RESUMEN
Biological effects of pulsed electrical stimulation (PES) on cells and tissues have been intensively studied with the aim of advancing their biomedical applications. These effects vary significantly depending on PES parameters, cell and tissue types, which can be attributed to the diverse variety of signaling pathways, ion channels, and epigenetic mechanisms involved. The development of new technology platforms, such as nanosecond pulsed electric fields (nsPEFs) with finely tuned parameters, have added further complexity. The present review systematically examines current research progress in various aspects of PES, from physical models to biological effects on cells and tissues, including voltage-sensing domains of voltage-gated channels, pore formation, intracellular components/organelles, and signaling pathways. Emphasis is placed on the complexity of PES parameters and inconsistency of induced biological effects, with the aim of exploring the underlying physical and cellular mechanisms of the physiological effects of electrical stimulation on cells. With chondrogenic differentiation of stem cells and cartilage regeneration as examples, the underlying mechanisms involved were reviewed and analyzed, hoping to move forward towards potential biomedical applications. Hopefully, the present review will inspire more interest in the wider clinical applications of PES and lay the basis for further comprehensive studies in this field.
Asunto(s)
Cartílago/fisiología , Condrocitos/citología , Regeneración , Animales , Cartílago/citología , Condrocitos/metabolismo , Condrogénesis , Estimulación Eléctrica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-ß) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-ß signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-ß-SMAD2/3 signaling.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Pulpa Dental/citología , Células Endoteliales/fisiología , Células Madre/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Benzamidas/farmacología , Células Cultivadas , Niño , Dioxoles/farmacología , Humanos , Masculino , Proteínas de la Membrana/farmacología , Fenotipo , Fosforilación , Transducción de Señal , Diente Primario/citología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND AND OBJECTIVE: The goal of periodontal therapy is to regenerate/reconstruct the damaged supporting tissues of diseased teeth and to facilitate recovery of their physiological functions. Combination of stem cell transplantation and gene therapy offers a viable method for accelerating periodontal repair and regeneration. In this study, the role of the ephrinB2/EphB4 signaling pathway in regulating osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and crosstalk between PDLSCs and pre-osteoblasts within co-culture was investigated through ephrinB2 transgenic expression in PDLSCs. MATERIAL AND METHODS: PDLSCs isolated from premolar teeth of teenage patients undergoing orthodontic treatment were transfected with transgenic (hEfnB2-GFP-Bsd) vector or empty vector (GFP-Bsd). Vector-PDLSCs, EfnB2-PDLSCs, MC3T3-E1 and co-cultures of vector-PDLSCs with MC3T3-E1, and EfnB2-PDLSCs with MC3T3-E1 were subjected to osteogenic induction. The osteogenic differentiation of EfnB2-PDLSCs, vector-PDLSCs and co-cultures were assessed by reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) assay and Alizarin-red S staining. Protein expression levels of ephrinB2, EphB4, phosphorylated ephrinB2 and EphB4 were analyzed by western blot, immunoprecipitation and co-immunoprecipitation assays. RESULTS: ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EfnB2-PDLSCs vs. vector-PDLSCs and with co-culture of EfnB2-PDLSCs and MC3T3-E1 vs. vector-PDLSCs and MC3T3-E1. Reverse transcription-polymerase chain reaction revealed that the expression of human odonto/osteogenic markers were significantly enhanced in EfnB2-PDLSCs compared to vector-PDLSCs, and that the expression of mouse odonto/osteogenic markers were significantly higher in co-culture of EfnB2-PDLSCs with MC3T3-E1 vs. vector-PDLSCs with MC3T3-E1. The EphB4 receptor was activated through phosphorylation during osteogenic differentiation. CONCLUSION: Our data indicate that transgenic expression of ephrinB2 in PDLSCs could promote osteogenic differentiation via stimulation of the phosphorylation of ephrinB2 and EphB4, which regulates cell communication between PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.
