The treatment of chronic wounds remains a major challenge in regenerative medicine due to prolonged healing times, susceptibility to infection, and underlying conditions like diabetes. Incorporating bioactive and antibacterial nanoparticles (NPs) into wound dressings can significantly enhance their mechanical properties, structural integrity, and functionality, improving stability, biocompatibility, and healing efficacy. However, conventional methods of loading NPs in polymer matrices often lead to uneven distribution and localized toxicity. To overcome these limitations, we employ a novel in situ synthesis of copper nanoparticles (CuNPs) using an encapsulation method via the self-assembled polymerization of dopamine (DOPA) or tannic acid (TA) within collagen/carboxymethyl cellulose (Coll/CMC) 3D freeze-dried scaffolds. When CuNPs are synthesized ex situ, both DOPA and TA act as reducing and encapsulating agents. However, in situ synthesis within Coll/CMC scaffolds results in TA functioning solely as a reducing agent, while DOPA serves both as a reducing agent and, through its polymerization into polydopamine, as a stabilizing agent. The polydopamine network enhances collagen fiber adhesion to CuNPs and stabilizes them via noncovalent interactions. Notably, the DOPA-in situ/Cu sample exhibited prolonged enzymatic stability for up to 7 days. X-ray microcomputed tomography confirmed the homogeneous distribution of CuNPs throughout the scaffold. Biological assays demonstrated the enhanced antibacterial efficacy of DOPA/TA-in situ/Cu samples against Staphylococcus aureus and MRSA, along with cytocompatibility with 3T3 fibroblasts. Future research should explore the in vivo application of these scaffolds and their potential in regenerative medicine for treating infected wounds.

• Publication type
• Journal Article MeSH

Using supramolecular self-assembled nanocomposite materials made from protein and polysaccharide components is becoming more popular because of their unique properties, such as biodegradability, hierarchical structures, and tunable multifunctionality. However, the fabrication of these materials in a reproducible way remains a challenge. This study presents a new evaporation-induced self-assembly method producing layered hydrogel membranes (LHMs) using tropocollagen grafted by partially deacetylated chitin nanocrystals (CO-g-ChNCs). ChNCs help stabilize tropocollagen's helical conformation and fibrillar structure by forming a hierarchical microstructure through chemical and physical interactions. The LHMs show improved mechanical properties, cytocompatibility, and the ability to control drug release using octenidine dihydrochloride (OCT) as a drug model. Because of the high synergetic performance between CO and ChNCs, the modulus, strength, and toughness increased significantly compared to native CO. The biocompatibility of LHM was tested using the normal human dermal fibroblast (NHDF) and the human osteosarcoma cell line (Saos-2). Cytocompatibility and cell adhesion improved with the introduction of ChNCs. The extracted ChNCs are used as a reinforcing nanofiller to enhance the performance properties of tropocollagen hydrogel membranes and provide new insights into the design of novel LHMs that could be used for various medical applications, such as control of drug release in the skin and bone tissue regeneration.

• MeSH
• Biocompatible Materials * chemistry   MeSH
• Chitin * chemistry   MeSH
• Fibroblasts MeSH
• Hydrogels * chemistry   MeSH
• Imines pharmacokinetics   MeSH
• Protein Conformation, alpha-Helical MeSH
• Delayed-Action Preparations * chemistry   MeSH
• Humans MeSH
• Mechanical Phenomena MeSH
• Membranes chemistry   MeSH
• Cell Line, Tumor MeSH
• Nanoparticles chemistry   MeSH
• Nanocomposites * chemistry   MeSH
• Pyridines pharmacokinetics   MeSH
• Tropocollagen * chemistry   MeSH
• Drug Liberation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biocompatible Materials * MeSH
• Chitin * MeSH
• Hydrogels * MeSH
• Imines MeSH
• Delayed-Action Preparations * MeSH
• octenidine MeSH Browser
• Pyridines MeSH
• Tropocollagen * MeSH

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.

• Keywords
• FGF2, chitosan, collagen, scaffold, skin regeneration, tissue engineering,
• Publication type
• Journal Article MeSH

BACKGROUND: Collagen-based scaffolds provide a promising option for the treatment of bone defects. One of the key parameters of such scaffolds consists of porosity, including pore size. However, to date, no agreement has been found with respect to the methodology for pore size evaluation. Since the determination of the exact pore size value is not possible, the comparison of the various methods applied is complicated. Hence, this study focuses on the comparison of two widely-used methods for the characterization of porosity-scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). METHODS: 7 types of collagen-based composite scaffold models were prepared by means of lyophilization and collagen cross-linking. Micro-CT analysis was performed in 3D and in 2D (pore size parameters were: major diameter, mean thickness, biggest inner circle diameter and area-equivalent circle diameter). Afterwards, pore sizes were analyzed in the same specimens by an image analysis of SEM microphotographs. The results were statistically evaluated. The comparison of the various approaches to the evaluation of pore size was based on coefficients of variance and the semi-quantitative assessment of selected qualities (e.g. the potential for direct 3D analysis, whole specimen analysis, non-destructivity). RESULTS: The pore size values differed significantly with respect to the parameters applied. Median values of pore size values were ranging from 20 to 490 µm. The SEM values were approximately 3 times higher than micro-CT 3D values for each specimen. The Mean thickness was the most advantageous micro-CT 2D approach. Coefficient of variance revealed no differences among pore size parameters (except major diameter). The semi-quantitative comparison approach presented pore size parameters in descending order with regard to the advantages thereof as follows: (1) micro-CT 3D, (2) mean thickness and SEM, (3) biggest inner circle diameter, major diameter and area equivalent circle diameter. CONCLUSION: The results indicated that micro-CT 3D evaluation provides the most beneficial overall approach. Micro-CT 2D analysis (mean thickness) is advantageous in terms of its time efficacy. SEM is still considered as gold standard for its widespread use and high resolution. However, exact comparison of pore size analysis in scaffold materials remains a challenge.

• Keywords
• Bone regeneration, Micro-CT, Pore size, Porosity, SEM, Scaffold,
• MeSH
• Collagen chemistry   MeSH
• Porosity MeSH
• X-Ray Microtomography MeSH
• Tissue Engineering * MeSH
• Tissue Scaffolds * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Collagen MeSH