Papers by Author: Masataka Sakane

Abstract: Regenos® is a unidirectional porous hydroxyapatite (UDPHAp) bone substitutes, and the most distinctive feature of UDPHAp is its interconnected porous structure. We used Regenos® for the bone defect while distal radius fracture surgery with metal fixation implant. At six months post-operation, CT scans revealed the Regenos® implant was uniformly composed of cortical bone adjacent to the trabecular bone. At one-year postoperatively, we collected a sample of the implanted Regenos® while metal fixation plate removal surgery for histological evaluation. Regenos® implant sample revealed the presence of ossified bone stained green with Villanueva Goldner stain. Our findings demonstrate that Regenos® is a useful bone substitution material in the clinical setting.

Abstract: To evaluate the osteoconductive potential of connected porous hydroxyapatite (HAp), we histologically analyzed the newly formed bone inside unidirectional porous HAp (Regenos^®, Kuraray, Japan; 75% porosity, n=17) and interconnected porous HAp (Neobone^®, Covalent Materials, Japan; 75% porosity, n=10) 26 weeks after their implantation as bone spacers between the split lumbar laminae of goats. As a control, non-connected porous HAp spacers (Apaceram^®, Pentax, Japan; 50% porosity, n=5) were used. After staining non-decalcified samples with Villanueva Goldner, changes in pore shape were evaluated microscopically and new bone formation in HAp spacers was quantitatively analyzed. In addition, blood vessel distribution was evaluated by hematoxylin and eosin staining. Changes in pore shape were observed in 76% of the Regenos^® spacers and 90% of the Neobone^® spacers but were not detected in the Apaceram^® spacers. Only limited new bone formation was observed in the Regenos^® and Neobone^® spacers, whereas vascular-like structures were detected in 82% of the Regenos^®, 70% of the Neobone^®, and 80% of the Apaceram^® spacers. The changes in pore shape were thought to have resulted from the low initial compression strength of the connected porous HAp, which may have limited the inherent osteoconductive potential of connected HAp. Our findings suggest that the maintenance of pore shape is required for promoting new bone formation in connected porous HAp when used as lamina spacers in spinal surgery.

Abstract: In the present study, we have newly developed an artificial bone substitute, which is unidirectional porous β-tricalcium phosphate (UDPTCP). The objective of this study was to examine the effects of high and low porosity substitutes on the balance between new bone formation and β-TCP absorption. Materials and MethodsSix male Japanese white rabbits (weight 3.1–3.5 kg, approximately 18– 21 weeks old) were used for this study. Intra-venous injection of pent barbiturate was administered and the both medial and lateral femoral condyle were exposed. A hole of 5 mm diameter was drilled to a depth of 12 mm in the metaphysis, perpendicular to the long axis of the femur. (Figure 1) Figure 1. Operation procedureIn the next step, a cylindrical UDPTCP test piece measuring 4.8 × 11 mm was implanted in the holes. Within the bone substitute, unidirectional pores ranging from 100 to 300 μm in diameter were made. This unique architecture fostered transmission of fluids and cells into the piece. In this case, the test piece was implanted into the bone perpendicular to the long axis of the femur, and the orientation of uni-directional pore was parallel to the long axis of femur. We prepared two different test pieces having low (69%) and high (74%) porosities. Half of the animals were sacrificed at 3 weeks after the operation and the remaining half at 6 weeks. After removal of the femoral condyle, the specimen was fixed in formalin and demineralized. Specimens were obtained from the central axis of the cylindrical piece as well as from the lateral or medial surfaces at a distance of 4 mm from midline. The histological samples were prepared for H&E and TRAP staining. Results and Discussion 　At 3 weeks interval, woven bone, which was formed along the wall of the substitute, could be observed by H&E staining in both low and high porosity substitutes (Figure 2a, 2b). In addition, there were osteoblast-like cells lining the newly formed bone surface with extensive capillary formation (Figure 3). At 6 weeks, the β-TCP walls had thinned and bone had matured in both the groups (Figure 4a, 4b). However, in the high-porosity group, β-TCP absorption tended to be more prominent (Figure 4). In addition, it was observed that at the center of the piece, β-TCP absorption was more prominent than that in the 4 mm-area obtained from the lateral or medial surfaces. At 3 and 6 weeks interval, activities of osteoclast-like multinuclear cells were seen on the surface of the pore wall as observed by TRAP staining. Figure 2a. Low porosity (69%) Figure 2b. High porosity (74%) Fig.2a and Fig.2b H&E staining (×12.5) after 3 weeks (center of the specimen)Figure 3. Formation of woven bone with osteoblast-like cells lining the low porosity specimen at 3 weeks. (H&E staining ×400) Figure 4a. Low porosity Figure 4b. High porosityFig.4a and Fig. 4b H&E staining at 6 weeks after implantation. In high porosity, dense-pink staining areas are located at peripheral in the field.Figure 5. TRAP-positive multinuclear cells (black arrow) were seen on the wall and in the capillaries.Conclusions　The UDPTCP implanted in the medullar canal of the femur was absorbed by multinuclear cells and quickly replaced by the newly formed bone. Our results are consistent with those of other studies using porous β-TCP [1]. In our preparation, porosity had certain effects on the balance between bone formation and　β-TCP absorption. Because of the unique architecture of unidirectional pores within the β-TCP specimen as well as easy formation of capillary network and access to osteoclasts may have accelerated absorption of the substitute. UDPTCP is very promising scaffolding material for bone regeneration. However, optimization of the porosity of UDPTCP in accordance with its application site is necessary before its clinical use. Reference[1] Naoki Kondo, Akira Ogose, Kunihiko Tokunaga, Tomoyuki Ito, Katsumitsu Arai, Naoko Kudo, Hikaru Inoue, Hiroyuki Irie, Naoto Endo: Bone formation and resorption of highly purified β-tricalcium phosphate in the rat femoral condyle. Biomaterials 26: 5600-5608, October 2005.

