Ayşe Eser Elçin

Evaluation of a biomimetic poly(ε-caprolactone)/β-tricalcium phosphate multispiral scaffold for bone tissue engineering: in vitro and in vivo studies.

In this study, the osteogenic potential of rat bone marrow mesenchymal stem cells (rBM-MSCs) on a biomimetic poly(ε-caprolactone)/β-tricalcium phosphate (PCL/β-TCP) composite scaffold composed of parallel concentric fibrous membranes was evaluated in vitro and in vivo. PCL/β-TCP composite membranes were prepared by electrospinning and characterized by x-ray diffraction, differential scanning calorimetry, Fourier transform-infrared spectroscopy, and scanning electron microscopy (SEM). rBM-MSCs were seeded on three-dimensional multispiral scaffolds prepared by the assembly of composite membranes. The cell-scaffold constructs were cultured in osteogenic medium for 4 weeks. Histochemical studies and biochemical assays confirmed the osteogenic differentiation of rBM-MSCs inside the scaffold by documenting the dense mineralized extracellular matrix formation starting from the second week of culture. In the in vivo part of the study, cell-scaffold constructs precultured for 7 days were implanted subcutaneously into the epigastric groin fascia of Wistar rats for a duration of 6 months. Ectopic bone-tissue like formation was documented by using computerized tomography, confocal laser microscopy, SEM, and histochemistry. In vivo findings indicated that the biomimetic multispiral scaffold seeded with rBM-MSCs supports the ectopic formation of new bone tissue in Wistar rats.

Silica coating of the pore walls of a microporous polycaprolactone membrane to be used in bone tissue engineering

Polycaprolactone/silica microporous hybrid membranes were produced in two steps: A microporous polycaprolactone membrane with an interconnected porosity of 80% was obtained via a freeze extraction procedure, then silica was formed by a sol-gel reaction inside the micropores using tetraethyl orthosilicate, TEOS, as silica precursor. It is shown that silica forms a thin coating layer homogeneously distributed over the pore walls when sol-gel reaction is catalyzed by hydrochloric acid, while it forms submicron spherical particles when using basic catalyzer. Some physical properties and the viability and osteoblastic differentiation of bone marrow rat mesenchymal stem cells cultured on pure and hybrid membranes were studied.

A comparative study on the in vitro cytotoxic responses of two mammalian cell types to fullerenes, carbon nanotubes and iron oxide nanoparticles.

Abstract The present study was designed to evaluate and compare the time- and dose-dependent cellular response of human periodontal ligament fibroblasts (hPDLFs), and mouse dermal fibroblasts (mDFs) to three different types of nanoparticles (NPs); fullerenes (C60), single walled carbon nanotubes (SWCNTs) and iron (II,III) oxide (Fe3O4) nanoparticles via in vitro toxicity methods, and impedance based biosensor system. NPs were characterized according to their morphology, structure, surface area, particle size distribution and zeta potential by using transmission electron microscopy, X-ray diffraction, Brunauer–Emmett–Teller, dynamic light scattering and zeta sizer analyses. The Mössbauer spectroscopy was used in order to magnetically characterize the Fe3O4 NPs. The hPDLFs and mDFs were exposed to different concentrations of the NPs (0.1, 1, 10, 50 and 100 μg/mL) for predetermined time intervals (6, 24 and 48 h) under controlled conditions. Subsequently, NP exposed cells were tested for viability, membrane leakage and generation of intracellular reactive oxygen species. Additional to in vitro cytotoxicity assays, the cellular responses to selected NPs were determined in real time using an impedance based biosensor system. Taken together, information obtained from all experiments suggests that toxicity of the selected NPs is cell type, concentration and time dependent.

Time-Resolved Fluorescence Resonance Energy Transfer [TR-FRET] Assays for Biochemical Processes

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is a fluorescence based technique which enables the analysis of molecular interactions in biochemical processes. Principle of TR-FRET is based on time-resolved fluorescence (TRF) measurement and fluorescence resonance energy transfer (FRET) between donor and acceptor molecules. To generate FRET signal, donor and acceptor molecules must show spectral overlap and should be in close proximity to each other and display suitable dipole orientation. The specific signal is acquired from molecules of interest via interactions of donor and acceptor molecules. TR-FRET technique is widely used for studying kinase assays, cellular signaling pathways, protein-protein interactions, DNA-protein interactions, and receptor-ligand binding. There are various propriety applications of TR-FRET. Two different sample protocols are summarized in this review.

Intraspinal Transplantation of Autologous neurogenically-Induced Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Paraplegic Dogs without Deep Pain Perception Secondary to Intervertebral Disk Disease

AIM To investigate the effects of neurogenically-induced autologous bone marrow-derived mesenchymal stem cells (NIBM-MSCs) in paraplegic dogs without deep pain perception (DPP) secondary to intervertebral disk disease (IVDD). MATERIAL AND METHODS Seven dogs which could not be improved neurologically with conventional treatment modalities were included in the study. All dogs were diagnosed by magnetic resonance imaging and surgically treated. Each dog received two times a suspension of autologous 5.0x106 NIBM-MSCs, which were positive to CNPase and MAP-2, as well as to GFAP and beta III tubulin into the spinal cord through the hemilaminectomy defect percutaneously, with a 21-day interval. RESULTS Two months after cell transplantation, there were no changes except for 1 gait score improvement for 1 of the cases. At the 4th month, gait score had improved 1 score in 5 cases, and one score progress was recorded in proprioception and nociception in 1 case. In eight months-followed up 4 cases were evaluated by the same parameter; gait score had improved in 3 cases, and propriception improved in 2 cases, and nociception improved in 3 cases. CONCLUSION Our findings suggest that utility of autologous NIBM-MSCs for cases with poor prognosis after IVDD can be a promising approach.

Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFβ1/BMP4

ABSTRACT Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three‐layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)‐like cells using a variety of inducer combinations. Among all, TGF&bgr;1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM‐like cell samples were compared to hASCs and to the positive control, human coronary artery‐smooth muscle cells (hCA‐SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM‐like cell characteristics. Evidence in transcriptome level was used to evaluate the TGF&bgr;1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells. HighlightsHuman adipose stem cells (hASCs) were isolated, characterized and cultured.Growth factor combinations were evaluated for their effectiveness in differentiation using IHC.hASCs were differentiated into smooth muscle (SM)‐like cells using TGF‐&bgr;1 and BMP4 combination.Microarray analysis was performed for hASCs, SM‐like cells and coronary artery‐SMCs.Microarray data was used to perform hierarchical clustering and interpretation of activated pathways.

Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold

This study evaluates the suitability of a macroporous three-dimensional chitosan/hydroxyapatite (CS/HA) composite as a bone tissue engineering scaffold using MC3T3-E1 cells. The CS/HA scaffold was produced by freeze-drying, and characterized by means of SEM and FTIR. In vitro findings demonstrated that CS/HA supported attachment and proliferation of cells, and stimulated extracellular matrix (ECM) production. Tissue biocompatibility and osteogenic capacity of the cell-laden constructs were evaluated in an ectopic Wistar rat model. In vivo results showed that the MC3T3-E1 cell-laden CS/HA was essentially histocompatible, promoted neovascularization and calcified matrix formation, and secreted osteoblast-specific protein. We conclude that the composite scaffold evaluated has potential for applications in bone regeneration.

Osteogenic differentiation of mesenchymal stem cells using hybrid nanofibers with different configurations and dimensionality

Novel, hybrid fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs aligned) and dimensionality (2-D vs 3-D environment) were used to control the overall behavior and the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADMSCs). Aligned nanofibers in both the 2-D and 3-D configurations are proved to be favored for osteodifferentiation. Morphologically, we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections; however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibers when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 days of culture in osteogenic medium were found on 3-D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favorable expression of osteogenic genes activity in 3-D environment particularly in aligned configuration.

The use of autologous neurogenically-induced bone marrow-derived mesenchymal stem cells for the treatment of paraplegic dogs without nociception due to spinal trauma.

The aim of this study was to investigate the effects of percutaneous transplanted autologous neurogenically-induced bone marrow-derived mesenchymal stem cells (NIBM-MSCs) in paraplegic dogs without deep pain perception (DPP) secondary to external spinal trauma. Thirteen client owned dogs that had failed in improvement neurologically at least 42 days after conservative management, decompression and decompression-stabilization were included in the study. Each dog received two doses of autologous 5.0 × 106 NIBM-MSCs suspension, which were positive to 2′,3′-Cyclic-nucleotide-3′-phosphodiesterase (CNPase) and Microtubule-associated protein 2 (MAP-2), as well as to Glial fibrillary acidic protein (GFAP) and beta III tubulin. The cells were injected into the spinal cord through the hemilaminectomy or laminectomy defects percutaneously with 21 days interval for 2 times. The results were evaluated using Texas Spinal Cord Injury Scale (TSCIS), somatosensory evoked potentials (SEP) and motor evoked potentials (MEP) at the admission time, cell transplantation procedures and during 2, 5, 7 and 12th months after the second cell transplantation. Improvement after cell transplantation in gait, nociception, proprioception, SEP and MEP results was observed in just 2 cases, and only gait score improvement was seen in 6 cases, and no improvement was recorded in 5 cases. All progresses were observed until 2nd month after the second cell transplantation, however, there was no improvement after this period. In conclusion, percutaneous transplantation of autologous NIBM-MSCs is a promising candidate modality for cases with spinal cord injury after spinal trauma and poor prognosis.

Nanofibrous silk fibroin/reduced graphene oxide scaffolds for tissue engineering and cell culture applications

Graphene and silk fibroin (SF) have been extensively investigated in the literature. Hybrid scaffolds of SF and graphene combine the properties of both of the materials and provide promising applications for tissue engineering purposes. In this study, reduced graphene oxide (RGO) (0.5%, 1.0% and 2.0% (w/v)) was incorporated into SF and fabricated into composite nanofibers through electrospinning. The fibers were characterized and analyzed by SEM, XRD, FTIR, TGA, circular dichroism analysis, contact angle measurements and tensile tests. Here, we document that the presence of RGO increases intermolecular forces between RGO and SF molecular chains in the SF matrix, which results in an increased silk II content. Upon the incorporation of RGO, thermal stability and mechanical properties of the fibers significantly improved. Furthermore, in-vitro findings showed that composite nanofibers supported cell viability and were hemocompatible. Finally, bone marrow mesenchymal stem cells were induced osteogenically on electrospun SF/RGO mats for 30days, which showed that the substrate supported osteogenic differentiation. In this study, a feasible method is proposed to generate biocompatible and versatile SF/RGO-composite nanofibers that can influence biomedical applications.