Asunto(s)
Efrina-B2/fisiología , Osteoblastos/fisiología , Osteogénesis/fisiología , Ligamento Periodontal/citología , Receptor EphB4/fisiología , Células Madre/fisiología , Western Blotting , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Técnicas de Cocultivo/métodos , Técnicas de Transferencia de Gen , Humanos , Inmunoprecipitación , Ligamento Periodontal/metabolismo , Ligamento Periodontal/fisiología , Transducción de Señal/fisiologíaRESUMEN
The purpose of this study was to evaluate the properties of a novel inorganic xenogenic bone substitute, calcinated antler cancellous bone (CACB). Physicochemical properties of CACB including surface morphology, phase composition, chemical bond structure, Ca/P ratio and porosity were characterized by scanning electron microscopy, X-ray diffraction spectroscopy, Fourier-transform infrared spectroscopy, inductively coupled plasma-atomic emission spectroscopy and nitrogen adsorption analysis, and were found to closely resemble calcinated human cancellous bone. The bone defect repair efficacy of CACB was evaluated in comparison with commercially available bone substitutes (Bio-Oss(®)) within rabbit mandible defects. The gross observation, micro-CT and histology analysis data demonstrated that CACB was efficacious for bone regeneration, and was comparable with Bio-Oss(®) bone substitute in inducing neovascularization and osteogenesis within the mandible defects. CACB can therefore serve as a safe, renewable, and sustainable source of bone graft material, but without the ethical issues pertaining to animal welfare.
Asunto(s)
Cuernos de Venado , Regeneración Ósea , Mandíbula/anatomía & histología , Modelos Biológicos , Animales , Microscopía Electrónica de Rastreo , Conejos , Tomografía Computarizada por Rayos X , Difracción de Rayos XRESUMEN
The objective of this study was to compare the osteogenic potential of human embryonic stem cells (hESCs) within two- and three-dimensional (2D and 3D) culture systems. hESCs of the H1 line (Wicell Inc., Madison, Wisc., USA) were induced to form embryoid bodies (EBs) through 5 days of suspension culture within non-adherent culture dishes. Following enzymatic dissociation, the EB-derived single cells were seeded on either novel 3D porous PLGA scaffolds or 2D culture dishes with the same total cell number. Osteogenic differentiation was induced through culture media supplemented with dexamethasone, L-ascorbic acid and beta-glycerophosphate. After 3 weeks of in vitro culture, quantitative and qualitative assays of osteogenic differentiation were conducted. Osteocalcin secretion and alkaline phosphatase (AP) activities were detected at significantly higher levels within 3D culture compared with the 2D system. Subsequently, the cell-scaffold constructs were implanted in iliac crest defects of immunosuppressed rabbits. After 4 weeks, the constructs were subsequently explanted and characterized by histology and X-ray analysis. Formation of new bone was detected within and around the implanted scaffolds. The results demonstrate that the osteogenic differentiation of human embryonic stem cells is enhanced in a 3D culture system compared to a 2D culture environment. Upon implantation in situ, the differentiating human embryonic stem cells can contribute positively to the repair and regeneration of bone defects.
Asunto(s)
Células Madre Embrionarias/citología , Osteogénesis/fisiología , Ingeniería de Tejidos/métodos , Fosfatasa Alcalina/metabolismo , Animales , Antígenos de Superficie/metabolismo , Ácido Ascórbico/farmacología , Regeneración Ósea , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Dexametasona/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Glicerofosfatos/farmacología , Humanos , Ilion/patología , Ilion/cirugía , Implantes Experimentales , Ácido Láctico/química , Masculino , Microscopía Confocal , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Conejos , Técnicas de Cultivo de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
The rising demand and increasing scarcity of donor oocytes in developed countries have led to some fertility clinics sourcing oocyte donors from abroad, particularly from poorer countries, in what is referred to as 'transnational' or 'international' oocyte donation. In a further new 'twist' to this scheme, frozen sperm of the recipient's male partner is exported abroad through courier mail and is used to fertilise donor oocytes in a foreign clinic to produce embryos, which are then cryopreserved and imported back by mail for transfer to the woman. There are numerous ethical concerns with regards to such means of procuring donor oocytes. First, there is an issue of exploiting economically underprivileged women in poorer countries and disproportionate gains on the part of medical doctors and fertility clinics. Second, there is a question of abdication of responsibility for the donor's welfare on the part of the fertility doctor who takes charge of the recipient's treatment abroad if oocyte donors were to develop severe ovarian hyperstimulation syndrome. Third, the issue of responsibility and accountability becomes even more contentious if congenital defects were to appear in offsprings born from transnational oocyte donation or in the case of transmission of communicable diseases such as hepatitis B, syphilis and AIDS to the recipient. Last, cost savings from the lower prescription price of fertility drugs in economically less-developed countries may not be passed down to the oocyte recipient but instead be exploited to boost the already substantial profit margin of fertility clinics and doctors.
