Tetsuya Tateishi

Corrosion resistance, mechanical properties, corrosion fatigue strength and cytocompatibility of new Ti alloys without Al and V

The effects of various metallic ions using various metallic powders on the relative growth ratio of fibroblasts L929 and osteoblasts MC3T3-E1 cells were carried out. Ti, Zr, Sn, Nb and Ta had evidently no effect on the relative growth ratios of cells. Otherwise, Al and V ions exhibit cytotoxicity from a concentration of > or = 0.2 ppm. This Al effect on cells tend to be stronger in medium containing small quantity of V ions (< or = 0.03 ppm). The new Ti-15%Zr-4%Nb-4%Ta-0.2%Pd alloy exhibited a higher corrosion resistance in physiological saline solution. The addition of 0.02%O and 0.05%N to Ti-Zr alloy improved the mechanical properties at room temperature and corrosion fatigue strength. The relative growth ratios for the new Ti alloy plate and the alloy block extraction were unity. Further, the relative growth ratios were almost unity for the new Ti alloy against apatite ceramic pins up to 10(5) wear cycles in Eagles MEM solution. However, there was a sharp decrease for Ti-6%Al-4%V ELI alloy from 3 x 10(4) wear cycles as V ion was released during wear into the wear test solution since the pH of the Eagles MEM increases with increasing wear cycles.

Preparation, solubility, and cytocompatibility of zinc-releasing calcium phosphate ceramics.

Zinc is an essential trace element with stimulatory effects on bone formation. Therefore, zinc was doped into beta-tricalcium phosphate to develop zinc-releasing biomaterials to promote bone formation. The zinc-doped beta-tricalcium phosphate, beta-tricalcium phosphate, and hydroxyapatite powders were mixed at a (Ca+Zn)/P molar ratio of 1.60, followed by sintering into a dense body at 1100 degrees C for 1 h. The sintered body was a composite ceramic consisting of zinc-doped beta-tricalcium phosphate and hydroxyapatite phases. The composite ceramic contained zinc oxide when the zinc content was higher than 1.20 wt %. The composite ceramic released zinc under pseudophysiological conditions. However, the release of calcium and phosphate decreased with an increase in zinc content in a range higher than 0.12 wt % owing to a decrease in solubility of the zinc-doped beta-tricalcium phosphate phase. Proliferation of osteoblastic MC3T3-E1 cells was significantly increased on the composite ceramic with a zinc content from 0.6 to 1.20 wt %, compared with those without zinc. When the zinc content was higher than 1.20 wt %, release of zinc from the zinc oxide caused cytotoxicity. Therefore, the zinc content of the composite ceramic must be <1.20 wt %.

Promotion of bone formation using highly pure porous β-TCP combined with bone marrow-derived osteoprogenitor cells

Beta-tricalcium phosphate (TCP) exhibits rapid degradation and weak mechanical properties, which has limited its application as bone graft substitutes, though it has good biocompatibility and osteoconductivity. We hypothesized that a composite of highly pure porous beta-TCP and bone marrow-derived osteoprogenitor cells (BMO) could improve bone formation, and slow down the degradation of beta-TCP. A highly pure porous beta-TCP with 75% porosity was fabricated. The pores averaged 200-400 microm in diameter, with interconnecting paths 100-200 microm. Blocks of beta-TCP 5 mm3 were combined with BMO, and incubated 2 weeks with (+) or without (-) osteogenic medium. They were then implanted into subcutaneous sites of syngeneic rats for 24 weeks. These composites were harvested at different time points. The alkaline phosphatase activity and bone osteocalcin content of the composites (+) were much higher than corresponding values in the composites (-) of the control group (p<0.01). Light microscopy revealed mature bone and lots of blood vessels only in the TCP/BMO composite (+). The amount of newly formed bone increased until week 24. Slow resorptive activity could be found. The mechanical parameters of the composites were much improved over those of dry beta-TCP blocks. These results showed that tissue engineering treatment on incubating the composites of beta-TCP and BMO cells in osteogenic medium results in a good osteogenic activity.

