This study investigated the effects of the three-dimensional (3-D) pore structure of a porous hydroxyapatite/collagen (HAp/Col) composite on their mechanical properties and in vivo tissue ingrowth. The unique 3-D pore structure, comprising unidirectionally interconnected pores, was fabricated by the unidirectional growth of ice crystals by using a cooling stage and a subsequent freeze-drying process. The unidirectional pores had a spindle-shaped cross section, and their size gradually increased from the bottom to the upper face. The porous composite showed an elastic property and anisotropic compressive strength for the pore directions. While the strength and modulus parallel to the pore axis were 1.3-and twofold higher than those of the porous composite with spherical pores formed randomly, the strength and modulus perpendicular to the pore axis showed the lowest values. The subcutaneous implantations revealed that when compared with the random pores, the unidirectional pores promote the ingrowth of the surrounding tissues into the pores.

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50-760 mmHg, 1-10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous β-tricalcium phosphate (β-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/β-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into β-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

Recent advances in tissue engineering techniques have allowed porous biomaterials to be combined with osteogenic cells for effective bone regeneration. We developed a simple low-pressure cell-loading method using only syringes and stopcocks, and examined the effect of this method on osteogenesis when applied to the combination of highly porous b-tricalcium phosphate (b-TCP) and fresh autologous bone marrow. Both block and granule b-TCP scaffolds were used to prepare implants in three different ways: without bone marrow as a control, with bone marrow that was allowed to penetrate spontaneously under atmospheric pressure (AP group), and with bone marrow that was seeded under low pressure (ULP group). These implants were transplanted into rabbit intramuscular sites, and the samples were examined biologically and histologically. The penetration efficiency of the block implants after marrow introduction was significantly higher in the ULP group than in the AP group. In the transplanted block samples, alkaline phosphatase activity was significantly higher in the ULP group at 2 weeks after implantation, and significantly more newly formed bone was observed in the ULP group at both 5 and 10 weeks compared with the AP group. Similar results were observed even in the experiment using b-TCP granules, which are smaller than the blocks and frequently used clinically. Because of its convenience and safety, this low-pressure method might be a novel, effective treatment to promote osteogenesis with bone marrow in clinical bone reconstruction surgeries. ß

To potentiate the bone formation capability of bone marrow stromal cell (BMSC)/beta-tricalcium phosphate (beta-TCP) constructs, we devised an autologous plasma-based construct. We tested its effectiveness and investigated the effects of its components on a monkey ectopic bone formation model. The autologous plasma (platelet-rich plasma, PRP, or platelet-poor plasma, PPP)/BMSC/beta-TCP construct (R group or P group) showed significantly more bone formation at 3 and 6 weeks after implantation than a conventional BMSC/beta-TCP construct using a culture medium (M group). There was no significant difference between the P and R groups. Moreover, the P group constructs with a 10-fold lower cell concentration yielded equivalent bone formation to the M group at 5 weeks after implantation. To elucidate the effect of fibrin and serum contained in the plasma, five constructs were prepared using the following cell vehicles: autologous serum + fibrinogen (0, 1, 4, or 16 mg/mL) or phosphate-buffered saline + fibrinogen (4 mg/mL). The serum + fibrinogen (4 mg/mL, physiological concentration of monkeys) construct showed the most abundant bone formation at 3 weeks after implantation, though at 5 weeks no statistical difference existed among the groups. Autologous plasma efficiently promoted osteogenesis of BMSCs/porous beta-TCP constructs, and both fibrin and serum proved to play significant roles in the mechanism.

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