Chitin scaffolds derived from the marine demosponge Aplysina fistularis stimulate the differentiation of dental pulp stem cells

The use of stem cells for tissue regeneration is a prominent trend in regenerative medicine and tissue engineering. In particular, dental pulp stem cells (DPSCs) have garnered considerable attention. When exposed to specific conditions, DPSCs have the ability to differentiate into osteoblasts and odontoblasts. Scaffolds are critical for cell differentiation because they replicate the 3D microenvironment of the niche and enhance cell adhesion, migration, and differentiation. The purpose of this study is to present the biological responses of human DPSCs to a purified 3D chitin scaffold derived from the marine demosponge Aplysina fistularis and modified with hydroxyapatite (HAp). Responses examined included proliferation, adhesion, and differentiation. The control culture consisted of the human osteoblast cell line, hFOB 1.19. Electron microscopy was used to examine the ultrastructure of the cells (transmission electron microscopy) and the surface of the scaffold (scanning electron microscopy). Cell adhesion to the scaffolds was determined by neutral red and crystal violet staining methods. An alkaline phosphatase (ALP) assay was used for assessing osteoblast/odontoblast differentiation. We evaluated the expression of osteogenic marker genes by performing ddPCR for ALP, RUNX2, and SPP1 mRNA expression levels. The results show that the chitin biomaterial provides a favorable environment for DPSC and hFOB 1.19 cell adhesion and supports both cell proliferation and differentiation. The chitin scaffold, especially with HAp modification, isolated from A. fistularis can make a significant contribution to tissue engineering and regenerative medicine

Unique Features of River Lamprey (<i>Lampetra fluviatilis</i>) Myogenesis

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates

Quantitative Phase Imaging Detecting the Hypoxia-Induced Patterns in Healthy and Neoplastic Human Colonic Epithelial Cells

Hypoxia is a frequent phenomenon during carcinogenesis and may lead to functional and structural changes in proliferating cancer cells. Colorectal cancer (CRC) is one of the most common neoplasms in which hypoxia is associated with progression. The aim of this study was to assess the optical parameters and microanatomy of CRC and the normal intestinal epithelium cells using the digital holotomography (DHT) method. The examination was conducted on cancer (HT-29, LoVo) and normal colonic cells (CCD-18Co) cultured in normoxic and hypoxic environments. The assessment included optical parameters such as the refractive index (RI) and dry mass as well as the morphological features. Hypoxia decreased the RI in all cells as well as in their cytoplasm, nucleus, and nucleoli. The opposite tendency was noted for spheroid-vesicular structures, where the RI was higher for the hypoxic state. The total volume of hypoxic CCD-18Co and LoVo cells was decreased, while an increase in this parameter was observed for HT-29 cells. Hypoxia increased the radius and cell volume, including the dry mass of the vesicular content. The changes in the optics and morphology of hypoxic cells may suggest the possibility of using DHT in the detection of circulating tumor cells (CTCs)

HMG-CoA Reductase Inhibitor, Simvastatin Is Effective in Decreasing Degree of Myocarditis by Inhibiting Metalloproteinases Activation

Background: Acute myocarditis often progresses to heart failure because there is no effective, etiology-targeted therapy of this disease. Simvastatin has been shown to be cardioprotective by decreasing matrix metalloproteinases’ (MMPs) activity. The study was designed to determine whether simvastatin inhibits MMPs activity, decreases the severity of inflammation and contractile dysfunction of the heart in experimental autoimmune myocarditis (EAM). Methods: Simvastatin (3 or 30 mg/kg/day) was given to experimental rats with EAM by gastric gavage for 21 days. Then transthoracic echocardiography was performed, MMPs activity and troponin I level were determined and tissue samples were assessed under a light and transmission electron microscope. Results: Hearts treated with simvastatin did not show left ventricular enlargement. As a result of EAM, there was an enhanced activation of MMP-9, which was significantly reduced in the high-dose simvastatin group compared to the low-dose group. It was accompanied by prevention of myofilaments degradation and reduction of severity of inflammation. Conclusions: The cardioprotective effects of simvastatin in the acute phase of EAM are, at least in part, due to its ability to decrease MMP-9 activity and subsequent decline in myofilaments degradation and suppression of inflammation. These effects were achieved in doses equivalent to therapeutic doses in humans

Identifying the Molecular Mechanisms and Types of Cell Death Induced by <i>bio</i>- and <i>pyr</i>-Silica Nanoparticles in Endothelial Cells

The term “nanosilica” refers to materials containing ultrafine particles. They have gained a rapid increase in popularity in a variety of applications and in numerous aspects of human life. Due to their unique physicochemical properties, SiO2 nanoparticles have attracted significant attention in the field of biomedicine. This study aimed to elucidate the mechanism underlying the cellular response to stress which is induced by the exposure of cells to both biogenic and pyrogenic silica nanoparticles and which may lead to their death. Both TEM and fluorescence microscopy investigations confirmed molecular changes in cells after treatment with silica nanoparticles. The cytotoxic activity of the compounds and intracellular RNS were determined in relation to HMEC-1 cells using the fluorimetric method. Apoptosis was quantified by microscopic assessment and by flow cytometry. Furthermore, the impact of nanosilica on cell migration and cell cycle arrest were determined. The obtained results compared the biological effects of mesoporous silica nanoparticles extracted from Urtica dioica L. and pyrogenic material and indicated that both types of NPs have an impact on RNS production causing apoptosis, necrosis, and autophagy. Although mesoporous silica nanoparticles did not cause cell cycle arrest, at the concentration of 50 μg/mL and higher they could disturb redox balance and stimulate cell migration

Naturally Formed Chitinous Skeleton Isolated from the Marine Demosponge Aplysina fistularis as a 3D Scaffold for Tissue Engineering

Tissue engineering (TE) is a field of regenerative medicine that has been experiencing a special boom in recent years. Among various materials used as components of 3D scaffolds, naturally formed chitinous materials seem to be especially attractive because of their abundance, non-toxic and eco-friendly character. In this study, chitinous skeleton isolated from the marine sponge Aplysina fistularis (phylum: Porifera) was used for the first time as a support for the cultivation of murine fibroblasts (Balb/3T3), human dermal fibroblasts (NHDF), human keratinocyte (HaCaT), and human neuronal (SH-SY5Y) cells. Characterization techniques such as ATR FTIR, TGA, and μCT, clearly indicate that an interconnected macro-porous, thermostable, pure α-chitin scaffold was obtained after alkali–acid treatment of air-dried marine sponge. The biocompatibility of the naturally formed chitin scaffolds was confirmed by cell attachment and proliferation determined by various microscopic methods (e.g., SEM, TEM, digital microscopy) and specific staining. Our observations show that fibroblasts and keratinocytes form clusters on scaffolds that resemble a skin structure, including the occurrence of desmosomes in keratinocyte cells. The results obtained here suggest that the chitinous scaffold from the marine sponge A. fistularis is a promising biomaterial for future research about tissues regeneration

Multimodal study of CHI3L1 inhibition and its effect on angiogenesis, migration, immune response and refractive index of cellular structures in glioblastoma

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721–0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721–0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour