Fibrin and Marine-Derived Agaroses for the Generation of Human Bioartificial Tissues: An Ex Vivo and In Vivo Study.

Development of an ideal biomaterial for clinical use is one of the main objectives of current research in tissue engineering. Marine-origin polysaccharides, in particular agaroses, have been widely explored as scaffolds for tissue engineering. We previously developed a biomaterial based on a combination of agarose with fibrin, that was successfully translated to clinical practice. However, in search of novel biomaterials with improved physical and biological properties, we have now generated new fibrin-agarose (FA) biomaterials using 5 different types of agaroses at 4 different concentrations. First, we evaluated the cytotoxic effects and the biomechanical properties of these biomaterials. Then, each bioartificial tissue was grafted in vivo and histological, histochemical and immunohistochemical analyses were performed after 30 days. Ex vivo evaluation showed high biocompatibility and differences in their biomechanical properties. In vivo, FA tissues were biocompatible at the systemic and local levels, and histological analyses showed that biointegration was associated to a pro-regenerative process with M2-type CD206-positive macrophages. These results confirm the biocompatibility of FA biomaterials and support their clinical use for the generation of human tissues by tissue engineering, with the possibility of selecting specific agarose types and concentrations for applications requiring precise biomechanical properties and in vivo reabsorption times.

Histological, Biomechanical, and Biological Properties of Genipin-Crosslinked Decellularized Peripheral Nerves.

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.

Comparison of Decellularization Protocols to Generate Peripheral Nerve Grafts: A Study on Rat Sciatic Nerves.

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.

Generation of a novel human dermal substitute functionalized with antibiotic-loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering.

BACKGROUND: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. 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-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK). RESULTS: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, 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.

Novel Desmin Mutation p.Glu401Asp Impairs Filament Formation, Disrupts Cell Membrane Integrity, and Causes Severe Arrhythmogenic Left Ventricular Cardiomyopathy/Dysplasia.

BACKGROUND: Desmin (DES) mutations cause severe skeletal and cardiac muscle disease with heterogeneous phenotypes. Recently, DES mutations were described in patients with inherited arrhythmogenic right ventricular cardiomyopathy/dysplasia, although their cellular and molecular pathomechanisms are not precisely known. Our aim is to describe clinically and functionally the novel DES-p.Glu401Asp mutation as a cause of inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia. METHODS: We identified the novel DES mutation p.Glu401Asp in a large Spanish family with inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia and a high incidence of adverse cardiac events. A full clinical evaluation was performed on all mutation carriers and noncarriers to establish clinical and genetic cosegregation. In addition, desmin, and intercalar disc-related proteins expression were histologically analyzed in explanted cardiac tissue affected by the DES mutation. Furthermore, mesenchymal stem cells were isolated and cultured from 2 family members with the DES mutation (1 with mild and 1 with severe symptomatology) and a member without the mutation (control) and differentiated ex vivo to cardiomyocytes. Then, important genes related to cardiac differentiation and function were analyzed by real-time quantitative polymerase chain reaction. Finally, the p.Glu401Asp mutated DES gene was transfected into cell lines and analyzed by confocal microscopy. RESULTS: Of the 66 family members screened for the DES-p.Glu401Asp mutation, 23 of them were positive, 6 were obligate carriers, and 2 were likely carriers. One hundred percent of genotype-positive patients presented data consistent with inherited arrhythmogenic cardiomyopathy/dysplasia phenotype with variable disease severity expression, high-incidence of sudden cardiac death, and absence of skeletal myopathy or conduction system disorders. Immunohistochemistry was compatible with inherited arrhythmogenic cardiomyopathy/dysplasia, and the functional study showed an abnormal growth pattern and cellular adhesion, reduced desmin RNA expression, and some other membrane proteins, as well, and desmin aggregates in transfected cells expressing the mutant desmin. CONCLUSIONS: The DES-p.Glu401Asp mutation causes predominant inherited left ventricular arrhythmogenic cardiomyopathy/dysplasia with a high incidence of adverse clinical events in the absence of skeletal myopathy or conduction system disorders. The pathogenic mechanism probably corresponds to an alteration in desmin dimer and oligomer assembly and its connection with membrane proteins within the intercalated disc.

Scleral surgical repair through the use of nanostructured fibrin/agarose-based films in rabbits.