Asunto(s)
Comercio , Donación de Oocito , Atención a la Salud , Femenino , Humanos , Internacionalidad , Responsabilidad Legal , Donación de Oocito/economía , Donación de Oocito/éticaRESUMEN
The rising demand for donor oocytes in developed countries has led to what is referred to as transnational or international oocyte donation, or the outsourcing of oocyte donation to poorer countries. In a further twist, frozen sperm from a recipient's partner can also be mailed to a foreign clinic to fertilize donor oocytes, and the resulting embryos are mailed back, cryopreserved, for transfer to the recipient. Among the numerous ethical concerns raised by this practice of mail order oocyte donation, the most obvious are that underprivileged women from poorer countries are often exploited; fertility physicians from richer counties abdicate responsibility for the welfare of donors; and responsibility could become an issue of contention if transmission of disease to the oocyte recipient or congenital defects in offspring born from such oocyte donation were to occur. Moreover, savings from utilizing donors from poorer countries ought to be shared with oocyte recipients.
Asunto(s)
Mercantilización , Países en Desarrollo , Donación de Oocito/ética , Superovulación/ética , Bioética , Criopreservación/ética , Femenino , Humanos , Cooperación Internacional , Donación de Oocito/métodos , Donación de Oocito/normas , Servicios Postales/ética , Técnicas Reproductivas Asistidas/economía , Técnicas Reproductivas Asistidas/éticaRESUMEN
A major challenge in the widespread application of hES (human embryonic stem) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of less than 5% as reported by previous studies. This study characterized cell death in frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly thawed hES colonies were placed in a 37 degrees C incubator, there was a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed down by keeping the freshly thawed hES colonies at 4 degrees C, with more than 90% of cells remaining viable after 90 min of incubation at 4 degrees C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 degrees C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.
Asunto(s)
Criopreservación/métodos , Células Madre Embrionarias , Apoptosis , Adhesión Celular , Muerte Celular , Supervivencia Celular , Células Cultivadas , Frío , Células Madre Embrionarias/citología , Humanos , Técnicas In Vitro , CinéticaRESUMEN
In recent years, much interest has been generated over the potential of human embryonic stem cells in transplantation medicine. The ground-breaking study of Fraidenraich and colleagues conclusively demonstrated that rescue of lethal cardiac defects in Id knockout mutant mouse embryos was not due to transplanted embryonic stem cells giving rise to functional new tissues within the defective embryonic heart. Instead, there is indirect evidence that the observed therapeutic effect was due to various secreted factors emanating from the transplanted cells. This therefore introduces the exciting prospect of utilizing human embryonic stem cells as "catalysts" to promote biological repair and regeneration in transplantation therapy. Nevertheless, the immunological barrier against allogenic transplantation, as well as the teratogenic potential of human embryonic stem cells poses major technical challenges. A possible strategy to overcome the immunological barrier may be to impose a temporary regimen of immunosuppressive drugs followed by their gradual withdrawal, once adequate tissue regeneration has been achieved. Other more novel alternatives include the use of microencapsulation to block interaction with the transplant recipient's immune system, and co-transplantation with bone marrow-derived mesenchymal stem cells, which have been demonstrated to possess immuno-suppressive properties. The teratogenic potential of human embryonic stem cells could possibly be alleviated by directing the differentiation of these cells to specific lineages prior to transplantation, or through mitotic inactivation. Co-transplantation with autologous adult stem cells may represent a novel strategy to further enhance the "catalytic" effects of human embryonic stem cells. The various factors secreted by human embryonic stem cells could then have a concentrated localized effect on relatively large numbers of co-transplanted autologous adult stem cells, which may in turn lead to enhanced repair and regeneration of the damaged tissue or organ. This new therapeutic strategy needs to rigorously investigated, in view of its potentially important clinical applications.
