Effect of amniotic membrane/collagen scaffolds on laryngeal cartilage repair.

Objectives: Laryngeal cartilage defects are a major problem that greatly impacts structural integrity and function. Cartilage repair is also a challenging issue. This study evaluated the efficacy of a collagen scaffold enveloped by amniotic membrane (AM/C) on laryngeal cartilage repair. Study Design: Experimental animal study. Methods: Fourteen Dutch rabbits were enrolled in the study. A 5 mm cartilage defect was created in the right and left thyroid lamina. The animals were divided into two groups randomly. Group 1 collagen scaffolds and group 2 AM/C were applied to the right side defects. Left side defects were not repaired, serving as control. Histologic evaluation was done 45 and 90 days following collagen and AM/C application with criteria of tissue and cell morphology, lacuna formation, vascularization, and inflammation. Results: Significant improvement in cartilage repair was observed in the AM/C side compared to the control side in all histologic criteria after 45 days (p<.05). After 90 days, cartilage repair improved in cell morphology, lacuna formation, and inflammation significantly (p<.05). Conclusion: The combination of amniotic membrane and collagen scaffolds provides a promising treatment modality for improving the repair of laryngeal cartilage defects. Level of Evidence: NA.

Potential advantages of genetically modified mesenchymal stem cells in the treatment of acute and chronic liver diseases.

Liver damage caused by toxicity can lead to various severe conditions, such as acute liver failure (ALF), fibrogenesis, and cirrhosis. Among these, liver cirrhosis (LC) is recognized as the leading cause of liver-related deaths globally. Unfortunately, patients with progressive cirrhosis are often on a waiting list, with limited donor organs, postoperative complications, immune system side effects, and high financial costs being some of the factors restricting transplantation. Although the liver has some capacity for self-renewal due to the presence of stem cells, it is usually insufficient to prevent the progression of LC and ALF. One potential therapeutic approach to improving liver function is the transplantation of gene-engineered stem cells. Several types of mesenchymal stem cells from various sources have been suggested for stem cell therapy for liver disease. Genetic engineering is an effective strategy that enhances the regenerative potential of stem cells by releasing growth factors and cytokines. In this review, we primarily focus on the genetic engineering of stem cells to improve their ability to treat damaged liver function. We also recommend further research into accurate treatment methods that involve safe gene modification and long-term follow-up of patients to increase the effectiveness and reliability of these therapeutic strategies.

Optimizing artificial meniscus by mechanical stimulation of the chondrocyte-laden acellular meniscus using ad hoc bioreactor.

BACKGROUND: Tissue engineering focuses on reconstructing the damaged meniscus by mimicking the native meniscus. The application of mechanical loading on chondrocyte-laden decellularized whole meniscus is providing the natural microenvironment. The goal of this study was to evaluate the effects of dynamic compression and shear load on chondrocyte-laden decellularized meniscus. MATERIAL AND METHODS: The fresh samples of rabbit menisci were decellularized, and the DNA removal was confirmed by histological assessments and DNA quantification. The biocompatibility, degradation and hydration rate of decellularized menisci were evaluated. The decellularized meniscus was injected at a density of 1 × 105 chondrocyte per scaffold and was subjected to 3 cycles of dynamic compression and shear stimuli (1 h of 5% strain, ± 25°shear at 1 Hz followed by 1 h rest) every other day for 2 weeks using an ad hoc bioreactor. Cytotoxicity, GAG content, ultrastructure, gene expression and mechanical properties were examined in dynamic and static condition and compared to decellularized and intact menisci. RESULTS: Mechanical stimulation supported cell viability and increased glycosaminoglycan (GAG) accumulation. The expression of collagen-I (COL-I, 10.7-folds), COL-II (6.4-folds), aggrecan (AGG, 3.2-folds), and matrix metalloproteinase (MMP3, 2.3-folds) was upregulated compared to the static conditions. Furthermore, more aligned fibers and enhanced tensile strength were observed in the meniscus treated in dynamic condition with no sign of mineralization. CONCLUSION: Compress and shear stimulation mimics the loads on the joint during walking and be able to improve cell function and ultrastructure of engineered tissue to recreate a functional artificial meniscus.

Engineered artificial articular cartilage made of decellularized extracellular matrix by mechanical and IGF-1 stimulation.

