Speeding up the production of clinical-grade skin substitutes using off-the-shelf decellularized self-assembled dermal matrices.

Patients with deep and extensive wounds need urgent skin coverage to re-establish the cutaneous barrier that prevents life-threatening infections and dehydration. However, the current clinically-available skin substitutes intended for permanent coverage are limited in number, and a trade-off between production time and quality must be made. Here, we report the use of decellularized self-assembled dermal matrices to reduce by half the manufacturing process time of clinical-grade skin substitutes. These decellularized matrices can be stored for over 18 months and recellularized with patients' cells in order to generate skin substitutes that show outstanding histological and mechanical properties in vitro. Once grafted in mice, these substitutes persist over weeks with high graft take, few contraction events, and high stem cell content. These next-generation skin substitutes constitute a substantial advancement in the treatment of major burn patients, combining, for the first time, high functionality, rapid manufacturability and easy handling for surgeons and healthcare practitioners. Future clinical trials will be conducted to assess the advantages of these substitutes over existing treatments. STATEMENT OF SIGNIFICANCE: The number of patients in need for organ transplantation is ever-growing and there is a shortage in tissue and organ donors. In this study, we show for the first time that we can preserve decellularized self-assembled tissues and keep them in storage. Then, in only three weeks we can use them to produce bilayered skin substitutes that have properties very close to those of the native human skin. These findings therefore represent a major step forward in the field of tissue engineering and organ transplantation, paving the way toward a universal off-the-shelf biomaterial for tissue reconstruction and surgery that will be beneficial for many clinicians and patients.

Peptide grafting on intraosseous transcutaneous amputation prostheses to promote sealing with skin cells: Potential to limit infections.

The long-term success of intraosseous transcutaneous amputation prostheses (ITAPs) mainly relies on dermal attachment of skin cells to the implant. Otherwise, bacteria can easily penetrate through the interface between the implant and the skin. Therefore, infection at this implant/skin interface remains a significant complication in orthopedic surgeries in which these prostheses are required. Two main strategies were investigated to prevent these potential infection problems which consist in either establishing a strong sealing at the skin/implant interface or on eradicating infections by killing bacteria. In this work, two adhesion peptides, either KRGDS or KYIGSR and one antimicrobial peptide, Magainin 2 (Mag 2), were covalently grafted via phosphonate anchor arms to the surface of the Ti6Al4V ELI (extra low interstitials) material, commonly used to manufacture ITAPs. X-ray photoelectron spectroscopy, contact angle, and confocal microscopy analyses enabled to validate the covalent and stable grafting of these three peptides. Dermal fibroblasts cultures on bare Ti6Al4V ELI surfaces and functionalized ones displayed a good cell adhesion and proliferation on all samples. However, cell spreading and viability appeared to be improved on grafted surfaces, especially for those conjugated with KRGDS and Mag 2. Moreover, the dermal sheet attachment, was significantly higher on surfaces functionalized with Mag 2 as compared to the other surfaces. Therefore, the surface functionalization with the antimicrobial peptide Mag 2 seems to be the best approach for the targeted application, as it could play a dual role, inducing a strong skin adhesion while limiting infections on Ti6Al4V ELI materials.

The Self-Assembled Skin Substitute History: Successes, Challenges, and Current Treatment Indications.

The self-assembled skin substitute (SASS) is an autologous bilayered skin substitute designed by our academic laboratory, the Laboratoire d'Organogenèse Expérimentale (LOEX) to offer definitive treatment for patients lacking donor sites (unwounded skin) to cover their burn wounds. This product shows skin-like attributes, such as an autologous dermal and epidermal layer, and is easily manipulable by the surgeon. Its development stems from the need for skin replacement in high total body surface area burned survivors presenting few donor sites for standard split-thickness skin grafting. This review aims to present the history, successes, challenges, and current therapeutic indications of this skin substitute. We review the product's development history, before discussing current production techniques, as well as clinical use. The progression observed since the initial SASS production technique described in 1999, up to the most recent technique expresses significant advances made in the technical aspect of our product, such as the reduction of the production time. We then explore the efficacy and benefits of SASS over existing skin substitutes and discuss the outcomes of a recent study focusing on the successful treatment of 14 patients. Moreover, an ongoing cross-Canada study is further assessing the product's safety and efficacy. The limitations and technical challenges of SASS are also discussed.

Cultured Autologous Corneal Epithelia for the Treatment of Unilateral Limbal Stem Cell Deficiency: A Case Series of 15 Patients.

Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27-79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60-98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.

