A human corneal endothelium equivalent constructed with acellular porcine corneal matrix.

Background & objectives: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. Methods: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. Results: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. Interpretation & conclusions: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.

Human saphenous vein organ culture under controlled hemodynamic conditions

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Elevated Contractile Responses to Acetylcholine in Organ Cultured Rabbit Carotid Artery

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum-free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.

Fitoflagelados potencialmente tóxicos y nocivos de costas del Pacífico mexicano / Potentially toxic and harmful phytoflagellates from the Mexican Pacific coasts

Los fitoflagelados son un grupo heterogéneo de flagelados autotróficos, heterotróficos y mixotróficos, con importancia ecológica para los niveles tróficos en diferentes ecosistemas. Los fitoflagelados en costas del Pacífico mexicano (y en Latinoamérica en términos generales) son virtualmente desconocidos, sólo se tienen pocos registros. El estudio de los fitoflagelados requiere de métodos complicados de recolección y análisis. Esta es, probablemente, la causa de la escasez de conocimiento de este grupo en áreas tropicales y subtropicales. Material recientemente recolectado a lo largo del Pacífico mexicano sirvió para el estudio de fitoflagelados marinos, incluyendo algunos tóxicos y potencialmente tóxicos. Se usaron muestras de plancton filtradas por gravedad y con bomba de vacío utilizando diferentes métodos de fijación y análisis. Se registran aquellas especies presentes o con posibilidad de estarlo que son potencialmente nocivas para el ecosistema marino pertenecientes a los Phyla Euglenophyta, Heterokontophyta y Haptophyta. Estas especies están distribuidas en el plancton, en aguas oceánicas y costeras

The phytoflagellates are a heterogeneous group of autotrophic, heterotrophic and mixothrophic flagellates of trophic importance in several ecosystems. As in the rest of Latin America, the phytoflagellates that occur in the Mexican Pacific coasts are virtually unknown except for a few records. Their study require complicated collection and analysis methods, a probable cause for the scarce knowledge of this group in tropical and subtropical areas. Material recently collected from various localities along the Mexican Pacific coasts was used to study phytoflagellates, including toxic and potentially toxic species. Plankton samples were treated by gravity and pump filtration, using different methods for fixation and analysis. The phyla Euglenophyta, Heterokontophyta and Haptophyta were found. They occur as plankton in oceanic and shallow coastal waters

Coral--polyhydroxybutrate composite scaffold for tissue engineering: prefabrication properties

In this study the surface properties of two particulate coral and polyhydroxybutrate (PHB) were studied in order to characterize them prior to use in composite production. Coral powder and PHB particle were evaluated using scanning electron microscopy and confocal laser scanning microscopy, to measure surface porosity and pores size. The results showed that coral powder has multiple pleomorphic micropores cross each others give appearance of micro-interconnectivity. Some pore reached to 18 microm with an average porosity of 70%. PHB revealed multiple different size pores extended to the depth, with an average some times reach 25 microm and porosity 45%. These findings demonstrate that both coral and PHB have excellent pores size and porosity that facilitate bone in growth, vascular invasion and bone development. We believe that incorporation of coral powder into PHB will make an excellent composite scaffold for tissue engineering.

Tissue-engineered bone via seeding bone marrow stem cell derived osteoblasts into coral: a rat model

In the present study, natural coral of porites species was used as scaffold combined with in vitro expanded bone marrow stem cell derived osteoblasts (BMSC-DO), to develop a tissue-engineered bone graft in a rat model. Coral was molded into the shape of rat mandible seeded with 5x10(6) /ml BMSC-DO subsequently implanted subcutaneously in the back of 5 week Sprague dawely rats for 3 months. Coral alone was implanted as a control. The implants were harvest and processed for gross inspection and histological observations. The results showed that newly bone grafts were successfully formed coral seeded with cells group showed smooth highly vascularized like bone tissue. Histological sections revealed mature bone formation and lots of blood vessel, the bone formation occurred in the manner resemble intramembraneous bone formation. This study demonstrates that coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal stem cells in tissue engineering.

Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.

Micropropagation of Sapindus mukorossi Gaertn.

Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of S. mukorossi on Murashige and Skoog (MS) medium supplemented with benzylamino purine (BAP) 0.4 microM or 0.8 microM alone. A combination of BAP and gibberellic acid (GA3) 0.4 microM and 2.8 microM produced elongated multiple shoots from both types of explants. Excised shoots were rooted on MS medium respectively with indole-3-butyric acid (IBA) 3.4 microM or 2.4 microM. The regenerated plantlets were successfully acclimatized and transferred to soil.