Cartilage engineering has the potential to overcome clinical deficiency in joint disorders. Decellularized extracellular matrix (dECM) has great biocompatibility and bioactivity and can be considered an appropriate natural scaffold for tissue engineering applications. Both insulin-like growth factor-1 (IGF-1) and mechanical compression stimulate the production of cartilage ECM, modulate mechanical properties, and gene expression. The current investigation aimed to fabricate a high-quality moldable artificial cartilage by exposing the chondrocytes in biomimicry conditions using cartilage dECM, IGF-1, and mechanical stimulations. In this study, an ad hoc bioreactor was designed to apply dynamic mechanical stimuli (10 % strain, 1 Hz) on chondrocyte-laden cartilage dECM-constructs with/without IGF-1 supplementation for 2 weeks, 3 h/day. Our data revealed that mechanical stimulation had no adverse effect on cell viability and proliferation. However, it elevated the expression of chondrogenic markers such as collagen type II (COL2A1), aggrecan (ACAN), and proteoglycan-4 (PRG-4), and reduced the expression of matrix metalloproteinase-3 (MMP-3). Mechanical stimulation also promoted higher newly formed glycosaminoglycan (GAG) and produced more aligned fibers that can be responsible for higher Young's modulus of the engineered construct. Even though IGF-1 demonstrated some extent of improvement in developing neocartilage, it was not as effective as mechanical stimulation. Neither IGF-1 nor compression elevated the collagen type I expression. Compression and IGF-1 showed a synergistic impact on boosting the level of COL2A1 but not the other factors. In conclusion, mechanical stimulation on moldable cartilage dECM can be considered a good technique to fabricate artificial cartilage with higher functionality.

Synergistic impact of platelet rich plasma-heparin sulfate with hydroxyapatite/zirconia on the osteoblast differentiation potential of adipose-derived mesenchymal stem cells.

3D porous hydroxyapatite (HA) has been reinforced by zirconia (ZrO2) coating and impregnation with a combination of platelet rich plasma (PRP) as a source of growth factors (GFs) and Heparin sulfate (HS) to sustain the release of GFs. Adipose mesenchymal stem cells (ADMSCs) were characterized by flow cytometry for CD (cluster of differentiation) 44, CD105, CD106, CD34 and CD144, along with checking the multipotency by differentiation into the adipocytes and osteoblasts. Then, they were cultured on the scaffold treated with and without osteogenic media on days 7, 14 and 21. Electron micrograph and PKH staining show that the ADMSCs have a fusiform phenotype in the absence of osteogenic induction. Cell viability assay shows a higher number of the viable cells on the PRP-containing scaffolds than PRP-free scaffolds on day 7. Colorimetric evaluation, quantitative RT-PCR and immunocytochemistry demonstrate that PRP and HS significantly elevate the alkaline phosphatase enzyme activity and also accelerate the production of both early and mid-osteogenic markers, including collagen I and osteopontin expression with and without osteogenic conditions. The PRP-HS also accelerates the expression of the late osteogenic marker, osteocalcin, in both mRNA and protein level expression with a peak on day 21. In conclusion, supplementation of HA/ZrO2 with PRP/HS has a synergistic impact on the ADMSCs, even in the absence of chemical induction. It seems that HA/ZrO2/PRP/HS scaffold provides a higher osteoconductive microenvironment for stem cell differentiation to osteoblasts.

Novel strategy of cartilage repairing via application of P. atlantica with stem cells and collagen.

Osteoarthritis (OA) is an inflammatory joint condition, still lacking effective treatments. Some factors consider as the main causes of OA, including biochemical, mechanical, and genetic factors. The growth of studies confirmed that modern medicine in combination with folk medicine regarding the arrival of reliable, efficient, and safe therapeutic products against OA. In the present study, the effects of various single and combinatorial treatments of knee articular cartilage, including stem cells, collagen, and P. atlantica hydroalcoholic leaves extract were investigated in a rat-induced OA model. On week 12 after OA confirmation, histopathology and radiography assessments were evaluated and the serum and synovial fluid levels of TAC, TNF-α, PEG2, MPO, MMP3, MMP13, and MDA were also measured. Combination therapy of OA-induced rats with hydroalcoholic extract of P. atlantic leaves, stem cells, and collagen considerably increased the efficacy of treatment as evidenced by increasing the TAC and lowering TNF-α, MPO, MMP3, and MMP13 compared to control group and even groups received single therapy. This is in agreement with a high amount of total phenolic compounds and antioxidant capacities of the hydroalcoholic extract of P. atlantic leaves. It is concluded that multifunctional agents targeting the pathophysiology of OA has exhibited significant therapeutic effects against OA.