Development of biodegradable porous scaffolds for tissue engineering

Abstract Three-dimensional biodegradable porous scaffolds play an important role in tissue engineering. A new method of preparing porous scaffolds composed of synthetic biodegradable polymers was developed by combining porogen leaching and freeze-drying techniques using preprepared ice particulates as the porogen material. The pore structures of the polymer sponges could be manipulated by controlling processing variables such as the size and weight fraction of the ice particulates and the polymer concentration. The synthetic polymer sponges were further hybridized with collagen microsponges to prepare biodegradable hybrid porous sponges of synthetic polymer and collagen. The collagen microsponges were formed in the pores of synthetic polymer sponges. The hybrid sponges exhibited the advantages of both the synthetic polymers and collagen. Hybrid sponges of synthetic polymer, collagen, and inorganic hydroxyapatite were developed by depositing hydroxyapatite particulates on the surfaces of the collagen microsponges in the synthetic polymer–collagen sponges. The use of synthetic polymer sponge as a mechanical skeleton facilitated the formation of these hybrid sponges into desired shapes, contributed good mechanical strength and handling, while the collagen and hydroxyapatite facilitated cell seeding and promoted cell interaction.

A biodegradable hybrid sponge nested with collagen microsponges.

A biodegradable hybrid sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and collagen was fabricated by forming microsponges of collagen in the pores of PLGA sponge. Observation of the PLGA-collagen hybrid sponge by scanning electron microscopy (SEM) showed that microsponges of collagen with interconnected pore structures were formed in the pores of PLGA sponge. The hybrid structure further was confirmed by scanning electron microscopy-electron probe microanalysis (SEM-EPMA), and elemental nitrogen was detected in the microsponges of collagen and on the pore surfaces of PLGA, but not in cross-sections of PLGA regions. The formation of collagen microsponges was dependent on collagen concentration, the effective range of which was from 0.1 to 1.5 (w/v) %. The mechanical strength of the hybrid sponge was higher than that of either PLGA or collagen sponges, in both dry and wet states. The wettability with water was improved by hybridization with collagen, which facilitated cell seeding in the hybrid sponge. Mouse fibroblast L929 cells attached well and spread on the surfaces of the microsponges of collagen in the hybrid sponge. The distribution of cells was spatially uniform throughout the hybrid sponge. Use of the PLGA sponge as a skeleton facilitated formation of the hybrid sponge into desired shapes with high mechanical strength while collagen microsponges contributed good cell interaction and hydrophilicity.

Stimulatory effect of zinc-releasing calcium phosphate implant on bone formation in rabbit femora

Although hydroxyapatite (HAP) and tricalcium phosphate (TCP) are currently used as bone graft substitutes or coatings on metallic prostheses because of their excellent biocompatibility and osteoconductivity, they do not stimulate bone formation or inhibit bone resorption. Zinc, an essential trace element in many animals, has a direct specific proliferative effect on osteoblastic cells and has a potent and selective inhibitory effect on osteoclastic bone resorption in vitro. Therefore, zinc-containing beta-tricalcium phosphate (ZnTCP) ceramics and composite ceramics of ZnTCP and HAP (ZnTCP/HAP) were implanted in the femora of New Zealand White rabbits for 4 weeks to promote bone formation. The implants were sintered ceramics with zinc contents of 0 (control), 0.063, 0.316 and 0.633 wt %. Histological and histomorphometrical investigation of the undecalcified sections revealed an increase by 51% (p =.0509) in the area of newly formed bone around the ZnTCP/HAP implants of 0. 316 Zn wt % compared with the control. Plasma zinc concentration was unchanged. An increased bone resorption on the endosteal surface was observed when ZnTCP and ZnTCP/HAP of 0.633 Zn wt % were implanted. To promote bone formation, the optimum zinc content of the calcium phosphate ceramics was therefore 0.316 wt %.

Corrosion resistance and corrosion fatigue strength of new titanium alloys for medical implants without V and Al

