Resveratrol's Impact on the Chondrogenic Reagents' Effects in Cell Sheet Cultures of Wharton's Jelly-Derived MSCs.

Human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation.

Multifrequency dielectric mapping of fixed mice colon tissues in cell culture media via scanning electrochemical microscopy.

Alternating current scanning electrochemical microscopy (AC-SECM) is a powerful tool for characterizing the electrochemical reactivity of surfaces. Here, perturbation in the sample is induced by the alternating current and altered local potential is measured by the SECM probe. This technique has been used to investigate many exotic a range of biological interfaces including live cells and tissues, as well as the corrosive degradation of various metallic surfaces, etc. In principle, AC-SECM imaging is derived from electrochemical impedance spectroscopy (EIS) which has been used for a century to describe interfacial and diffusive behaviour of molecules in solution or on a surface. Increasingly bioimpedance centric medical devices have become an important tool to detect evolution of tissue biochemistry. Predictive implications of measuring electrochemical changes within a tissue is one of the core concepts in developing minimally invasive and smart medical devices. In this study, cross sections of mice colon tissue were used for AC-SECM imaging. A 10 micron sized platinum probe was used for two-dimensional (2D) tan δ mapping of histological sections at a frequency of 10 kHz, Thereafter, multifrequency scans were performed at 100 Hz, 10 kHz, 300 kHz, and 900 kHz. Loss tangent (tan δ) mapping of mice colon revealed microscale regions within a tissue possessing a discrete tan δ signature. This tan δ map may be an immediate measure of physiological conditions in biological tissues. Multifrequency scans highlight subtle changes in protein or lipid composition as a function of frequency which was recorded as loss tangent maps. Impedance profile at different frequencies could also be used to identify optimal contrast for imaging and extracting the electrochemical signature specific for a tissue and its electrolyte.

IBD disease-modifying therapies: insights from emerging therapeutics.

Inflammatory bowel disease (IBD) pathogenesis is associated with gut mucosal inflammation, epithelial damage, and dysbiosis leading to a dysregulated gut mucosal barrier. However, the extent and underlying mechanisms remain largely unknown. Current treatment regimens have focused mainly on treating IBD symptoms; however, such treatment strategies do not address mucosal epithelial repair, barrier homeostasis, or intestinal dysbiosis. Although attempts have been made to identify new therapeutic modalities to enhance gut barrier functions, these are at an early developmental stage and have not been wholly successful. We review conventional therapies, the possible relevant role of gut barrier-protecting agents, and biomaterial strategies relating to combination therapies that may pave the way towards developing new therapeutic approaches for IBD.

Photobiomodulation in 3D tissue engineering.

SIGNIFICANCE: The method of photobiomodulation (PBM) has been used in medicine for a long time to promote anti-inflammation and pain-resolving processes in different organs and tissues. PBM triggers numerous cellular pathways including stimulation of the mitochondrial respiratory chain, alteration of the cytoskeleton, cell death prevention, increasing proliferative activity, and directing cell differentiation. The most effective wavelengths for PBM are found within the optical window (750 to 1100 nm), in which light can permeate tissues and other water-containing structures to depths of up to a few cm. PBM already finds its applications in the developing fields of tissue engineering and regenerative medicine. However, the diversity of three-dimensional (3D) systems, irradiation sources, and protocols intricate the PBM applications. AIM: We aim to discuss the PBM and 3D tissue engineered constructs to define the fields of interest for PBM applications in tissue engineering. APPROACH: First, we provide a brief overview of PBM and the timeline of its development. Then, we discuss the optical properties of 3D cultivation systems and important points of light dosimetry. Finally, we analyze the cellular pathways induced by PBM and outcomes observed in various 3D tissue-engineered constructs: hydrogels, scaffolds, spheroids, cell sheets, bioprinted structures, and organoids. RESULTS: Our summarized results demonstrate the great potential of PBM in the stimulation of the cell survival and viability in 3D conditions. The strategies to achieve different cell physiology states with particular PBM parameters are outlined. CONCLUSIONS: PBM has already proved itself as a convenient and effective tool to prevent drastic cellular events in the stress conditions. Because of the poor viability of cells in scaffolds and the convenience of PBM devices, 3D tissue engineering is a perspective field for PBM applications.

Quercetin conjugated fluorescent nitrogen-doped carbon dots for targeted cancer therapy application.