Preclinical Evaluation of BMP-9-Treated Human Bone-like Substitutes for Alveolar Ridge Preservation following Tooth Extraction.

The success of dental implant treatment after tooth extraction is generally maximized by preserving the alveolar ridge using cell-free biomaterials. However, these treatments can be associated with inflammatory reactions, leading to additional bone volume loss hampering dental implant positioning. Our group developed a self-assembled bone-like substitute constituted of osteogenically induced human adipose-derived stromal/stem cells (hASCs). We hypothesized that a bone morphogenetic protein (BMP) supplementation could improve the in vitro osteogenic potential of the bone-like substitute, which would subsequently translate into enhanced alveolar bone healing after tooth extraction. ASCs displayed a better osteogenic response to BMP-9 than to BMP-2 in monolayer cell culture, as shown by higher transcript levels of the osteogenic markers RUNX2, osterix (OSX/SP7), and alkaline phosphatase after three and six days of treatment. Interestingly, BMP-9 treatment significantly increased OSX transcripts and alkaline phosphatase activity, as well as pro-angiogenic angiopoietin-1 gene expression, in engineered bone-like substitutes after 21 days of culture. Alveolar bone healing was investigated after molar extraction in nude rats. Microcomputed tomography and histological evaluations revealed similar, or even superior, global alveolar bone preservation when defects were filled with BMP-9-treated bone-like substitutes for ten weeks compared to a clinical-grade biomaterial, with adequate gingival re-epithelialization in the absence of resorption.

Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.

BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

Fibronectin grafting to enhance skin sealing around transcutaneous titanium implant.

Intraosseous transcutaneous amputation prosthesis is a new approach in orthopedic implants that overcomes socket prosthesis problems. Its long-term performance requires a tight skin-implant seal to prevent infections. In this study, fibronectin (Fn), a widely used adhesion protein, was adsorbed or grafted onto titanium alloy. Fn grafting was performed using two different linking arms, dopamine/glutaric anhydride or phosphonate. The characterization of Fn-modified surfaces showed that Fn grating via phosphonate has led to the highest amount of Fn cell-binding site (RGD, arginine, glycine, and aspartate) available on the surface. Interestingly, cell culture studies revealed a strong correlation between the amount of available RGD ligands and cellular behavior, since enhanced proliferation and spreading of fibroblasts were noticed on Fn-grafted surfaces via phosphonate. In addition, an original in vitro mechanical test, inspired from the real situation, to better predict clinical outcomes after implant insertion, has been developed. Tensile test data showed that the adhesion strength of a bio-engineered dermal tissue was significantly higher around Fn-grafted surfaces via phosphonate, as compared to untreated surfaces. This study sheds light on the importance of an appropriate selection of the linking arm to tightly control the spatial conformation of biomolecules on the material surface, and consequently cell interactions at the interface tissue/implant.

In Vitro Prevascularization of Self-Assembled Human Bone-Like Tissues and Preclinical Assessment Using a Rat Calvarial Bone Defect Model.

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Biofabrication and preclinical evaluation of a large-sized human self-assembled skin substitute.

Severe skin burns are widely treated using split-thickness skin autografts. However, the accessibility of the donor site may be limited depending on the size of the injured surface. As an alternative to skin autografts, our laboratory is clinically investigating a model of human self-assembled skin substitute (SASS) with a standard size of 35 cm2. For the management of extensive skin wounds, multiple grafts are required to cover the entire wound bed. Even if SASSs could provide an adequate and efficient treatment, in some cases, the long-term follow-up of the skin graft site reveals the appearance of marks at the junction between SASSs. This study aims to produce a large-sized self-assembled skin substitute (L-SASS; 289 cm2) and evaluate its preclinical potential for skin wound coverage. The L-SASSs and SASSs shared similar contraction behavior on an agar surface, thickness, and epidermal differentiation in vitro. After grafting, similar histological results were obtained for skin substitutes produced with both methods. Hence, the self-assembly approach of tissue engineering is a scaffold-free method that allows the production of living skin substitutes in a large format.

Human Organ-Specific 3D Cancer Models Produced by the Stromal Self-Assembly Method of Tissue Engineering for the Study of Solid Tumors.

