Retinoic acid and taurine enhance differentiation of the human bone marrow stem cells into cone photoreceptor cells and retinal ganglion cells.

Degeneration and apoptotic death of the photoreceptor cell-layer of retina are a major cause of irreversible blindness in the development era. The stem cell replacement therapy is one of the strategies for the retinal repairing. In addition, exogenous signals critically contribute to the direction of lineage decisions that causes the fate-restricted photoreceptor progenitors from stem cell progeny in culture. It has been found that epidermal growth factor (EGF), taurine, and retinoic acid (RA) initially act in the instructive as well as lineage-restricted way in the progenitor lineage for producing neuroretinal cells or photoreceptor like cells from stem cell. The study aims to investigate the effect of RA and taurine in differentiation of the human bone marrow stem cell into cone photoreceptors cells and retinal ganglion cells. Mesenchymal stem cell was derived from human bone marrow of the term delivery. Therefore, the cultured cells have been treated with Dulbecco's modified Eagle's medium (DMEM)/high glucose (H+ ). After the four-cell passage, basal medium was replaced with DMEM/F12 complemented with 50 µmol/L taurine, RA (1 µM) and EGF (1 µg/ml). Subsequently cellular change morphology was detected following 7 and 14 days. Then, gene expression of neuroretinal and photoreceptor cell biomarkers (CRX, OTX2, PKC-α, recoverin, and Rho) were examined by quantitative polymerase chain reaction (Q-PCR). Also, cells were cultured, fixed, and then immunocytochemical analyzed. Primary antibodies included CRX and Rho. Cellular morphology demonstrated spindle elongated morphology. Taurine alone and combination of RA upregulate neuroretinal and photoreceptor cell biomarkers in messenger RNA and protein levels but along with EGF have not significant effect. Our data showed that taurine combination with RA can differentiate bone marrow mesenchymal stem cells into neuroretinal or photoreceptor like cells in vitro that can offer an attractive treatment ground for transplantation in the cell-replacement therapy for some forms of the retinal degeneration.

Bone regeneration in rat using polycaprolactone/gelatin/epinephrine scaffold.

Solid supports like the extracellular matrix network are necessary for bone cell attachment and start healing in the damaged bone. Scaffolds which are made of different materials are widely used as a supportive structure in bone tissue engineering. In the current study, a 3D polycaprolactone/gelatin bone scaffold was developed by blending electrospinning and freeze-drying techniques for bone tissue engineering. To improve the efficiency of the scaffold, different concentrations of epinephrine (EP) due to its effect on bone healing were loaded. Fabricated scaffolds were characterized by different tests such as surface morphology, FTIR, porosity, compressive strength, water contact angle, and degradation rate. The interaction between prepared scaffolds and blood and cells was evaluated by hemolysis, and MTT test, respectively, and bone healing was evaluated by a rat calvaria defect model. Based on the results, the porosity of scaffolds was about 75% and by adding EP, mechanical strength decreased while due to the hydrophilic properties of it, degradation rate increased. In vivo and in vitro studies showed the best cell proliferation and bone healing were in PCL/gelatin/EP1% treated group. These results showed the positive effect of fabricated scaffold on osteogenesis and bone healing and the possibility of using it in clinical trials.

Preparation and characterization of poly(ethylene oxide)/zinc oxide nanofibrous scaffold for chronic wound healing applications.

