Efficient Wound Healing Using a Synthetic Nanofibrous Bilayer Skin Substitute in Murine Model.

Treatment of full-thickness skin wounds with minimal scarring and complete restoration of native tissue properties still exists as a clinical challenge. A bilayer skin substitute was fabricated by coating human amniotic membrane (AM) with electrospun silk fibroin nanofibers, and its in vivo biological behavior was studied using murine full-thickness skin wound model. Donut-shaped silicon splints were utilized to prevent wound contraction in mouse skin and simulate re-epithelialization, which is the normal path of human wound healing. Skin regeneration using the bilayer scaffold was compared with AM and untreated defect after 30 d. Tissue samples were taken from healed wound areas and investigated through histopathological and immunohistochemical staining to visualize involucrin (IVL), P63, collagen I, CD31, and vascular endothelial growth factor. In addition, mRNA expression of IVL, P63, interleukin-6, and cyclooxygenase-2 was studied. The application of bilayer scaffold resulted in the best epidermal and dermal regeneration, demonstrated by histopathological examination and molecular analysis. In regenerated wounds of the bilayer scaffold group, the mRNA expression levels of inflammatory markers (interleukin-6 and cyclooxygenase-2) were downregulated, and the expression pattern of keratinocyte markers (IVL and P63) at both mRNA and protein levels was more similar to native tissue in comparison with AM and no-treatment groups. There was no significant difference in the expression level of collagen I, CD31, and vascular endothelial growth factor among different groups. Conclusively, these promising results serve as a supporting evidence for proceeding to clinical phase to examine the capacity of this bilayer scaffold for human skin regeneration.

Correction to: "Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocyes during 36 weeks in rabbit model.

The published online of the original version contains mistakes.

Down-regulation of miR-122 after transplantation of mesenchymal stem cells in acute liver failure in mice model.

This study investigated the correlation between the hepatic level of miR-122 and the extent of liver tissue regeneration in CCl4 induced liver injury mice model following transplantation of menstrual blood-(MenSCs) and bone marrow-derived stem cells (BMSCs). Hepatic miR-122 levels were significantly up-regulated following administration of CCl4 (P < 0.01). The significant positive correlations were observed between hepatic miR-122 and biochemical serum markers and the severity of liver injury in histopathological assessments (P < 0.01). Following stem cell therapy, all cell treated groups showed a significant down-regulation in miR-122 that was significantly correlated with improvement in histopathological features and biochemical markers (P < 0.01). Furthermore, the hepatic level of miR-122 was lower in the MenSCs-treated group compared with the BMSCs-treated group (P < 0.01) and in HPL cells-treated groups in reference to undifferentiated cells-treated groups (P < 0.05). These data suggest that miR-122 could be used as a potential predictor of outcome of liver injury after mesenchymal stem cell transplantation.

Bilayer Amniotic Membrane/Nano-fibrous Fibroin Scaffold Promotes Differentiation Capability of Menstrual Blood Stem Cells into Keratinocyte-Like Cells.

The skin provides a dynamic barrier separating and protecting human body from the exterior world, and then immediate repair and rebuilding of the epidermal barrier is crucial after wound and injury. Wound healing without scars and complete regeneration of skin tissue still remain as a clinical challenge. The demand to engineer scaffolds that actively promote regeneration of damaged areas of the skin has been increased. In this study, menstrual blood-derived stem cells (MenSCs) have been induced to differentiate into keratinocytes-like cells in the presence of human foreskin-derived keratinocytes on a bilayer scaffold based on amniotic membrane and silk fibroin. Based on the findings, newly differentiated keratinocytes from MenSCs successfully expressed the keratinocytes specific markers at both mRNA and protein levels judged by real-time PCR and immunostaining techniques, respectively. We could show that the differentiated cells over bilayer composite scaffolds express the keratinocytes specific markers at higher levels when compared with those cultured in conventional 2D culture system. Based on these findings, bilayer amniotic membrane/nano-fibrous fibroin scaffold represents an efficient natural construct with broad applicability to generate keratinocytes from MenSCs for stem cell-based skin wounds healing and regeneration.

