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Extensive bone loss is still a major problem in orthopedics. A number of different therapeutic approaches have been developed and proposed, but so far none have proven to be fully satisfactory. We used a new tissue engineering approach to treat four patients with large bone diaphysis defects and poor therapeutic alternatives. To obtain implantable three-dimensional (3D) living constructs, cells isolated from the patients' bone marrow stroma were expanded in culture and seeded onto porous hydroxyapatite (HA) ceramic scaffolds designed to match the bone deficit in terms of size and shape. During the surgical session, an Ilizarov apparatus or a monoaxial external fixator was positioned on the patient's affected limb and the ceramic cylinder seeded with cells was placed in the bone defect. Patients were evaluated at different postsurgery time intervals by conventional radiographs and computed tomography (CT) scans. In one patient, an angiographic evaluation was performed at 6.5 years follow-up. In this study we analyze the long-term outcome of these patients following therapy. No major complications occurred in the early or late postoperative periods, nor were major complaints reported by the patients. No signs of pain, swelling, or infection were observed at the implantation site. Complete fusion between the implant and the host bone occurred 5 to 7 months after surgery. In all patients at the last follow-up (6 to 7 years postsurgery in patients 1 to 3), a good integration of the implants was maintained. No late fractures in the implant zone were observed. The present study shows the long-term durability of bone regeneration achieved by a bone engineering approach. We consider the obtained results very promising and propose the use of culture-expanded osteoprogenitor cells in conjunction with porous bioceramics as a real and significant improvement in the repair of critical-sized long bone defects.

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We conclude that FGF2 maintains human BMSC in an immature state allowing their 'in vitro' expansion. Expanded cells retain the chondro- osteogenic potential. Interestingly, the chondrogenic potential of BMSC 'in vitro' is directly related to their ability to form bone 'in vivo'. BMSC expanded 'ex vivo' are presently being proposed for cell therapy of bone defects. 'In vitro' chondrogenesis may be regarded as a rapid prediction assay to assess cell ability to form bone after 'in vivo' transplant.

Bone-marrow stromal cells can differentiate into multiple mesenchymal lineages including cartilage and bone. When these cells are seeded in high-density 'pellet culture', they undergo chondrogenesis and form a tissue that is morphologically and biochemically defined as cartilage. Here, we show that dual chondro-osteogenic differentiation can be obtained in the same micromass culture of human bone-marrow stromal cells. Human bone-marrow stromal cells were pellet cultured for 4 weeks in chondro-inductive medium. Cartilage 'beads' resulting from the micromass culture were then subcultured for further 1-3 weeks in osteo-inductive medium. This resulted in the formation of a distinct mineralized bony collar around hyaline cartilage. During the chondrogenesis phase, type I collagen and bone sialoprotein were produced in the outer portion of the cartilage bead, which, upon subsequent exposure to beta-glycerophosphate, mineralized and accumulated extracellular bone sialoprotein and osteocalcin. Our modification of the pellet culture system results in the formation of a chondro-osseous 'organoid' structurally reminiscent of pre-invasion endochondral rudiments, in which a bony collar forms around hyaline cartilage. The transition from a cell culture to an organ culture dimension featured by our system provides a suitable model for the dissection of molecular determinants of endochondral bone formation, which unfolds in a precisely defined spatial and temporal frame