BACKGROUND: Skin, the first barrier to pathogens, loses its integrity and function after an injury. The presence of an antibacterial dressing at the wound site may prevent bacterial invasion and also improve the healing process. OBJECTIVES: The current study aimed to fabricate a biomimetic membrane with antibacterial properties for healing chronic wounds. MATERIAL AND METHODS: The membranes, fabricated through electrospinning, are comprised of poly(ethylene oxide) (PEO) and zinc oxide nanoparticles (ZnO-NPs) as the main biomaterial and antibacterial agent, respectively. Antibacterial activity, cell attachment and viability were tested to evaluate the biological properties of the membranes. The optimal cell compatible concentration of ZnO-NPs was determined for further studies. In vitro characterization of the membranes was performed to confirm their suitable properties for wound healing. RESULTS: The antibacterial PEO/ZnO-NP membrane containing 2% of nanoparticles showed no cell toxicity, and human fibroblast cells were able to adhere and proliferate on the scaffold. The in vitro results from the tensile test, wettability, porosity, and protein adsorption revealed appropriate properties of the membrane as a scaffold for skin tissue engineering. CONCLUSIONS: Synthetic polymers have been widely used for tissue engineering applications. The proper characteristics of PEO nanofibers, including a high ratio of surface/volume, moderate hydrophilicity and good mechanical properties, make this polymer interesting for skin regeneration. The results demonstrate the potential of the antibacterial PEO/ZnO-NP membrane to be used as an engineered scaffold to improve the wound healing process.

Development of new nanofibrous nerve conduits by PCL-Chitosan-Hyaluronic acid containing Piracetam-Vitamin B12 for sciatic nerve: A rat model.

Peripheral nerve injury is a critical condition that can disrupt nerve functions. Despite the progress in engineering artificial nerve guidance conduits (NGCs), nerve regeneration remains challenging. Here, we developed new nanofibrous NGCs using polycaprolactone (PCL) and chitosan (CH) containing piracetam (PIR)/vitamin B12(VITB12) with an electrospinning method. The lumen of NGCs was coated by hyaluronic acid (HA) to promote regeneration in sciatic nerve injury. The NGCs were characterized via Scanning Electron Microscopy (SEM), Fourier transform infrared (FTIR), tensile, swelling, contact angle, degradation, and drug release tests. Neuronal precursor cell line (PCL12 cell) and rat mesenchymal stem cells derived from bone marrow (MSCs) were seeded on the nanofibrous conduits. After that, the biocompatibility of the NGCs was evaluated by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, and SEM images. The SEM demonstrated that PCL/CH/PIR/VITB12 NGCs had nonaligned, interconnected, smooth fibers. The mechanical properties of these NGCs were similar to rat sciatic nerve. These conduits had an appropriate swelling and degradation rate. The In Vitro studies exhibited favorable biocompatibility of the PCL/CH/PIR/VITB12 NGCs towards PC12 cells and MSCs. The in vitro studies exhibited favorable biocompatibility of the PCL/CH/PIR/VIT B12 NGCs towards MSCs and PC12 cells. To analyze functional efficacy, NGCs were implanted into a 10 mm Wistar rat sciatic nerve gap and bridged the proximal and distal stump of the defect. After three months, the results of sciatic functional index (55.3 ± 1.8), hot plate latency test (5.6 ± 0.5 s), gastrocnemius muscle wet weight-loss (38.57 ± 1.6 %) and histopathological examination using hematoxylin-eosin (H&E) /toluidine blue/ Anti-Neurofilament (NF200) staining demonstrated that the produced conduit recovered motor and sensory functions and had comparable nerve regeneration compared to the autograft that can be as the gold standard to bridge the nerve gaps.

Fabrication of functional and nano-biocomposite scaffolds using strontium-doped bredigite nanoparticles/polycaprolactone/poly lactic acid via 3D printing for bone regeneration.

