Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

INTRODUCTION: Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. METHODS: 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. RESULTS: Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. CONCLUSIONS: Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. STATEMENT OF SIGNIFICANCE: The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation.

Face Graft Scaffold Production in a Rat Model.

BACKGROUND: As a route toward face bioengineering, the authors previously reported the production of a complete scaffold by perfusion-decellularization of a porcine ear subunit graft and partial recellularization. To extend the scaffold to the whole face and to down-scale it, they applied their findings to a rodent hemifacial graft model. METHODS: After the animals were killed, seven full-thickness rat hemiface grafts were harvested with the common carotid artery and the external jugular vein as a pedicle, and cannulated. Grafts were decellularized by a detergent-based protocol: either by perfusion through the common carotid artery, or by mechanical agitation. After decellularization, samples were analyzed for DNA quantification and histology by hematoxylin and eosin, Masson trichrome, Sirius red, or Safranin O staining. Vascular tree patency was assessed by microangiographic computed tomography after contrast injection. Cell-friendly extracellular matrix was assessed by seeding of human adipose-derived stem cells and vital staining after 7 days of culture. RESULTS: Decellularization was effective in both groups, with a cell clearance at all levels, with the exception of cartilage areas in the agitation-treated groups. Microscopic assessment found a well-preserved extracellular matrix in both groups. Vascular contrast was found in all regions of the scaffolds. After the animals were killed, seeded cells were found viable and well distributed on all scaffolds. CONCLUSIONS: The authors successfully decellularized face grafts in a rodent model, with a preserved vascular tree. Perfusion-decellularization led to better and faster results compared with mechanical agitation but is not mandatory in this model. The rat face is an interesting scaffold model for further recellularization studies, in the final goal of human face bioengineering.

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.