Junzo Tanaka

SELF-ORGANIZATION MECHANISM IN A BONE-LIKE HYDROXYAPATITE/COLLAGEN NANOCOMPOSITE SYNTHESIZED IN VITRO AND ITS BIOLOGICAL REACTION IN VIVO

When bone is lost due to injury and/or illness, the defects are generally filled with natural bone because artificial bone materials have problems of bioaffinity. However, natural bone also has supply and infection problems. If an artificial material has the same biological properties as bone, it can replace natural bone for grafting. We synthesized a hydroxyapaite (HAp) and collagen (Col) composite by a simultaneous titration coprecipitation method using Ca(OH)2, H3PO4 and porcine atelocollagen as starting materials. The composite obtained showed a self-organized nanostructure similar to bone assembled by the chemical interaction between HAp and Col. The consolidated composite by a cold isostatic pressure of 200 MPa indicated a quarter of the mechanical strength of bone. It also indicated the same biological properties as grafted bone: The material was resorbed by phagocytosis of osteoclast-like cells and conducted osteoblasts to form new bone in the surrounding area. This HAp/Col composite having similar nanostructure and composition can replace autologous bone grafts.

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 and microstructure analysis of chitosan/hydroxyapatite nanocomposites

Chitosan/hydroxyapatite (HAp) composites with a homogeneous nanostructure have been prepared by a co-precipitation method. According to TEM observations, HAp crystallites in the composites formed elliptic aggregations 230 nm in length and 50 nm in width. The typical length of the aggregations corresponded approximately to that of a chitosan molecule. The size of the constituent HAp crystallites was found to be predominantly 30 nm in length and 10 nm in width, and the c-axes were well aligned in parallel with the chitosan molecules in the respective aggregations. The growth of the HAp crystallites is considered to occur at nucleation sites, most probably forming the complexes with amino groups on chitosan with calcium ions. The compact composites obtained have been found to be mechanically flexible, and this flexibility has been improved further by heating at 120 degrees C for 20 min in an autoclave with saturated steam pressure.

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas.

Type I collagens were extracted from fish scales of Pagrus major and Oreochromis niloticas as a possible underutilized resource for medical materials. The fish scales were demineralized with EDTA and digested by pepsin. The resultant type I collagens contained more than 33.6% of glycine as the most abundant amino acid. The denaturation temperatures of the collagens from P. major and O. niloticas were 303 and 308K, respectively, both of which were relatively lower than that of porcine dermis collagen (314K). CD spectra indicated that the denaturation temperatures were dependent on the amount of hydroxyproline, rather than proline residues. Raman spectra also indicated that the relative intensities of Raman lines at 879 and 855cm(-1) assigned to Hyp and Pro rings were changed due to the contents of the imino acids. Significantly, the content of sulphur-containing methionine was higher in the fish scales than in porcine dermis. The enthalpy and entropy estimated from thermal analyses could be correlated to amino acid sequences (Gly-Pro-Hyp) of type I collagens and the number of methionine amino acid residues.

High temperature cubic boron nitride P-N junction diode made at high pressure

A p-n junction diode of cubic boron nitride was made by growing an n-type crystal epitaxially on a p-type seed crystal at a pressure of 55 kilobars and a temperature of about 1700�C. A temperature-difference solvent method was used for the crystal growth, and beryllium and silicon were doped as acceptors and donors, respectively. Formation of the p-n junction was clearly confirmed at 1 bar by rectification characteristics and by existence of a space charge layer of the junction as observed by electron beam induced current measurement. This diode operated at 530�C.

Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1-2 microm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (approximately 90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5-0.6 microm in length and 0.1-0.2 microm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.

Ultraviolet light‐emitting diode of a cubic boron nitride pn junction made at high pressure

Injection luminescence in the ultraviolet is observed from a cubic boron nitride pn junction diode made at high pressure. Microscopic observation and spectroscopic studies show that the light emission occurs near the junction region only in the forward‐bias condition. The spectra extend from ∼215 nm to the red, having a few peaks mainly in the ultraviolet.

Application of Perfusion Culture System Improves in Vitro and in Vivo Osteogenesis of Bone Marrow-Derived Osteoblastic Cells in Porous Ceramic Materials

Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue.

Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites☆

To control the mechanical properties and biodegradability of self-organized hydroxyapatite/collagen (HAp/Col) nanocomposites, cross-linkage was introduced into the composites with glutaraldehyde (GA). The HAp/Col composite suspensions, prepared by a simultaneous titration method and aged for 3h, were cross-linked with the reagents for 10min under vigorous stirring. The precipitates obtained were filtrated and compacted by dehydration under a uniaxial pressure. The particle size distribution, 3-point bending strength, contained water amount and swelling ratio of the composites were examined as a function of cross-linkage amount; the biodegradability was estimated by animal tests using rabbits. As regards the cross-linked composites, no long-rage alignment of HAp crystals along collagen molecules was found with a transmission electron microscope, suggesting that the cross-linking reagents suppressed their long-range self-organization mechanism. The 3-point bending strength increased with the GA content and took a maximal value at 1.35mmol/g(col). The animal tests indicated no toxicity and osteoclastic resorption with good osteoconductivity. The resorption rate was decreased with increasing GA concentration. These results suggest that GA cross-linkage controls mechanical properties and resorption rate without reducing high biocompatibility of the composite.

Transplantation of cultured bone cells using combinations of scaffolds and culture techniques.

The transplantation of cultured bone cells is expected to become a candidate for bone regeneration therapy. For the clinical application of this therapy, there remain several problems to be overcome, for example, the improvements of scaffolds and culture techniques. In this review article, two kinds of porous ceramics, a novel sintered porous hydroxyapatite and a porous beta-tricalcium phosphate (TCP), as well as a collagen-phosphosphoryn sponge are introduced as new scaffolds for bone regeneration. The former two ceramic scaffolds proved to be applicable for bone regeneration therapy. The collagen-phosphophoryn sponge proved to have bone formation ability in vivo. Moreover, for the application of this therapy to the regeneration of large bone defects, we improved the culture method by applying a low-pressure system and a perfusion system. Both culture systems accelerated the formation of bone in vivo in this transplantation model. Combinations of the scaffolds and culture techniques might be considered when designing therapeutic strategies.