Scleral defects can result as a consequence of trauma, infectious diseases or cancer and surgical repair with allogeneic scleral grafts can be required. However, this method has limitations and novel alternatives are needed. Here, the efficacy of acellular nanostructured fibrin-agarose hydrogel-based substitutes (NFAH) in the repair of scleral defects in rabbits was studied. For this, scleral defects of 5-mm diameter were made on 18 adult-male New Zealand rabbits and repaired with acellular NFAH, NFAH crosslinked with genipin (NFAH-GP) or glutaraldehyde (NFAH-GA), allogeneic scleral grafts as control (C-CTR) or not repaired (negative control N-CTR) (n = 3 each). Macroscopic and histological analyses were performed after 40-days. Macroscopy confirmed the repair of all defects in a comparable manner than the C-CTR. Histology showed no degradation nor integration in C-CTR while NFAH-GP and NFAH-GA biomaterials were encapsulated by connective and inflammatory tissues with partial biodegradation. The NFAH were fully biodegraded and replaced by a loose connective tissue and sclera covering the defects. This in vivo study demonstrated that the NFAH are a promising biocompatible and pro-regenerative alternative to the use of allogeneic cadaveric grafts. However, large defects and long-term studies are needed to demonstrate the potential clinical usefulness of these substitutes.

Distribution of extracellular matrix molecules in human uterine tubes during the menstrual cycle: a histological and immunohistochemical analysis.

The uterine tube (UT) is an important and complex organ of the women's reproductive system. In general, the anatomy and basic histology of this organ are well-known. However, the composition and function of the extracellular matrix (ECM) of the UT is still poorly understood. The ECM is a complex supramolecular material produced by cells which is commonly restricted to the basement membrane and interstitial spaces. ECM molecules play not only a structural role, they are also important for cell growth, survival and differentiation in all tissues. In this context, the aim of this study was to evaluate the deposition and distribution of type I and III collagens and proteoglycans (decorin, biglycan, fibromodulin and versican) in human UT during the follicular and luteal phases by using histochemical and immunohistochemical techniques. Our results showed a broad synthesis of collagens (I and III) in the stroma of the UT. The analysis by regions showed, in the mucosa, a specific distribution of versican and fibromodulin in the epithelial surface, whereas decorin and fibromodulin were observed in the lamina propria. Versican and decorin were found in the stroma of the muscular layer, whereas all studied proteoglycans were identified in the serosa. Curiously, biglycan was restricted to the wall of the blood vessels of the serosa and muscular layers. Furthermore, there was an immunoreaction for collagens, decorin, versican and fibromodulin in the UT peripheral nerves. The differential distribution of these ECM molecules in the different layers of the UT could be related to specific structural and/or biomechanical functions needed for the oviductal transport, successful fertilization and early embryogenesis. However, further molecular studies under physiological and pathological conditions are still needed to elucidate the specific role of each molecule in the human UT.

Combination of melatonin and rapamycin for head and neck cancer therapy: Suppression of AKT/mTOR pathway activation, and activation of mitophagy and apoptosis via mitochondrial function regulation.

Head and neck squamous cell carcinoma (HNSCC) clearly involves activation of the Akt mammalian target of rapamycin (mTOR) signalling pathway. However, the effectiveness of treatment with the mTOR inhibitor rapamycin is often limited by chemoresistance. Melatonin suppresses neoplastic growth via different mechanisms in a variety of tumours. In this study, we aimed to elucidate the effects of melatonin on rapamycin-induced HNSCC cell death and to identify potential cross-talk pathways. We analysed the dose-dependent effects of melatonin in rapamycin-treated HNSCC cell lines (Cal-27 and SCC-9). These cells were treated with 0.1, 0.5 or 1 mmol/L melatonin combined with 20 nM rapamycin. We further examined the potential synergistic effects of melatonin with rapamycin in Cal-27 xenograft mice. Relationships between inhibition of the mTOR pathway, reactive oxygen species (ROS), and apoptosis and mitophagy reportedly increased the cytotoxic effects of rapamycin in HNSCC. Our results demonstrated that combined treatment with rapamycin and melatonin blocked the negative feedback loop from the specific downstream effector of mTOR activation S6K1 to Akt signalling, which decreased cell viability, proliferation and clonogenic capacity. Interestingly, combined treatment with rapamycin and melatonin-induced changes in mitochondrial function, which were associated with increased ROS production, increasing apoptosis and mitophagy. This led to increase cell death and cellular differentiation. Our data further indicated that melatonin administration reduced rapamycin-associated toxicity to healthy cells. Overall, our findings suggested that melatonin could be used as an adjuvant agent with rapamycin, improving effectiveness while minimizing its side effects.

