Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration.

BACKGROUND: Due to loss of peripheral nerve structure and/or function resulting from trauma, accidents, and other causes, peripheral nerve injuries continue to be a major clinical problem. These injuries can cause partial or total loss of sensory, motor, and autonomic capabilities as well as neuropathic pain. PNI affects between 13 and 23 out of every 100,000 people annually in developed countries. Regeneration of damaged nerves and restoration of function after peripheral nerve injury remain significant therapeutic challenges. Although autologous nerve graft transplantation is a viable therapy option in several clinical conditions, donor site morbidity and a lack of donor tissue often hinder full functional recovery. Biomimetic conduits used in tissue engineering to encourage and direct peripheral nerve regeneration by providing a suitable microenvironment for nerve ingrowth are only one example of the cutting-edge methods made possible by this field. Many innate extracellular matrix (ECM) structures of different tissues can be successfully mimicked by nanofibrous scaffolds. Nanofibrous scaffolds can closely mimic the surface structure and morphology of native ECMs of many tissues. METHODS: In this study, we have produced bilayer nanofibrous nerve conduit based on poly-lactic acid/polyurethane/multiwall carbon nanotube (PLA/PU/MWCNT), for application as composite scaffolds for static nerve tissue engineering. The contact angle was indicated to show the hydrophilicity properties of electrospun nanofibers. The SEM images were analyzed to determine the fiber's diameters, scaffold morphology, and endometrial stem cell adhesion. Moreover, MTT assay and DAPI staining were used to show the viability and proliferation of endometrial stem cells. RESULTS: The constructed bilayer PLA/PU/MWCNT scaffolds demonstrated the capacity to support cell attachment, and the vitality of samples was assessed using SEM, MTT assay, and DAPI staining technique. CONCLUSIONS: According to an in vitro study, electrospun bilayer PLA/PU/MWCNT scaffolds can encourage the adhesion and proliferation of human endometrial stem cells (hEnSCs) and create the ideal environment for increasing cell survival.

The Effect of Fetal Bovine Acellular Dermal Matrix Seeded with Wharton's Jelly Mesenchymal Stem Cells for Healing Full-Thickness Skin Wounds.

The treatment of full-thickness skin wounds is a problem in the clinical setting, as they do not heal spontaneously. Extensive pain at the donor site and a lack of skin grafts limit autogenic and allogeneic skin graft availability. We evaluated fetal bovine acellular dermal matrix (FADM) in combination with human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) to heal full-thickness skin wounds. FADM was prepared from a 6-month-old trauma-aborted fetus. WJ-MSCs were derived from a human umbilical cord and seeded on the FADM. Rat models of full-thickness wounds were created and divided into three groups: control (no treatment), FADM, and FADM-WJMSCs groups. Wound treatment was evaluated microscopically and histologically on days 7, 14, and 21 post-surgery. The prepared FADM was porous and decellularized with a normal range of residual DNA. WJ-MSCs were seeded and proliferated on FADM effectively. The highest wound closure rate was observed in the FADM-WJMSC group on days 7 and 14 post-surgery. Furthermore, this group had fewer inflammatory cells than other groups. Finally, in this study, we observed that, without using the differential cell culture media of fibroblasts, the xenogeneic hWJSCs in combination with FADM could promote an increased rate of full-thickness skin wound closure with less inflammation.

Mesenchymal Stromal Cells and their EVs as Potential Leads for SARSCoV2 Treatment.

In December 2019, a betacoronavirus was isolated from pneumonia cases in China and rapidly turned into a pandemic of COVID-19. The virus is an enveloped positive-sense ssRNA and causes a severe respiratory syndrome along with a cytokine storm, which is the main cause of most complications. Therefore, treatments that can effectively control the inflammatory reactions are necessary. Mesenchymal Stromal Cells and their EVs are well-known for their immunomodulatory effects, inflammation reduction, and regenerative potentials. These effects are exerted through paracrine secretion of various factors. Their EVs also transport various molecules such as microRNAs to other cells and affect recipient cells' behavior. Scores of research and clinical trials have indicated the therapeutic potential of EVs in various diseases. EVs also seem to be a promising approach for severe COVID-19 treatment. EVs have also been used to develop vaccines since EVs are biocompatible nanoparticles that can be easily isolated and engineered. In this review, we have focused on the use of Mesenchymal Stromal Cells and their EVs for the treatment of COVID-19, their therapeutic capabilities, and vaccine development.

