Effect of cell density on formation of three-dimensional cartilaginous constructs using fibrin & human osteoarthritic chondrocytes

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

Plant population and row spacing on biomass sorghum yield performance / Espaçamento entrelinhas e população de plantas no desempenho produtivo do sorgo biomassa

ABSTRACT: Biomass sorghum is one of the most promising crops for the production of electricity through the burning in high-pressure boilers, due to its high calorific value, high yield, seed propagation, short cycle, and to the possibility of full mechanization of its agricultural processes. However, there is still a lack of information about its cultural practices. To this end, this research aimed to evaluate the influence of row spacing and plant population on the yield performance of biomass sorghum. The experimental design was a randomized block, in factorial scheme of 4 x 4, with four row spacings (0.5, 0.7, 0.9 and 1.1m), and four plant populations (80,000; 100,000; 120,000 and 140,000 plants ha-1), with three replications. The characteristics evaluated were: plant height, stem diameter, number of leaves, number of tillers per plant, fresh weight per plant and biomass. Total biomass yield was greatly influenced by the row spacing, showing a sharp reduction when row spacing increased, in the two years of study, changing from 180.27 to 114.42t ha-1 in the 2012/13 crop year, and from 146.50 to 102.56t ha-1 in the 2013/14 crop year, for 0.5 and 1.1m between rows, respectively. The lowest yields observed in the second year of the study were due to unfavorable weather conditions in the period.

RESUMO: O sorgo biomassa é uma das culturas mais promissoras para a produção de energia elétrica, através da queima da planta inteira em caldeiras de alta pressão, devido ao seu alto poder calorífico, alta produtividade, propagação por sementes, ciclo curto e por possibilitar mecanização completa de seus processos agrícolas. Contudo, ainda há uma carência de informações acerca do seu manejo cultural. Para tanto, esta pesquisa objetivou avaliar a influência do espaçamento entrelinhas e da população de plantas no desempenho produtivo do sorgo biomassa. Os experimentos foram instalados em Sete Lagoas/MG, na Embrapa Milho e Sorgo, durante duas safras consecutivas. O delineamento experimental utilizado foi em blocos ao acaso, em esquema fatorial 4 x 4, sendo 4 espaçamentos entrelinhas (0,5; 0,7; 0,9 e 1,1m) e 4 populações de plantas (80.000; 100.000; 120.000 e 140.000 plantas ha-1) e utilizadas três repetições. As características avaliadas foram: altura da planta, diâmetro do colmo, número de folhas, número de perfilhos por planta, massa fresca por planta e biomassa total por área cultivada. A produtividade de biomassa total foi influenciada pelo espaçamento entrelinhas, apresentando uma forte redução com o aumento do espaçamento entrelinhas para os dois anos de estudos, passando de 180,27 para 114,42t ha-1, na safra 2012/13, e de 146,50 para 102,56t ha-1, na safra 2013/14, para espaçamentos entrelinhas de 0,5 e 1,1m, respectivamente. As menores produtividades observadas no segundo ano de estudo foram devido às condições climáticas desfavoráveis no período.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-gamma in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

Espaçamento e manejo de restos culturais para o controle de Sclerotinia sclerotiorum em feijoeiro / Plant spacing and management of crop residues for control of Sclerotinia sclerotiorum in bean

A manutenção da camada de palha na superfície do solo e o incremento no espaçamento entrelinhas têm sido difundidos como métodos para o controle de mofo branco. O objetivo deste trabalho foi avaliar a alteração no espaçamento entrelinhas e o manejo da cobertura do solo para o controle de Sclerotinia sclerotiorum na cultura do feijoeiro. O delineamento experimental foi blocos casualizados, em esquema fatorial 2x3, constituído de seis tratamentos com quatro repetições. O primeiro fator foi o manejo de restos culturais, com dois níveis de manejo (com cobertura e sem cobertura de solo). O segundo fator foi o espaçamento entre linhas, com três níveis (20, 40 e 60cm). As variáveis analisadas foram a área abaixo da curva de progresso (AACPD), incidência de mofo branco, números de escleródios, massa de 100 grãos e produtividade. De acordo com os resultados, não ocorreu interação significativa (P<0,05) entre o manejo dos restos culturais e os espaçamentos. Os espaçamentos de 40 e 60cm entre linhas foram significativamente superiores a 20cm para a redução número de escleródios de S. sclerotiorum, incidência de mofo branco e AACPD. Os parâmetros produtivos não foram afetados significativamente pela variação dos espaçamentos e dos restos culturais.

