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In vitro evaluation of macrophages polarization in dermo-epidermal skin substitutes

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Summary:Due to chronic wounds, caused by different organism disbalances such as prolonged microbial infections or high immune cells response, a lot of people are suffering and need proper treatment. How- ever, traditional debridement techniques are non-selective and can be painful. Modern cellular dressings, such as Apligraf®, Dermagraft®, and Theraskin®, are becoming increasingly important in chronic wound research, but they have disadvantages such as higher costs, more complicated commercialization, and lower wound healing rates comparing with non-cell-based products. To overcome these limitations, dermo-epidermal skin substitutes (DESs), based on cellular electrospun fibers and freeze-dried sponges using chitosan and fish gelatin cross-linked with glutaraldehyde, were produced and cultured with keratinocytes (HaCaT), fibroblasts (HFFF2), and monocytes/macrophages (THP-1). To determine the most suitable DESs for skin applications, fiber and sponge scaffolds were evaluated through physico- chemical assay by ATR-FTIR, morphology assay by SEM, mechanical assay, thermal stability and swelling assays. The viability of cells in scaffolds was studied using resazurin, PicoGreen, and Live/Dead assays. The evaluation of THP-1 cells polarization was carried out using immunofluores- cence microscopy. The results demonstrated that fibers were more stable than sponges during thermal degradation, but sponges absorb and retain more liquid than fibers. Following, both fiber and sponge scaffolds did not show any cytotoxicity on HaCaT and HFFF2 cells. Additionally, fiber DESs had strat- ified epidermal layer but promoted more in pro-inflammatory M1 THP-1 cells, while sponge DESs had incorrect stratified epidermis, located in pores, but promoted the equal activity of native M0, pro-in- flammatory M1, and pro-healing M2 THP-1 cells. In conclusion, the produced electrospun and freeze- dried DESs show not suitable mechanical but promise biological properties for skin applications, poten- tially promoting formation of epidermis and dermis layers. DESs, composed with same chemicals but different morphology, promote different macrophage polarization. Future research is necessary to opti- mize DES construction and use other methods for more specific evaluation of macrophages polarization.
Main Authors:Fomichev, Ilya
Subject:chronic wounds tissue engineering electrospinning freeze-drying skin substitutes gelatin
Year:2024
Country:Portugal
Document type:master thesis
Access type:open access
Associated institution:Universidade Nova de Lisboa
Language:English
Origin:Repositório Institucional da UNL

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