Bone tissue engineering is a field to manufacture scaffolds for bone defects that cannot repair without medical interventions. Ceramic nanoparticles such as bredigite have importance roles in bone regeneration. We synthesized a novel strontium (Sr) doped bredigite (Bre) nanoparticles (BreSr) and then developed new nanocomposite scaffolds using polycaprolactone (PCL), poly lactic acid (PLA) by the 3D-printing technique. Novel functional nanoparticles were synthesized and characterized using field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and energy dispersive spectroscopy (EDS: map). The nanoparticles were uniformly distributed in the polymer matrix composites. The 3D- printed scaffolds were investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), attenuated total reflection-fourier transform infrared (ATR-FTIR), degradation rate porosity, mechanical tests, apatite formation and cell culture. Degradation rate and mechanical strength were increased in the PLA/PCL/Bre-5%Sr nanocopmposite scaffolds. Hydroxyapatite crystals were also created on the scaffold surface in the bioactivity test. The scaffolds supported viability and proliferation of human osteoblasts. Gene expression and calcium deposition in the samples containing nanoparticles indicated statistical different than the scaffolds without nanoparticles. The nanocomposite scaffolds were implanted into the critical-sized calvarial defects in rat for 3 months. The scaffolds containing Bre-Sr ceramic nanoparticles exhibited the best potential to regenerate bone tissue.

Isolation and Characterization of Crab Haemolymph Exosomes and Its Effects on Breast Cancer Cells (4T1).

OBJECTIVE: The use of animal or plant exosomes in cancer treatment is promising because of their easy access and low cost. Freshwater crabs are used in traditional Iranian medicine to treat cancer. This study aims to determine the anti-cancer properties of exosomes removed from freshwater crabs on a breast cancer cell line (4T1) compared to bone marrow mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: In this experimental study, crab haemolymph exosomes were isolated via the precipitation method and characterised by electron microscopy, dynamic light scattering (DLS), and Western blot analysis. The protein concentration and total antioxidant capacity of these exosomes were determined by bicinchoninic acid (BCA) and cupric reducing antioxidant capacity (CUPRAC). The 4T1 cells and BMSCs were treated with exosomes and we assessed the cell survival by the resazurin and MTT assays. The level of nitric oxide (NO) secretion from the 4T1 cells was determined after treatment with the exosomes. RESULTS: Electron microscopy, DLS and Western blot for CD63 confirmed that the isolated exosomes were <100 nm in size and expressed CD63. The total antioxidant capacity in these exosomes was 1.003 µM/ml and the protein concentration was 650 mg/ml. Resazurin and MTT assay results showed a decrease in survival of the 4T1 cells (P≤0.001) after treatment with the exosomes compared to cell growth in the exosome-treated BMSCs. CONCLUSION: Crab haemolymph contains protein-rich exosomes with antioxidant activities that can have anti-cancer effects on 4T1 cells. These exosomes may be proposed for breast cancer therapeutics.

Evaluating the effects of vacuum on the microstructure and biocompatibility of bovine decellularized pericardium.

The aim of this study was evaluating the effects of vacuum on microstructure and biocompatibility of bovine decellularized pericardium. So the bovine pericardia were decellularized and then the vacuum was applied for two periods of time; 90 and 180 min. DNA, glucose amino glycan, collagen and elastin content assay, scanning electron microscopy (SEM) examination, hematoxylin and eosin (H&E) and Masson's trichrome stainings performed to evaluate microstructure of tissues. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, subcutaneous implantation, and tensile test were used to assay biocompatibility and mechanical properties of decellularized tissues. The results showed that applying vacuum reduced residual DNA significantly. Vacuum after 180 min reduced more residual DNA. There were no significant differences in the content of glucose amino glycan (GAG), collagen, and elastin between the vacuumed and control groups. SEM examination was revealed that vacuum for 180 min increased pore size and porosity more than 90 min and control groups. H&E and Masson's trichrome stainings revealed extracellular matrix preservation after decellularization in all groups. Cell viability was increased in vacuumed samples significantly after 72 h in vaccumed samples. H&E staining and tensile test after implantation of tissues were showed less inflammation in the vacuum applied tissues and increased durability. The vacuum increased DNA removal, pore size, porosity, and biocompatibility in vitro and in vivo and durability of bovine decellularized pericardium in vivo. Considering the important role of time, more studies should be performed to optimize time, intensity, and method of application of vacuum in decellularization of different tissues as well as bovine pericardium.

