moderately changed from amorphous-like nanocrystalline to crystalline with the increase of PAM. The dry body of HAp/PAM nanocomposite cracked after the immersion test in water, but the cross-linked sample using glutaraldehyde did not crack. The macrocomposite of HAp/GEL nanocomposite and HAp/PAM nanocomposite showed good toughness, but cracked after the immersion test in water. The cross-linked macrocomposite sample did not crack after the immersion test in water.

Structural Analysis of Al2TiO5 at Room Temperature and at 600 °C by DV-Xα Approach (II)

Electronic structures of Al2TiO5 at room temperature and at 600 °C were investigated by using discrete variational Xα molecular orbital methods. The aluminum titanate having pseudobrookite isomorphous structure, M2 3+Ti4+O5 (in which M3+ may be Al3+) crystallizes in space group Bbmm. At room temperature, the reported expansion coefficients are 9.8 × 10−6, 20.6 × 10−6, and −1.4 × 10−6 °C−1 along the a, b, and c axes, respectively. At 600 °C the reported reduced lattice constants are a0 = 9.481, b0 = 9.738, and c0 = 3.583 A. By using the positional and isotropic thermal parameters for Al2TiO5 DV-Xα molecular orbital energy was calculated. With the addition of Mg and/or Si into Al or Ti site in Al2TiO5 cluster, variations of the calculated chemical bonds were coordinated with the lattice structure in order to understand the single crystal thermal expansion and inter-atomic separations in Al2TiO5 at room temperature and 600 °C. The regular configurations of the coordination polyhedral about the metal ions with the temperature increase were calculated with the degree of the disorder in the metal sites.

Morphology Development of HAp Crystallites in GEL Matrix

The crystal morphology of hydroxyapatite [HAp] phase in gelatin [GEL] matrices was investigated with the condition of a GEL precursor treatment in an aqueous solution of H₃PO₄ at 37-80℃. Needle-shaped nanocomposite particles were prepared through a dynamic reaction during a coprecipitation process using a phosphoric GEL solution. Various types of mineralized morphology appeared with a phosphorylated condition of the GEL solution. HAp/GEL nanocomposite slurries showed the existence of an octacalcium phosphate [OCP] phase during the process.

Molecular Orbital Calculation on the Configuration of Hydroxyl Group in Hexagonal Hydroxyapatite

The possible configurations of hydroxyl group in hexagonal hadroxyapatite were identified through molecular orbital calculation. The molecular orbital interaction between O and H in hydroxyl column was analyzed using charge variation and Bond Overlap Population (BOP). We supposed 5 kinds of O-H bond configurations as cluster types of Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ. Mullikens population analysis was applied to evaluate ionic charges of O, H, P, and Ca ions, and BOPs (Bond Overlap Populations) in order to discuss the bond strength change by the atomic arrangement. The stability of each O-H bond configuration was analyzed using bond overlap and ionic charge.