Sustainable whey proteins-nanostructured zinc oxide-based films for the treatment of chronic wounds: New insights from biopharmaceutical studies.

Chronic wounds represent silent epidemic affecting a large portion of the world population, especially the elders; in this context, the development of advanced bioactive dressings is imperative to accelerate wound healing process, while contrasting or preventing infections. The aim of the present work was to provide a deep characterization of the functional and biopharmaceutical properties of a sustainable thin and flexible films, composed of whey proteins alone (WPI) and added with nanostructured zinc oxide (WPZ) and intended for the management of chronic wounds. The potential of whey proteins-based films as wound dressings has been confirmed by their wettability, hydration properties, elastic behavior upon hydration, biodegradation propensity and, when added with nanostructured zinc oxide, antibacterial efficacy against both Gram-positive and Gram-negative pathogens, i.e. Staphylococcus aureus and Escherichia coli. In-vitro experiments, performed on normal human dermal fibroblasts, confirmed film cytocompatibility, also revealing the possible role of Zn2+ ions in promoting fibroblast proliferation. Finally, in-vivo studies on rat model confirmed film suitability to act as wound dressing, since able to ensure a regular healing process while providing effective protection from infections. In particular, both films WPI and WPZ are responsible for the formation in the wound bed of a continuous collagen layer similar to that of healthy skin.

Hep3Gel: A Shape-Shifting Extracellular Matrix-Based, Three-Dimensional Liver Model Adaptable to Different Culture Systems.

Drug-induced hepatotoxicity is a leading cause of clinical trial withdrawal. Therefore, in vitro modeling the hepatic behavior and functionalities is not only crucial to better understand physiological and pathological processes but also to support drug development with reliable high-throughput platforms. Different physiological and pathological models are currently under development and are commonly implemented both within platforms for standard 2D cultures and within tailor-made chambers. This paper introduces Hep3Gel: a hybrid alginate-extracellular matrix (ECM) hydrogel to produce 3D in vitro models of the liver, aiming to reproduce the hepatic chemomechanical niche, with the possibility of adapting its shape to different manufacturing techniques. The ECM, extracted and powdered from porcine livers by a specifically set-up procedure, preserved its crucial biological macromolecules and was embedded within alginate hydrogels prior to crosslinking. The viscoelastic behavior of Hep3Gel was tuned, reproducing the properties of a physiological organ, according to the available knowledge about hepatic biomechanics. By finely tuning the crosslinking kinetics of Hep3Gel, its dualistic nature can be exploited either by self-spreading or adapting its shape to different culture supports or retaining the imposed fiber shape during an extrusion-based 3D-bioprinting process, thus being a shape-shifter hydrogel. The self-spreading ability of Hep3Gel was characterized by combining empirical and numerical procedures, while its use as a bioink was experimentally characterized through rheological a priori printability evaluations and 3D printing tests. The effect of the addition of the ECM was evident after 4 days, doubling the survival rate of cells embedded within control hydrogels. This study represents a proof of concept of the applicability of Hep3Gel as a tool to develop 3D in vitro models of the liver.

Toward 3D-Bioprinted Models of the Liver to Boost Drug Development.

The main problems in drug development are connected to enormous costs related to the paltry success rate. The current situation empowered the development of high-throughput and reliable instruments, in addition to the current golden standards, able to predict the failures in the early preclinical phase. Being hepatotoxicity responsible for the failure of 30% of clinical trials, and the 21% of withdrawal of marketed drugs, the development of complex in vitro models (CIVMs) of liver is currently one of the hottest topics in the field. Among the different fabrication techniques, 3D-bioprinting is emerging as a powerful ally for their production, allowing the manufacture of three-dimensional constructs characterized by computer-controlled and customized geometry, and inter-batches reproducibility. Thanks to these, it is possible to rapidly produce tailored cell-laden constructs, to be cultured within static and dynamic systems, thus reaching a further degree of personalization when designing in vitro models. This review highlights and prioritizes the most recent advances related to the development of CIVMs of the hepatic environment to be specifically applied to pharmaceutical research, with a special focus on 3D-bioprinting, since the liver is primarily involved in the metabolism of drugs.

New System Delivering Microwaves Energy for Inducing Subcutaneous Fat Reduction: In - Vivo Histological and Ultrastructural Evidence.

