International Journal of Molecular Sciences

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17 pages, 2576 KiB

Open AccessArticle

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs

by Sarah Grossi, Annalisa Grimaldi, Terenzio Congiu, Arianna Parnigoni, Giampiero Campanelli and Paola Campomenosi

Int. J. Mol. Sci. 2021, 22(2), 526; https://doi.org/10.3390/ijms22020526 - 7 Jan 2021

Cited by 1 | Viewed by 1906

Abstract

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices [...] Read more.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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17 pages, 4858 KiB

Open AccessArticle

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role

by Nicolò Baranzini, Laura Pulze, Marcella Reguzzoni, Rossella Roncoroni, Viviana Teresa Orlandi, Gianluca Tettamanti, Francesco Acquati and Annalisa Grimaldi

Int. J. Mol. Sci. 2020, 21(24), 9722; https://doi.org/10.3390/ijms21249722 - 19 Dec 2020

Cited by 1 | Viewed by 2243

Abstract

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech’s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid [...] Read more.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech’s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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24 pages, 43222 KiB

Open AccessArticle

The 3D Pattern of the Rainbow Trout (Oncorhynchus mykiss) Enterocytes and Intestinal Stem Cells

by Nicole Verdile, Rolando Pasquariello, Tiziana A. L. Brevini and Fulvio Gandolfi

Int. J. Mol. Sci. 2020, 21(23), 9192; https://doi.org/10.3390/ijms21239192 - 2 Dec 2020

Cited by 8 | Viewed by 3048

Abstract

We previously showed that, according to the frequency and distribution of specific cell types, the rainbow trout (RT) intestinal mucosa can be divided in two regions that form a complex nonlinear three-dimensional (3D) pattern and have a different renewal rate. This work had [...] Read more.

We previously showed that, according to the frequency and distribution of specific cell types, the rainbow trout (RT) intestinal mucosa can be divided in two regions that form a complex nonlinear three-dimensional (3D) pattern and have a different renewal rate. This work had two aims. First, we investigated whether the unusual distribution of cell populations reflects a similar distribution of functional activities. To this end, we determined the protein expression pattern of three well-defined enterocytes functional markers: peptide transporter 1 (PepT1), sodium–glucose/galactose transporter 1 (SGLT-1), and fatty-acid-binding protein 2 (Fabp2). Second, we characterized the structure of RT intestinal stem-cell (ISC) niche and determined whether the different proliferative is accompanied by a different organization and/or extension of the stem-cell population. We studied the expression and localization of well-characterized mammal ISC markers: LGR5, HOPX, SOX9, NOTCH1, DLL1, and WNT3A. Our results indicate that morphological similarity is associated with similar function only between the first portion of the mid-intestine and the apical part of the complex folds in the second portion. Mammal ISC markers are all expressed in RT, but their localization is completely different, suggesting also substantial functional differences. Lastly, higher renewal rates are supported by a more abundant ISC population. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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24 pages, 5649 KiB

Open AccessArticle

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges

by Laura Pulze, Terenzio Congiu, Tiziana A. L. Brevini, Annalisa Grimaldi, Gianluca Tettamanti, Paola D’Antona, Nicolò Baranzini, Francesco Acquati, Federico Ferraro and Magda de Eguileor

Int. J. Mol. Sci. 2020, 21(15), 5400; https://doi.org/10.3390/ijms21155400 - 29 Jul 2020

Cited by 16 | Viewed by 3965

Abstract

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of [...] Read more.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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Review

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26 pages, 2165 KiB

Open AccessReview

Current Advances in 3D Tissue and Organ Reconstruction

by Georgia Pennarossa, Sharon Arcuri, Teresina De Iorio, Fulvio Gandolfi and Tiziana A. L. Brevini

Int. J. Mol. Sci. 2021, 22(2), 830; https://doi.org/10.3390/ijms22020830 - 15 Jan 2021

Cited by 31 | Viewed by 5600

Abstract

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with [...] Read more.

