Injectable hydrogels in central nervous system: Unique and novel platforms for promoting extracellular matrix remodeling and tissue engineering.

Repairing central nervous system (CNS) is difficult due to the inability of neurons to recover after damage. A clinically acceptable treatment to promote CNS functional recovery and regeneration is currently unavailable. According to recent studies, injectable hydrogels as biodegradable scaffolds for CNS tissue engineering and regeneration have exceptionally desirable attributes. Hydrogel has a biomimetic structure similar to extracellular matrix, hence has been considered a 3D scaffold for CNS regeneration. An interesting new type of hydrogel, injectable hydrogels, can be injected into target areas with little invasiveness and imitate several aspects of CNS. Injectable hydrogels are being researched as therapeutic agents because they may imitate numerous properties of CNS tissues and hence reduce subsequent injury and regenerate neural tissue. Because of their less adverse effects and cost, easier use and implantation with less pain, and faster regeneration capacity, injectable hydrogels, are more desirable than non-injectable hydrogels. This article discusses the pathophysiology of CNS and the use of several kinds of injectable hydrogels for brain and spinal cord tissue engineering, paying particular emphasis to recent experimental studies.

Enhanced sciatic nerve regeneration with fibrin scaffold containing human endometrial stem cells and insulin encapsulated chitosan particles: An in vivo study.

BACKGROUND: Based on recent advances in tissue engineering and stem cell therapy in nervous system diseases treatments, this study aimed to investigate sciatic nerve regeneration using human endometrial stem cells (hEnSCs) encapsulated fibrin gel containing chitosan nanoparticle loaded by insulin (Ins-CPs). Stem cells and also Insulin (Ins), which is a strong signaling molecule in peripheral nerve regeneration, play an important role in neural tissue engineering. METHODS: The fibrin hydrogel scaffold containing insulin loaded chitosan particles was synthesized and characterized. Release profiles of insulin from hydrogel was determined through UV-visible spectroscopy. Also, human endometrial stem cells encapsulated in hydrogel and its cell biocompatibility were assigned. Furthermore, the sciatic nerve crush injury was carried out and prepared fibrin gel was injected at the crush injury site by an 18-gage needle. Eight and twelve weeks later, the recovery of motor and sensory function and histopathological evaluation were assessed. RESULTS: The in vitro experiments showed that the insulin can promote hEnSCs proliferation within a certain concentration range. Animals' treatment confirmed that developed fibrin gel containing Ins-CPs and hEnSCs significantly improves motor function and sensory recovery. Hematoxylin and Eosin (H&E) images provided from cross-sectional and, longitudinal-sections of the harvested regenerative nerve showed that regenerative nerve fibers had been formed and accompanied with new blood vessels in the fibrin/insulin/hEnSCs group. CONCLUSION: Our results demonstrated that the prepared hydrogel scaffolds containing insulin nanoparticles and hEnSCs could be considered as a potential biomaterial aimed at regeneration of sciatic nerves.

Advanced approaches to regenerate spinal cord injury: The development of cell and tissue engineering therapy and combinational treatments.

Spinal cord injury (SCI) is a central nervous system (CNS) devastate event that is commonly caused by traumatic or non-traumatic events. The reinnervation of spinal cord axons is hampered through a myriad of devices counting on the damaged myelin, inflammation, glial scar, and defective inhibitory molecules. Unfortunately, an effective treatment to completely repair SCI and improve functional recovery has not been found. In this regard, strategies such as using cells, biomaterials, biomolecules, and drugs have been reported to be effective for SCI recovery. Furthermore, recent advances in combinatorial treatments, which address various aspects of SCI pathophysiology, provide optimistic outcomes for spinal cord regeneration. According to the global importance of SCI, the goal of this article review is to provide an overview of the pathophysiology of SCI, with an emphasis on the latest modes of intervention and current advanced approaches for the treatment of SCI, in conjunction with an assessment of combinatorial approaches in preclinical and clinical trials. So, this article can give scientists and clinicians' clues to help them better understand how to construct preclinical and clinical studies that could lead to a breakthrough in spinal cord regeneration.

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.

Enhanced sciatic nerve regeneration by poly-L-lactic acid/multi-wall carbon nanotube neural guidance conduit containing Schwann cells and curcumin encapsulated chitosan nanoparticles in rat.