The corrosion resistance and the corrosion fatigue strength of Ti-15Zr-4Nb-4Ta-0.2Pd-0.2O-0.05N and Ti-15Sn-4Nb-2Ta-0.2Pd-0.2O alloys were compared with those of Ti-6Al-4 V extra low interstitial (ELI), Ti-6Al-2Nb-1Ta, pure Ti grade 2 and β type Ti-15%Mo-5Zr-3Al alloys. Anodic polarization and corrosion fatigue testings were performed in various physiological saline solutions at 310 K. The corrosion fatigue test was carried out under the condition of a tension to tension mode with a sine wave at a stress ratio of 0.1 and at a frequency of 10 Hz. The tensile properties of these alloys were measured at room temperature. The change in current density was small up to passivity zone in 1 wt.% lactic acid, PBS(−), calf serum and eagles MEM + fetal bovine serum solutions except 5 wt.% HCl. The current density of Ti-15Zr-4Nb-4Ta-0.2Pd-0.2O-0.05N alloy at potential up to 5 volt tend to be lower than that of Ti-6Al-4V ELI. Otherwise passive current density of the β type Ti-15Mo-SZr-3Al alloy was higher than that of α + β type alloys. The passive films formed on Ti-15Zr-4Nb-4Ta-0.2Pd-0.2O alloy in the calf serum consisted mainly of TiO2, ZrO2, Nb2O5, Ta2O5 and Pd or PdO as demonstrated using X-ray photoelectron spectroscopy. The cycle to failure of Ti-15Zr-4Nb-4Ta-0.2Pd-0.2O-0.05N and Ti-15Sn-4Nb-4Ta-0.2Pd-0.2O alloys annealed at 973 K for 7.2 ks increased with decreasing applied maximum stress. The fatigue strength at 108 cycles in those alloys was about 600 MPa. The fatigue strength of Ti-6Al-2Nb-1Ta alloy at 108 cycles was about 700 MPa. The fatigue strength of β type Ti-15Mo-5Zr-3Al alloy at 107 cycles was lower than that of α + β type alloys.

Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture

Monolayer cell cultures and cartilage tissue fragments have been used to examine the effects of hydrostatic fluid pressure (HFP) on the anabolic and catabolic functions of chondrocytes. In this study, bovine articular chondrocytes (bACs) were grown in porous three‐dimensional (3‐D) collagen sponges, to which constant or cyclic (0.015 Hz) HFP was applied at 2.8 MPa for up to 15 days. The effects of HFP were evaluated histologically, immunohistochemically, and by quantitative biochemical measures. Metachromatic matrix accumulated around the cells within the collagen sponges during the culture period. There was intense intracellular, pericellular, and extracellular immunoreactivity for collagen type II throughout the sponges in all groups. The incorporation of [35S]‐sulfate into glycosaminoglycans (GAGs) was 1.3‐fold greater with constant HFP and 1.4‐fold greater with cyclic HFP than in the control at day 5 (P < 0.05). At day 15, the accumulation of sulfated‐GAG was 3.1‐fold greater with constant HFP and 2.7‐fold with cyclic HFP than the control (0.01). Quantitative immunochemical analysis of the matrix showed significantly greater accumulation of chondroitin 4‐sulfate proteoglycan (C 4‐S PG), keratan sulfate proteoglycan (KS PG), and chondroitin proteoglycan (chondroitin PG) than the control (P < 0.01). With this novel HFP culture system, 2.8 MPa HFP stimulated synthesis of cartilage‐specific matrix components in chondrocytes cultured in porous 3‐D collagen sponges. J. Cell. Physiol. 193: 319–327, 2002.

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.

TISSUE ENGINEERING OF CARTILAGE USING A HYBRID SCAFFOLD OF SYNTHETIC POLYMER AND COLLAGEN

A biodegradable hybrid scaffold of synthetic polymer, poly (DL-lactic-co-glycolic acid) (PLGA), and naturally derived polymer, collagen, was prepared by forming collagen microsponges in the pores of PLGA sponge. This was then used as the three-dimensional scaffold for tissue engineering of bovine articular cartilage, both in vitro and in vivo. In vitro studies show that hybridization with collagen facilitated cell seeding in the sponge and raised seeding efficiency. Chondrocytes adhered to the collagen microsponges, where they proliferated and secreted extracellular matrices with time, filling the space within the sponge. Hematoxylin and eosin staining revealed that most of the chondrocytes after 4 weeks of culture, and almost all cell types after 6 weeks of culture, maintained their phenotypically rounded morphology. While new tissue formed, the scaffold degraded and lost almost 36.9% of its original weight after 10 weeks. Subcutaneous implantation studies in nude mice demonstrated more homogeneous tissue formation in hybrid sponge than in PLGA sponge. The new tissue formed maintained the original shape of the hybrid sponge. The synthetic PLGA sponge, serving as a skeleton, facilitated easy formation into desired shapes and provided appropriate mechanical strength to define the ultimate shape of engineered tissue. Incorporation of collagen microsponges facilitated cell seeding and homogeneous cell distribution and created a favorable environment for cellular differentiation. The hybrid sponge could therefore represent a promising candidate as a three-dimensional scaffold for articular cartilage tissue engineering.