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

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60mm of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications

Mycelium-based biomaterials as smart devices for skin wound healing

Introduction: Recently, mycelia of Ganoderma lucidum and Pleurotus ostreatus, edible fungi, have been characterized in vitro as self-growing biomaterials for tissue engineering since they are constituted of interconnected fibrous networks resembling the dermal collagen structure.Aim: This work aims to investigate the biopharmaceutical properties of G. lucidum and P. ostreatus mycelia to prove their safety and effectiveness in tissue engineering as dermal substitutes.Methods: The mycelial materials were characterized using a multidisciplinary approach, including physicochemical properties (morphology, thermal behavior, surface charge, and isoelectric point). Moreover, preclinical properties such as gene expression and in vitro wound healing assay have been evaluated using fibroblasts. Finally, these naturally-grown substrates were applied in vivo using a murine burn/excisional wound model.Conclusions: Both G. lucidum and P. ostreatus mycelia are biocompatible and able to safely and effectively enhance tissue repair in vivo in our preclinical model

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

Ultrastructural features of human sperm cells cryopreserved by different methods

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied

Expression of p63 transcription factor in ectoderm-derived oral tissues.

The p63 gene encodes six splice variants expressed with transactivating or dominant-negative activities. Animal studies with p63 -/- mutants have suggested that p63 is important for proper development of several organs, including tooth and salivary gland. Moreover, mutations of p63 have been detected in patients affected by ectrodactyly, ectodermal dysplasia and facial clefts. To clarify the role of p63 in craniofacial development, we have studied the localization of p63 protein in human and rat oral tissues using immunohistochemistry. p63 immunostaining was identified in the enamel organ, oral epithelium and developing salivary glands. All compartments of the enamel organ were immunolabelled, whereas only basal and some suprabasal cells of the oral epithelium were stained. Ectomesenchyme-derived cells, including pulp cells, odontoblasts, bone cells and chondrocytes, were negative. The staining pattern was identical in human and rat tissues. These data lend support to the hypothesis that p63 is involved in growth and differentation of ectoderm-derived oral tissues and may be useful to clarify molecular and developmental aspects observed in animal knockout experiments and human syndromes related to p63 gene alteration