Generation of biocompatible and biomimetic tissue-like biomaterials is crucial to ensure the success of engineered substitutes in tissue repair. Natural biomaterials able to mimic the structure and composition of native extracellular matrices typically show better results than synthetic biomaterials. The aim of this study was to perform an in vivo time-course biocompatibility analysis of fibrin-agarose tissue-like hydrogels at the histological, imagenological, hematological and biochemical levels. Tissue-like hydrogels were produced by a controlled biofabrication process allowing the generation of biomechanically and structurally stable hydrogels. The hydrogels were implanted subcutaneously in 25 male Wistar rats and evaluated after 1, 5, 9 and 12 weeks of in vivo follow-up. At each period of time, animals were analyzed using magnetic resonance imaging (MRI), hematological analyses and histology of the local area in which the biomaterials were implanted, along with major vital organs (liver, kidney, spleen and regional lymph nodes). MRI results showed no local or distal alterations during the whole study period. Hematology and biochemistry showed some fluctuation in blood cells values and in some biochemical markers over the time. However, these parameters were progressively normalized in the framework of the homeostasis process. Histological, histochemical and ultrastructural analyses showed that implantation of fibrin-agarose scaffolds was followed by a progressive process of cell invasion, synthesis of components of the extracellular matrix (mainly, collagen) and neovascularization. Implanted biomaterials were successfully biodegraded and biointegrated at 12 weeks without any associated histopathological alteration in the implanted zone or distal vital organs. In summary, our in vivo study suggests that fibrin-agarose tissue-like hydrogels could have potential clinical usefulness in engineering applications in terms of biosafety and biocompatibility.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells.

Background: Treatment of patients affected by severe burns is challenging, especially due to the high risk ofPseudomonasinfection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonasantibiotics: sodium colistimethate (SCM) and amikacin (AMK).Results: Results show that the novel tissue-like substitutes have strong antibacterial effect onPseudomonascultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs.Conclusions: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.

The myelin sheath is a lipoprotein-rich, multilayered structure capable of increasing conduction velocity in central and peripheral myelinated nerve fibers. Due to the complex structure and composition of myelin, various histological techniques have been developed over the centuries to evaluate myelin under normal, pathological or experimental conditions. Today, methods to assess myelin integrity or content are key tools in both clinical diagnosis and neuroscience research. In this review, we provide an updated summary of the composition and structure of the myelin sheath and discuss some histological procedures, from tissue fixation and processing techniques to the most used and practical myelin histological staining methods. Considering the lipoprotein nature of myelin, the main features and technical details of the different available methods that can be used to evaluate the lipid or protein components of myelin are described, as well as the precise ultrastructural techniques.