Objectives: Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. Background: Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. Methods: To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs’ ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results: Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4⁺ T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. Discussion: We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.

Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off‐the‐shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three‐dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self‐renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

We showed that the chemokine receptor C‐X‐C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP‐2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP‐2 expression was retained in adult articular cartilage. GCP‐2 loss‐of‐function inhibited extracellular matrix production. GCP‐2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo . To exploit the chondrogenic activity of GCP‐2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP‐2 haptotactic gradient on endothelia. This mutated version (GCP‐2‐T) had reduced capacity to induce transendothelial migration in vitro and in vivo , without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra‐articular adenoviral overexpression of GCP‐2‐T, but not wild‐type GCP‐2, reduced pain and cartilage loss in instability‐induced osteoarthritis in mice. We suggest that GCP‐2‐T may be used for disease modification in osteoarthritis.