Evaluation of viscoelastic parameters and photo-based assessment of newly developed dermal substitutes modified with thermostabilized fibroblast growth factor 2.

BACKGROUND: The purpose of dermal substitutes is to mimic the basic properties of the extracellular matrix of human skin. The application of dermal substitutes to the defect reduces the formation of hypertrophic scars and improves the scar quality. This study aims to develop an original dermal substitute enriched with stable fibroblast growth factor 2 (FGF2-STAB®) and test it in an animal model. METHODS: Dermal substitutes based on collagen/chitosan scaffolds or collagen/chitosan scaffolds with nanofibrous layer were prepared and enriched with FGF2-STAB® at concentrations of 0, 0.1, 1.0, and 10.0 µg ‧ cm-2. The performance of these dermal substitutes was tested in vivo on artificially formed skin defects in female swine. The outcomes were evaluated using cutometry at 3 and 6 months. In addition, visual appearance was assessed based on photos of the scars at 1-month, 3-month and 6-month follow-ups using Yeong scale and Visual Analog Scale. RESULTS: The dermal substitute was fully integrated into all defects and all wounds healed successfully. FGF2-STAB®-enriched matrices yielded better results in cutometry compared to scaffolds without FGF2. Visual evaluation at 1, 3, and 6 months follow-ups detected no significant differences among groups. The FGF2-STAB® effectiveness in improving the elasticity of scar tissues was confirmed in the swine model. This effect was independently observed in the scaffolds with nanofibres as well as in the scaffolds without nanofibres. CONCLUSION: The formation of scars with the best elasticity was exhibited by addition 1.0 µg ‧ cm-2of FGF2-STAB® into the scaffolds, although it had no significant effect on visual appearance at longer follow-ups. This study creates the basis for further translational studies of the developed product and its progression into the clinical phase of the research.

Accelular nanofibrous bilayer scaffold intrapenetrated with polydopamine network and implemented into a full-thickness wound of a white-pig model affects inflammation and healing process.

Treatment of complete loss of skin thickness requires expensive cellular materials and limited skin grafts used as temporary coverage. This paper presents an acellular bilayer scaffold modified with polydopamine (PDA), which is designed to mimic a missing dermis and a basement membrane (BM). The alternate dermis is made from freeze-dried collagen and chitosan (Coll/Chit) or collagen and a calcium salt of oxidized cellulose (Coll/CaOC). Alternate BM is made from electrospun gelatin (Gel), polycaprolactone (PCL), and CaOC. Morphological and mechanical analyzes have shown that PDA significantly improved the elasticity and strength of collagen microfibrils, which favorably affected swelling capacity and porosity. PDA significantly supported and maintained metabolic activity, proliferation, and viability of the murine fibroblast cell lines. The in vivo experiment carried out in a domestic Large white pig model resulted in the expression of pro-inflammatory cytokines in the first 1-2 weeks, giving the idea that PDA and/or CaOC trigger the early stages of inflammation. Otherwise, in later stages, PDA caused a reduction in inflammation with the expression of the anti-inflammatory molecule IL10 and the transforming growth factor ß (TGFß1), which could support the formation of fibroblasts. Similarities in treatment with native porcine skin suggested that the bilayer can be used as an implant for full-thickness skin wounds and thus eliminate the use of skin grafts.

Effect of Polymeric Nanoparticles with Entrapped Fish Oil or Mupirocin on Skin Wound Healing Using a Porcine Model.

The utilization of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) with entrapped fish oil (FO) loaded in collagen-based scaffolds for cutaneous wound healing using a porcine model is unique for the present study. Full-depth cutaneous excisions (5 × 5 cm) on the pig dorsa were treated with pure collagen scaffold (control, C), empty PLGA NPs (NP), FO, mupirocin (MUP), PLGA NPs with entrapped FO (NP/FO) and PLGA NPs with entrapped MUP (NP/MUP). The following markers were evaluated on days 0, 3, 7, 14 and 21 post-excision: collagen, hydroxyproline (HP), angiogenesis and expressions of the COX2, EGF, COL1A1, COL1A3, TGFB1, VEGFA, CCL5 and CCR5 genes. The hypothesis that NP/FO treatment is superior to FO alone and that it is comparable to NP/MUP was tested. NP/FO treatment increased HP in comparison with both FO alone and NP/MUP (day 14) but decreased (p < 0.05) angiogenesis in comparison with FO alone (day 3). NP/FO increased (p < 0.05) the expression of the CCR5 gene (day 3) and tended (p > 0.05) to increase the expressions of the EGF (day 7, day 14), TGFB1 (day 21) and CCL5 (day 7, day 21) genes as compared with NP/MUP. NP/FO can be suggested as a suitable alternative to NP/MUP in cutaneous wound treatment.

Healing and Angiogenic Properties of Collagen/Chitosan Scaffolds Enriched with Hyperstable FGF2-STAB® Protein: In Vitro, Ex Ovo and In Vivo Comprehensive Evaluation.

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.