Characterization of Macroporous Polycaprolactone/Silk Fibroin/Gelatin/Ascorbic Acid Composite Scaffolds and In Vivo Results in a Rabbit Model for Meniscus Cartilage Repair.

OBJECTIVE: Meniscus injuries in the inner avascular zone have weak intrinsic self-healing capacity and often progress to osteoarthritis. This study focused on evaluating the effects of polycaprolactone/silk fibroin/gelatin/ascorbic acid (PCL/SF/Gel/AA) composite scaffolds seeded with adipose-derived mesenchymal stem cells (ASCs), in the meniscus repair. DESIGN: To this end, composite scaffolds were cross-linked using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride. Scaffolds were then characterized by scanning electron microscope, mechanical tests, total antioxidant capacity, swelling, and toxicity tests. RESULTS: The PCL/SF/Gel/AA scaffolds exhibited suitable mechanical properties. Furthermore, vitamin C rendered them the highest antioxidant capacity. The PCL/SF/Gel/AA scaffolds also showed good biocompatibility and proliferation for chondrocytes. Moreover, the PCL/SF/Gel/AA scaffold seeded with allogeneic ASCs was engrafted in New Zealand rabbits who underwent unilateral punch defect in the medial meniscus of the right knee. After 2 months postimplantation, macroscopic and histologic studies for new meniscus cartilage were performed. CONCLUSIONS: Our results indicated that the PCL/SF/Gel/AA composite scaffolds seeded with allogeneic ASCs could successfully improve meniscus healing in damaged rabbits.

Macroporous scaffold surface modified with biological macromolecules and piroxicam-loaded gelatin nanofibers toward meniscus cartilage repair.

Meniscus cartilage has poor self-healing capacity in the inner zone and its damage leads to articular cartilage degeneration. Here we have developed hybrid constructs using polycaprolactone (PCL) and polyurethane (PU) surface modified by gelatin (G), chitosan (C), and hyaluronic acid (H) biomacromolecules and piroxicam-loaded gelatin nanofibers (PCL/PU/GCH/P). The surface of constructs was crosslinked using EDC and NHS. The scaffolds were investigated by SEM, FTIR spectroscopy, swelling test, degradation rate, mechanical tests, and in vitro piroxicam release assay. Furthermore, the cell-seeded scaffolds were evaluated by SEM, viability assay, dapi staining, cell migration, proliferation, and gene expression of chondrocytes within these scaffolds. Finally, the animal study was performed in a rabbit model. Chondrocyte and rabbit adipose-derived mesenchymal stem cells (ASCs) from the infrapatellar fat pad (Hoffa's fat pad) were used. Swelling and degradation rate were increased in the modified scaffolds. Tensile and compressive Young's modulus also were near to human native meniscus tissue. The highest expression level of chondrocyte marker genes was observed for the PCL/PU/GCH scaffold. A significant regeneration was obtained in rabbits treated with ASCs-loaded PCL/PU/GCH/P scaffold after 3 months. The surface-modified scaffolds with or without ASCs could successfully accelerate meniscus regeneration and exhibit potential application in meniscus tissue engineering.

Chondrocytes Proliferation of Patients with Cartilage Lesions in Their Own Body for Use in Cartilage Tissue Engineering: Hypotheses on a New Approach for the Proliferation of Autologous Chondrocytes.

Osteoarthritis is one of the most common chronic diseases, which have involved 250 million people around the world. One of the challenges in the field of cartilage tissue engineering is to provide an adequate source of chondrocytes to prevent changes in gene expression profile as a result of multiple passages.We hypothesized that by creating a low invasive lesion by scalpel or shear laser in the outer ear cartilage and stimulation of wound healing process, hyperplasia occurs and will provide an appropriate number of autologous chondrocytes for extraction and use in articular cartilage tissue engineering. Also, due to the effect of platelet-rich plasma and biomechanical forces in stimulating and accelerating of the repair process, these two factors can be used to achieve more desirable results.We describe a new approach to proliferate chondrocytes in the body. To evaluate this idea, various techniques of gene expression at the level of RNA or protein and animal experiments for histological studies can be used. Also, flowcytometry technique can be used to determine the cell viability and counting them.The use of autologous cell sources with minimal changes in gene expression profile can be promising in tissue engineering products.

