Decellularization of the Porcine Ear Generates a Biocompatible, Nonimmunogenic Extracellular Matrix Platform for Face Subunit Bioengineering.

OBJECTIVE: The purpose of this study was to assess whether perfusion-decellularization technology could be applied to facial grafts. BACKGROUND: Facial allotransplantation remains an experimental procedure. Regenerative medicine techniques allow fabrication of transplantable organs from an individual's own cells, which are seeded into extracellular matrix (ECM) scaffolds from animal or human organs. Therefore, we hypothesized that ECM scaffolds also can be created from facial subunits. We explored the use of the porcine ear as a clinically relevant face subunit model to develop regenerative medicine-related platforms for facial bioengineering. METHODS: Porcine ear grafts were decellularized and histologic, immunologic, and cell culture studies done to determine whether scaffolds retained their 3D framework and molecular content; were biocompatible in vitro and in vivo, and triggered an anti-MHC immune response from the host. RESULTS: The cellular compartment of the porcine ear was completely removed except for a few cartilaginous cells, leaving behind an acellular ECM scaffold; this scaffold retained its complex 3D architecture and biochemical components. The framework of the vascular tree was intact at all hierarchical levels and sustained a physiologically relevant blood pressure when implanted in vivo. Scaffolds were biocompatible in vitro and in vivo, and elicited no MHC immune response from the host. Cells from different types remained viable and could even differentiate at the scale of a whole-ear scaffold. CONCLUSIONS: Acellular scaffolds were produced from the porcine ear, and may be a valuable platform to treat facial deformities using regenerative medicine approaches.

Bioengineering a Human Face Graft: The Matrix of Identity.

OBJECTIVE: During the last decade, face allotransplantation has been shown to be a revolutionary reconstructive procedure for severe disfigurements. However, offer to patients remains limited due to lifelong immunosuppression. To move forward in the field, a new pathway in tissue engineering is proposed. BACKGROUND: Our previously reported technique of matrix production of a porcine auricular subunit graft has been translated to a human face model. METHODS: 5 partial and 1 total face grafts were procured from human fresh cadavers. After arterial cannulation, the specimens were perfused using a combined detergent/polar solvent decellularization protocol. Preservation of vascular patency was assessed by imaging, cell and antigen removal by DNA quantification and histology. The main extracellular matrix proteins and associated cytokines were evaluated. Lip scaffolds were cultivated with dermal, muscle progenitor and endothelial cells, either on discs or in a bioreactor. RESULTS: Decellularization was successful in all facial grafts within 12 days revealing acellular scaffolds with full preservation of innate morphology. Imaging demonstrated a preservation of the entire vascular tree patency. Removal of cells and antigens was confirmed by reduction of DNA and antigen markers negativation. Microscopic evaluation revealed preservation of tissue structures as well as of major proteins. Seeded cells were viable and well distributed within all scaffolds. CONCLUSIONS: Complex acellular facial scaffolds were obtained, preserving simultaneously a cell-friendly extracellular matrix and a perfusable vascular tree. This step will enable further engineering of postmortem facial grafts, thereby offering new perspectives in composite tissue allotransplantation.

Decellularization of Massive Bone Allografts By Perfusion: A New Protocol for Tissue Engineering.

In terms of large bone defect 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. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-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 and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated 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 human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Intermittent Surface Oxygenation Results in Similar Mitochondrial Protection and Maintenance of Aerobic Metabolism as Compared to Continuous Oxygenation during Hypothermic Machine Kidney Machine Perfusion.

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.

Periosteum and fascia lata: Are they so different?

Introduction: The human fascia lata (HFL) is used widely in reconstructive surgery in indications other than fracture repair. The goal of this study was to compare microscopic, molecular, and mechanical properties of HFL and periosteum (HP) from a bone tissue engineering perspective. Material and Methods: Cadaveric HP and HFL (N = 4 each) microscopic morphology was characterized using histology and immunohistochemistry (IHC), and the extracellular matrix (ECM) ultrastructure assessed by means of scanning electron microscopy (SEM). DNA, collagen, elastin, glycosaminoglycans, major histocompatibility complex Type 1, and bone morphogenetic protein (BMP) contents were quantified. HP (N = 6) and HFL (N = 11) were submitted to stretch tests. Results: Histology and IHC highlighted similarities (Type I collagen fibers and two-layer organization) but also differences (fiber thickness and compaction and cell type) between both tissues, as confirmed using SEM. The collagen content was statistically higher in HFL than HP (735 vs. 160.2 µg/mg dry weight, respectively, p < 0.0001). On the contrary, DNA content was lower in HFL than HP (404.75 vs. 1,102.2 µg/mg dry weight, respectively, p = 0.0032), as was the immunogenic potential (p = 0.0033). BMP-2 and BMP-7 contents did not differ between both tissues (p = 0.132 and p = 0.699, respectively). HFL supported a significantly higher tension stress than HP. Conclusion: HP and HFL display morphological differences, despite their similar molecular ECM components. The stronger stretching resistance of HFL can specifically be explained by its higher collagen content. However, HFL contains many fewer cells and is less immunogenic than HP, as latter is rich in periosteal stem cells. In conclusion, HFL is likely suitable to replace HP architecture to confer a guide for bone consolidation, with an absence of osteogenicity. This study could pave the way to a bio-engineered periosteum built from HFL.