In this work, we report the development of nitrogen-doped carbon dots (NDCDs) as a drug carrier using quercetin (QC) as a model drug for anti-cancer drug delivery application. NDCDs were prepared by a simple hydrothermal method using Luffa acutangula as a carbon source. The characterization of QC-NDCDs was done by UV-vis spectroscopy, fluorescence spectroscopy, zeta potential measurements, high-resolution transmission electron microscopy (HR-TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and Raman spectroscopy. The as-synthesized NDCDs have a small particle size with hydroxyl and nitrogen-containing groups (pyridinic and amide groups), enhancing the fluorescence properties, and were obtained in a good quantum yield (14%). Furthermore, the in vitro alamarBlue® assay revealed that the NDCDs-QC conjugate was nontoxic to colon cancer cells. This NDCDs-QC conjugate is able to kill cancer cells in the NDCDs-QC form compared to free QC as confirmed by in vitro MTT assay results. Thus, the developed NDCDs conjugate can be used as a promising drug delivery and bio-imaging vehicle in cancer therapy.

3D or not 3D: a guide to assess cell viability in 3D cell systems.

Cell viability is the primary integrative parameter used for various purposes, particularly when fabricating tissue equivalents (e.g., using bioprinting or scaffolding techniques), optimizing conditions to cultivate cells, testing chemicals, drugs, and biomaterials, etc. Most of the conventional methods were originally designed for a monolayer (2D) culture; however, 2D approaches fail to adequately assess a tissue-engineered construct's viability and drug effects and recapitulate the host-pathogen interactions and infectivity. This study aims at revealing the influence of particular 3D cell systems' parameters such as the components' concentration, gel thickness, cell density, etc. on the cell viability and applicability of standard assays. Here, we present an approach to achieving adequate and reproducible results on the cell viability in 3D collagen- and fibrin-based systems using the Live/Dead, AlamarBlue, and PicoGreen assays. Our results have demonstrated that a routine precise analysis of 3D systems should be performed using a combination of at least three methods based on different cell properties, e.g. the metabolic activity, proliferative capacity, morphology, etc.

Inflammation-specific targeted carriers for local drug delivery to inflammatory bowel disease.

Delivering drugs directly to the inflamed intestinal sites to treat inflammatory bowel disease (IBD), particularly Crohn's and ulcerative colitis, is highly challenging. Recent advances in colitis therapy medications are expanding opportunities for improving local on-site drug availability by minimising the associated systemic side-effects. Drug delivery with targeted carrier systems has shown the potential to increase site-specificity, stability, and therapeutic efficacy. Herein, we report the development of a strong anionic charged inflammation targeted nanocarriers (IT-NCs) loaded with an immunosuppressant model drug. This system showed preferential adhesion on a charge-modified surface in vitro, and in both dextran sulfate sodium (DSS) and TNBS colitis mice in vivo models. IT-NCs showed improved colitis phenotype therapeutic efficacy in both animal models compared to free drug. Furthermore, ex vivo study of colon tissue biopsies from patients with colitis revealed that IT-NCs adhered preferentially to inflamed biopsies compared to normal. Together, our results suggest that IT-NCs have promising therapeutic potential as delivery carriers' in colitis management.

Modulation of Gut Barrier Functions in Ulcerative Colitis by Hyaluronic Acid System.

The active stages of intestinal inflammation and the pathogenesis of ulcerative colitis are associated with superficial mucosal damage and intermittent wounding that leads to epithelial barrier defects and increased permeability. The standard therapeutic interventions for colitis have focused mainly on maintaining the remission levels of the disease. Nonetheless, such treatment strategies (using anti-inflammatory, immunomodulatory agents) do not address colitis' root cause, especially the mucosal damage and dysregulated intestinal barrier functions. Restoration of barrier functionality by mucosal healing or physical barrier protecting strategies shall be considered as an initial event in the disease suppression and progression. Herein, a biphasic hyaluronan (HA) enema suspension, naïve-HA systems that protect the dysregulated gut epithelium by decreasing the inflammation, permeability, and helping in maintaining the epithelial barrier integrity in the dextran sodium sulfate-induced colitis mice model is reported. Furthermore, HA-based system modulates intestinal epithelial junctional proteins and regulatory signaling pathways, resulting in attenuation of inflammation and mucosal protection. The results suggest that HA-based system can be delivered as an enema to act as a barrier protecting system for managing distal colonic inflammatory diseases, including colitis.

Inhomogeneities in PNIPAM Aqueous Solutions: The Inside View by Spin Probe EPR Spectroscopy.

