Vojtěch Pavlík

Therapies based on mesenchymal stromal cells (MSCs) have high potential in the field of regenerative medicine due mainly to their immunomodulatory properties. However, their clinical translation is hampered by a lack of sufficiently standardised potency tests. Since macrophages constitute key mediators of the effects of MSCs, macrophage-based assays potentially provide a relevant in vitro tool for the evaluation of the activity of MSC products. This study involved the coculturing of canine adipose-derived mesenchymal stromal cells (ASCs) with macrophages derived from human THP-1 and U937 monocyte cell lines, murine RAW264.7 macrophages and primary human macrophages. The M2 polarisation was assessed following stimulation with IL-4/IL-13 in THP-1 and U937 macrophages. The mRNA expression of the pro- and anti-inflammatory markers was analysed using qPCR. The ASC transwell coculture altered the LPS-induced inflammatory mRNA expression in a strongly model- and marker-dependent manner. The U937-derived macrophages exhibited the most consistent suppression of the tested inflammatory transcripts and the RAW264.7 cells provided a practical readout for selected inflammatory markers, whereas the THP-1 macrophages evinced the suppression of TNFA but not IL1B or PTGS2 under the selected stimulation conditions. IL-4/IL-13 induced moderate but statistically non-significant changes in IL10 and TGFB1 in the U937-derived macrophages but no reproducible response in the THP-1-derived macrophages. In a subsequent U937 coculture experiment, ASC-derived paracrine factors altered selected M2-associated transcripts at specific time points. The results thus provided support for macrophage-based transcriptional readouts as an early-stage tool for comparing responder macrophage models and detecting the selected anti-inflammatory paracrine effects of canine ASCs; the U937 cells were found to be particularly suitable for the study of inflammatory polarisation and the RAW264.7 cells for the purpose of standardised screening.

The research of new structures and solid forms from natural polymers intended for applications in the field of wound dressings has been carried out intensively in recent years. However, often promising research results face further technological challenges in the form of subsequent development necessary for advancing new materials into clinical studies and further into real production. The results of this work follow our laboratory feasibility study and aim to present a high-quality material with repeatedly confirmed parameters produced with the aid of an already developed automated pilot-scale device. A material with the structure of a nonwoven fabric (NWF) composed of short fragments of hyaluronic acid (HA) fibers, i.e. staples, is prepared in sequential steps by fiber formation through precipitation of an aqueous HA solution, fiber shortening, deposition into a planar layer, and drying. The active substance, Octenidine dihydrochloride (OCT), is applied onto the already dried NWFs at the end of the production cycle. OCT is an antiseptic substance with a bactericidal effect against a broad spectrum of gram-positive and gram-negative bacteria, which is homogeneously incorporated into the NWFs prepared in this way using an ink-jet nozzle positionally controlled in 2D. The main results of this work show that a relatively complicated and multistep process was successfully transferred and upscaled to an automated pilot-scale level. The newly developed pilot-scale unit is optimally controlled in important process parameters and is capable of repeatable production of antibacterial NWFs composed purely of HA with an area density of (49.0 ± 6.6) g·m-2 and an areal OCT content of (5.2 ± 0.4) µg·cm-2. The required material composition, stability, and cleanliness of production, final sterilization, and biological efficacy were demonstrated by in-vitro tests of antibacterial efficacy and cytotoxicity.

Therapies based on mesenchymal stromal cells (MSCs) have high potential in the field of regenerative medicine due mainly to their immunomodulatory properties. However, their clinical translation is hampered by a lack of sufficiently standardised potency tests. Since macrophages comprise key mediators of the effects of MSCs, macrophage-based assays potentially provide a relevant in vitro tool for the evaluation of the activity of MSC products. This study involved the coculturing of canine adipose-derived mesenchymal stem cells (ASCs) with macrophages derived from human THP-1 and U937 monocyte cell lines, murine RAW264.7 macrophages and primary human macrophages. The M2 polarisation was assessed following stimulation with IL-4/IL-13. The mRNA expression of the pro- and anti-inflammatory markers was analysed applying qPCR. The ASC secretome acted to reduce the pro-inflammatory mRNA expression across all the macrophage models, albeit with a certain degree of model–dependent variability. Only the U937 macrophages responded consistently to the M2-polarising stimuli, while the RAW264.7 cells provided practical advantages in terms of routine screening. The results thus provided support for the application of macrophage-based potency assays as a suitable platform for the testing of MSC products; the U937 cells were found to be particularly suitable for the study of polarisation and the RAW264.7 cells for standardised screening.

Canine adipose-derived mesenchymal stem cells (cASC) are promising for regenerative veterinary medicine due to their immunomodulatory and reparative capacities. Three-dimensional (3D) culturing provides a more physiologically relevant environment than conventional two-dimensional (2D) monolayers, enhancing paracrine activity and therapeutic potential of mesenchymal stem cells (MSC). This study investigates the production and biological characterization of cASC secretome generated under hypoxic conditions with platelet lysate (PLT) supplementation, either in a 2D culture or in a stirred-tank 3D culture. Secretomes obtained from 3D cultures were compared with those from 2D cultures prepared under identical hypoxic and PLT-supplemented conditions. Quantitative analyses revealed enhanced secretion of key factors, including monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), in 3D-derived secretomes. Functional in vitro assays demonstrated superior anti-inflammatory, pro-migratory, and antifibrotic effects of the 3D secretome, evidenced by nuclear factor kappa B (NF-κB) inhibition, increased fibroblast migration, and modulation of extracellular matrix gene expression. Additionally, the bioreactor system enabled consistent secretome production with reproducible biological activity. These findings indicate that 3D bioreactor cultivation under hypoxia with PLT supplementation can generate a biologically active secretome from canine adipose-derived stem cells, providing a promising basis for further exploration in veterinary regenerative applications.

Cellular processes such as tissue regeneration, inflammation, and migration require the proteolysis of the extracellular matrix and the proteolytic activation of signaling molecules. A widely used and accessible technique for studying these processes is gelatin zymography, particularly for investigating matrix metalloproteinases (MMPs), though it is not limited to them. This method is favored for its simplicity, low cost, and robustness. Despite certain limitations, it remains a preferred approach for the initial investigation of complex samples. Here, we present a protocol applicable to various sample sources, including proteases from human cell lines and bacteria isolated from chronic wounds. We also explore changes in protease activity within exudates from human chronic wounds, a challenging analysis for more complex techniques. Additionally, we emphasize the potential to extend the basic protocol to study the conditions under which proteases are active.

Exposure to the sun affects the skin and may eventually result in UV‐induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV‐related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT‐PCR, confocal microscopy and LC–MS/MS we compared the naturally sun‐exposed (SE), sun‐protected, experimentally repeatedly UVA + UVB‐exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV‐irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis.

All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

Photoaged skin exhibits signs of inflammation, DNA damage and changes in morphology that are visible at the macroscopic and microscopic levels. Photoaging also affects the extracellular matrix (ECM) including hyaluronan (HA), the main polysaccharide component thereof. HA is a structurally simple but biologically complex molecule that serves as a water‐retaining component and provides both a scaffold for a number of the proteins of the ECM and the ligand for cellular receptors. The study provides an overview of the literature concerning the changes in HA amount, size and metabolism, and the potential role of HA in photoaging. We also suggest novel HA contributions to photoaging based on our knowledge of the role of HA in other pathological processes, including the senescence and inflammation‐triggered ECM reorganization. Moreover, we discuss potential direct or indirect intervention to mitigate photoaging that targets the hyaluronan metabolism, as well as supplementation.