BACKGROUND: Recently, it has been developed a new technology for the reduction of subcutaneous adipose tissue through a non-invasive treatment by microwaves. The main objective of the present study is to demonstrate the feasibility of utilising a non-invasive, localised microwaves (MW) device to induce thermal modifications into subcutaneous adipose tissue only by a controlled electromagnetic field that heats up fat preferentially. This device is provided with a special handpiece appropriately cooled, directly contacting the cutaneous surface of the body, which provides a calibrated energy transfer by microwaves. AIM: In this paper, microscopic and ultrastructural modifications of subcutaneous adipose tissue induced by microwaves irradiation are evaluated. METHODS: Our experimental plan was designed for collecting biopsy samples, for each skin region treated with a single irradiation session, 1) before treatment (control), 2) immediately after treatment, 3) after 6 hrs, 4) after 1 month, 5) after 2 months. Bioptic samples from each step were processed for light microscopy and transmission electron microscopy. At the same time, each region where biopsies were collected was subjected to ultrasound examination. Recorded images permitted to evaluate the thickness of different layers as epidermis, dermis, hypodermis, connective fasciae, until to muscle layer, and related modifications induced by treatment. RESULTS: In every biopsy collected at different time-steps, epidermis and superficial dermis appeared not modified compared to control. Differently, already in the short-term biopsies, in the deep dermis and superficial hypodermis, fibrillar connective tissue appeared modified, showing reduction and fragmentation of interlobular collagen septa. The most important adipose tissue modifications were detectable following 1 month from treatment, with a significant reduction of subcutaneous fat, participating both the lysis of many adipocytes and the related phagocytic action of monocytes/macrophages on residuals of compromised structures of adipocytes. In the samples collected two months following treatment, the remnants of adipose tissue appeared normal, and macrophages were completely absent. CONCLUSIONS: Ultrasound, microscopic and ultrastructural evidence are supporting significant effectiveness of the new device treatment in the reduction of subcutaneous fat. In this paper, the possible mechanisms involved in the activation of the monocytes/macrophages system responsible for the removal of adipocytes residuals have also been discussed.

Early Regenerative Modifications of Human Postmenopausal Atrophic Vaginal Mucosa Following Fractional CO2 Laser Treatment.

BACKGROUND: Postmenopausal women experience undesired symptoms that adversely affect their quality of life. In the recent years, a specific 12 - week fractional CO2 laser treatment has been introduced, with highly significant relief of symptoms. AIM: The aim of this paper is the identification of the early modifications of structural components of atrophic vaginal mucosa induced by laser irradiation, which is responsible for the restorative processes. MATERIAL AND METHODS: We investigated by microscopical, ultrastructural and biochemical methods the modifications of the structural components of postmenopausal atrophic vaginal mucosa tissues after 1 hour following a single fractional laser CO2 application. RESULTS: In one hour, the mucosal epithelium thickens, with the maturation of epithelial cells and desquamation at the epithelial surface. In the connective tissue, new papillae indenting the epithelium with newly formed vessels penetrating them, new thin fibrils of collagen III are also formed in a renewed turnover of components due to the increase of metalloproteinase - 2. Specific features of fibroblasts support stimulation of their activity responsible of the renewal of the extracellular matrix, with an increase of mechanical support as connective tissue and stimulation of growth and maturation to epithelium thanks to new vessels and related factors delivered. CONCLUSION: We found the activation of regenerative mechanisms expressed both in the connective tissue - with the formation of new vessels, new papillae, and new collagen - and in the epithelium with the associated thickening and desquamation of cells at the mucosal surface.

Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds.

Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

Halloysite and chitosan oligosaccharide nanocomposite for wound healing.

