A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: In vivo bone formation showing proof of concept

In an effort to produce clinically relevant volumes of tissue-engineered bone products, we report a direct perfusion bioreactor system. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cc) of small sized macroporous biphasic calcium phosphate scaffolds (BCP, 2–6 mm). Cell load and cell distribution were shown using methylene blue block staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate viability of the present cells. After 19 days of cultivation, the scaffolds were covered with a viable, homogeneous cell layer. The hybrid structures became interconnected and a dense layer of extracellular matrix was present as visualized by environmental scanning electron microscopy (ESEM). ESEM images showed within the extracellular matrix sphere like structures which were identified as calcium phosphate nodules by energy dispersive X-ray analysis (EDX). On line oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption can be used to estimate GBMSC population doubling times during growth in this bioreactor system. Implantation of hybrid constructs, which were proliferated dynamically, showed bone formation in nude mice after 6 weeks of implantation. On the basis of our results we conclude that a direct perfusion bioreactor system is capable of producing clinically relevant volumes of tissue-engineered bone in a bioreactor system which can be monitored on line during cultivation and show bone formation after implantation in nude mice.