The main aim of this study was to improve the efficacy of peripheral nerve regeneration by an artificial neural guidance conduit (NGC) as a carrier to transplant allogeneic Schwann cells (SCs) and curcumin encapsulated chitosan nanoparticles (nanocurcumin). The conduit was prepared by poly-L-lactic acid (PLLA) and surface-modified multi-wall carbon nanotubes (mMWCNT) and filled with SCs and nanocurcumin. SCs play an important role in the regeneration of injured peripheral nerve and controlled curcumin release can decrease SCs apoptosis, and enhance the regeneration and functional recovery of injured peripheral nerves. The mechanical properties, contact angle, and cell biocompatibility experiments showed that the optimized concentration of mMWCNT inside PLLA wall of conduits was 0.15 wt%. The drug release experiments showed slower release of curcumin from nanocurcumin samples compared to nanocurcumin encapsulated inside NGC wrapped fibrin gel sample. It was found that simultaneous using of both SCs and curcumin inside NGC had a significant role in sciatic nerve regeneration in vivo. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following treatment by SCs and nanocurcumin compared to negative control group. Histological evaluation also revealed a significant decrease in the number of vessels in fibrin groups compared to positive control group. The results showed that there was no significant difference between the reaction time and sciatic functional index (SFI) values of rats with injured sciatic nerve treated by NGC/SCs/nanocurcumin sample and autograft sample. In conclusion, our results strongly showed that PLLA/mMWCNT nanofibrous conduit filled with fibrin gel containing SCs and nanocurcumin is a proper strategy for improving nerve regeneration after a nerve transaction in the rat.

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 silk fibroin/decellularized extract of Wharton's jelly hydrogel intended for cartilage tissue engineering.

A hybrid hydrogel was obtained from decellularized extract from Wharton's jelly (DEWJ) and silk fibroin (SF) and characterized for cartilage tissue engineering. Wharton's jelly was used due to its similarity with articular cartilage in extracellular matrix composition. Also, silk fibroin has good mechanical properties which make this construct appropriate for cartilage repair. Decellularization of Wharton's jelly was verified by DAPI staining, DNA quantification, and PCR analysis. Then, the biochemical composition of DEWJ was determined by ELISA kits for total proteins, collagens, sulfated glycosaminoglycans (sGAG), and transforming growth factor ß1 (TGF-ß1). After fabricating pure SF and SF/DEWJ hybrid hydrogels, their physical and mechanical properties were characterized by FESEM, Fourier-transform infrared spectroscopy (FTIR) and rheological assays (amplitude and frequency sweeps). Furthermore, cell viability and proliferation were assessed by MTT assay. The results have shown that DEWJ in hybrid hydrogels enhances mechanical properties of the construct relative to pure SF hydrogels. Also, this extract at its 40% concentration in culture media and 20% or 40% concentrations in SF/DEWJ hybrid hydrogels significantly increases population of the cells compared to control and pure SF hydrogel after 7 days. In conclusion, this study proposes the potential of SF/DEWJ hybrid hydrogels for cartilage 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.

Comparing the Effect of Silybin and Silybin Advanced™ on Viability and HER2 Expression on the Human Breast Cancer SKBR3 Cell Line by no Serum Starvation.

The polyphenol silybin has anti-oxidant and anti-cancer properties. The poor bioavailability of some polyphenols (flavonoids, and terpenoids) can be improved by binding them to phosphatidylcholine (phytosome technology). Many studies have focused on the most common phytosome, silybin-phosphatidylcholine, particularly for its hepatoprotective effects. However, in recent years, studies have also been conducted to determine its anti-cancer effect. Considering that the serum starvation should not be used for studies that are not focused on cell cycle arrest, we studied the effect of silybin-phosphatidylcholine from Silybin Advanced™ in 1:2 ratio (one part silybin bound to two parts phosphatidylcholine) on HER2 gene expression on the SKBR3 breast cancer cell line which were cultured in complete medium (not serum deprivation). The results were compared with our previous study of silybin on HER2 expression on SKBR3 cells. An MTT test was used to determine concentrations for cell treatment, and the gene expression was defined by real-time RT-PCR. Outcomes showed significant concentration- and time-dependent cell growth inhibitory effects of silybin, and silybin-phosphatidylcholine and HER2 down regulation on SKBR3 cells. Silybin-phosphatidylcholine concentrations had a much larger inhibitory and HER2 down regulate effect on cell growth than the same silybin concentrations on SKBR3 cells.

The Comparison of The Effects of Silybin and Silybin-Phosphatidylcholine on Viability and ESR Expression in Human Breast Cancer T47D Cell Line.

OBJECTIVE: Silybin is a polyphenol with anti-oxidant and anti-cancer properties. The poor bioavailability of some polyphenols can be improved by binding to phosphatidylcholine. In recent years, studies have been conducted to evaluate the anti-cancer effect of silybin. We studied the effect of silybin and silybin-phosphatidylcholine on ESR1 and ESR2 gene expression and viability in the T47D breast cancer cell line. MATERIALS AND METHODS: In this experimental study, a 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide test (MTT test) was used to determine doses for cell treatment, and the gene expression was analyzed by real-time reverse transcriptase-polymerase chain reaction (real-time RT- PCR). RESULTS: Significant dose- and time-dependent cell growth inhibitory effects of silybin and silybin-phosphatidylcholine along with ESR1 down-regulation were observed in T47D cells. In contrast to ESR1, the T47D cell line showed negligible ESR2 expression. CONCLUSION: This study suggests that silybin and silybin-phosphatidylcholine down-regulate ESR1 in ER(+)breast cancers. Results also show that in the T47D cell line, silybindown-regulation of ESR1 compared with silybin.