Abstract: Several drug delivery carriers have reported on local delivery of paclitaxel (PTX), but their effects on intraosseous cancer model are not well known. This study was conducted to clarify the therapeutic effects of our newly developed PTX-loaded HAp-alginate composite beads. Cytotoxic activity was assessed on rat’s mammary adenocarcinoma by cell proliferation assay using WST-1 reagent. Antitumor activity was assessed by 8-week-old rat female Fischer 344 rats of metastatic spine cancer. Twenty-three rats were divided into 3 groups: Group 1 (n = 7) and Group 2 (n = 8) was treated with the PTX-loaded HAp-alginate beads using strontium ions and barium ions, respectively. Group 3 (n = 8) was administered with drug-free HAp-alginate beads. We checked disease-free time and survival time among 3 groups. The HAp-alginate beads containing 2.4wt% of PTX showed significant cytotoxic activity on CRL-1666 cells. The effects were decreased with time during 72 h. The animals treated with 2.4wt% of PTX-loaded HAp-alginate beads showed 40% increase in the disease-free time and 25% increase in survival time. Our studies suggest that newly developed HAp-alginate beads can be a candidate carrier of PTX to bone.

Abstract: Unidirectional porous hydroxyapatite (UDPHAp) was developed which has microstructure in that cross sectionally oval pores 100 ~ 300µm in diameter penetrate through the material, and that is suitable for osteogenesis and angiogenesis.The porosity of the UDPHAp was 75 % and the compression strength was 14 MPa. A cortical bone defect was made at proximal tibia of Japanese white rabbit, and a trapezoidal prisms shaped UDPHAp was implanted. By histlogical evaluation, 2 weeks after implantation, new bone and new capillary was observed inside UDPHAp. Twelve weeks after implantation, new bone formation was observed in 41.6 % of the porous area. The results of this study suggest a great possibility of utilizing it in actual clinical setting as a bone substitution.

Abstract: Rigid hydroxyapatite (HAp)-alginate beads were prepared as drug delivery carriers for an anti-cancer drug, paclitaxel (Taxol). Paclitaxel was loaded into the HAp microparticle in process of a spray-drying technique. The HAp-alginate beads including paclitaxel were obtained by a droplet method into barium solution as ionic cross-linkage and dehydration. Cross-sectional analyses indicated the homogeneity of HAp microparticles and barium ions inside the bead. The ratio of alginate to HAp in the beads dominated both mechanical and swelling properties. Drug-release experiment demonstrated the sustained release of paclitaxel from the beads cross-linked with barium ion for 7 days.

Abstract: Osteoclasts isolated from rabbits were cultured on zinc-containing tricalcium phosphate (ZnTCP) disks with zinc contents of 0.316 and 0.633 wt%, and on β-tricalcium phosphate (TCP) disks with nearly identical porosities, grain sizes and surface roughnesses. ZnTCPs directly suppressed the resorption activity of mature osteoclasts by enhancing apoptosis. We hypothesized that resorbing osteoclasts attached to ZnTCP locally accumulate zinc ions within the space defined by the clear zone during resorption, which in turn leads to apoptosis induction, even though the change in chemical composition of the culture medium is very small.

Abstract: Hydroxyapatite (HAp)-alginate gels were developed as drug delivery carriers of the anti-cancer drug, water-insoluble paclitaxel (Taxol). The spray-drying technique was employed for loading the paclitaxel into spherical HAp microparticles with 1 to 20 μm in diameter. The microparticles loaded with 2.4 or 7.3wt% of paclitaxel were then mixed with sodium alginate, which was followed by Ca2+-mediated gelation. The compressive strength of the HAp-alginate gels and the release of paclitaxel from the gels in a medium were investigated in vitro. The alginate matrix was effective for the achievement of controlled release of anti-cancer drugs.

Abstract: Porous ceramics of hydroxyapatite was fabricated utilizing the crystal growth of thin ice columns parallel to one another in gelatin gel containing hydroxyapatite nanoparticles. The obtained ceramics possessed unidirectional pore channels with a porosity of around 75% and showed compressive strength of up to 13.1 MPa. As control materials, porous hydroxyapatite ceramics with a directionless pore structure were also fabricated by isotropic freezing and compared with the unidirectional samples regarding compressive strength and tissue reaction in vivo. Although the porosity and pore size distribution were similar, the compressive strength and new bone formation ability of the unidirectional samples were significantly greater than those of the random structured porous ceramics.

Abstract: A FGF-2-apatite composite layer (FGF-AP layer) was formed on the surface of Ti screws in a supersaturated calcium phosphate solution supplemented with FGF-2. By an in vitro study using fibroblastic NIH3T3 cells, it was confirmed that FGF-2 was immobilized in the layer without complete denaturation although the composite layer was formed at 37°C. When Ti screws with the FGF-AP layer were percutaneously implanted in the proximal tibial metaphysis of 16 rabbits, no osteomyelitis was observed in any rabbits although a FGF-2-free AP layer allowed osteomyelitis in some cases in our previous study. These results suggest that a FGF-AP layer formed on Ti screws is useful for resisting bacterial infection during external fixations.