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1). METHODS: Human adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were grown in either normal medium alone (control group), the medium supplemented with AICAR (1 mM) and NAM (5 mM), or in the presence of both for 5 weeks to P10. Cell proliferation, differentiation capacity, level of apoptosis and autophagy, morphological changes, total cellular reactive oxygen species (ROS), and activity of mTORC1 and AMPK were compared among different treatment groups. RESULTS: MSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of ß-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy. CONCLUSIONS: Selective inhibition of mTORC1 by AICAR and NAM boosts autophagy, retains MSCs' self-renewal and multi-lineage differentiation capacity, and postpones senescence-associated changes after prolonged in vitro culture. Additionally, co-administration of AICAR and NAM shows an additive or probably a synergistic effect on cellular senescence.

Successful Management of a Suspected Case of Meperidine-Induced Anaphylaxis in Cesarean Delivery.

INTRODUCTION: Meperidine is known as the gold standard drug for shivering after spinal anesthesia (SA). This drug has been used widely and safely during the Cesarean Section (CS). CASE PRESENTATION: This case report presents an anaphylaxis reaction to a single intravenous dose of 25 mg meperidine, aiming to control shivering during CS under SA a few minutes after surgical incision. CONCLUSIONS: The condition was well managed with timely intervention. This rare fetal reaction to meperidine is worthy of reporting to make the medical team aware of the potential risks of anaphylaxis due to many routine safe drugs.

Concomitant use of mesenchymal stem cells and neural stem cells for treatment of spinal cord injury: A combo cell therapy approach.

TREATMENT: with neural stem cells (NSCs) provides a hope to recover the neural damage and compensate for the lost neural structures for restoration of interrupted neural communications above and below the site of injury. However, cell-based therapy approach suffers from many biological barriers and technical caveats which severely hamper the prognosis. The biochemically-rich microenvironment at the site of spinal cord injury (SCI), the continuing neuro-degenerative process and infiltrating immune cells offer a serious barrier to the donor cells. We hypothesized that mesenchymal stem cells (MSCs) concomitantly delivered with NSCs would significantly enhance the effectiveness of cell-based therapy for SCI. In a rodent model of SCI (n = 15 animals/group), MSCs labeled with PKH67 (green fluorescence dye) were delivered on day1 after SCI whereas the same animals were treated with NSCs during the subacute phase on day3 (group-5). In comparison with untreated control (group-1), sham group (without cell treatment; group-2), MSCs alone (group-3) and NSCs alone treated animals (group-4), the combined cell treated animals (group-5) showed significantly higher homing of cells at the site of injury during in vivo imaging. Caspase-3 activity was lower in group-5 (P < 0.05 vs all groups) with concomitant reduction in the pro-inflammatory cytokines IL-1ß and IL-6 (P < 0.05 vs all groups). All cell therapy groups showed significant improvement in neurological function as compared to group-2, however, it was highest in group-5 (P < 0.05 vs all groups). In conclusion, combined treatment with (NSCs + MSCs) enhances NSCs survival and functional recovery in SCI and is superior to the treatment with either of NSCs or MSCs alone.

Nicorandil potentiates sodium butyrate induced preconditioning of neurons and enhances their survival upon subsequent treatment with H2O2.

BACKGROUND: Extensive loss of donor neural stem cell (NSCs) due to ischemic stress and low rate of differentiation at the site of cell graft are two of the major issues that hamper optimal outcome in NSCs transplantation studies. Given that histone deacetylases (HDACs) modulate various cellular processes by deacetylating histones and non-histone proteins, we hypothesized that combined treatment with small molecules, sodium butyrate (NaB; a known HDAC inhibitor) and nicorandil, will enhance the rate neuronal differentiation of NSCs besides their preconditioning to resist oxidative stress. METHODS: NSCs derived from 14-day old Sprague Dawley rat ganglion eminence were characterized for tri-lineage differentiation. Treatment with 1 mM NaB significantly changed their culture characteristics while continuous treatment for 10 days enhanced their neural differentiation. NaB treatment also preconditioned the cells for their resistance to oxidative stress. RESULTS: The highest rate of neural differentiation and preconditioning effect was achieved when the NSCs were treated concomitantly with NaB and nicorandil. Cell proliferation assay showed that concomitant treatment with NaB and nicorandil retarded their rate of proliferation. CONCLUSION: These data conclude that preconditioning of NSCs with NaB and nicorandil effectively enhances their differentiation capacity besides preconditioning the cells to support their survival under ischemic conditions.