Asunto(s)
Embrión de Mamíferos/citología , Regeneración , Células Madre/citología , Trasplante de Células , HumanosRESUMEN
Postslaughter processing of sow carcasses results in the ovaries being exposed to temperatures of 41.3 to 42.1 degrees C within a 30-min time frame. This study investigated whether the maturational and developmental competence of the recovered germinal vesicle stage oocytes could be compromised by post-slaughter processing. The results showed that the in vitro maturation rates of GV stage oocytes exposed to elevated temperature did not significantly differ from the corresponding controls (74.1 vs. 75.8%). Immunocytochemical staining revealed that elevated temperature did not adversely affect metaphase II spindle formation but resulted in extensive disruption of oocyte cytoskeletal organization. This, in turn, had a detrimental effect on parthenogenetic development compared with the corresponding nonheat-treated controls (cleavage rate = 27.7 vs. 65.3%, P < 0.01; blastulation rate = 6.7 vs. 20.6%, P < 0.01). Hence, transient exposure to elevated temperature during slaughter did not have any detrimental effects on nuclear maturation per se, but it did result in extensive cytoskeletal damage, which in turn drastically decreased the developmental competence.
Asunto(s)
Calor , Oocitos/crecimiento & desarrollo , Porcinos/fisiología , Animales , Núcleo Celular/fisiología , Citoesqueleto/fisiología , Femenino , Meiosis , Metafase/fisiología , Oocitos/citología , Folículo Ovárico/fisiología , Factores de TiempoRESUMEN
The relatively large size (300-400 microm) and fragile semi-permeable membrane of microcapsules makes them particularly prone to cryodamage. This study investigated slow-cooling protocols for the cryopreservation of microcapsules. Instead of a programmable freezing-machine, slow cooling was carried out directly within a -80 degrees C refrigerator. A range of increasing cryoprotectant (DMSO and EG) concentrations with slow cooling was investigated. The results showed that 2.8 M (20% v/v) DMSO and 2.7 M (15% v/v) EG were optimal for microcapsule cryopreservation, resulting in approximately 55-60% of the microcapsules remaining intact, with a relatively high post-thaw cell viability of 80-85%. Post-thaw cell viability and microcapsule integrity were consistently higher at equivalent molarities of DMSO compared to EG. Hence, all subsequent studies utilized only DMSO. Post-thaw cell viability upon slow cooling with 2.8 M (20% v/v) DMSO was significantly improved in the presence of 0.25 M sucrose (> 95%), but there was no enhancement in microcapsule integrity. Neither post-thaw cell viability nor microcapsule integrity was improved with multi-step exposure and removal of sucrose, compared to a single-step protocol. There was also no improvement in either post-thaw cell viability or microcapsule integrity in the presence of 20% (w/v) Ficoll. Hence, the optimal condition for microcapsule cryopreservation by slow-cooling is with 2.8 M (20% v/v) DMSO and 0.25 M sucrose.
Asunto(s)
Criopreservación/métodos , Citoprotección , Riñón/citología , Animales , Cápsulas , Chlorocebus aethiops , Crioprotectores , Composición de Medicamentos/métodos , Membranas Artificiales , Factores de TiempoAsunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas de la Matriz Extracelular/metabolismo , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Agrecanos , Animales , Carbohidratos/análisis , Proteínas de la Matriz Extracelular/análisis , Ácido Hialurónico/metabolismo , Técnicas In Vitro , Cinética , Lectinas Tipo C , Ligandos , Unión Proteica , Proteoglicanos/metabolismoRESUMEN
Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl(2), MnCl(2) and CaCl(2)) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10 mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca(2+) and Mn(2+), FA-HA (830 kDa) was more compact than in Mg(2+), Na(+) or K(+). These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl(2) (36 nm) than in NaCl (43 nm). The permeability of more concentrated solutions of HA (<10 mg/ml) to FITC-dextran tracers (2000 kDa) was higher in CaCl(2). The properties of HA in urea (up to 6 M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain-chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA(20-26)) also had no effect on the self diffusion of high-molecular-mass FA-HA (830 kDa) solutions, or on dextran tracer diffusion, showing that there were no chain-chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.
Asunto(s)
Ácido Hialurónico/química , Colorantes Fluorescentes , Soluciones , Espectrometría de FluorescenciaRESUMEN
Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.