Comparative restoration of acute liver failure by menstrual blood stem cells compared with bone marrow stem cells in mice model.

BACKGROUND AIMS: The application of menstrual blood stem cells (MenSCs) in regenerative medicine is gaining increasing attention. The aim of this study was to investigate the therapeutic potential of MenSCs compared with bone marrow-derived stem cells (BMSCs) in an animal model of CCl4-induced acute hepatic failure. METHODS: Injured Balb/C mice were divided into multiple groups and received MenSCs, BMSCs or hepatocyte progenitor-like (HPL) cells derived from these cells. RESULTS: Tracking of green fluorescent protein-labeled cells showed homing of cells in injured areas of the liver. In addition, the liver engraftment of MenSCs was shown by immunofluorescence staining using anti-human mitochondrial antibody. Microscopically examination, periodic acid-Schiff and Masson's trichrome staining of liver sections demonstrated the considerable liver regeneration post-cell therapy in all groups. Assessment of serum parameters including aspartate aminotransferase, alanine aminotransferase, total bilirubin, urea and cholesterol at day 7 exhibited significant reduction, such that this downward trend continued significantly until day 30. The restoration of liver biochemical markers, changes in mRNA levels of hepatic markers and the suppression of inflammatory markers were more significant in the MenSC-treated group compared with the BMSC-treated group. On the other hand, HPL cells in reference to undifferentiated cells had better effectiveness in the treatment of the acute liver injury. CONCLUSIONS: Our data show that MenSCs may be considered an appropriate alternative stem cell population to BMSCs for treatment of acute liver failure.

In vitro differentiation of menstrual blood stem cells into keratinocytes: A potential approach for management of wound healing.

The skin wounds caused by insults should be treated immediately to restore the functions and integrity. Recent studies suggest that stem cells-based therapies may be applicable in wound healing. Newly defined menstrual blood-derived stem cells (MenSCs) show high rate of cell proliferation and trans-differentiation potency to various cell types. However, MenSCs potential to generate keratinocyte for future therapeutic use of skin lesions has been remained to investigate. We cultivated MenSCs in the presence of isolated foreskin derived-keratinocytes using an indirect co-culture system and evaluated efficiency of this protocol to generate keratinocytes using immunofluorescent staining and Real Time PCR technique. Our results showed that differentiated keratinocytes express epidermal/keratinocytes lineage specific markers such as K14, p63, and involucrin at both mRNA and protein levels. Immunofluorescent staining showed the expression of involucrin and K14 in differentiated cells in contrast to undifferentiated cells. Moreover, mRNA expression levels of K14 (11.1 folds, p = 0.001), p63 (10.23 folds, p = 0.001), and involucrin (2.94 folds, p = 0.001) were higher in differentiated MenSCs compared to non-cocultured cells. Therefore, we firstly presented evidence about differentiation capability of MenSCs into epidermal/keratinocytes lineage. Considering the advantages of MenSCs such as great accessibility, these stem cells are promising for stem cells-based therapies of skin defects.

Comparative Immunophenotypic Characteristics, Proliferative Features, and Osteogenic Differentiation of Stem Cells Isolated from Human Permanent and Deciduous Teeth with Bone Marrow.

To find out differences and similarities in phenotypic, proliferative, and trans-differentiation properties of stem cells isolated from pulp of deciduous (SHEDs) and permanent (DPSCs) teeth with human bone marrow stem cells (BMSCs), we examined the expression of mesenchymal and embryonic stem cell markers in relation to the proliferation and osteogenic differentiation potentials of these cells. In this way, after isolating SHEDs, DPSCs, and BMSCs, cell proliferation was evaluated and population doubling time was calculated accordingly. Expression patterns of mesenchymal, hematopoietic, and embryonic stem cell markers were assessed followed by examining differentiation potential toward osseous tissue through alizarin red staining and qRT-PCR. Based on the results, the proliferation rates of SHEDs and DPSCs were significantly higher than that of BMSCs (P < 0.0001). High expression of mesenchymal stem cell markers and weak expression of hematopoietic markers were observed in all the three groups. The mean expression of OCT-4 was significantly higher in SHEDs and DPSCs (P = 0.028), while the expression of SSEA-4 was lower (P = 0.006) compared to BMSCs. Osteogenic differentiation potential of SHEDs was greater than DPSCs; however, it was lower than that of BMSCs. Conclusively, the distinctive immunophenotyping, proliferation rate, and differentiation pattern of SHEDs and DPSCs discriminate these cells from BMSCs. Furthermore, dissimilarity in differentiation potential is evidence implying that SHEDs might be more primitive stem cell population compared to DPSCs.

Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocytes during 36 weeks in rabbit model.

The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies.

Fabrication and characterization of nano-fibrous bilayer composite for skin regeneration application.

Full thickness wound healing with minimal scarring and complete restoration of normal skin properties still remains as a clinical challenge. In this study, a bilayer skin substitute has been fabricated to biomimic the microstructure of natural extracellular matrix of the skin. Human amniotic membrane (HAM) and silk fibroin nano-fibers were combined to produce bilayer construct, which was further treated and characterized. HAM was obtained from healthy mothers and de-epithelized by means of fine enzymatic method to preserve the extracellular structure. Fibroin protein was extracted from fresh Bombyx mori cocoons and transformed to uniform nano-fiberous structure, which was used as a coating layer on the de-epithelized membrane. Surface modification through oxygen plasma treatment was attempted to further induce hydrophilicity. Subsequently, scaffolds were fully characterized in terms of morphology, mechanical properties, hydrophilicity and cell culture response. Histological and immunohistological staining demonstrated localization of fibronectin, cell denudation and structural integrity of HAM after de-epithelization. Scanning electron microscopy images showed bead-free silk fibroin nano-fibers with the average diameter of 250nm. Water contact angle of bilayer scaffolds reduced dramatically to 26.34° after oxygen plasma treatment, which is correlated with more hydrophilic surface. Due to fibroin nano-fiber coating, mechanical properties of HAM improved significantly. Tensile Young's modulus and tensile strength increased from 16.14MPa and 68.46MPa to 25.69MPa and 108.03MPa, respectively. 14days in vitro cultivation of mouse embryonic fibroblasts on the scaffolds revealed that bilayer scaffolds are able to support cell attachment and proliferation. Plasma-etched scaffolds provided the best niche for cell-matrix crosstalk by allowing cells to penetrate beneath the pores and to integrate in fibers direction. The obtained results suggest that the presented nano-fibrous bilayer composite based on HAM is a potential substitute for skin regeneration application.

Comparative evaluation of in vivo biocompatibility and biodegradability of regenerated silk scaffolds reinforced with/without natural silk fibers.

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration. spjba;30/6/793/FIG10885328215601925F1fig1-0885328215601925Figure 1.Illustrative summary of the main methods and findings.RS: regenerated silk; RSF: regenerated fibroin/ silk fiber composite scaffolds; H&E: Hematoxylin and eosin; COX-1: Cyclooxygenase.

Gene expression pattern of some classes of cytochrome P-450 and glutathione S-transferase enzymes in differentiated hepatocytes-like cells from menstrual blood stem cells.

Recently, valuable characteristics of menstrual blood stem cells (MenSCs) have impelled scientists to take its advantages for cell therapy of different diseases including liver disorders. In this study, we examined messenger RNA (mRNA) expression levels of phases I and II drug metabolizing enzymes including glutathione S-transferase (GST) and cytochrome P-450 (CYP) in differentiated hepatocyte-like cells from MenSCs. The isolated MenSCs were characterized and differentiated into hepatocyte-like cells using hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum-free culture media. After primary characterization of hepatocyte markers, mRNA expression of GSTA1, GSTA2, GSTP1, CYP3A4, and CYP7A1 was assessed in differentiated cells in reference to undifferentiated cells using real-time PCR. Based on immunofluorescent staining and real-time PCR data, the differentiated MenSCs could express functional hepatocyte markers at mRNA and/or protein levels suggesting development of hepatocyte-like cells from MenSCs. Moreover, the expression levels of GSTA1, GSTA2, and CYP3A4 mRNA were upregulated in differentiated cells compared to undifferentiated cells. The expression of CYP7A1 gene was also remarkable on the last day of differentiation process. However, the expression level of GSTP1 did not exhibit statistically significant change during differentiation (P = 0.6). Based on accumulative data, MenSCs could be viewed as an accessible population of stem cells with differentiation ability into drug-metabolizing hepatocyte-like cells.

