De novo reconstruction of functional bone by tissue engineering in the metatarsal sheep model.

Large bone defects are still a challenge to orthopedic surgeons. In this study, a massive bone defect with a clinically relevant volume was efficiently reconstructed by transplanting an engineered bone in which mesenchymal stem cells (MSCs) expanded in autologous serum (AS) were combined with a porous scaffold. In the first step, we established that the way in which the MSCs are distributed over the scaffold affects the ultimate bone-forming ability of the transplant: constructs consisting of a natural coral scaffold and a pseudo-periosteal layer of MSCs surrounding the implant (coral-MSC3D) formed significantly more bone than constructs in which the MSCs were distributed throughout the implant (p = 0.01). However, bone healing occurred in only one sheep, owing to the high resorption rate of natural coral scaffold. To overcome this problem, constructs in which MSCs were combined with a porous coralline-based hydroxyapatite (CHA) scaffold having the same architecture as natural coral but a lower resorption rate were prepared. After their implantation, these constructs were found to have the same osteogenic potential as autologous bone grafts in terms of the amount of newly formed bone present at 4 months (p = 0.89) and to have been completely replaced by newly formed, structurally competent bone within 14 months. Nevertheless, although the rate of bone healing was strikingly improved when CHA-MSC3D constructs were used (five of seven animals healed) as compared with the coral-MSC3D construct (one of seven healed), it was still less satisfactory than that obtained with autografts (five of five healed).

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

Tissue-engineered bone regeneration.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.