The methods to control white mold of bean include a layer of straw on the soil surface and the increase in row spacing, aiming to provide disadvantage to the pathogen. The objective of this paper was to assess the change in row spacing and the presence or absence of ground cover (straw) for control of Sclerotinia sclerotiorum in common bean. The experimental design was randomized blocks in factorial scheme 2x3, consisting of six treatments with four replications. The first factor was the management of crop residues, with two levels of management (with and without coverage of soil). The second factor was the spacing between lines, with three levels (20, 40 and 60cm). The variables were the area below the disease progress curve (AUDPC), incidence of white mold, sclerotia numbers, weight of 100 grains and yield. There was no significant interaction (P<0.05) between the management of crop residues and spacing. The spacing between rows 40 and 60cm were significantly better than 20cm to reduce the number of sclerotia of S. sclerotiorum, white mold incidence and AUDPC. The yield and weight of 100 grains were not affected by variations in the spacing and ground cover.

Culture of Rat Retinal Ganglion Cells / 华中科技大学学报（医学）（英德文版）

This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro.The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers:inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers (inner layer,outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15％ FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells (RGCs％) was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7),were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ).Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15％ FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed:(1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs％ was (19.9±1.2)％,(0.5±0.2)％,and (6.2±1.7)％ respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant (P＜0.05); (2)fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs％,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that:(1) RGCs％ in the inner layer retina was higher than that in the outer layer retina;(2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.

Fitomorfologia e produção de cultivares de trigo duplo propósito em diferentes manejos de corte e densidades de semeadura / Phytomorphology and production of dual purpose wheat with different cutting regimes and seeding density

A diversificação de cultivares e finalidades da cultura do trigo podem alterar as indicações de manejo no sistema de integração lavoura-pecuária. Dessa forma, este trabalho tem como objetivo verificar o desempenho das cultivares de trigo duplo propósito em diferentes densidades de semeadura, com e sem corte. As cultivares de trigo utilizados foram: 'BRS Figueira', 'BRS Guatambu', 'BRS Tarumã' e 'BRS Umbu', e as densidades de semeadura utilizadas foram de 50, 200, 350, 500 e 650 sementes por metro quadrado. As variáveis fitomorfológicas e de produção possuem um desempenho diferenciado conforme a cultivar, o manejo e a densidade de semeadura. A cultivar 'Figueira' possui o maior potencial produtivo dentro dos limites de densidades estudados quanto à produção de massa seca, em relação aos demais cultivares. A produção de grãos teve interação significativa entre as cultivares e o manejo (com e sem corte). As cultivares com maiores produções de grãos foram, respectivamente, 'Figueira', 'Umbu', 'Tarumã' e 'Guatambu' no manejo sem corte, já no sistema com corte a cultivar 'Tarumã' foi a que obteve o maior rendimento de grãos, diferenciando-se das demais. A maioria das correlações altas e significativas ocorreu nos manejos com e sem corte. O corte diminuiu todos os valores dos componentes de rendimento, bem como a produção de grãos de todos as cultivares.

The diversification of cultivars and purposes of wheat crop may alter the information management system of crop-livestock integration. Therefore, this research has the objective to verify the performance of dual purpose wheat with different cutting regimes and seeding density. The whet cultivate used were: 'BRS Figueira', 'BRS Guatambu', 'BRS Tarumã' and 'BRS Umbu'. The seeding density was of 50, 200, 350, 500 and 650 seeds for square meter. The phitomorfological and production variables have a different performance depending on as cultivar, management and seeding density. The cultivar 'Figueira' has the highest productive potential yield within the range of densities analyzed for dry mater production in relation to other cultivars The grain yield had significant interaction between cultivars and management (with and without cutting). The cultivars with higher grain yields were, respectively, 'Figueira', 'Umbu', 'Tarumã' and 'Guatambu' in managing blunt, already in the court system the cultivar 'Tarumã' was the one that had the highest grain yield, differing from the others. The most high and significant correlations occurred in plots with and without cutting. The court dropped all values of yield components, and grain yield of all cultivars.