Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

Studio dei meccanismi di fotodegradazione del benzil 4-idrossibenzoato (benzilparabene)

: Photodegradation is an important mechanism for removal of many organic pollutants of natural waters. This thesis reports and discusses the results of a study of the photochemical reaction in an aqueous solution of an important component of many cosmetic formulations and products for personal hygiene, benzyl paraben (4-hydroxybenzoic acid benzyl ester). This compound, and more generally, parabens, concern for the possible effects on the endocrine system of humans. I made a preliminary characterization of the photophysical behavior of benzyl paraben: has led to the determination of fluorescence quantum yields (0.33%), phosphorescence (69%) and triplet excited state formation by sensitized delayed fluorescence (65%). I then conducted experiments to photodegradation under different experimental conditions with the aim to characterize the speed of the process and products and to propose a hypothesis for the mechanism of reaction. The experiments concerning the direct photolysis with UV-Vis in three different environments (air, oxygen and argon), photolysis with NUV-Vis in the presence of a photosensitizer (two have been tested, humic acid and the Rose Bengal) and photocatalysis with TiO2. In each case, were analyzed both the evolution of the kinetic reaction intermediates and products formed. Have also been conducted a few experiments to test the possible involvement of hydroxyl radical in the reaction, using two probes specific chemical (hydrogen peroxide, known source of OH radicals, and alcohol terbutilico known quencher of OH radicals). For kinetic determinations of solution samples taken at various times of treatment were analyzed by a simple measurement of absorbance at 256 nm and by HPLC-UV . The first method has often provided unreliable results due to contributions to the absorbance measurement due to photo products of the reaction, it was possible to eliminate interference in the second method of investigation. With HPLC-UV have been possible to follow not only the disappearance of the reagent but also the formation and eventual disappearance of intermediates and reaction products. A purely qualitative and preliminary characterization of these products was performed by HPLC-MS and HPLC-MS/MS using an ESI ionization source in negative mode. The main results indicate that, in experiments of direct photolysis, molecular oxygen slows the photodegradation of benzyl paraben, suggesting a mechanism of reaction through the singlet excited state produces the reactive species of benzyl paraben in the triplet state. This species undergoes a break and / or 'other of the two CO bonds of the ester group,-C (= O)-OC-forming free radicals which, through different reaction paths may explain the origin of products postulates. The results of experiments conducted in the presence of hydrogen peroxide and alcohol terbutilico agree to exclude a major involvement of the OH radical in the degradation of benzylparaben induced by direct photolysis. I also checked for radiation in the NUV-Vis benzyl paraben is stable but undergoes degradation in the presence of a photo sensitizer, humic acid is that the Rose Bengal, although with different kinetic efficiency and course. Finally, I made an experiment of photocatalysis with TiO2 for determining the kinetic of benzyl paraben and the structure of products. The kinetics suggest that the degradation of benzyl paraben occurs through two distinct processes, one of the first order, probably on the surface of the photo catalyst, the other zero-order, with the reactive species which diffuse in solution. This hypothesis is in agreement with the results of the analysis of total organic carbon, TOC. Among the reaction products were identified as phenol and other intermediate advanced stages of oxidation such as acids unsaturated maleic / fumaric

Label-Free Multiphoton Microscopy: Much More Than Fancy Images

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy

Label-Free Multiphoton Microscopy: Much More Than Fancy Images

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy

A Novel Hybrid Membrane for Urinary Conduit Substitutes Based on Small Intestinal Submucosa Coupled with Two Synthetic Polymers

Among the urinary tract’s malignancies, bladder cancer is the most frequent one: it is at the tenth position of most common cancers worldwide. Currently, the gold standard therapy consists of radical cystectomy, which results in the need to create a urinary diversion using a bowel segment from the patient. Nevertheless, due to several complications associated with bowel resection and anastomosis, which significantly affect patient quality of life, it is becoming extremely important to find an alternative solution. In our recent work, we proposed the decellularized porcine small intestinal submucosa (SIS) as a candidate material for urinary conduit substitution. In the present study, we create SIS-based hybrid membranes that are obtained by coupling decellularized SIS with two commercially available polycarbonate urethanes (Chronoflex AR and Chronoflex ARLT) to improve SIS mechanical resistance and impermeability. We evaluated the hybrid membranes by means of immunofluorescence, two-photon microscopy, FTIR analysis, and mechanical and cytocompatibility tests. The realization of hybrid membranes did not deteriorate SIS composition, but the presence of polymers ameliorates the mechanical behavior of the hybrid constructs. Moreover, the cytocompatibility tests demonstrated a significant increase in cell growth compared to decellularized SIS alone. In light of the present results, the hybrid membrane-based urinary conduit can be a suitable candidate to realize a urinary diversion in place of an autologous intestinal segment. Further efforts will be performed in order to create a cylindrical-shaped hybrid membrane and to study its hydraulic behavior

Characterization of a decellularized rat larynx: comparison between microscopy techniques

No effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues

Hybrid Materials for Vascular Applications: A Preliminary In Vitro Assessment

The production of biomedical devices able to appropriately interact with the biological environment is still a great challenge. Synthetic materials are often employed, but they fail to replicate the biological and functional properties of native tissues, leading to a variety of adverse effects. Several commercial products are based on chemically treated xenogeneic tissues: their principal drawback is due to weak mechanical stability and low durability. Recently, decellularization has been proposed to bypass the drawbacks of both synthetic and biological materials. Acellular materials can integrate with host tissues avoiding/mitigating any foreign body response, but they often lack sufficient patency and impermeability. The present paper investigates an innovative approach to the realization of hybrid materials that combine decellularized bovine pericardium with polycarbonate urethanes. These hybrid materials benefit from the superior biocompatibility of the biological tissue and the mechanical properties of the synthetic polymers. They were assessed from physicochemical, structural, mechanical, and biological points of view; their ability to promote cell growth was also investigated. The decellularized pericardium and the polymer appeared to well adhere to each other, and the two sides were distinguishable. The maximum elongation of hybrid materials was mainly affected by the pericardium, which allows for lower elongation than the polymer; this latter, in turn, influenced the maximum strength achieved. The results confirmed the promising features of hybrid materials for the production of vascular grafts able to be repopulated by circulating cells, thus, improving blood compatibility

A New Decellularization Protocol of Porcine Aortic Valves Using Tergitol to Characterize the Scaffold with the Biocompatibility Profile Using Human Bone Marrow Mesenchymal Stem Cells

The most common aortic valve diseases in adults are stenosis due to calcification and regurgitation. In pediatric patients, aortic pathologies are less common. When a native valve is surgically replaced by a prosthetic one, it is necessary to consider that the latter has a limited durability. In particular, current bioprosthetic valves have to be replaced after approximately 10 years; mechanical prostheses are more durable but require the administration of permanent anticoagulant therapy. With regard to pediatric patients, both mechanical and biological prosthetic valves have to be replaced due to their inability to follow patients' growth. An alternative surgical substitute can be represented by the acellular porcine aortic valve that exhibits less immunogenic risk and a longer lifespan. In the present study, an efficient protocol for the removal of cells by using detergents, enzyme inhibitors, and hyper- and hypotonic shocks is reported. A new detergent (Tergitol) was applied to replace TX-100 with the aim to reduce toxicity and maximize ECM preservation. The structural integrity and efficient removal of cells and nuclear components were assessed by means of histology, immunofluorescence, and protein quantification; biomechanical properties were also checked by tensile tests. After decellularization, the acellular scaffold was sterilized with a standard protocol and repopulated with bone marrow mesenchymal stem cells to analyze its biocompatibility profile