Conceptions of learning factors in postgraduate health sciences master students: a comparative study with non-health science students and between genders.

BACKGROUND: The students' conceptions of learning in postgraduate health science master studies are poorly understood. The aim of this study was to compare the factors influencing conceptions of learning in health sciences and non-health sciences students enrolled in postgraduate master programs in order to obtain information that may be useful for students and for future postgraduate programs. METHODS: A modified version of the Learning Inventory Conception Questionnaire (COLI) was used to compare students' conception learning factors in 131 students at the beginning of their postgraduate studies in health sciences, experimental sciences, arts and humanities and social sciences. RESULTS: The present study demonstrates that a set of factors may influence conception of learning of health sciences postgraduate students, with learning as gaining information, remembering, using, and understanding information, awareness of duty and social commitment being the most relevant. For these students, learning as a personal change, a process not bound by time or place or even as acquisition of professional competences, are less relevant. According to our results, this profile is not affected by gender differences. CONCLUSIONS: Our results show that the overall conceptions of learning differ among students of health sciences and non-health sciences (experimental sciences, arts and humanities and social sciences) master postgraduate programs. These finding are potentially useful to foster the learning process of HS students, because if they are metacognitively aware of their own conception or learning, they will be much better equipped to self-regulate their learning behavior in a postgraduate master program in health sciences.

Encapsulation of human elastic cartilage-derived chondrocytes in nanostructured fibrin-agarose hydrogels.

The generation of elastic cartilage substitutes for clinical use is still a challenge. In this study, we investigated the possibility of encapsulating human elastic cartilage-derived chondrocytes (HECDC) in biodegradable nanostructured fibrin-agarose hydrogels (NFAH). Viable HECDC from passage 2 were encapsulated in NFAH and maintained in culture conditions. Constructs were harvested for histochemical and immunohistochemical analyses after 1, 2, 3, 4 and 5 weeks of development ex vivo. Histological results demonstrated that it is possible to encapsulate HECDC in NFAH, and that HECDC were able to proliferate and form cells clusters expressing S-100 and vimentin. Additionally, histochemical and immunohistochemical analyses of the extracellular matrix (ECM) showed that HECDC synthetized different ECM molecules (type I and II collagen, elastic fibers and proteoglycans) in the NFAH ex vivo. In conclusion, this study suggests that NFAH can be used to generate biodegradable and biologically active constructs for cartilage tissue engineering applications. However, further cell differentiation, biomechanical and in vivo studies are still needed.

Cell viability and proliferation capability of long-term human dental pulp stem cell cultures.

BACKGROUND AIMS: Evaluation of cell viability is one of the most important steps of the quality control process for therapeutic use of cells. The aim of this study was to evaluate the long-term cell viability profile of human dental pulp stem cell (hDPSC) subcultures (beyond 10 passages) to determine which of these passages are suitable for clinical use and to identify the cell death processes that may occur in the last passages. METHODS: Four different cell viability assays were combined to determine the average cell viability levels at each cell passage: trypan blue exclusion test, water-soluble tetrazolium 1 (WST-1), LIVE/DEAD Viability/Cytotoxicity Kit and electron probe x-ray microanalysis (EPXMA). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase 4 and BCL7C Western blotting, and cell proliferation was analyzed by WST-1 and proliferating cell nuclear antigen protein detection. RESULTS: hDPSCs showed high average cell viability levels from passages 11-14, with adequate cytoplasmic and mitochondrial functionality at these subcultures. A non-significant trend to decreased cell proliferation was found from passages 16-20. EPXMA and TUNEL analyses suggested that a pre-apoptotic process could be activated from passages 15-20 (P < 0.001), with a correlation with caspase 4 and BCL7C expression. CONCLUSIONS: hDPSCs corresponding to passages 11-14 show adequate cell function, proliferation and viability. These cells could be considered as potentially useful for clinical applications.

Motivational component profiles in university students learning histology: a comparative study between genders and different health science curricula.

BACKGROUND: The students' motivation to learn basic sciences in health science curricula is poorly understood. The purpose of this study was to investigate the influence of different components of motivation (intrinsic motivation, self-determination, self-efficacy and extrinsic -career and grade- motivation) on learning human histology in health science curricula and their relationship with the final performance of the students in histology. METHODS: Glynn Science Motivation Questionnaire II was used to compare students' motivation components to learn histology in 367 first-year male and female undergraduate students enrolled in medical, dentistry and pharmacy degree programs. RESULTS: For intrinsic motivation, career motivation and self-efficacy, the highest values corresponded to medical students, whereas dentistry students showed the highest values for self-determination and grade motivation. Genders differences were found for career motivation in medicine, self-efficacy in dentistry, and intrinsic motivation, self-determination and grade motivation in pharmacy. Career motivation and self-efficacy components correlated with final performance in histology of the students corresponding to the three curricula. CONCLUSIONS: Our results show that the overall motivational profile for learning histology differs among medical, dentistry and pharmacy students. This finding is potentially useful to foster their learning process, because if they are metacognitively aware of their motivation they will be better equipped to self-regulate their science-learning behavior in histology. This information could be useful for instructors and education policy makers to enhance curricula not only on the cognitive component of learning but also to integrate students' levels and types of motivation into the processes of planning, delivery and evaluation of medical education.

Histological characterization of the human scapholunate ligament.

The scapholunate interosseous ligament (SLIL) plays a fundamental role in stabilizing the wrist bones, and its disruption is a frequent cause of wrist arthrosis and disfunction. Traditionally, this structure is considered to be a variety of fibrocartilaginous tissue and consists of three regions: dorsal, membranous and palmar. Despite its functional relevance, the exact composition of the human SLIL is not well understood. In the present work, we have analyzed the human SLIL and control tissues from the human hand using an array of histological, histochemical and immunohistochemical methods to characterize each region of this structure. Results reveal that the SLIL is heterogeneous, and each region can be subdivided in two zones that are histologically different to the other zones. Analysis of collagen and elastic fibers, and several proteoglycans, glycoproteins and glycosaminoglycans confirmed that the different regions can be subdivided in two zones that have their own structure and composition. In general, all parts of the SLIL resemble the histological structure of the control articular cartilage, especially the first part of the membranous region (zone M1). Cells showing a chondrocyte-like phenotype as determined by S100 were more abundant in M1, whereas the zone containing more CD73-positive stem cells was D2. These results confirm the heterogeneity of the human SLIL and could contribute to explain why certain zones of this structure are more prone to structural damage and why other zones have specific regeneration potential. RESEARCH HIGHLIGHTS: Application of an array of histological analysis methods allowed us to demonstrate that the human scapholunate ligament is heterogeneous and consists of at least six different regions sharing similarities with the human cartilage, ligament and other anatomical structures.

A Novel In Vitro Pathological Model for Studying Neural Invasion in Non-Melanoma Skin Cancer.

Neural Invasion (NI) is a key pathological feature of cancer in the colonization of distant tissues, and its underlying biological mechanisms are still scarcely known. The complex interactions between nerve and tumor cells, along with the stroma, make it difficult to reproduce this pathology in effective study models, which in turn has limited the understanding of NI pathogenesis. In this study, we have designed a three-dimensional model of NI squamous cell carcinoma combining human epidermoid carcinoma cells (hECCs) with a complete peripheral nerve segment encapsulated in a fibrine-agarose hydrogel. We recreated two vital processes of NI: a pre-invasive NI model in which hECCs were seeded on the top of the nerve-enriched stroma, and an invasive NI model in which cancer cells were immersed with the nerve in the hydrogel. Histological, histochemical and immunohistochemical analyses were performed to validate the model. Results showed that the integration of fibrin-agarose advanced hydrogel with a complete nerve structure and hECCs successfully generated an environment in which tumor cells and nerve components coexisted. Moreover, this model correctly preserved components of the neural extracellular matrix as well as allowing the proliferation and migration of cells embedded in hydrogel. All these results suggest the suitability of the model for the study of the mechanisms underlaying NI.

Comparison of Printable Biomaterials for Use in Neural Tissue Engineering: An In Vitro Characterization and In Vivo Biocompatibility Assessment.