Coil to globule transition in poly(N-isopropylacrylamide) aqueous solutions was studied using spin probe continuous-wave electronic paramagnetic resonance (CW EPR) spectroscopy with an amphiphilic TEMPO radical as a guest molecule. Using Cu(II) ions as the "quencher" for fast-moving radicals in the liquid phase allowed obtaining the individual spectra of TEMPO radicals in polymer globule and observing inhomogeneities in solutions before globule collapsing. EPR spectra simulations confirm the formation of molten globules at the first step with further collapsing and water molecules coming out of the globule, making it denser.

Mechanical properties of cell sheets and spheroids: the link between single cells and complex tissues.

Cell aggregates, including sheets and spheroids, represent a simple yet powerful model system to study both biochemical and biophysical intercellular interactions. However, it is becoming evident that, although the mechanical properties and behavior of multicellular structures share some similarities with individual cells, yet distinct differences are observed in some principal aspects. The description of mechanical phenomena at the level of multicellular model systems is a necessary step for understanding tissue mechanics and its fundamental principles in health and disease. Both cell sheets and spheroids are used in tissue engineering, and the modulation of mechanical properties of cell constructs is a promising tool for regenerative medicine. Here, we review the data on mechanical characterization of cell sheets and spheroids, focusing both on advances in the measurement techniques and current understanding of the subject. The reviewed material suggest that interplay between the ECM, intercellular junctions, and cellular contractility determines the behavior and mechanical properties of the cell aggregates.

Recent advances and prospects of hyaluronan as a multifunctional therapeutic system.

Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG), cell-surface-associated biopolymer and is the key component of tissue extracellular matrix (ECM). Along with remarkable physicochemical properties, HA also has multifaceted biological effects that include but not limited to ECM organization, immunomodulation, and various cellular processes. Environmental cues such as tissue injury, infection or cancer change downstream signaling functionalities of HA. Unlike native HA, the fragments of HA have diversified effects on inflammation, cancer, fibrosis, angiogenesis and autoimmune response. In this review, we aim to discuss HA as a therapeutic delivery system development process, source, biophysical-chemical properties, and associated biological pathways (especially via cell surface receptors) of native and fragmented HA. We also tried to address an overview of the potential role of HA (native HA vs fragments) in the modulation of inflammation, immune response and various cancer targeting delivery applications. This review will also highlight the HA based therapeutic systems, medical devices and future perspectives of various biomedical applications were discussed in detail.

Scaffold-free cell-based tissue engineering therapies: advances, shortfalls and forecast.

Cell-based scaffold-free therapies seek to develop in vitro organotypic three-dimensional (3D) tissue-like surrogates, capitalising upon the inherent capacity of cells to create tissues with efficiency and sophistication that is still unparalleled by human-made devices. Although automation systems have been realised and (some) success stories have been witnessed over the years in clinical and commercial arenas, in vitro organogenesis is far from becoming a standard way of care. This limited technology transfer is largely attributed to scalability-associated costs, considering that the development of a borderline 3D implantable device requires very high number of functional cells and prolonged ex vivo culture periods. Herein, we critically discuss advancements and shortfalls of scaffold-free cell-based tissue engineering strategies, along with pioneering concepts that have the potential to transform regenerative and reparative medicine.

Potential immuno-nanomedicine strategies to fight COVID-19 like pulmonary infections.

COVID-19, coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic. At the time of writing this (October 14, 2020), more than 38.4 million people have become affected, and 1.0 million people have died across the world. The death rate is undoubtedly correlated with the cytokine storm and other pathological pulmonary characteristics, as a result of which the lungs cannot provide sufficient oxygen to the body's vital organs. While diversified drugs have been tested as a first line therapy, the complexity of fatal cases has not been reduced so far, and the world is looking for a treatment to combat the virus. However, to date, and despite such promise, we have received very limited information about the potential of nanomedicine to fight against COVID-19 or as an adjunct therapy in the treatment regimen. Over the past two decades, various therapeutic strategies, including direct-acting antiviral drugs, immunomodulators, a few non-specific drugs (simple to complex), have been explored to treat Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), influenza, and sometimes the common flu, thus, correlating and developing specific drugs centric to COVID-19 is possible. This review article focuses on the pulmonary pathology caused by SARS-CoV-2 and other viral pathogens, highlighting possible nanomedicine therapeutic strategies that should be further tested immediately.

Toward Developing Immunocompetent Diabetic Foot Ulcer-on-a-Chip Models for Drug Testing.

