Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases.

Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review.

Electron Beam Powder Bed Fusion of Ti-48Al-2Cr-2Nb Open Porous Scaffold for Biomedical Applications: Process Parameters, Adhesion, and Proliferation of NIH-3T3 Cells.

Titanium aluminide (TiAl)-based intermetallics, especially Ti-48Al-2Cr-2Nb, are a well-established class of materials for producing bulky components using the electron beam powder bed fusion (EB-PBF) process. The biological properties of Ti-48Al-2Cr-2Nb alloy have been rarely investigated, specifically using complex cellular structures. This work investigates the viability and proliferation of NIH-3T3 fibroblasts on Ti-48Al-2Cr-2Nb dodecahedral open scaffolds manufactured by the EB-PBF process. A process parameter optimization is carried out to produce a fully dense part. Then scaffolds are produced and characterized using different techniques, including scanning electron microscopy and X-ray tomography. In vitro viability tests are performed with NIH-3T3 cells after incubation for 1, 4, and 7 days. The results show that Ti-48Al-2Cr-2Nb represents a promising new entry in the biomaterial field.

Surface Properties of a Biocompatible Thermoplastic Polyurethane and Its Anti-Adhesive Effect against E. coli and S. aureus.

Thermoplastic polyurethane (TPU) is a polymer used in a variety of fields, including medical applications. Here, we aimed to verify if the brush and bar coater deposition techniques did not alter TPU properties. The topography of the TPU-modified surfaces was studied via AFM demonstrating no significant differences between brush and bar coater-modified surfaces, compared to the un-modified TPU (TPU Film). The effect of the surfaces on planktonic bacteria, evaluated by MTT assay, demonstrated their anti-adhesive effect on E. coli, while the bar coater significantly reduced staphylococcal planktonic adhesion and both bacterial biofilms compared to other samples. Interestingly, Pearson's R coefficient analysis showed that Ra roughness and Haralick's correlation feature were trend predictors for planktonic bacterial cells adhesion. The surface adhesion property was evaluated against NIH-3T3 murine fibroblasts by MTT and against human fibrinogen and human platelet-rich plasma by ELISA and LDH assay, respectively. An indirect cytotoxicity experiment against NIH-3T3 confirmed the biocompatibility of the TPUs. Overall, the results indicated that the deposition techniques did not alter the antibacterial and anti-adhesive surface properties of modified TPU compared to un-modified TPU, nor its bio- and hemocompatibility, confirming the suitability of TPU brush and bar coater films in the biomedical and pharmaceutical fields.

Effect of Low Copper Doping on the Optical, Cytocompatible, Antibacterial, and SARS-CoV-2 Trapping Properties of Calcium Phosphate Glasses.

Calcium phosphate glasses (CPGs) are acquiring great importance in the biomedical field because of their thermomechanical and bioresorbable properties. In this study, optically transparent copper (1 mol %)-doped calcium phosphate glasses (CPGs_Cu) were prepared through the melt-quenching method, and their biocompatibility and antibacterial and antiviral properties were evaluated and compared with undoped CPGs. Biocompatibility was evaluated on murine fibroblast NIH-3T3 cells as a preliminary study of cytocompatibility. The in vitro tests were performed through indirect and direct cytotoxicity analyses by MTT and Alamar Blue assays and supported by electron microscopy observations. Microbiological analyses were performed against the most common Gram-negative and Gram-positive pathogens that cause nosocomial infections: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and the methicillin-resistant Staphylococcus aureus strain. In addition, the bioglass samples were exposed to SARS-CoV-2 to assess their effects on viral survival. The obtained results assessed the biocompatibility of both bioglass types and their ability to reduce the viral load and trap the virus. In addition, Cu2+-doped bioglass was found to be antibacterial despite its low content (1 mol %) of copper, making this a promising candidate material for biomedical applications, e.g., surgery probes, drug delivery, and photodynamic therapy.

The nano-revolution in the diagnosis and treatment of endometriosis.

Endometriosis is a painful gynecological disease with a high prevalence, affecting millions of women worldwide. Innovative, non-invasive treatments, and new patient follow-up strategies are needed to deal with the harmful social and economic effects. In this scenario, considering the recent, very promising results already reported in the literature, a commitment to new research in the field of nanomedicine is urgently needed. Study findings clearly show the potential of this approach in both the diagnostic and therapeutic phases of endometriosis. Here, we offer a brief review of the recent exciting and effective applications of nanomedicine in both the diagnosis and therapy of endometriosis. Special emphasis will be placed on the emerging theranostic application of nanoproducts, and the combination of phototherapy and nanotechnology as new therapeutic modalities for endometriosis. The review will also provide interested readers with a guide to the selection process and parameters to consider when designing research into this type of approach.

