Evaluation of human dental stem cell growth characteristics and cellular morphological changes in response to extracellular matrix nanotopography

Objetivo: Nanotopografia e fatores extracelulares solúveis estão presentes nas célulastroncoda polpa dentária Seu efeito na sobrevivência e diferenciação de células-tronco dentárias ainda não foi estabelecido. Nosso objetivo foi analisar os papéis individuais e combinados da nanotopografia e do soro (fatores solúveis) da matriz extracelular (MEC) no crescimento, potencial de diferenciação e características morfológicas das células-tronco da polpa dentária humana. Este estudo avaliou e comparou a resposta detais células-tronco a diferentes estímulos ambientais – nanofibras, soro e meios condicionados. Materiais e métodos: Neste estudo, nanofibras de PLLA fabricadas foram usadas como meio biomimético estrutural in vitro da nanotopografia nativa encontrada na polpa de células-tronco/MEC in vivo. O soro e o meio condicionado foram utilizados como o imitador in vitro dos fatores solúveis aos quais as células-tronco são expostas in vivo. As células-tronco foram cultivadas na presença e ausência de nanofibras de ácido poli-L lático biodegradáveis e soro. As características de crescimento das células-tronco da polpa dentária humana foram avaliadas em termos de viabilidade celular e tempo de duplicação no intervalo de cada passagem. As alterações morfológicas celulares foram estudadas usando microscopia invertida e coloração H&E. Como segunda parte do estudo, as células em todas as condições de cultura foram expostas ao Meio Condicionado para Polpa Dentária (MCPD) por um curto período de 3 dias. As características de crescimento e as alterações morfológicas das células foram avaliadas após a exposição curta ao MCPD. Além disso, a microscopia eletrônica de varredura foi utilizada para o estudo morfológico das células-tronco em nanofibras, expostas aos meios condicionados. As células diferenciadas foram analisadas por TR-PCR quantitativa quanto à expressão neurogênica e odontogênica dos genes RUNX2, osteopontina e β-tubulina III. Resultados: As células-tronco dentárias apresentaram melhor sobrevida e proliferação na presença de nanofibras e soro. A ausência de nanofibras ou soro alterou a sobrevivência e proliferação de células-tronco de forma significativa e indicou diferenciação. Além disso, observou-se que, após a exposição curta ao MCPD, a presença de nanofibras e soro de PLLA favoreceu maior potencial de diferenciação odontogênica e neurogênica, sem alterações morfológicas características da diferenciação terminal. Conclusão: Células-tronco da polpa dentária humana são capazes de detectar sinais geométricos em nanoescala de seu microambiente. Nanotopografia e fatores solúveis da matriz extracelular afetam as células-tronco. Estudos adicionais são essenciais para identificar os principais caminhos que desempenham um papel vital nessas interações.Objective: Nanotopography and soluble extracellular factors are present in the dental stem cell niche in the pulp. Their effect on dental stem cell survival and differentiation is yet to be established. We aimed to analyze the individual and combined roles of extracellular matrix (ECM) nanotopography and serum (soluble factors) on the growth, differentiation potential, and morphological characteristics of the human dental pulp stem cells (hDPSC). This study aimed to evaluate and compare the hDPSC response to different environmental cues – nanofibers, serum, and conditioned media. Materials and methods: In this study, fabricated PLLA nanofibers were used as the in vitro structural biomimetic of the native nanotopography found in the in vivo ECM/stem cell niche. Serum and conditioned media were used as the in vitro mimic of the soluble factors to which stem cells get exposed in vivo. hDPSC were grown in the presence and absence of biodegradablepoly-L-lactic-acid nanofibers and serum. The growth characteristics of hDPSC were assessed in terms of cell viability and doubling time at the interval of every passage. Cellular morphological changes were studied using inverted microscopy and H&E. As the second part of the study, hDPSC in all culture conditions were exposed to Dental Pulp Conditioned Media (DPCM) for a short duration of 3 days. After transient exposure to DPCM, the growth characteristics and the morphological changes of hDPSC were assessed. In addition, scanning electron microscopy was used for the morphological study of hDPSC on nanofibers, exposed to conditioned media. The differentiated cells were analyzed by qRT-PCR for neurogenic and odontogenic expression of RUNX2, osteopontin, and β-tubulin III genes. Results: hDPSC showed better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also indicated differentiation. In addition, it was observed that after transient exposure to DPCM, the presence of both PLLA nanofiber and serum favoured higher odontogenic and neurogenic differentiation potential, without characteristic morphological changes of terminal differentiation. Conclusion: hDPSC has the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the extracellular matrix both affect hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions. The hDPSC demonstrated better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also showed changes indicative of differentiation. The results were compared and analyzed using GraphPad Prism 5 Software. hDPSC possess the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the Extracellular matrix together influence the fate of hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions

Development of an antidiabetic formulation (ADJ6) and its inhibitory activity against α-amylase and α-glucosidase

AbstractThere has recently been much advancement in the diagnosis, treatment, and research of metabolic disorders, especially diabetes. Current research around the world is focused on finding an alternative source of treatment from natural resources for diabetic management, apart from the available synthetic medicines. The present study is a preliminary study of a polyherbal formulation using edible natural resources and an assessment of its antidiabetic activity. The formulation was screened for its phytochemical constituents, total phenols, flavonoids, and vitamin C content. It was also analyzed for its inhibitory effect against the digestive enzymes α-amylase and α-glucosidase, compared with the standard drug acarbose. The formulation showed the presence of major constituents such as steroids, cardiac glycosides, phenols, flavonoids, and saponins. It also had a high level of phenols (340 ± 2.5 mg/g), flavonoids (235.4 ± 8.3 mg/g), and vitamin C (470.8 ± 16.6 mg/g), and showed a half-maximal inhibitory concentration (IC50) value of 0.41 ± 0.03 mg/mL and 0.51 ± 0.01 mg/mL for amylase and glucosidase, respectively. The results showed that ADJ6 had a significant inhibitory activity on α-amylase and α-glucosidase; however, its inhibitory activity was less than that of acarbose. The plants that are formulated in ADJ6 possess potent antidiabetic activity. Thus, we found that ADJ6 is a potent lead for effective diabetic management; however, an evaluation of the formulation must be illustrated using an in vivo model

Biodegradable nanofiber coated human umbilical cord as nerve scaffold for sciatic nerve regeneration in albino Wistar rats

Background: Human umbilical cord (hUC) is encompassed by a mucoid connective tissue called Wharton’s jelly (WJ), made of hyaluronic acid, collagen, and stromal cells to support the blood vessels of hUC. This study was aimed to determine the in vitro neuronal differentiation of Wharton’s jelly-derived Mesenchymal stem cells (WJMSCs), and in vivo axonal regeneration potential of nanofiber coated human Wharton’s jelly as a neuronal graft after sciatic nerve injury in immunosuppressed Albino Wistar rats. Materials and methods: WJMSCs could be differentiated to neuron-like cells by inducing with neuronic supplementing media. The test animal’s axotomized nerves were implanted with trimmed human umbilical cord devoid of vascularity and nanocoated with electro-spun PLLA nanofibers. The control animals were bridged with native sciatic nerve reversed and sutured. Post-surgical functional recovery was studied by walking track, pinprick, muscle weight, and sweating quantification. At the end of the 4th week, the animals were euthanized, and Magneto Neuro graph (MNG) was performed. The explanted grafts were quantified by immunohistochemistry for immuno-rejection, neural scarring, neural adhesion axon regeneration, fiber diameter, myelin thickness, and g-ratio. The sciatic function index (SFI) values were similar by walking track analysis for both the test and control groups. Results:  The animals had functional and sensation recovery by the end of two weeks. No mortality, signs of inflammation, and acute immune rejection were observed post-surgery. Conclusions: The hUCWJ devoid of vascular elements can be a perfect peripheral nerve graft, and we hypothesis that the cryopreserved hUC could be an ideal resource for axonal regeneration in the future

Trans-differentiation of human mesenchymal stem cells generates functional hepatospheres on poly(l-lactic acid)-co-poly(ε-caprolactone)/collagen nanofibrous scaffolds

Journal of Materials Chemistry B1323972-398

Coronary artery to pulmonary artery communications in pulmonary atresia with ventricular septal defect