Decellularized vascularized bone grafts: A preliminary in vitro porcine model for bioengineered transplantable bone shafts.

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.

Delivering siRNA Compounds During HOPE to Modulate Organ Function: A Proof-of-concept Study in a Rat Liver Transplant Model.

BACKGROUND: Apoptosis contributes to the severity of ischemia-reperfusion injury (IRI), limiting the use of extended criteria donors in liver transplantation (LT). Machine perfusion has been proposed as a platform to administer specific therapies to improve graft function. Alternatively, the inhibition of genes associated with apoptosis during machine perfusion could alleviate IRI post-LT. The aim of the study was to investigate whether inhibition of an apoptosis-associated gene (FAS) using a small interfering RNA (siRNA) approach could alleviate IRI in a rat LT model. METHODS: In 2 different experimental protocols, FASsiRNA (500 µg) was administered to rat donors 2 h before organ procurement, followed by 22 h of static cold storage, (SCS) or was added to the perfusate during 1 h of ex situ hypothermic oxygenated perfusion (HOPE) to livers previously preserved for 4 h in SCS. RESULTS: Transaminase levels were significantly lower in the SCS-FASsiRNA group at 24 h post-LT. Proinflammatory cytokines (interleukin-2, C-X-C motif chemokine 10, tumor necrosis factor alpha, and interferon gamma) were significantly decreased in the SCS-FASsiRNA group, whereas the interleukin-10 anti-inflammatory cytokine was significantly increased in the HOPE-FASsiRNA group. Liver absorption of FASsiRNA after HOPE session was demonstrated by confocal microscopy; however, no statistically significant differences on the apoptotic index, necrosis levels, and FAS protein transcription between treated and untreated groups were observed. CONCLUSIONS: FAS inhibition through siRNA therapy decreases the severity of IRI after LT in a SCS protocol; however the association of siRNA therapy with a HOPE perfusion model is very challenging. Future studies using better designed siRNA compounds and appropriate doses are required to prove the siRNA therapy effectiveness during liver HOPE liver perfusion.

Development of vascularized nerve scaffold using perfusion-decellularization and recellularization.

INTRODUCTION: Vascularized nerve grafts (VNG) may offer an advantage in peripheral nerve regeneration by avoiding ischemic damage and central necrosis observed in non-VNG, particularly for the treatment of large and long nerve defects. However, surgical complexity, donor site morbidity and limited nerve availability remain important drawbacks for the clinical use of VNG. Here we explore the potential of perfusion-decellularization for bioengineering a VNG to be used in peripheral nerve reconstruction. METHODS: Porcine sciatic nerves were surgically procured along with their vascular pedicle attached. The specimens were decellularized via perfusion-decellularization and preservation of the extracellular matrix (ECM), vascular patency and tissue cytokine contents were examined. Scaffold reendothelialization was conducted with porcine aortic endothelial cells in a perfusion-bioreactor. RESULTS: Morphologic examination of decellularized VNG and analysis of the DNA content demonstrated cell clearance whereas ECM content and structures of the nerve fascicles were preserved. Using 3D micro-computed tomography imaging we observed optimal vasculature preservation in decellularized scaffolds, down to the capillary level. Cytokine quantification demonstrated measurable levels of growth factors after decellularization. Endothelial cell engraftment of the large caliber vessels was observed in reendothelialized scaffolds. CONCLUSIONS: In this study we provide evidence that perfusion-decellularization can be used to create vascularized nerve scaffolds in which the vasculature and the ECM component are well preserved. As compared to non-vascularized conduits, engineered vascularized nerve scaffolds may represent an ideal approach for promoting better nerve regeneration in larger nerve defect reconstructions.

Nose and Lip Graft Variants: A Subunit Anatomical Study.

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.

Single-Artery Human Ear Graft Procurement: A Simplified Approach.

In the field of experimental facial vascularized composite tissue allotransplantation, a human auricular subunit model, pedicled on both superficial temporal and posterior auricular arteries, was described. Clinical cases of extensive auricular replantation, however, suggested that a single artery could perfuse the entire flap. In this study, variants of this single-pedicle approach have been studied, aiming to develop a more versatile replantation technique, in which the question of venous drainage has also been addressed. For arterial perfusion study, the authors harvested 11 auricular grafts, either on a single superficial temporal artery pedicle (n = 3) or a double superficial temporal and posterior auricular artery pedicle (n = 8). The authors then proceeded to selective barium injections, in the superficial temporal, posterior auricular, or both superficial temporal and posterior auricular arteries. Arteriograms were acquired with a micro-computed tomographic scan and analyzed on three-dimensionally reconstructed images. Venous drainage was investigated in eight hemifaces, carefully dissected after latex injection. Observations showed a homogenous perfusion of the whole auricle in all arterial graft variants. Venous drainage was highly variable, with either a dominant superficial temporal vein (37.5 percent), dominant posterior auricular vein (12.5 percent), or co-dominant trunks (50 percent). The authors demonstrated that auricular subunit vascularized composite tissue allotransplantation can be performed on a single artery, relying on the dynamic intraauricular anastomoses between superficial temporal artery and posterior auricular branches. Potentially, this vascular versatility is prone to simplify the subunit harvest and allows various strategies for pedicle selection. Venous drainage, however, remains inconstant and thus the major issue when considering auricular transplantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.