The ad hoc chemical design of random PBS-based copolymers influences the activation of cardiac differentiation while altering the HYPPO pathway target genes in hiPSCs.

Cardiac tissue engineering is a cutting-edge technology aiming to replace irreversibly damaged cardiac tissue and restore contractile functionality. However, cardiac tissue engineering porous and perfusable scaffolds to enable oxygen supply in vitro and eventually promote angiogenesis in vivo are still desirable. Two fully-aliphatic random copolymers of poly(butylene succinate) (PBS), poly(butylene succinate/Pripol), P(BSBPripol), and poly(butylene/neopentyl glycol succinate), P(BSNS), containing two different subunits, neopentyl glycol and Pripol 1009, were successfully synthesized and then electrospun in tridimentional fibrous mats. The copolymers show different thermal and mechanical behaviours as result of their chemical structure. In particular, copolymerization led to a reduction in crystallinity and consequently PBS stiffness, reaching values of elastic modulus very close to those of soft tissues. Then, to check the biological suitability, human induced Pluripotent Stem Cells (hiPSCs) were directly seeded on both PBS-based copolymeric scaffolds. The results confirmed the ability of both the scaffolds to sustain cell viability and to maintain their stemness during cell expansion. Furthermore, gene expression and immunofluorescence analysis showed that P(BSBPripol) scaffold promoted an upregulation of the early cardiac progenitor and later-stage markers with a simultaneously upregulation of HYPPO pathway gene expression, crucial for mechanosensing of cardiac progenitor cells. These results suggest that the correct ad-hoc chemical design and, in turn, the mechanical properties of the matrix, such as substrate stiffness, together with surface porosity, play a critical role in regulating the behaviour of cardiac progenitors, which ultimately offers valuable insights into the development of novel bio-inspired scaffolds for cardiac tissue regeneration.

Combined Effects of HA Concentration and Unit Cell Geometry on the Biomechanical Behavior of PCL/HA Scaffold for Tissue Engineering Applications Produced by LPBF.

This experimental study aims at filling the gap in the literature concerning the combined effects of hydroxyapatite (HA) concentration and elementary unit cell geometry on the biomechanical performances of additively manufactured polycaprolactone/hydroxyapatite (PCL/HA) scaffolds for tissue engineering applications. Scaffolds produced by laser powder bed fusion (LPBF) with diamond (DO) and rhombic dodecahedron (RD) elementary unit cells and HA concentrations of 5, 30 and 50 wt.% were subjected to structural, mechanical and biological characterization to investigate the biomechanical and degradative behavior from the perspective of bone tissue regeneration. Haralick's features describing surface pattern, correlation between micro- and macro-structural properties and human mesenchymal stem cell (hMSC) viability and proliferation have been considered. Experimental results showed that HA has negative influence on scaffold compaction under compression, while on the contrary it has a positive effect on hMSC adhesion. The unit cell geometry influences the mechanical response in the plastic regime and also has an effect on the cell proliferation. Finally, both HA concentration and elementary unit cell geometry affect the scaffold elastic deformation behavior as well as the amount of micro-porosity which, in turn, influences the scaffold degradation rate.

Human Ovarian Follicular Fluid Mesenchymal Stem Cells Express Osteogenic Markers When Cultured on Bioglass 58S-Coated Titanium Scaffolds.

Recent studies have reported that stem cells (human follicular fluid mesenchymal stem cells or hFF-MSCs) are present in ovarian follicular fluid (hFF) and that they have a proliferative and differentiative potential which is similar to that of MSCs derived from other adult tissue. These mesenchymal stem cells, isolated from human follicular fluid waste matter discarded after retrieval of oocytes during the IVF process, constitute another, as yet unutilized, source of stem cell materials. There has been little work on the compatibility of these hFF-MSCs with scaffolds useful for bone tissue engineering applications and the aim of this study was to evaluate the osteogenic capacity of hFF-MSCs seeded on bioglass 58S-coated titanium and to provide an assessment of their suitability for bone tissue engineering purposes. Following a chemical and morphological characterization with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), cell viability, morphology and expression of specific osteogenic markers were examined after 7 and 21 days of culture. The hFF-MSCs seeded on bioglass and cultured with osteogenic factors, when compared with those seeded on tissue culture plate or on uncoated titanium, exhibited enhanced cell viability and osteogenic differentiation, as reflected by increased calcium deposition and increased ALP activity with expression and production of bone-related proteins. Taken together, these results demonstrate that MSCs from human follicular fluid waste materials can be easily cultured in titanium scaffolds coated with bioglass, having osteoinductive properties. This process has significant potential for regenerative medicine applications and indicates that hFF-MSCs may be a valid alternative to hBM-MSC cells in experimental models in bone tissue engineering.

