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.
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