The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment

: Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure

Getting everyone to agree on gene signatures for murine macrophage polarization in vitro.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4)

Supercritical impregnation of mesoglycan into calcium alginate aerogel for wound healing

Mesoglycan (MSG) was impregnated onto calcium alginate aerogel (CAA) to produce an innovative topical device to be applied on wounds. The main purpose of the composite material is to favor the re‐epithelialization through a direct and immediate action, also offering a protective barrier to ensure a favorable and sterilized environment for the wound healing process. Impregnation kinetics and isotherms were determined at 18 MPa and 40 and 60 °C. Composite systems were analyzed using Field Emission Scanning Electron Microscopy (FESEM), Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD), Fourier Transform Infrared (FT-IR) and UV–vis spectroscopies. A functional assay of in vitro wound healing confirmed the pro-migratory role of the composite system MSG/CAA, since an increase of the migration rate on fibroblast (BJ) cells and of the covered distance for keratinocytes (HaCaT) was observed with MSG impregnated on CAA compared to non-treated cells, CAA and pure MSG

Mesoglycan induces keratinocyte activation by triggering syndecan‐4 pathway and the formation of the annexin A1/S100A11 complex

Wound healing is a dynamic process comprising multiple events, such as inflammation, re‐epithelialization, and tissue remodeling. Re‐epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re‐epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non‐healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re‐epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan‐4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new prohealing drug for skin wound care

IFNγ+LPS up-regulated DEGs identified by RRA and fold-change analysis.

(A) On the left, the heatmap of the thirteen datasets showing the top 20 most robust genes upregulated is depicted. Value in the boxes represents fold-change and shade of blue represents RRA score. On the right, the corresponding average RRA score and fold-change. (B) On the left, the heatmap of the thirteen datasets showing the top 20 most upregulated genes which also present a significant RRA score. Value in the boxes represents fold-change and shade of blue represents RRA score. On the right, the corresponding average RRA score and fold-change.</p

UPSTREAM regulators analysis.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4).</div

PCL/Mesoglycan Devices Obtained by Supercritical Foaming and Impregnation

In this work, a one-shot process for the simultaneous foaming of polycaprolactone (PCL) and impregnation of mesoglycan (MSG) into the porous structure was successfully attempted. Supercritical carbon dioxide plays the role of the foaming agent with respect to PCL and of the solvent with respect to MSG. The main objective is to produce an innovative topical device for application on skin lesions, promoting prolonged pro-resolving effects. The obtained device offers a protective barrier to ensure a favorable and sterilized environment for the wound healing process. The impregnation kinetics revealed that a pressure of 17 MPa, a temperature of 35 °C, and a time of impregnation of 24 h assured a proper foaming of PCL in addition to the impregnation of the maximum amount of MSG; i.e., 0.22 mgMSG/mgPCL. After a preliminary study conducted on PCL granules used as brought, the MSG impregnation was performed at the optimized process conditions also on a PCL film, produced by compression molding, with the final goal of producing medical patches. Comparing the dissolution profiles in phosphate buffered saline solution (PBS) of pure MSG and MSG impregnated on foamed PCL, it was demonstrated that the release of MSG was significantly prolonged up to 70 times. Next, we performed functional assays of in vitro wound healing, cell invasion, and angiogenesis to evaluate the biological effects of the PCL-derived MSG. Interestingly, we found the ability of this composite system to promote the activation of human keratinocytes, fibroblasts, and endothelial cells, as the main actors of tissue regeneration, confirming what we previously showed for the MSG alon

IL-4 up-regulated DEGs identified by RRA and fold-change analysis.

(A) On the left, the heatmap of the seventeen datasets showing the top 20 most robust genes upregulated is depicted. Value in the boxes represents fold-change and shade of blue represents RRA score. On the right, the corresponding average RRA score and fold-change. (B) On the left, the heatmap of the seventeen datasets showing the top 20 most upregulated genes which also present a significant RRA score. Value in the boxes represents fold-change and shade of blue represents RRA score. On the right, the corresponding average RRA score and fold-change.</p