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.

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.

Correction: Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

[This corrects the article DOI: 10.1371/journal.pone.0136217.].

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

BACKGROUND: Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential. RESULTS: We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers. CONCLUSIONS: These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.