Efficient generation of functional hepatocyte-like cells from menstrual blood-derived stem cells.

In recent years, the advantages of menstrual blood-derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum-supplemented or serum-free culture media, were also investigated. The sequential differentiation was monitored by real-time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT-4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin-18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte-specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte-like cells. We recommend a complementary serum-free differentiation protocol for enrichment of in vitro production of functional MenSC-derived hepatocyte-like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases.

Comparative evaluation of differentiation potential of menstrual blood- versus bone marrow-derived stem cells into hepatocyte-like cells.

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

Differentiation potential of menstrual blood- versus bone marrow-stem cells into glial-like cells.

Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells that have been interested for cell therapy of neurodegenerative diseases. In this study, we showed conversion of menstrual blood stem cells (MenSCs) into clonogenic neurosphere- like cells (NSCs), which can be differentiated into glial-like cells. Moreover, differentiation potential of MenSCs into glial lineage was compared with bone marrow stem cells (BMSCs). Differentiation potential of individual converted NSCs derived from MenSCs or BMSCs into glial-like cells was investigated using immunofluorescence staining and real-time polymerase chain reaction.The fibroblastic morphology of both MenSCs and BMSCs was turned into NSCs shape during first step of differentiation. NSCs derived from both BMSCs and MenSCs expressed higher levels of Olig-2 and Nestin markers compared to undifferentiated cells. The expression levels of myelin basic protein (MBP) mRNA up regulated only in BMSCs-NSCs no in MenSCs-NSCs. However, outgrowth of individual NSCs derived from both MenSCs and BMSCs into glial-like cells led to significant up regulation of glial fibrillary acidic protein,Olig-2 and MBP at mRNA and protein level accompanied with down regulation of Nestin protein.This is the first study demonstrating that MenSCs can be converted to NSCs with differentiation ability into glial-like cells. Accumulative data show different expression pattern of glial markers in differentiated MenSCs compared to BMSCs. The comparable differentiation potential, more accessibility and no invasive technique for sample collection of MenSCs in comparison with BMSCs introduce MenSCs as an apt, consistent and safe alternative to BMSCs for cell therapy of neurodegenerative diseases.

Proliferation and chondrogenic differentiation potential of menstrual blood- and bone marrow-derived stem cells in two-dimensional culture.

Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells. In this study, we investigated the chondrogenic differentiation potential of menstrual blood-derived stem cells (MenSCs) compared with that of bone marrow-derived stem cells (BMSCs) in two-dimensional culture. Following characterization of isolated cells, the potential for chondrogenic differentiation of MenSCs and BMSCs was evaluated by immunocytochemical and molecular experiments. MenSCs were strongly positive for mesenchymal stem cell markers in a manner similar to that of BMSCs. In contrast to BMSCs, MenSCs exhibited marked expression of OCT4, and higher proliferative capacity. Differentiated MenSCs showed strong immunoreactivity to a monoclonal antibody against Collagen type 2, in a pattern similar to BMSCs. Accumulation of proteoglycans in differentiated MenSCs was also comparable with that in differentiated BMSCs. However, the mRNA expression patterns as judged by RT-PCR of chondrogenic markers such as Collagen 2A1, Collagen 9A1 and SOX9 in MenSCs were different from those in BMSCs. Given these findings, MenSCs appear to be a unique stem cell population with higher proliferation than and comparable chondrogenic differentiation ability to BMSCs in two-dimensional culture. Much quantitative studies at the molecular level may elucidate the reasons for the observed differences in MenSCs and BMSCs.