Engineering axially vascularized bone in the sheep arteriovenous-loop model.

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)-loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2), were harvested and directly autotransplanted in combination with ß-tricalcium phosphate-hydroxyapatite (ß-TCP-HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP-2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the ß-TCP-HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro-computed tomography (µCT) imaging confirmed the dense vascularization arising from the AV-loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP-2 stimulation in about 12 weeks in the sheep AV-loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies.

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model.

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and ß-tricalcium phosphate/hydroxyapatite (ß-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29(+), CD44(+) and CD166(+) after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a ß-TCP/HA matrix comparable to the application of 60 µg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with ß-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.