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In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

This investigation describes the production and characterization of calcium phosphate scaffolds with defined and reproducible porous macro-architectures and their preliminary in vitro and in vivo bone-tissue-engineered response. Fugitive wax molds were designed and produced using a rapid prototyping technique. An aqueous hydroxyapatite slurry was cast in these molds. After sintering at 1250°C and then cleaning, dimensional and material characterizations of the scaffolds were performed. The resulting scaffolds represented the design, and their dimensions were remarkably consistent. A texture inherent to the layer-bylayer production of the mold was impressed onto the vertical surfaces of the scaffolds. The surface roughness (R a ) of the textured surfaces was significantly greater than that of the nontextured surfaces. Material analyses revealed a ␤-TCP phase in addition to hydroxyapatite for the molded ceramics. Non-molded control ceramics exhibited only hydroxyapatite. Thirty scaffolds were seeded with cultureexpanded goat bone-marrow stromal cells (BMSCs) and implanted subcutaneously in nude mice for 4 or 6 weeks. Histology revealed mineralized bone formation in all the scaffolds for both implantation periods. After 4 weeks, bone was present primarily as a layer on scaffold surfaces. After 6 weeks, the surface bone formation was accompanied by bone budding from the surface and occasional bridging of pores. This budding and bridging bone formation almost always was associated with textured scaffold surfaces. However, the area percentage of bone in pores was similar for the 4-and 6-week implantation periods.

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