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.

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.

Synergistic effect of bovine platelet lysate and various polysaccharides on the biological properties of collagen-based scaffolds for tissue engineering: Scaffold preparation, chemo-physical characterization, in vitro and ex ovo evaluation.

Crosslinked 3D porous collagen-polysaccharide scaffolds, prepared by freeze-drying, were modified with bovine platelet lysate (BPL) and evaluated in terms of chemical, physical and biological properties. Natural antibacterial polysaccharides like chitosan, chitin/chitosan-glucan complex and calcium salt of oxidized cellulose (CaOC) incorporated in collagen scaffolds affected not only chemo-physical properties of the composite scaffolds but also improved their biological properties, especially when BPL was presented. Lipophilic BPL formed microspheres in porous scaffolds while reduced by half their swelling ratio. The resistance of collagen sponges to hydrolytic degradation in water depended strongly on chemical crosslinking varying from 60 min to more than one year. According to in-vitro tests, chemically crosslinked scaffolds exhibited a good cellular response, cell-matrix interactions, and biocompatibility of the material. The combination of collagen with natural polysaccharides confirmed a significant positive synergistic effect on cultivation of cells as determined by MTS assay and PicoGreen method, as well as on angiogenesis evaluated by ex ovo Chick Chorioallantoic Membrane (CAM) assay. Contrary, modification only by BLP of pure collagen scaffolds exhibited decreased biocompatibility in comparison to unmodified pure collagen scaffold. We propose that the newly developed crosslinked collagen sponges involving bioactive additives could be used as scaffold for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

Accurate micro-computed tomography imaging of pore spaces in collagen-based scaffold.

In this work we have used X-ray micro-computed tomography (µCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a µCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for µCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag(+)) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and µCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.