Bioengineering of skin has been significantly explored, ranging from the use of traditional cell culture systems to the most recent organ-on-a-chip (OoC) technology that permits skin modeling on physiological scales among other benefits. This article presents key considerations for developing physiologically relevant immunocompetent diabetic foot ulcer (DFU) models. Diabetic foot ulceration affects hundreds of millions of individuals globally, especially the elderly, and constitutes a major socioeconomic burden. When DFUs are not treated and managed in a timely manner, 15-50% of patients tend to undergo partial or complete amputation of the affected limb. Consequently, at least 40% of such patients die within 5 years postamputation. Currently, therapeutic strategies are actively sought and developed. However, present-day preclinical platforms (animals and in vitro models) are not robust enough to provide reliable data for clinical trials. Insights from published works on immunocompetent skin-on-a-chip models and bioengineering considerations, presented in this article, can inform researchers on how to develop robust OoC models for testing topical therapies such as growth factor-based therapies for DFUs. We propose that immunocompetent DFU-on-a-chip models should be bioengineered using diseased cells derived from individuals; in particular, the pathophysiological contribution of macrophages in diabetic wound healing, along with the typical fibroblasts and keratinocytes, needs to be recapitulated. The ideal model should consist of the following components: diseased cells embedded in reproducible scaffolds, which permit endogenous "diseased" extracellular matrix deposition, and the integration of the derived immunocompetent DFU model onto a microfluidic platform. The proposed DFU platforms will eventually facilitate reliable and robust drug testing of wound healing therapeutics, coupled with reduced clinical trial failure rates. Impact statement Current animal and cell-based systems are not physiologically relevant enough to retrieve reliable results for clinical translation of diabetic foot ulcer (DFU) therapies. Organ-on-a-chip (OoC) technology offers desirable features that could finally enable the vision of modeling DFU for pathophysiological studies and drug testing at a microscale. This article brings together the significant recent findings relevant to developing a minimally functional immunocompetent DFU-on-a-chip model, as wound healing cannot occur without a proper functioning immune response. It looks feasible in the future to recapitulate the stagnant inflammation in DFU (thought to impede wound healing) using OoC, diseased cells, and an endogenously produced extracellular matrix.

A high molecular weight hyaluronic acid biphasic dispersion as potential therapeutics for interstitial cystitis.

Interstitial cystitis (IC) is a progressive bladder disease characterized by increased urothelial permeability, inflammation of the bladder with abdominal pain. While there is no consensus on the etiology of the disease, it was believed that restoring the barrier between urinary solutes and (GAG) urothelium would interrupt the progression of this disease. Currently, several treatment options include intravesical delivery of hyaluronic acid (HA) and/or chondroitin sulfate solutions, through a catheter to restore the urothelial barrier, but have shown limited success in preclinical, clinical trials. Herein we report for the first time successful engineering and characterization of biphasic system developed by combining cross-linked hyaluronic acid and naïve HA solution to decrease inflammation and permeability in an in vitro model of interstitial cystitis. The cross-linking of HA was performed by 4-arm-polyethyeleneamine chemistry. The HA formulations were tested for their viscoelastic properties and the effects on cell metabolism, inflammatory markers, and permeability. Our study demonstrates the therapeutic effects of different ratios of the biphasic system and reports their ability to increase the barrier effect by decreasing the permeability and alteration of cell metabolism with respect to relative controls. Restoring the barrier by using biphasic system of HA therapy may be a promising approach to IC.

Tissue engineering using a combined cell sheet technology and scaffolding approach.

Cell sheet technology has remained quite popular among tissue engineering techniques over the last several years. Meanwhile, there is an apparent trend in modern scientific research towards combining different approaches and strategies. Accordingly, a large body of work has arisen where cell sheets are used not as separate structures, but in combination with scaffolds as supporting constructions. The aim of this review is to analyze the intersection of these two vast areas of tissue engineering described in the literature mainly within the last five years. Some practical and technical details are emphasized to provide information that can be useful in research design and planning. The first part of the paper describes the general issues concerning the use of combined technology, its advantages and limitations in comparison with those of other tissue engineering approaches. Next, the detailed literature analysis of in vivo studies aimed at the regeneration of different tissues is performed. A significant part of this section concerns bone regeneration. In addition to that, other connective tissue structures, including articular cartilage and fibrocartilage, ligaments and tendons, and some soft tissues are discussed. STATEMENT OF SIGNIFICANCE: This paper describes the intersection of two technologies used in designing of tissue-engineered constructions for regenerative medicine: cell sheets as extracellular matrix-rich structures and supporting scaffolds as essentials in tissue engineering. A large number of reviews are devoted to each of these scientific problems. However, the solution of complex problems of tissue engineering requires an integrated approach that includes both three-dimensional scaffolds and cell sheets. This manuscript serves as a description of advantages and limitations of this method, its use in regeneration of bones, connective tissues and soft tissues and some other details.