Injectable nanocomposite hydrogels as an emerging platform for biomedical applications: A review.

Hydrogels have attracted much attention for biomedical and pharmaceutical applications due to the similarity of their biomimetic structure to the extracellular matrix of natural living tissues, tunable soft porous microarchitecture, superb biomechanical properties, proper biocompatibility, etc. Injectable hydrogels are an exciting type of hydrogels that can be easily injected into the target sites using needles or catheters in a minimally invasive manner. The more comfortable use, less pain, faster recovery period, lower costs, and fewer side effects make injectable hydrogels more attractive to both patients and clinicians in comparison to non-injectable hydrogels. However, it is difficult to achieve an ideal injectable hydrogel using just a single material (i.e., polymer). This challenge can be overcome by incorporating nanofillers into the polymeric matrix to engineer injectable nanocomposite hydrogels with combined or synergistic properties gained from the constituents. This work aims to critically review injectable nanocomposite hydrogels, their preparation methods, properties, functionalities, and versatile biomedical and pharmaceutical applications such as tissue engineering, drug delivery, and cancer labeling and therapy. The most common natural and synthetic polymers as matrices together with the most popular nanomaterials as reinforcements, including nanoceramics, carbon-based nanostructures, metallic nanomaterials, and various nanosized polymeric materials, are highlighted in this review.

Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel.

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Defining the role of 17ß-estradiol in human endometrial stem cells differentiation into neuron-like cells.

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17ß-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17ß-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17ß-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17ß-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering.

Microtubule stabilizer epothilone B as a motor neuron differentiation agent for human endometrial stem cells.

Microtubule-stabilizing agents (MSAs), until now, have primarily been considered for their anti-proliferative effects in the setting of cancer. However, recent studies have revealed that one particular MSA, epothilone B (EpoB), can promote axonal regeneration after traumatic spinal cord injuries (SCI) even in the presence of inhibitor molecules such as neurite outgrowth inhibitor-A (Nogo-A). On the basis of the importance of having an efficient motor neuron (MN) differentiation protocol for stem cell therapy and the attention of MSAs for SCI treatment, our study investigated the effect of EpoB on human endometrial stem cells (hEnSCs) differentiation into MN-like cells. hEnSCs were isolated and characterized by flow cytometry. The hEnSC cell viability was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To mimic the in vivo inhibitory environment, hEnSCs were also differentiated in the presence of Nogo-A. After 15 days of differentiation, the expressions of MN-markers were evaluated by real-time reverse-transcriptase polymerase chain reaction and immunofluorescence. According to the MTT assay results, three doses (1, 5, and 10 nM) of EpoB were selected to evaluate their effect on MN-differentiation. All selected doses can increase the efficacy of hEnSCs differentiation into MN-like cells. In particular, the 10 nM EpoB dosage was shown to increase the axon elongation, cell alignment, and upregulation of these MN-markers compared with other doses. EpoB can improve MN differentiation from hEnSC and potentially provide a unique route for neuronal replacement in the setting of SCI.

A facile two step heat treatment strategy for development of bioceramic scaffolds for hard tissue engineering applications.