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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14 pages, 6650 KiB

Open AccessReview

How to Build and to Protect the Neuromuscular Junction: The Role of the Glial Cell Line-Derived Neurotrophic Factor

by Serena Stanga, Marina Boido and Pascal Kienlen-Campard

Int. J. Mol. Sci. 2021, 22(1), 136; https://doi.org/10.3390/ijms22010136 - 24 Dec 2020

Cited by 13 | Viewed by 4320

Abstract

The neuromuscular junction (NMJ) is at the crossroad between the nervous system (NS) and the muscle. Following neurotransmitter release from the motor neurons (MNs), muscle contraction occurs and movement is generated. Besides eliciting muscle contraction, the NMJ represents a site of chemical bidirectional [...] Read more.

The neuromuscular junction (NMJ) is at the crossroad between the nervous system (NS) and the muscle. Following neurotransmitter release from the motor neurons (MNs), muscle contraction occurs and movement is generated. Besides eliciting muscle contraction, the NMJ represents a site of chemical bidirectional interplay between nerve and muscle with the active participation of Schwann cells. Indeed, signals originating from the muscle play an important role in synapse formation, stabilization, maintenance and function, both in development and adulthood. We focus here on the contribution of the Glial cell line-Derived Neurotrophic Factor (GDNF) to these processes and to its potential role in the protection of the NMJ during neurodegeneration. Historically related to the maintenance and survival of dopaminergic neurons of the substantia nigra, GDNF also plays a fundamental role in the peripheral NS (PNS). At this level, it promotes muscle trophism and it participates to the functionality of synapses. Moreover, compared to the other neurotrophic factors, GDNF shows unique peculiarities, which make its contribution essential in neurodegenerative disorders. While describing the known structural and functional changes occurring at the NMJ during neurodegeneration, we highlight the role of GDNF in the NMJ–muscle cross-talk and we review its therapeutic potential in counteracting the degenerative process occurring in the PNS in progressive and severe diseases such as Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). We also describe functional 3D neuromuscular co-culture systems that have been recently developed as a model for studying both NMJ formation in vitro and its involvement in neuromuscular disorders. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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12 pages, 3050 KiB

Open AccessReview

Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering

by Hidekazu Sekine and Teruo Okano

Int. J. Mol. Sci. 2021, 22(1), 92; https://doi.org/10.3390/ijms22010092 - 23 Dec 2020

Cited by 14 | Viewed by 3511

Abstract

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct [...] Read more.

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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27 pages, 6217 KiB

Open AccessReview

3D Cell Printing of Tissue/Organ-Mimicking Constructs for Therapeutic and Drug Testing Applications

by Jongmin Kim, Jeong Sik Kong, Wonil Han, Byoung Soo Kim and Dong-Woo Cho

Int. J. Mol. Sci. 2020, 21(20), 7757; https://doi.org/10.3390/ijms21207757 - 20 Oct 2020

Cited by 28 | Viewed by 4676

Abstract

The development of artificial tissue/organs with the functional maturity of their native equivalents is one of the long-awaited panaceas for the medical and pharmaceutical industries. Advanced 3D cell-printing technology and various functional bioinks are promising technologies in the field of tissue engineering that [...] Read more.

The development of artificial tissue/organs with the functional maturity of their native equivalents is one of the long-awaited panaceas for the medical and pharmaceutical industries. Advanced 3D cell-printing technology and various functional bioinks are promising technologies in the field of tissue engineering that have enabled the fabrication of complex 3D living tissue/organs. Various requirements for these tissues, including a complex and large-volume structure, tissue-specific microenvironments, and functional vasculatures, have been addressed to develop engineered tissue/organs with native relevance. Functional tissue/organ constructs have been developed that satisfy such criteria and may facilitate both in vivo replenishment of damaged tissue and the development of reliable in vitro testing platforms for drug development. This review describes key developments in technologies and materials for engineering 3D cell-printed constructs for therapeutic and drug testing applications. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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17 pages, 1104 KiB

Open AccessReview

Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application

by Kamila Białkowska, Piotr Komorowski, Maria Bryszewska and Katarzyna Miłowska

Int. J. Mol. Sci. 2020, 21(17), 6225; https://doi.org/10.3390/ijms21176225 - 28 Aug 2020

Cited by 166 | Viewed by 12009

Abstract

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells [...] Read more.

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications. Full article

(This article belongs to the Special Issue Thinking in 3D: From Molecules to Organisms)

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