Halloysite is a natural nanotubular clay mineral (HNTs, Halloysite Nano Tubes) chemically identical to kaolinite and, due to its good biocompatibility, is an attractive nanomaterial for a vast range of biological applications. Chitosan oligosaccharides are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine, that accelerate wound healing by enhancing the functions of inflammatory and repairing cells. The aim of the work was the development of a nanocomposite based on HNTs and chitosan oligosaccharides, to be used as pour powder to enhance healing in the treatment of chronic wounds. A 1:0.05 wt ratio HTNs/chitosan oligosaccharide nanocomposite was obtained by simply stirring the HTNs powder in a 1% w/w aqueous chitosan oligosaccharide solution and was formed by spontaneous ionic interaction resulting in 98.6% w/w HTNs and 1.4% w/w chitosan oligosaccharide composition. Advanced electron microscopy techniques were considered to confirm the structure of the hybrid nanotubes. Both HTNs and HTNs/chitosan oligosaccharide nanocomposite showed good in vitro biocompatibility with normal human dermal fibroblasts up to 300µg/ml concentration and enhanced in vitro fibroblast motility, promoting both proliferation and migration. The HTNs/chitosan oligosaccharide nanocomposite and the two components separately were tested for healing capacity in a murine (rat) model. HTNs/chitosan oligosaccharide allowed better skin reepithelization and reorganization than HNTs or chitosan oligosaccharide separately. The results suggest to develop the nanocomposite as a medical device for wound healing. STATEMENT OF SIGNIFICANCE: The present work is focused on the development of halloysite and chitosan oligosaccharide nanocomposite for wound healing. It considers a therapeutic option for difficult to heal skin lesions and burns. The significance of the research considers two fundamental aspects: the first one is related to the development of a self-assembled nanocomposite, formed by spontaneous ionic interaction, while the second one is related to the possibility to find an effective treatment for cutaneous non healing lesions. The characterization of this hybrid system involves a multidisciplinary approach considering integrated techniques of solid state investigation and advanced electron microscopies, and in vitro/in vivo models to understand biocompatibility and proliferation properties (enhancement of in vitro fibroblast motility, proliferation and migration, and of in vivo burn healing), to understand safety and effectiveness of the developed nanocomposite.

Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration.

The production of stable suspensions of strontium-substituted hydroxyapatite (Sr-HA) nanopowders, as Sr ions vector for bone tissue regeneration, was carried out in the present work. Sr-HA nanopowders were synthesized via aqueous precipitation methods using Sr2+ amount from 0 to 100mol% and were characterized by several complementary techniques such as solid-state Nuclear Magnetic Resonance spectroscopy, X-ray diffraction, Infrared spectroscopy, N2 physisorption and Transmission Electron Microscopy. The substitution of Ca2+ with Sr2+ in HA is always isomorphic with gradual evolution between the two limit compositions (containing 100% Ca and 100% Sr), this pointing out the homogeneity of the synthesized nanopowders and the complete solubility of strontium in HA lattice. Strontium addition is responsible for an increasing c/a ratio in the triclinic unit cell. A significant variation of the nanopowders shape and dimension is also observed, a preferential growth along the c-axis direction being evident at higher strontium loads. Modifications in the local chemical environment of phosphate and hydroxyl groups in the apatite lattice are also observed. Stable suspensions were produced by dispersing the synthesized nanopowders in bovine serum albumin. Characterization by Dynamic Light Scattering and ζ-potential determination allowed to show that Ca2+→Sr2+ substitution influences the hydrodynamic diameter, which is always twice the particles size determined by TEM, the nanoparticles being always negatively charged as a result from the albumin rearrangement upon the interaction with nanoparticles surface. The biocompatibility of the suspensions was studied in terms of cell viability, apoptosis, proliferation and morphology, using osteosarcoma cell line SAOS-2. The data pointed out an increased cell proliferation for HA nanoparticles containing larger Sr2+ load, the cells morphology remaining essentially unaffected.

Application of DoE approach in the development of mini-capsules, based on biopolymers and manuka honey polar fraction, as powder formulation for the treatment of skin ulcers.

The aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components in the treatment of chronic skin ulcers. In particular pectin (PEC)/chitosan glutamate (CS)/hyaluronic acid (HA) mini-capsules were obtained by inverse ionotropic gelation in presence of calcium chloride and subsequently freeze-dried. Optimization of unloaded (blank) formulation was performed using DoE approach. In a screening phase, the following three factors were investigated at two levels: CS (0.5-1% w/w), PEC (0.5-1% w/w) and HA (0.3-0.5% w/w) concentrations. For the optimization phase a "central composite design" was used. The response variables considered were: particle size, buffer (PBS) absorption and mechanical resistance. In a previously work two different MH fractions were investigated, in particular MH fraction 1 (Fr1), rich in polar substances (sugars, methylglyoxal (MGO), dicarbonyl compounds, …), was able to enhance human fibroblasts in vitro proliferation. In the present work, the loading of MH Fr1 into mini-capsules of optimized composition determined a significant increase in cell proliferation in comparison with the unloaded ones. Loaded particles showed antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes; they were also able to improve wound healing in vivo on a rat wound model.