Evaluation of vacuum washing in the removal of SDS from decellularized bovine pericardium: method and device description.

AIMS: The aim of this study was to present a new method for removing Sodium dodecyl sulfate (SDS) detergent from decellularized bovine pericardium using vacuum. MATERIALS AND METHODS: The cows' pericardia were collected and decellularized. The samples were incubated with SDS1% for 48 h at 40 °C. To perform vacuum washing (VW: negative pressure was used to wash and remove detergents), every decellularized tissue was cut in 75mm diameter and fixed via a stainless-steel ring with 60mm diameter in the center of filtration Buchner Funnel which was connected to glass filtration flask The system was connected to a vacuum pump by a hose, and a negative pressure of -100 mmHg was applied for 15 min. Then, the samples were shaken and washed at 40-rpm in 100 ml of distilled water for 45 min. This process was repeated for samples of each group (6 times for sample VW6h, 12 times for sample VW12h, and 24 times for sample VW24h). At the end of every cycle, the effluent was collected to take a sample for SDS measurement. The normal washing (NW) group containing distilled water (NWd) and PBS (Phosphate buffered saline) (NWp) were used to wash and remove detergents. SDS measurements, MTT Assay, histological and tensile test, to compare two methods were used. RESULTS: The highest SDS in the effluent was in groups VW12h and VW24h (P ≤ 0.001) and the lowest residual SDS in scaffold was in two groups of VW12h and VW24h (P ≤ 0.001). MTT assay showed that cell survival in the VW12h and VW24h groups was higher than other groups and there' was no significant difference between cell survival in the VW12h and VW24h groups. Histological study showed destruction of tissue in the VW24h group. The results of the tensile test were shown that the native group had the highest module and the lowest amount was the VW24h sample which was reported with P ≤ 0.001 significance for all groups. CONCLUSION: VW12h can be used as an effective method for SDS removal from decellularized pericardium which morphologically demonstrated a good structure in ECM.

Fibrin gel as a scaffold for photoreceptor cells differentiation from conjunctiva mesenchymal stem cells in retina tissue engineering.

Stem cell-based therapies are attraction approaches for regenerative medicine for treating retinal diseases. One of the limitations in cell therapy is cell death following post-injection whit preventing functional integration with retinal tissue. Fibrin gel, a bio-polymeric material with excellent biocompatibility, provides numerous advantages as a tissue engineering scaffold and a stem cell carrier. Therefore, current research is focusing on developing fibrin hydrogel scaffolds to protect stem cells during delivery and to stimulate endogenous regeneration through interactions of transplanted stem cells and retinal tissue. In this study fibrin gel was used as hydrogel scaffold for immobilization of cells. The structural characteristics of fibrin gel scaffold were examined with SEM. Rheological properties of fibrin gel were measured by rheometer and biodegradation rate of fibrin were assayed for 2 weeks. After isolation of stem cells CJMSCs, the cells were differentiated into photoreceptor-like cells by exposing with taurin for 14 days in tissue culture plate (TCP group) and fibrin hydrogel (3 D group). The attachment of cells was analyzed with SEM and MTT. The expression of rhodopsin, PKC, CRX, recoverin, peripherin, nestin and RPE65 as photoreceptor-like cell markers was evaluated by immunocytochemistry and quantitative real-time PCR (RT-PCR) in TCP and 3 D groups. The results of SEM analysis showed CJMSCs were well attached in fibrin gels and there were good integrity between cells and scaffold. The elastic modulus and constant degradation of the gel contributes to the growth and proliferation of cells. There was no toxicity effect of fibrin hydrogel on cells and the viability of cultured cells was higher in 3 D fibrin gels in comparison with TCP groups. After 2 weeks, the expression of rhodopsin, PKC, CRX, peripherin, recoverin, nestin and RPE65 as special markers of photoreceptor cells were detected by Real time PCR and immunofluorescence that these expressions in 3 D groups were higher than TCP groups. In conclusion, our findings showed that application of readily available sources of adult stem cells like human conjunctiva stem cells encapsulated in fibrin gel could be interesting strategy to enhance photoreceptor progenitor cell numbers for repair and regeneration of retina disease such as photoreceptor injury.

