Introduction: Durable reconstruction of critical size bone defects is still a surgical challenge despite the availability of numerous autologous and substitute bone options. In this paper, we have investigated the possibility of creating a living bone allograft, using the perfusion/decellularization/recellularization (PDR) technique, which was applied to an original model of vascularized porcine bone graft.Materials and Methods: 11 porcine bone forelimbs, including radius and ulna, were harvested along with their vasculature including the interosseous artery and then decellularized using a sequential detergent perfusion protocol. Cellular clearance, vasculature, extracellular matrix (ECM), and preservation of biomechanical properties were evaluated. The cytocompatibility and in vitro osteoinductive potential of acellular extracellular matrix were studied by static seeding of NIH-3T3 cells and porcine adipose mesenchymal stem cells (pAMSC), respectively.Results: The vascularized bone grafts were successfully decellularized, with an excellent preservation of the 3D morphology and ECM microarchitecture. Measurements of DNA and ECM components revealed complete cellular clearance and preservation of ECM’s major proteins. Bone mineral density (BMD) acquisitions revealed a slight, yet non-significant, decrease after decellularization, while biomechanical testing was unmodified. Cone beam computed tomography (CBCT) acquisitions after vascular injection of barium sulphate confirmed the preservation of the vascular network throughout the whole graft. The non-toxicity of the scaffold was proven by the very low amount of residual sodium dodecyl sulfate (SDS) in the ECM and confirmed by the high live/dead ratio of fibroblasts seeded on periosteum and bone ECM-grafts after 3, 7, and 16 days of culture. Moreover, cell proliferation tests showed a significant multiplication of seeded cell populations at the same endpoints. Lastly, the differentiation study using pAMSC confirmed the ECM graft’s potential to promote osteogenic differentiation. An osteoid-like deposition occurred when pAMSC were cultured on bone ECM in both proliferative and osteogenic differentiation media.Conclusion: Fully decellularized bone grafts can be obtained by perfusion decellularization, thereby preserving ECM architecture and their vascular network, while promoting cell growth and differentiation. These vascularized decellularized bone shaft allografts thus present a true potential for future in vivo reimplantation. Therefore, they may offer new perspectives for repairing large bone defects and for bone tissue engineering.

Background: In the field of vascularized composite tissue allotransplantation, the surgical design of facial subunit grafts is an evolving concept. The purpose of the present article is to study the possibility of dividing the historical nose and lip face transplant into several morphologic and functional subunit grafts, depending on their respective supply. Methods: This study was conducted in 20 adult cadavers. The facial artery and its branches were dissected bilaterally in 16 fresh and four embalmed heads. Nasolabial perfusion was assessed by selective injection of methylene blue and eosin (n = 2) or India ink (n = 2) in the superior labial and distal facial arteries. Dynamic perfusion through the distal facial artery was illustrated by fluoroscopy (n = 3). Three nose–upper lip grafts were harvested and injected with barium sulfate for microangiography computed tomographic analysis. Finally, three isolated nasal and bilabial grafts were procured and their vascular patency assessed by fluoroscopy. Results: The distal facial artery can perfuse the entire nose, septum, and upper lip, without any contribution of the superior labial artery. A dense anastomotic network indeed exists between the respective distal rami of both vessels. Furthermore, the exclusion of the superior labial artery from the harvested nasal subunit allowed safe bilabial subunit procurement, from the same specimen. Conclusions: The authors’ results demonstrate the feasibility of harvesting nasal and labial subunits, in an isolated or a combined manner. These results can find applications in subunit autologous replantation, allotransplantation, allogenic face partial retransplantation, and the emerging field of vascularized composite tissue engineering.

Background: In terms of large bone defects reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Material and methods: 72 porcine bones were used to characterize the efficiency of our NaOH-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology & Immunohistochemistry) and quantitative (fluoroscopic absorbance & ELISA) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Results: Comparatively to non-treated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the HLA antigenic load of the graft by more than 50%. The majority of measured growth factors are still present in the same amount into the decellularized bones compared to the non-treated bones. Histology and immunohistochemistry show that the bones were cell-compatible, non-cytotoxic and capable of inducing osteoblastic differentiation of MSCs. Conclusion: Our decellularization/perfusion protocol allowed to create decellularized long bone grafts models thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (n = 12); (2) 22-h HMP + continuous membrane oxygenation (n = 6); and (3) 22-h HMP + continuous surface oxygenation (n = 7). Brief perfusate O₂ uploading before kidney perfusion was either obtained by direct bubble (groups 1, 3) or by membrane (group 2) oxygenation. Bubble oxygenation during minimum 15 min was as efficient as membrane oxygenation in achieving supraphysiological perfusate pO₂ levels before kidney perfusion. Metabolic tissue analysis (i.e., lactate, succinate, ATP, NADH, and FMN) during and at the end of the preservation period demonstrated similar mitochondrial protection between all study groups. Short bubble and subsequent intermittent surface oxygenation of the perfusate of an HMP-kidney might be an effective and cheap preservation strategy to protect mitochondria, eliminating the need/costs of a membrane oxygenator and oxygen source during transport.