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

Best practices for enhancing surgical research: a perspective from the Canadian Association of Chairs of Surgical Research

Summary: The Canadian Association of Chairs of Surgical Research was created in 2014, with representation from every departmental surgical research committee across Canada, to establish Canadian surgical research as a beacon for health care innovation and to propose solutions for the daily challenges facing surgeon-researchers. Our key mandate has been to identify challenges for surgeons and scientists performing research to prevent further erosion of this vital area of activity that benefits patients, health care service providers and Canadian society. This article outlines the findings of a nationwide survey sent to all members of departments of surgery across Canada, seeking input on current threats and potential solutions. The results suggest that surgical research in Canada is experiencing a decline in funding and an increase in challenges affecting research productivity of academic surgeons, such as pressures to be clinically active, unpredictable surgical schedules, growing administrative demands, and increasing complexity of patient populations. Although surgeons are productive in their research endeavours, institutional changes and sharing of best practices are needed to ensure sustainable growth of research programs.

Isolation and Culture of Human Dermal Microvascular Endothelial Cells.

Primary endothelial cells are needed for angiogenesis studies, and more particularly in the field of tissue engineering, to engineer pre-vascularized tissues. Investigations often use human umbilical vein endothelial cells due to their extensive characterization, but also because they are easy to obtain and isolate. An alternative is the use of human dermal microvascular endothelial cells, more representative of adult skin angiogenesis and vascularization processes. This chapter presents a detailed methodology to isolate and culture microvascular endothelial cells from skin biopsies based on enzymatic digestion and mechanical extraction.

Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice.

Organs are needed for the long-term replacement of diseased or wounded tissues. Various technologies based on cells seeded in synthetic or biomaterial scaffolds, or scaffold-free methods have been developed in order to produce substitutes that mimic native organs and tissues. For cell-based approaches, the use of living allogeneic fibroblasts could potentially lead to the production of "off-the-shelf" bioengineered organs/tissues. However, questions remain regarding the outcome of allogeneic grafts in terms of persistence of allogeneic cells, tolerance and the host immune reaction against the tissue after implantation. To evaluate graft tolerance of engineered-tissues containing non-autologous fibroblasts, tissue-engineered skin substitutes (TESs) produced with syngeneic, allogeneic or xenogeneic fibroblasts associated with syngeneic, allogeneic or xenogeneic epithelial cells were grafted in mice as primary and secondary grafts. The immune response was evaluated by histological analysis and immunodetection of M2 macrophages, CD4- and CD8-positive T cells, 15, 19, 35 and 56 days after grafting. Tissue-engineered skin composed of non-autologous epithelial cells were rejected. In contrast, TESs composed of non-autologous fibroblasts underlying syngeneic epithelial cells were still present 56 days after grafting. This work shows that TES composed of non-autologous fibroblasts and autologous epithelial cells are not rejected after grafting. STATEMENT OF SIGNIFICANCE: We found that tissue-engineered skin substitutes produced by a scaffold-free cell-based approach from allogeneic fibroblasts and autologous epithelial cells are not rejected after grafting and allow for the permanent coverage of a full-thickness skin wounds. In the field of tissue engineering, these findings open the possibility of selecting a human fibroblastic or stromal cell population based on its biological properties and adequate biosafety, banking it, in order to produce "ready-to-use" bioengineered organs/tissues that could be grafted to any patient without eliciting immune reaction after grafting. Our results can be generalized to any organs produced from fibroblasts. Thus, it is a great step with multiple applications in tissue engineering and transplantation.

Cell Seeding on UV-C-Treated 3D Polymeric Templates Allows for Cost-Effective Production of Small-Caliber Tissue-Engineered Blood Vessels.

There is a strong clinical need to develop small-caliber tissue-engineered blood vessels for arterial bypass surgeries. Such substitutes can be engineered using the self-assembly approach in which cells produce their own extracellular matrix (ECM), creating a robust vessel without exogenous material. However, this approach is currently limited to the production of flat sheets that need to be further rolled into the final desired tubular shape. In this study, human fibroblasts and smooth muscle cells were seeded directly on UV-C-treated cylindrical polyethylene terephthalate glycol-modified (PETG) mandrels of 4.8 mm diameter. UV-C treatment induced surface modification, confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis, was necessary to ensure proper cellular attachment and optimized ECM secretion/assembly. This novel approach generated solid tubular conduits with high level of cohesion between concentric cellular layers and enhanced cell-driven circumferential alignment that can be manipulated after 21 days of culture. This simple and cost-effective mandrel-seeded approach also allowed for endothelialization of the construct and the production of perfusable trilayered tissue-engineered blood vessels with a closed lumen. This study lays the foundation for a broad field of possible applications enabling custom-made reconstructed tissues of specialized shapes using a surface treated 3D structure as a template for tissue engineering.

Specialized Living Wound Dressing Based on the Self-Assembly Approach of Tissue Engineering.

