The Combination of Synoeca-MP Antimicrobial Peptide with IDR-1018 Stimulates Proliferation, Migration, and the Expression of Pro-Regenerative Genes in Both Human Skin Cell Cultures and 3D Skin Equivalents.

In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14.

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 µM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

3D Cell Printing of Perfusable Vascularized Human Skin Equivalent Composed of Epidermis, Dermis, and Hypodermis for Better Structural Recapitulation of Native Skin.

Although skin cell-printing has exhibited promises for fabrication of functional skin equivalents, existing skin models through 3D cell printing are still composed of dermal and epidermal layers. However, a key hope for printing skin is to improve structural complexity of human skin over conventional construction, enabling the precise localization of multiple cell types and biomaterials. Here, the complexity of skin anatomy is increased using 3D cell printing. A novel printing platform is suggested for engineering a matured perfusable vascularized 3D human skin equivalent composed of epidermis, dermis, and hypodermis. The skin model is evaluated using functional markers representing each region of epidermis, dermis, and hypodermis to confirm tissue maturation. It is hypothesized that the vascularized dermal and hypodermal compartments that provide a more realistic microenvironment can promote cross-talks with the epidermal compartment, producing better recapitulation of epidermal morphogenesis. Skin stemness in epithelial tissue is investigated. These findings reveal that the full-thickness skin has more similarities to the native human skin compared with the dermal and epidermal skin model, indicating that it better reflects the actual complexity of native human skin. It is envisioned that it offers better predictive and reliable in vitro platform for investigation of mechanisms of pathological research and skin disease modeling.