Immunomodulatory and Anti-inflammatory effect of Neural Stem/Progenitor Cells in the Central Nervous System.

Neuroinflammation is a critical event that responds to disturbed homeostasis and governs various neurological diseases in the central nervous system (CNS). The excessive inflammatory microenvironment in the CNS can adversely affect endogenous neural stem cells, thereby impeding neural self-repair. Therapies with neural stem/progenitor cells (NSPCs) have shown significant inhibitory effects on inflammation, which is mainly achieved through intercellular contact and paracrine signalings. The intercellular contact between NSPCs and immune cells, the activated CNS- resident microglia, and astrocyte plays a critical role in the therapeutic NSPCs homing and immunomodulatory effects. Moreover, the paracrine effect mainly regulates infiltrating innate and adaptive immune cells, activated microglia, and astrocyte through the secretion of bioactive molecules and extracellular vesicles. However, the molecular mechanism involved in the immunomodulatory effect of NSPCs is not well discussed. This article provides a systematic analysis of the immunomodulatory mechanism of NSPCs, discusses efficient ways to enhance its immunomodulatory ability, and gives suggestions on clinical therapy.

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.

Haralick's texture analysis to predict cellular proliferation on randomly oriented electrospun nanomaterials.

Using a computer vision approach we have extracted the Haralick's texture features of randomly oriented electrospun nanomaterials in order to predict the proliferative behavior of cells which were subsequently seeded onto the nanosurfaces.

Early Osteogenic Marker Expression in hMSCs Cultured onto Acid Etching-Derived Micro- and Nanotopography 3D-Printed Titanium Surfaces.

Polyetheretherketone (PEEK) titanium composite (PTC) is a novel interbody fusion device that combines a PEEK core with titanium alloy (Ti6Al4V) endplates. The present study aimed to investigate the in vitro biological reactivity of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) to micro- and nanotopographies produced by an acid-etching process on the surface of 3D-printed PTC endplates. Optical profilometer and scanning electron microscopy were used to assess the surface roughness and identify the nano-features of etched or unetched PTC endplates, respectively. The viability, morphology and the expression of specific osteogenic markers were examined after 7 days of culture in the seeded cells. Haralick texture analysis was carried out on the unseeded endplates to correlate surface texture features to the biological data. The acid-etching process modified the surface roughness of the 3D-printed PTC endplates, creating micro- and nano-scale structures that significantly contributed to sustaining the viability of hBM-MSCs and triggering the expression of early osteogenic markers, such as alkaline phosphatase activity and bone-ECM protein production. Finally, the topography of 3D-printed PTC endplates influenced Haralick's features, which in turn correlated with the expression of two osteogenic markers, osteopontin and osteocalcin. Overall, these data demonstrate that the acid-etching process of PTC endplates created a favourable environment for osteogenic differentiation of hBM-MSCs and may potentially have clinical benefit.

Gold Nanoparticles Contact with Cancer Cell: A Brief Update.

The fine-tuning of the physicochemical properties of gold nanoparticles has facilitated the rapid development of multifunctional gold-based nanomaterials with diagnostic, therapeutic, and therapeutic applications. Work on gold nanoparticles is increasingly focusing on their cancer application. This review provides a summary of the main biological effects exerted by gold nanoparticles on cancer cells and highlights some critical factors involved in the interaction process (protein corona, tumor microenvironment, surface functionalization). The review also contains a brief discussion of the application of gold nanoparticles in target discovery.

Biomechanical performances of PCL/HA micro- and macro-porous lattice scaffolds fabricated via laser powder bed fusion for bone tissue engineering.

The present experimental study aims to extend know-how on resorbable polycaprolactone/hydroxyapatite (PCL/HA, 70/30 wt%) scaffolds, produced by Laser Powder Bed Fusion (LPBF) technology, to geometrically complex lattice structures and micro porous struts. Using optimized LPBF printing parameters, micro- and macro-porous scaffolds for bone tissue regeneration were produced by regularly repeating in space Diamond (DO) and Rhombic Dodecahedron (RD) elementary unit cells. After production, scaffolds were submitted to structural, mechanical, and biological characterization. The interaction of scaffolds with human Mesenchymal Stem Cells (hMSCs) allowed studying the degradative processes of the PCL matrix. Biomechanical performances and biodegradation of scaffolds were compared to literature results and bone tissue data. Mechanical compression test, biological viability up to 4 days of incubation and degradation rate evidenced strong dependence of scaffold behavior on unit cell geometry as well as on global geometrical features.

Gold Nanoparticles: Can They Be the Next Magic Bullet for Multidrug-Resistant Bacteria?