There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air⁻liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.

Tissue-engineered 3D melanoma model with blood and lymphatic capillaries for drug development.

While being the rarest skin cancer, melanoma is also the deadliest. To further drug discovery and improve clinical translation, new human cell-based in vitro models are needed. Our work strives to mimic the melanoma microenvironment in vitro as an alternative to animal testing. We used the self-assembly method to produce a 3D human melanoma model exempt of exogenous biomaterial. This model is based on primary human skin cells and melanoma cell lines while including a key feature for tumor progression: blood and lymphatic capillaries. Major components of the tumor microenvironment such as capillaries, human extracellular matrix, a stratified epidermis (involucrin, filaggrin) and basement membrane (laminin 332) are recapitulated in vitro. We demonstrate the persistence of CD31+ blood and podoplanin+/LYVE-1+ lymphatic capillaries in the engineered tissue. Chronic treatment with vemurafenib was applied to the model and elicited a dose-dependent response on proliferation and apoptosis, making it a promising tool to test new compounds in a human-like environment.

Biomimetic Tissue-Engineered Bone Substitutes for Maxillofacial and Craniofacial Repair: The Potential of Cell Sheet Technologies.

Maxillofacial defects are complex lesions stemming from various etiologies: accidental, congenital, pathological, or surgical. A bone graft may be required when the normal regenerative capacity of the bone is exceeded or insufficient. Surgeons have many options available for bone grafting including the "gold standard" autologous bone graft. However, this approach is not without drawbacks such as the morbidity associated with harvesting bone from a donor site, pain, infection, or a poor quantity and quality of bone in some patient populations. This review discusses the various bone graft substitutes used for maxillofacial and craniofacial repair: allografts, xenografts, synthetic biomaterials, and tissue-engineered substitutes. A brief overview of bone tissue engineering evolution including the use of mesenchymal stem cells is exposed, highlighting the first clinical applications of adipose-derived stem/stromal cells in craniofacial reconstruction. The importance of prevascularization strategies for bone tissue engineering is also discussed, with an emphasis on recent work describing substitutes produced using cell sheet-based technologies, including the use of thermo-responsive plates and the self-assembly approach of tissue engineering. Indeed, considering their entirely cell-based design, these natural bone-like substitutes have the potential to closely mimic the osteogenicity, osteoconductivity, osteoinduction, and osseointegration properties of autogenous bone for maxillofacial and craniofacial reconstruction.

Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis.

This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3). After stacking of tissue layers and tissue maturation at the air-liquid interface (stage 4), the 3D construct containing the lymphatic microvascular network can be analyzed by microscopy (stage 5). Lymphatic vasculature generated by this approach exhibits the major cellular and ultrastructural features of native in vivo human dermal lymphatic microvasculature and is stable over many weeks. The protocol for generating a 3D construct takes 6 weeks to complete, and it requires experience in cell culture techniques. The system described here offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function in a human cell context.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

In Vivo Evaluation and Imaging of a Bilayered Self-Assembled Skin Substitute Using a Decellularized Dermal Matrix Grafted on Mice.

As time to final coverage is the essence for better survival outcome in severely burned patients, we have continuously strived to reduce the duration for the preparation of our bilayered self-assembled skin substitutes (SASS). These SASS produced in vitro by the self-assembly approach have a structure and functionality very similar to native skin. Recently, we have shown that a decellularized dermal matrix preproduced by the self-assembly approach could be used as a template to further obtain self-assembled skin substitute using a decellularized dermal template (SASS-DM) in vitro. Thus, the production period with patient cells was then reduced to about 1 month. Herein, preclinical animal experiments have been performed to confirm the integration and evolution of such a graft and compare the maturation of SASS and SASS-DM in vivo. Both tissues, reconstructed from adult or newborn cells, were grafted on athymic mice. Green fluorescent protein-transfected keratinocytes were also used to follow grafted tissues weekly for 6 weeks using an in vivo imaging system (IVIS). Cell architecture and differentiation were studied with histological and immunofluorescence analyses at each time point. Graft integration, macroscopic evolution, histological analyses, and expression of skin differentiation markers were similar between both skin substitutes reconstructed from either newborn or adult cells, and IVIS observations confirmed the efficient engraftment of SASS-DM. In conclusion, our in vivo graft experiments on a mouse model demonstrated that the SASS-DM had equivalent macroscopic, histological, and differentiation evolution over a 6-week period, when compared with the SASS. The tissue-engineered SASS-DM could improve clinical availability and advantageously shorten the time necessary for the definitive wound coverage of severely burned patients.