Successful Reconstruction of Nerve Defects Using Distraction Neurogenesis with a New Experimental Device.

INTRODUCTION: Repair of peripheral nerve injuries is an intensive area of challenge and research in modern reconstructive microsurgery. Intensive research is being carried out to develop effective alternatives to the standard nerve autografting, avoiding its drawbacks. The aim of the study was to evaluate the effectiveness of a newly designed mechanical device for the reconstruction of the sciatic nerve in rats in comparison to nerve autografting and to assess the pain during the period of distraction neurogenesis. METHODS: Fourteen Sprague Dawley rats were used and randomly assigned into 2 groups with 7 rats in each group; group A (Nerve Autografting group) in which a 10-mm segment of the sciatic nerve was resected and rotated 180 degrees, then primary end-to-end neurorrhaphy was performed in the reverse direction; group B (Nerve Lengthening group) in which the mechanical device was inserted after surgical resection of 10 mm of the sciatic nerve, then secondary end-to-end neurorrhaphy was performed after completing the nerve lengthening. Thirteen weeks later, assessment of the functional sciatic nerve recovery using static sciatic index (SSI) was performed. Furthermore, fourteen weeks after the nerve resection, assessment of the nerve regeneration with electrophysiological study and histological analysis were performed. Also, gastrocnemius wet weight was measured. For pain assessment in group B, Rat Grimace Scale (RGS) score was used. RESULTS: Significantly better functional recovery rate (using the SSI) was reported in the nerve lengthening group in comparison to autografting group. Also, a statistically significant higher nerve conduction velocity was detected in the nerve lengthening group. On histological analysis of the distal nerve section at 3 mm distal to the nerve repair site, significant myelin sheath thickness was detected in the nerve lengthening group. DISCUSSION: Distraction neurogenesis with the new experimental device is a reliable therapeutic method for the reconstruction of nerve defects.

The biological effects of quadripolar radiofrequency sequential application: a human experimental study.

OBJECTIVE: An experimental study was conducted to assess the effectiveness and safety of an innovative quadripolar variable electrode configuration radiofrequency device with objective measurements in an ex vivo and in vivo human experimental model. BACKGROUND DATA: Nonablative radiofrequency applications are well-established anti-ageing procedures for cosmetic skin tightening. METHODS: The study was performed in two steps: ex vivo and in vivo assessments. In the ex vivo assessments the radiofrequency applications were performed on human full-thickness skin and subcutaneous tissue specimens harvested during surgery for body contouring. In the in vivo assessments the applications were performed on two volunteer patients scheduled for body contouring surgery at the end of the study. The assessment methods were: clinical examination and medical photography, temperature measurement with thermal imaging scan, and light microscopy histological examination. RESULTS: The ex vivo assessments allowed for identification of the effective safety range for human application. The in vivo assessments allowed for demonstration of the biological effects of sequential radiofrequency applications. After a course of radiofrequency applications, the collagen fibers underwent an immediate heat-induced rearrangement and were partially denaturated and progressively metabolized by the macrophages. An overall thickening and spatial rearrangement was appreciated both in the collagen and elastic fibers, the latter displaying a juvenile reticular pattern. A late onset in the macrophage activation after sequential radiofrequency applications was appreciated. CONCLUSIONS: Our data confirm the effectiveness of sequential radiofrequency applications in obtaining attenuation of the skin wrinkles by an overall skin tightening.

Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR-669a downregulation has been linked to the severe DCM progression displayed by Sgcb-null dystrophic mice. However, the impact of long-term overexpression of miR-669a on muscle structure and functionality of the dystrophic heart is yet unknown. METHODS AND RESULTS: Here, we demonstrate that intraventricular delivery of adeno-associated viral (AAV) vectors induces long-term (18 months) miR-669a overexpression and improves survival of Sgcb-null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR-669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long-term miR-669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. CONCLUSIONS: Our findings provide the first evidence of long-term beneficial impact of AAV-mediated miRNA therapy in a transgenic model of severe, chronic MD-associated DCM.

Growth and stratification of epithelial cells in minimal culture conditions.