In 2017 the World Health Organization (WHO) announced a list of the 12 multidrug-resistant (MDR) families of bacteria that pose the greatest threat to human health, and recommended that new measures should be taken to promote the development of new therapies against these superbugs. Few antibiotics have been developed in the last two decades. Part of this slow progression can be attributed to the surge in the resistance acquired by bacteria, which is holding back pharma companies from taking the risk to invest in new antibiotic entities. With limited antibiotic options and an escalating bacterial resistance there is an urgent need to explore alternative ways of meeting this global challenge. The field of medical nanotechnology has emerged as an innovative and a powerful tool for treating some of the most complicated health conditions. Different inorganic nanomaterials including gold, silver, and others have showed potential antibacterial efficacies. Interestingly, gold nanoparticles (AuNPs) have gained specific attention, due to their biocompatibility, ease of surface functionalization, and their optical properties. In this review, we will focus on the latest research, done in the field of antibacterial gold nanoparticles; by discussing the mechanisms of action, antibacterial efficacies, and future implementations of these innovative antibacterial systems.

Targeting the "Sweet Side" of Tumor with Glycan-Binding Molecules Conjugated-Nanoparticles: Implications in Cancer Therapy and Diagnosis.

Nanotechnology is in the spotlight of therapeutic innovation, with numerous advantages for tumor visualization and eradication. The end goal of the therapeutic use of nanoparticles, however, remains distant due to the limitations of nanoparticles to target cancer tissue. The functionalization of nanosystem surfaces with biological ligands is a major strategy for directing the actions of nanomaterials specifically to tumor cells. Cancer formation and metastasis are accompanied by profound alterations in protein glycosylation. Hence, the detection and targeting of aberrant glycans are of great value in cancer diagnosis and therapy. In this review, we provide a brief update on recent progress targeting aberrant glycosylation by functionalizing nanoparticles with glycan-binding molecules (with a special focus on lectins and anti-glycan antibodies) to improve the efficacy of nanoparticles in cancer targeting, diagnosis, and therapy and outline the challenges and limitations in implementing this approach. We envision that the combination of nanotechnological strategies and cancer-associated glycan targeting could remodel the field of cancer diagnosis and therapy, including immunotherapy.

Topological, Mechanical and Biological Properties of Ti6Al4V Scaffolds for Bone Tissue Regeneration Fabricated with Reused Powders via Electron Beam Melting.

Cellularized scaffold is emerging as the preferred solution for tissue regeneration and restoration of damaged functionalities. However, the high cost of preclinical studies creates a gap between investigation and the device market for the biomedical industry. In this work, bone-tailored scaffolds based on the Ti6Al4V alloy manufactured by electron beam melting (EBM) technology with reused powder were investigated, aiming to overcome issues connected to the high cost of preclinical studies. Two different elementary unit cell scaffold geometries, namely diamond (DO) and rhombic dodecahedron (RD), were adopted, while surface functionalization was performed by coating scaffolds with single layers of polycaprolactone (PCL) or with mixture of polycaprolactone and 20 wt.% hydroxyapatite (PCL/HA). The mechanical and biological performances of the produced scaffolds were investigated, and the results were compared to software simulation and experimental evidence available in literature. Good mechanical properties and a favorable environment for cell growth were obtained for all combinations of scaffold geometry and surface functionalization. In conclusion, powder recycling provides a viable practice for the biomedical industry to strongly reduce preclinical costs without altering biomechanical performance.

In Vitro Production of Calcified Bone Matrix onto Wool Keratin Scaffolds via Osteogenic Factors and Electromagnetic Stimulus.

Pulsed electromagnetic field (PEMF) has drawn attention as a potential tool to improve the ability of bone biomaterials to integrate into the surrounding tissue. We investigated the effects of PEMF (frequency, 75 Hz; magnetic induction amplitude, 2 mT; pulse duration, 1.3 ms) on human osteoblast-like cells (SAOS-2) seeded onto wool keratin scaffolds in terms of proliferation, differentiation, and production of the calcified bone extracellular matrix. The wool keratin scaffold offered a 3D porous architecture for cell guesting and nutrient diffusion, suggesting its possible use as a filler to repair bone defects. Here, the combined approach of applying a daily PEMF exposure with additional osteogenic factors stimulated the cells to increase both the deposition of bone-related proteins and calcified matrix onto the wool keratin scaffolds. Also, the presence of SAOS-2 cells, or PEMF, or osteogenic factors did not influence the compression behavior or the resilience of keratin scaffolds in wet conditions. Besides, ageing tests revealed that wool keratin scaffolds were very stable and showed a lower degradation rate compared to commercial collagen sponges. It is for these reasons that this tissue engineering strategy, which improves the osteointegration properties of the wool keratin scaffold, may have a promising application for long term support of bone formation in vivo.