Coronary artery to pulmonary artery communications (CAPAC) are an important source of pulmonary blood flow in approximately 10% of patients with pulmonary atresia and ventricular septal defect (PA-VSD). A diligent look for these abnormal communications is important to prevent perioperative complications and achieve a complete repair. We present two cases of PA-VSD with CAPAC, one in a 7-year-old child and the other in a 33-year-old adult. The method of occlusion of these communications could be either surgical or catheter based

Genotoxicity assessment of antidiabetic formulation (ADPHF6) in human lymphocytes by single cell gel electrophoresis (comet assay) - an in vitro study

Levels of Reactive Oxygen Species (ROS) molecules during aerobic metabolism are often regulated by unique endogenous antioxidant system. During hyperglycaemic condition, accumulation of excess fatty acids & glucose in adipose tissue (Wright Jr E., 2006) results in increased levels of ROS. When ROS molecules overwhelms the cells antioxidant defence system, it ends up in cellular oxidative stress; which in turn is reported to cause oxidative DNA damage & intervene damage to macromolecules & cellular membranes (Ahmad et al., 2013). Our novel anti-hyperglycaemic polyherbal formulation (ADPHF6) had already illustrated significant inhibitory activity against α-amylase & α-glucosidase enzymes and also scavenging free radicals (in vitro models). The present study demonstrates the protective effect of formulation against H2O2 induced DNA damage in human lymphocytes by Single Cell Gel Electrophoresis (SCGE) assay. Experimental procedures were approved by Institutional Human Ethics Committee of Frontier Lifeline Hospital, Chennai, India (FLL/IEC/02/2014). Peripheral human lymphocytes were isolated (Duthie et.al, 2002) and subjected for Cell viability by Trypan blue exclusion method. The alkaline SCGE assay was carried out to determine the level of DNA damage in ADPHF6 treated cells with minor modifications from Singh et al., 1988. Frosted microscopic slides were pre-coated with 1% NMA followed by 1% LMA and incubated for 15 min at 15-20o C. 100 μL of freshly prepared cell suspension (2 x 104 cells) was mixed with 0.5% LMA & casted on microscopic slide. The cells were immersed in lysing solution for 2 hours at 4O C and washed in TBE buffer for 5 min at RT. All the slides were treated with fresh alkaline solution for 20 minutes for expression of alkali-labile damage. Electrophoresis was performed at 24 V for 20 min at RT. Slides were washed in neutralizing buffer for 5 min at RT. All the groups were stained with Acridine Orange (20µg/ml) & Propidium Iodide (20µg/ml) and visualized under microscope (Nikon Eclipse 80i). Reaction mixture of Lymphocytes (suspended in RPMI-1640) with H2O2 (50 µM) served as Positive Control (PC); Lymphocytes in RPMI-1640 served as Vehicle control; Lymphocytes with H2O2 (50 µM) & Quercetin (50 mM) served as Standard Control. Lymphocytes treated with ADPHF6 aqueous extract (50 µg/ml) & H2O2 (50 µM) in various concentrations served as Test group. In untreated group (Vehicle control), % of tail DNA found to be null. Cells treated with 50 µM H2O2 (Positive control) showed maximum % of tail DNA (>90%) compared to Standard control group (< 30%). ADPHF6 treated cells (50 µg/ml) with 50 µM H2O2 showed significant decrease in % of tail DNA (< 20%). Though mammalian cells are equipped with impressive range of antioxidant enzymes & molecules, but in oxidative stress conditions these agents fails to normalize the redox status of cell. Various scientific investigations in recent times have stated that natural based antioxidants proved to be an alternative therapy for ROS mediated effects. To support this hypothesis, findings from our present study based on SCGE assay clearly suggests that, our anti-hyperglycaemic polyherbal formulation possesses strong antioxidant property in scavenging Free Radical molecules which restore the redox homeostasis in cells. Further studies are required to confirm the potential role of ADPHF6 against Type II Diabetes Mellitus involving in vivo models

Anomalous muscle bundle in the right atrium; Implication to trans atrial device closure

Intracavitary muscle bands or aberrant bands have been well described in all four chambers of the heart but rarely seen thick muscular band crossing right atrium. We report a case of devisable secundum atrial septal defect with an intra-atrial anomalous muscular band, crossing right atrial wall to the rim of the secundum atrial septal defect warranting surgical closure. Keywords: Atrial septal defect, Anomalous tendon, Device closur