Biological risk management is required in modern tissue engineering. Particular attention should be paid to the culture medium and the scaffold used. In this perspective, it is important to define minimal culture conditions which allow proper growth and differentiation of epithelial cells in vitro. We propose a simple experimental system which permits the generation of three-dimensional epidermal constructs using a collagen layer as a scaffold mimicking the entire dermal tissue and without the need of any feeder layer. Although showing significant differences compared to natural epidermis, these epidermal constructs appear useful to study keratinocyte differentiation and epidermis histogenesis.

Dysplastic histogenesis of cartilage growth plate by alteration of sulphation pathway: a transgenic model.

Mutations in the diastrophic dysplasia sulphate transporter (dtdst) gene causes different forms of chondrodysplasia in the human. The generation of a knock-in mouse strain with a mutation in dtdst gene provides the basis to study developmental dynamics in the epiphyseal growth plate and long bone growth after impairment of the sulphate pathway. Our microscopical and histochemical data demonstrate that dtdst gene impairment deeply affects tissue organization, matrix structure, and cell differentiation in the epiphyseal growth plate. In mutant animals, the height of the growth plate was significantly reduced, according to a concomitant decrease in cell density and proliferation. Although the pathway of chondrocyte differentiation seemed complete, alteration in cell morphology compared to normal counterparts was detected. In the extracellular matrix, it we observed a dramatic decrease in sulphated proteoglycans, alterations in the organization of type II and type X collagen fibers, and premature onset of mineralization. These data confirm the crucial role of sulphate pathway in proteoglycan biochemistry and suggest that a disarrangement of the extracellular matrix may be responsible for the development of dtdts cartilage dysplasia. Moreover, we corroborated the concept that proteoglycans not only are structural components of the cartilage architecture, but also play a dynamic role in the regulation of chondrocyte growth and differentiation.

Generation of human epidermal constructs on a collagen layer alone.

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.

Cell kinetic analysis in artificial skin using immunochemical methods.

Cell kinetic studies provide important information on histogenesis in vivo and in vitro. In this regard, specific antibodies to cell cycle-related antigens have been raised and characterized, thus permitting the study of cell kinetics using immunochemical methods. Recent advances in culture technology permitted the generation of human skin equivalents in vitro. We here provide detailed practical procedures for the study of epidermal cell kinetics in a model of artificial skin using immunohistochemistry and flow cytometry. The combined application of both techniques allows a precise detection of tissue growth sites and a quantitative assessment of cell growth. Moreover, simultaneous analysis of differentiation markers and proliferation antigens may be useful to understand molecular mechanisms that regulate tissue growth and development.

Microscopic, immunocytochemical, and ultrastructural properties of peri-implant mucosa in humans.

BACKGROUND: Microscopic and immunocytochemical studies have demonstrated both similarities and differences between the gingival mucosa and the peri-implant mucosa restored around dental implants. METHODS: This study was performed on 10 samples of peri-implant mucosa from 10 patients who had undergone implant treatment 16 to 18 months before. Microscopic, ultrastructural, and immunocytochemical investigations were performed to characterize the epithelial and connective layers of the peri-implant mucosa. RESULTS: All specimens showed the morphologic characteristics of well restored tissues. The immunocytochemical reactions used to detect cytokeratins demonstrated that the restored peri-implant mucosa had a non-keratinized epithelium. The apical part of the inner epithelium was a few cell layers thick, like the corresponding junctional epithelium, but it stained positively with the markers for the cytokeratins expressed by the stratified epithelia. Ultrastructurally the keratinocytes adjacent to the implant displayed nuclei containing a rich cytoplasm with filaments and specialized intercellular junctions right up to the more superficial strata. Immunocytochemical reactions and ultrastructural observations demonstrated the presence of non-keratinocyte cells; i.e., Langerhans cells, melanocytes, and Merkel cells. Several cells were found to be proliferating by immunoreaction with mAb anti-PCNA, while immunoreactions with mAbs to detect von Willebrand factor, CD34, and vascular endothelial growth factor marked the well-developed networks of blood and lymphatic vessels in the connective tissue. S-100 and protein gene product 9.5 positive nerve fibers were marked. Immunocytochemical reactions with mAbs anti-vimentin, anti-laminin were also performed. CONCLUSIONS: Our results demonstrated that all the epithelial and connective components of the mucosa are involved in the substantial regrowth of the peri-implant tissue and subsequently in the success of the implant.