Anti-inflammatory Effects of Atorvastatin in Human Glioblastoma Spheroids Cultured in a Three-dimensional Model: Possible Relevance to Glioblastoma Treatment.

Glioblastoma multiform (GBM) is a primary malignant brain tumor with a few therapeutic targets available for it. The interaction between the immune system and glioma is an important factor that could lead to novel therapeutic approaches to fight glioma. In this study, we investigated in vitro anti-inflammatory and apoptotic activity of atorvastatin in different concentrations 1, 5, and 10 µM on glioma spheroid cells cultured in a three-dimensional model in fibrin gel that indicate the complex in vivo microenvironment better than a simple two-dimensional cell culture. A mechanistic insight into the role of IL-17RA, TRAF3IP2, and apoptotic genes in progression of glioma could provide an important way for therapy of malignant tumors with manipulation of this inflammatory axis. To reach for these aims, after 24 and 48 h exposure with different concentrations of atorvastatin, caspase-8, caspase-3, Bcl-2, TRAF3IP2, and IL-17RA gene expression were assayed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cell cycle assay were used for evaluating the cell apoptosis and proliferation. The results showed that atorvastatin has anti-inflammatory and apoptotic effects against glioma spheroids. Atorvastatin induced the expression of caspase-3 and caspase-8 and downregulated the expression of Bcl-2, TRAF3IP2, and IL-17RA especially at 10 µM concentration. These effects are dose dependent. The most likely mechanisms are the inhibition of inflammation by IL-17RA interaction with TRAF3IP2 and NF-κB signaling pathway. Finally, these results suggest that atorvastatin could be used as an anti-cancer agent for glioblastoma treatment.

Retina tissue engineering by conjunctiva mesenchymal stem cells encapsulated in fibrin gel: Hypotheses on novel approach to retinal diseases treatment.

BACKGROUND: Retinitis pigmentosa (RP) and age related macular degeneration (AMD) are two retinal diseases that progress by photoreceptor cells death. In retinal transplantation studies, stem and progenitor cells inject into the sub retinal space or vitreous and then these cells can be migrate to the site of retinal degeneration and locate in the host retina and restitute vision. PRESENTATION OF THE HYPOTHESIS: Our hypothesis suggests that using human conjunctiva stem cells (as the source for increasing the number of human stem cells progenitor cells in retina dysfunction diseases) with fibrin gel and also assessing its relating in vitro (cellular and molecular processes) and in vivo (vision tests and pathology) could be a promising strategy for treatment of AMD and RP disorders. TESTING THE HYPOTHESIS: In this idea, we describe a novel approach for retina tissue engineering with differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells in fibrin gel with induction medium contain taurine. For assessment of differentiation, immunocytochemistry and real time PCR are used for the expression of Rhodopsin, RPE65, Nestin as differentiated photoreceptor cell markers in 2D and 3D culture. The results show that fibrin gel will offer a proper 3D scaffold for CJMSCs derived photoreceptor cell-like cells. IMPLICATIONS OF THE HYPOTHESIS: Application of immune-privileged, readily available sources of adult stem cells like human conjunctiva stem cells with fibrin gel would be a promising strategy to increase the number of photoreceptor progenitor cells and promote involuntary angiogenesis needed in retina layer repair and regeneration.