Differentiation Potential of Breast Milk-Derived Mesenchymal Stem Cells into Hepatocyte-Like Cells.

Human breast milk stem cells (hBSCs) contain a population of cells with the ability to differentiate into various cell lineages for cell therapy applications. The current study examined the differentiation potential of hBSCs into hepatocytes-like cells. The cells were isolated from the breast milk and were treated with hepatogenic medium containing hepatocyte growth factor, insulin-like growth factor and dexamethasone for 7 days subsequently; Oncostatin M was added to the culture media. RT-PCR and immunocytochemistry were performed to detect the hepatogenic markers. The glycogen storage and the ability of the cells to absorb and release indocynanin green were also tested. The data showed that most of the differentiated cells formed cell aggregates after the 30th day, with more cells accumulated to form spheroids. RT-PCR revealed the expression of the hepatic nuclear factor, albumin, cytokeratin 18 and 19, cytochrome P2B6, glucose-6-phospahtase and claudin. The functional assays also showed glycogen storage and omission of indicynine green. Our study demonstrated hBSCs are novel population that can differentiate into hepatocyte-like cells.

Fabrication and characterization of platelet-rich plasma scaffolds for tissue engineering applications.

Platelet-Rich Plasma (PRP), as a rich source of growth factor, can form a fibrin gel that recapitulates the extracellular matrix of the tissues. The aim of this study was to evaluate the effects of different concentrations of CaCl2 on the PRP scaffold structure which in turn could change the cell's behavior. PRP was mixed with 2.5, 5 and 10% (w/v) CaCl2. Then, the tensile strength, biodegradability and water content of the scaffolds were evaluated. We also performed immunostaining for assessment of the actin stress fiber orientation and SEM for detecting the cell phenotype and physical properties of the fibers. Cell viability, attachment and migration were also evaluated. The highest cell attachment and short term proliferation rate was observed on the scaffolds with 2.5% CaCl2. The cells cultured on the scaffold with higher CaCl2 concentration had fusiform phenotype with few cell processes and parallel arrangement of stress fibers while those cultured on the other scaffolds were fibroblast-like with more processes and net-like stress fibers. The scaffolds with 10% CaCl2 demonstrated the highest osmolarity (358.75±4.99mOsmole), fiber thickness (302.1±54.3nm), pore size (332.1±118.9nm2) and the longest clotting time (12.2±0.776min) compared with the other scaffolds. Water content, branching angle, porosity, orientation and tensile strength did not change by gelation with different CaCl2 concentrations. In conclusion, the cell shape, viability and proliferation were modified by culturing on the PRP scaffolds prepared with various concentrations of CaCl2, and as a result, the scaffolds showed different physical and biological properties.

Partial replacement of left hemidiaphragm in dogs by either cryopreserved or decellularized heterograft patch.

OBJECTIVES: Large diaphragmatic defects are still a challenging issue for reconstruction using either synthetic prosthesis or bioprosthesis. To evaluate the possibility of using diaphragm allograft as a natural bioprosthesis in humans, we conducted a two-group study and compared cryopreserved and decellularized diaphragmatic heterograft patched in a canine model. METHODS: At the end of organ harvesting from a human donor, the left hemidiaphragm was taken to the laboratory in phosphate-buffered saline solution. The next step was freezing the grafts at -80°C, and preserving them for up to 2 months in Group 1. It was subjected to a detergent-enzymatic method (containing sodium deoxycholate/DNase lavations) of decellularization for 25 cycles in Group 2. Through left thoracotomy in the eighth intercostal space, cryopreserved patches in six dogs and decellularized patches in five dogs replaced the diaphragm. During the follow-up, sonography was done in all animals, but three and two dogs in Group 1 and 2 underwent computed tomography (CT) scan, respectively. The animals were euthanized after 6 months. RESULTS: There was no mortality. Sonography showed only motion impairment of the patches in all cases. In Group 1, CT scan showed mild atelectasis and scattered infiltration in the left lower lobe, fibrotic bands and minimal fluid collection under the diaphragm. In Group 2, CT scan showed scattered fibrotic bands and mild to moderate elevation of the left hemidiaphragm. There was no evidence of gross disruption and complete healing of the suture line. Necropsy in both groups showed patches were completely replaced with a dense fibrous tissue. In Group 1, focal calcification was noticeable in every case and foreign body-type granulomas were clearly seen all over the grafted tissue. Histology in Group 2 animals showed less inflammatory cell infiltration and scattered foreign body granulomas in comparison with the cryopreserved patch graft. CONCLUSIONS: The gross healing process in the decellularized heterograft is similar to the cryopreserved diaphragm but with fewer inflammatory cells and foreign body granulomas on histology. Both of them can be used instead of bioprostheses with regard to the fact that the decellularized patch technique is more complex and expensive. It is recommended to compare them with commercial bioprostheses.

