Small extracellular vesicles (SEVs) offer a promising strategy for tissue regeneration, yet their short lifetime at the injured tissue limits their efficacy. Here, we show that kinetics of SEV delivery impacts tissue regeneration at tissue, cellular, and molecular levels. We show that multiple carefully timed applications of SEVs had superior regeneration than a single dose of the same total concentration of SEVs. Importantly, diabetic and nondiabetic wounds treated with a single time point dose of an injectable light-triggerable hydrogel containing SEVs demonstrated a robust increase in closure kinetics relative to wounds treated with a single or multiple doses of SEVs or platelet-derived growth factor BB, an FDA-approved wound regenerative therapy. The pro-healing activity of released SEVs was mediated at the tissue/cell level by an increase in skin neovascularization and re-epithelization and at the molecular level by an alteration in the expression of 7 miRNAs at different times during wound healing. This includes an alteration of has-miR-150-5p, identified here to be important for skin regeneration.

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Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues.

IntroductionHuman adipose tissue-derived stem cells (hASCs) are attractive cells for therapeutic applications and are currently being evaluated in multiple clinical trials. Prior to their clinical application, hASCs must be expanded ex vivo to obtain the required number of cells for transplantation. Fetal bovine serum is the supplement most widely used for cell culture, but it has disadvantages and it is not safe for cell therapy due to the risks of pathogen transmission and immune reaction. Furthermore, the cell expansion poses a risk of accumulating genetic abnormalities that could lead to malignant cell transformation. In this study, our aim was to evaluate the proliferation pattern as well as the resistance to spontaneous transformation of hASCs during expansion in a xeno-free culture condition.MethodshASCs were expanded in Dulbecco’s modified Eagle’s medium supplemented with pooled allogeneic human serum or fetal bovine serum to enable a side-by-side comparison. Cell viability and differentiation capacity toward the mesenchymal lineages were assessed, along with immunophenotype. Ki-67 expression and the proliferation kinetics were investigated. The expression of the transcription factors c-FOS and c-MYC was examined with Western blot, and MYC, CDKN2A, ERBB2 and TERT gene expression was assessed with quantitative PCR. Senescence was evaluated by β-gal staining. Karyotype analysis was performed and tumorigenesis assay in vivo was also evaluated.ResultsThe hASCs expanded in medium with pooled allogeneic human serum did not show remarkable differences in morphology, viability, differentiation capacity or immunophenotype. The main difference observed was a significantly higher proliferative effect on hASCs cultured in pooled allogeneic human serum. There was no significant difference in C-FOS expression; however, C-MYC protein expression was enhanced in pooled allogeneic human serum cultures compared to fetal bovine serum cultures. No difference was observed in MYC and TERT mRNA levels. Moreover, the hASCs presented normal karyotype undergoing senescence, and did not form in vivo tumors, eliminating the possibility that spontaneous immortalization of hASCs had occurred with pooled allogeneic human serum.ConclusionsThis complete characterization of hASCs cultivated in pooled allogeneic human serum, a suitable xeno-free approach, shows that pooled allogeneic human serum provides a high proliferation rate, which can be attributed for the first time to C-MYC protein expression, and showed cell stability for safe clinical applications in compliance with good manufacturing practice.

Human adipose-derived stem cells (hASCs) are currently a point of focus for bone tissue engineering applications. However, the ex vivo expansion of stem cells before clinical application remains a challenge. Fetal bovine serum (FBS) is largely used as a medium supplement and exposes the recipient to infections and immunological reactions. In this study, we evaluated the osteogenic differentiation process of hASCs in poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) scaffolds with the osteogenic medium supplemented with pooled allogeneic human serum (aHS). The hASCs grown in the presence of FBS or aHS did not show remarkable differences in morphology or immunophenotype. The PHB-HV scaffolds, which were developed by the freeze-drying technique, showed an adequate porous structure and mechanical performance as observed by micro-computed tomography, scanning electron microscopy (SEM), and compression test. The three-dimensional structure was suitable for allowing cell colonization, which was revealed by SEM micrographs. Moreover, these scaffolds were not toxic to cells as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The differentiation capacity of hASCs seeded on scaffolds was confirmed by the reduction of the proliferation, the alkaline phosphatase (AP) activity, expression of osteogenic gene markers (AP, collagen type I, Runx2, and osteocalcin), and the expression of bone markers, such as osteopontin, osteocalcin, and collagen type I. The osteogenic capacity of hASCs seeded on PHB-HV scaffolds indicates that this scaffold is adequate for cell growth and differentiation and that aHS is a promising supplement for the in vitro expansion of hASCs. In conclusion, this strategy seems to be useful and safe for application in bone tissue engineering.

Bioactive glass/polymer composites are promising materials for bone tissue engineering. The present research group has developed porous hybrid scaffolds comprised of 50% polyvinyl alcohol/50% bioactive glass with a 70%SiO(2)-30%CaO composition. Prior studies have also shown the adequate structural and mechanical behavior of these scaffolds. As such, the present study investigates the in vitro and in vivo osteogenic potential of the scaffold, using mesenchymal stem cells (MSC) from the bone marrow of female rats. MTT, alkaline phosphatase activity, collagen secretion and Von Kossa staining were conducted to evaluate the differentiation ability of MSC in an osteogenic medium. The in vitro results indicate an increase in both cell proliferation and osteogenic differentiation when the hybrid material is present. Von Kossa staining showed a progressive increase in mineralization nodules, coupled with time differentiation. For the in vivo evaluation, three groups were studied: (1) group implanted with the hybrid scaffold, (2) group implanted with scaffold colonized by non-differentiated MSC and (3) group implanted with scaffold colonized by differentiated MSC. The scaffolds were subcutaneously implanted on the back of Wistar rats for 1-8 weeks, and histological and histomorphometric analyses were performed. The tissue ingrowth proved to be higher in the groups colonized by MSC in the first week. In the second week, only the hybrid colonized by differentiated MSC presented a larger percentage of connective tissue. In the third, fourth and eighth weeks, all groups presented 70% of the hybrid scaffold filled with tissue. However, only the group with differentiated MSC presented some form of osteoid tissue, indicating that the hybrid scaffold with differentiated MSC does indeed present osteogenic potential.

The combinatorial delivery of miRNAs holds great promise to modulate cell activity in the context of angiogenesis. Yet, the delivery of multiple miRNAs with spatiotemporal control remains elusive. Here, we report a plasmonic nanocarrier to control the release of two microRNAs. The nanocarrier consists of gold nanorods modified with single-stranded DNA for hybridization with complementary DNA-conjugated microRNAs. DNA strands with distinct melting temperatures enable the independent release of each microRNA with a near-infrared laser using the same wavelength but different powers. Tests in human outgrowth endothelial cells (OECs) indicate that this system can be used to silence different targets sequentially and, by doing so, to modulate cell activity with spatiotemporal resolution. Finally, using an in vivo acute wound healing animal model, it is demonstrated that the order by which each miRNA was released in transplanted OECs significantly impacted the wound healing kinetics.