Nervous system traumatic injuries are prevalent in our society, with a significant socioeconomic impact. Due to the highly complex structure of the neural tissue, the treatment of these injuries is still a challenge. Recently, 3D printing has emerged as a promising alternative for producing biomimetic scaffolds, which can lead to the restoration of neural tissue function. The objective of this work was to compare different biomaterials for generating 3D-printed scaffolds for use in neural tissue engineering. For this purpose, four thermoplastic biomaterials, ((polylactic acid) (PLA), polycaprolactone (PCL), Filaflex (FF) (assessed here for the first time for biomedical purposes), and Flexdym (FD)) and gelatin methacrylate (GelMA) hydrogel were subjected to printability and mechanical tests, in vitro cell-biomaterial interaction analyses, and in vivo biocompatibility assessment. The thermoplastics showed superior printing results in terms of resolution and shape fidelity, whereas FD and GelMA revealed great viscoelastic properties. GelMA demonstrated a greater cell viability index after 7 days of in vitro cell culture. Moreover, all groups displayed connective tissue encapsulation, with some inflammatory cells around the scaffolds after 10 days of in vivo implantation. Future studies will determine the usefulness and in vivo therapeutic efficacy of novel neural substitutes based on the use of these 3D-printed scaffolds.

Genipin crosslinking promotes biomechanical reinforcement and pro-regenerative macrophage polarization in bioartificial tubular substitutes.

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

Histochemical and Immunohistochemical Methods for the Identification of Proteoglycans.

Proteoglycans (PGs) are non-fibrillar extracellular matrix (ECM) molecules composed by a protein core and glycosaminoglycan (GAG) chains. These molecules are present in all tissues playing essential structural, biomechanical, and biological roles. In addition, PGs can regulate cell behavior due to their versatility and ability to interact with other ECM molecules, growth factors, and cells. The distribution of PGs can be evaluated by histochemical and immunohistochemical methods. Histochemical methods aimed to provide a useful overview of the presence and distribution pattern of certain groups of PGs. In contrast, immunohistochemical procedures aimed the identification of highly specific target molecules. In this chapter we described Alcian Blue, Safranin O, and Toluidine Blue histochemical methods for the screening of PGs in tissue sections. Finally, we describe the immunohistochemical procedures for specific identification of PGs (decorin, biglycan, and versican) in formaldehyde-fixed and paraffin-embedded tissues.

Tissue Fixation and Processing for the Histological Identification of Lipids.

Lipids are a heterogeneous group of substances characterized by their solubility in organic solvents and insolubility in water. Lipids can be found as normal components of different tissues and organs, and they can be affected by several pathological conditions. The histochemical identification of lipids plays an important role in the histopathological diagnosis and research, but successful staining depends on adequate fixation and processing of the tissue. Here we describe methods to fix, cryoprotect, and process tissue samples for the histochemical identification of lipids in frozen or paraffin-embedded tissues.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe four histological methods for the evaluation of myelin on peripheral nerve tissue sections. The first method combines the Luxol fast blue (LFB) technique with a modified Picrosirius staining contrasted with Harris hematoxylin, called MCOLL. This method simultaneously stains myelin, collagen fibers, and cell nuclei, thus giving an integrated overview of the histology, collagen network, and myelin content of the tissue in paraffin-embedded or cryosectioned samples. Secondly, we describe the osmium tetroxide method, which provides a permanent positive reaction for myelin as well as other lipids present in the tissue. The third method is the immunofluorescence-based detection of myelin proteins that allows to combine information about their expression status with other proteins of interest. Finally, the FluoroMyelin™ stains enable a fast detection of the myelin content that can be easily implemented in immunofluorescence staining panels for cryosectioned tissues. Together, this chapter provides a variety of methods to accurately identify myelin in different experimental approaches.

Frontal Fibrosing Alopecia: An Observational Single-Center Study of 306 Cases.

(1) Background: Frontal fibrosing alopecia (FFA) is a scarring alopecia that predominantly affects postmenopausal women; (2) Methods: A retrospective, observational, single-center study was conducted in the Hospital General Universitario in Ciudad Real, Spain, including all patients diagnosed with FFA between 2010 and 2021; (3) Results: A total of 306 patients (296 women and 10 men) were included in our study. The mean age of onset was 59.5 years. The severity of this disease was evenly distributed between mild (147 patients) and severe (149 patients) forms. There was a positive, statistically significant, medium correlation between the severity of the disease and its time of progression. Moreover, hypothyroidism was present in 70 patients (22.9%) and classic signs of concomitant lichen planopilaris were observed in just 30 patients (9.8%), while other forms of lichen planus were uncommon. The estimated prevalence in our population is 0.15% and the incidence is 15.47 new cases per 100,000 inhabitants; (4) Conclusions: The time of progression was positively correlated with the severity of FFA. However, the presence of clinical signs, such as inflammatory trichoscopic signs, was not associated with the progression of this condition.