The Preventive Effects of Neural Stem Cells and Mesenchymal Stem Cells Intra-ventricular Injection on Brain Stroke in Rats.

INTRODUCTION: Stroke is one of the most important causes of disability in developed countries and, unfortunately, there is no effective treatment for this major problem of central nervous system (CNS); cell therapy may be helpful to recover this disease. In some conditions such as cardiac surgeries and neurosurgeries, there are some possibilities of happening brain stroke. Inflammation of CNS plays an important role in stroke pathogenesis, in addition, apoptosis and neural death could be the other reasons of poor neurological out come after stroke. In this study, we examined the preventive effects of the neural stem cells (NSCs) and mesenchymal stem cells (MSCs) intra-ventricular injected on stroke in rats. AIM: The aim of this study was to investigate the preventive effects of neural and MSCs for stroke in rats. MATERIALS AND METHODS: The MSCs were isolated by flashing the femurs and tibias of the male rats with appropriate media. The NSCs were isolated from rat embryo ganglion eminence and they cultured NSCs media till the neurospheres formed. Both NSCs and MSCs were labeled with PKH26-GL. One day before stroke, the cells were injected into lateral ventricle stereotactically. RESULTS: During following for 28 days, the neurological scores indicated that there are better recoveries in the groups received stem cells and they had less lesion volume in their brain measured by hematoxylin and eosin staining. Furthermore, the activities of caspase-3 were lower in the stem cell received groups than control group and the florescent microscopy images showed that the stem cells migrated to various zones of the brains. CONCLUSION: Both NSCs and MSCs are capable of protecting the CNS against ischemia and they may be good ways to prevent brain stroke consequences situations.

Origins of the breast milk-derived cells; an endeavor to find the cell sources.

Fresh human breast milk consists of a heterogeneous population of cells that may offer a non-invasive source of cells for therapeutic proposes. The aims of this study were to characterize the breast milk-derived cells cultured in vitro. To do this, the cells from human breast milk were cultured and the expression of the CD markers along with the embryonic stem cell markers, endothelial and luminal mammary epithelial cell markers was evaluated by flow cytometry and immunofluorescence. The presence of fetal microchimerism among the isolated cells was also determined by the presence of SRY gene. They were also differentiated into adipocytes and osteoblasts. The results showed that a remarkable number of cells expressed the mesenchymal stem cell (MSC) markers such as CD90, CD44, CD271, and CD146. A subpopulation of the human breast milk-derived cells (HBMDC) also expressed the embryonic stem cell markers, such as TRA 60-1, Oct4, Nanog and Sox2 but not SSEA1 or 4. The frequencies of the cells which expressed the endothelial, hematopoietic cell markers were negligible. SRY gene was not detected in the breast milk isolated cells. A subpopulation of the cells also expressed cytokeratin 18, the marker of luminal mammary epithelial cells. These cells showed the capability to differentiate into adipocytes and osteoblasts. In conclusion, these finding highlighted the presence of cells with various sources in the breast milk. Different stem cells including MSCs or embryonic stem cell-like cell along with the exfoliated cells from luminal epithelial cells were found among the isolated cells. The breast milk-derived stem cells might be considered as a non-invasive source of the stem cells for therapeutic purpose.

Differentiation of human breast-milk stem cells to neural stem cells and neurons.

Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing fibroblast growth factor (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF) 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for ß-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.