In the present study, a two-step sintering (TSS) method has been used to improve the mechanical properties, biocompatibility, drug release, and osteogenesis abilities of hardystonite (HT) ceramic scaffolds for tissue engineering and drug delivery applications. The average particle size of HT scaffold is kept lower than 80 nm and is reached higher than 130 nm by using two-step and conventional sintering methods, respectively. The compressive strengths of the prepared nanocrystalline HT scaffolds were found to be significantly higher than those of the micro-structure HT and currently available hydroxyapatite scaffolds. A comparative analysis of cell viability and live/dead staining of human mesenchymal stem cells (hMSCs) in nano- and micro-structured HT scaffolds and their drug release potentiation was carried out. The results showed that the nano-structured HT scaffolds have higher cell viability, biocompatibility and longer-term doxorubicin (DOX) release potential than the micro-structured ones. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analyses showed that the expression of adhesion and differentiation supporting genes were significantly higher in nano-structured HT scaffolds as compared to the micro-structured ones. The results of qRT-PCR also showed that the mRNA expression level of ERK1/2 and P38 MAPK from hMSCs were significantly higher in nano-structured HT scaffolds than the micro-structured ones. These results potentially open new aspects for using nano-structured scaffolds in bone tissue engineering applications.

Preparation of fibrin gel scaffolds containing MWCNT/PU nanofibers for neural tissue engineering.

Compared to the peripheral nervous system, in the central nervous system (CNS) disorders, neurons are less able to regenerate and reconstruct the neural tissue. Tissue engineering is considered as a promising approach for neural regeneration and restoring neurologic function after CNS injuries. Nanofibrous hydrogels have been recently used as three-dimensional (3D) scaffolds for tissue engineering applications. In this kind of composites, hydrogels are incorporated with fibers to enhance their poor mechanical properties. Furthermore, introducing meshes within hydrogels can result in composites associated with advantages of both components. In the present study, we have prepared 3D nanofibrous hydrogel scaffolds based on fibrin/polyurethane/multiwall carbon nanotube (fibrin/PU/MWCNT), for application as composite scaffolds for neural tissue engineering. The fabricated fibrin/PU/MWCNT hydrogel scaffolds were characterized and their ability to support cell attachment and viability was assessed in comparison with fibrin hydrogel. Scanning electron microscopy (SEM) analysis was performed to examine the microstructural features of scaffolds. The rate of biodegradation and rheological properties of scaffolds were also investigated. After isolation of human endometrial stem cells (hEnSCs), they were cultured into the scaffolds, then their attachment and viability were assessed through SEM analysis, MTT assay and DAPI staining. Based on the results, the viability and proliferation of hEnSCs in the fibrin/PU/MWCNT hydrogels were higher than those in fibrin hydrogels. Therefore, our novel fabricated fibrin/PU/MWCNT hydrogel is able to support cell proliferation and can be used as a scaffold to provide an appropriate microenvironment for enhancing cell viability. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 802-814, 2019.

The stability evaluation of mesenchymal stem cells differentiation toward endothelial cells by chemical and mechanical stimulation.

Adipose-derived mesenchymal stem cells (ADSCs) are adult multipotent cells able to differentiate into several cell lineages. Vascular endothelial growth factor (VEGF) and the shear stress associated with blood flow are considered as the most important chemical and mechanical cues that play major roles in endothelial differentiation. However, the stability of endothelial-specific gene expression has not been completely addressed yet. ADSCs in passage 3 were cultured inside the tubular silicon tubes and then exposed to VEGF or shear stress produced in a perfusion bioreactor. To investigate the differentiation, the expression levels of Flk-1, von Willebrand factor (vWF), and vascular endothelial-cadherin (VE-cadherin) were studied using Real-Time PCR. For studying the endothelial differentiation stability, mRNA levels of the genes were evaluated in certain time intervals after completion of the tests so as to determine whether the expression level of each gene in different time points was stable and remained constant or not. Application of VEGF and shear stress caused an elevation in endothelial cells' specific genes. Although there are some changes following the days after application of mechanical and chemical stimuli, the gene expression results depicted significantly higher gene expression between sequential chemically and mechanically incited groups. In conclusion, stress alone can be a differentiating factor, by itself. Our results verified the efficient stable differentiation ability of the chemical and mechanical factors.