Beyond 2D: effects of photobiomodulation in 3D tissue-like systems.

SIGNIFICANCE: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. AIM: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. APPROACH: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. RESULTS: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. CONCLUSIONS: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.

An Orally Administrated Hyaluronan Functionalized Polymeric Hybrid Nanoparticle System for Colon-Specific Drug Delivery.

There is a pressing clinical need for advanced colon-specific local drug delivery systems that can provide major advantages in treating diseases associated with the colon, such as inflammatory bowel disease (IBD) and colon cancer. A precise colon targeted drug delivery platform is expected to reduce drug side effects and increase the therapeutic response at the intended disease site locally. In this study, we report the fabrication of hyaluronan (HA) functionalized polymeric hybrid nanoparticulate system (Cur-HA NPs) by using curcumin as a model fluorescent drug. The Cur-HA NPs were about 200-300 nm in size, -51.3 mV overall surface charge after HA functionalization, with 56.0% drug released after 72 h in simulated gastrointestinal fluids. The Cur-HA NPs did not exhibit any cytotoxicity by AlamarBlue, PicoGreen and Live/Dead assays. Following the Cur-HA NPs use on HT-29 monolayer cell cultures demonstrating, the efficacy of HA functionalization increases cellular interaction, uptake when compared to uncoated nanoparticulate system. These findings indicate that HA functionalized nano-hybrid particles are effective in delivering drugs orally to the lower gastrointestinal tract (GIT) in order to treat local colonic diseases.

From Aggregates to Porous Three-Dimensional Scaffolds through a Mechanochemical Approach to Design Photosensitive Chitosan Derivatives.

The crustacean processing industry produces large quantities of waste by-products (up to 70%). Such wastes could be used as raw materials for producing chitosan, a polysaccharide with a unique set of biochemical properties. However, the preparation methods and the long-term stability of chitosan-based products limit their application in biomedicine. In this study, different scale structures, such as aggregates, photo-crosslinked films, and 3D scaffolds based on mechanochemically-modified chitosan derivatives, were successfully formed. Dynamic light scattering revealed that aggregation of chitosan derivatives becomes more pronounced with an increase in the number of hydrophobic substituents. Although the results of the mechanical testing revealed that the plasticity of photo-crosslinked films was 5⁻8% higher than that for the initial chitosan films, their tensile strength remained unchanged. Different types of polymer scaffolds, such as flexible and porous ones, were developed by laser stereolithography. In vivo studies of the formed structures showed no dystrophic and necrobiotic changes, which proves their biocompatibility. Moreover, the wavelet analysis was used to show that the areas of chitosan film degradation were periodic. Comparing the results of the wavelet analysis and X-ray diffraction data, we have concluded that degradation occurs within less ordered amorphous regions in the polymer bulk.

Bioresponsive drug delivery systems in intestinal inflammation: State-of-the-art and future perspectives.

Oral colon-specific delivery systems emerged as the main therapeutic cargos by making a significant impact in the field of modern medicine for local drug delivery in intestinal inflammation. The site-specific delivery of therapeutics (aminosalicylates, glucocorticoids, biologics) to the ulcerative mucus tissue can provide prominent advantages in mucosal healing (MH). Attaining gut mucosal healing and anti-fibrosis are main treatment outcomes in inflammatory bowel disease (IBD). The pharmaceutical strategies that are commonly used to achieve a colon-specific drug delivery system include time, pH-dependent polymer coating, prodrug, colonic microbiota-activated delivery systems and a combination of these approaches. Amongst the different approaches reported, the use of biodegradable polysaccharide coated systems holds great promise in delivering drugs to the ulcerative regions. The present review focuses on major physiological gastro-intestinal tract challenges involved in altering the pharmacokinetics of delivery systems, pathophysiology of MH and fibrosis, reported drug-polysaccharide cargos and focusing on conventional to advanced disease responsive delivery strategies, highlighting their limitations and future perspectives in intestinal inflammation therapy.