The Use of Layered Freeform Fabrication Technologies to Produce Tissue Engineering Scaffolds for Skull Patches

Congenital skull defects in infants are difficult to correct using metal plates due to the growth of the skull. Tissue engineering of bone patches could be the answer to help such patients. Custom scaffolds have been designed based on Computed Tomography (CT) images of the patient’s skull. An in-house developed single screw extruder, casting and a commercial laser cutter has been evaluated in the fabrication of pure polycaprolactone (PCL) scaffolds as well as PCL mixed with hydroxyapatite (HA) scaffolds. Evaluation criteria for each process included the ability to maintain an optimal pore size for cells to proliferate, inclusion of micro surface properties for cell adhesion, incorporation of hydroxyapatite, and ability to maintain desired shape. The mechanical properties of the fabricated scaffolds will be presented in this paper as well as initial cell seeding results with human adipose-derived adult stem (hADAS) cells.Mechanical Engineerin

Distribuição de potássio em gotejamento com fertirrigação em diferentes pontos de injeção na linha principal

[EN] The purpose of this research was to evaluate the K2O distribution uniformity by surface drip irrigation at Universitat Politecnica de Valencia, Valencia, Spain (39º 29′ N, 0º 23′ W, 20 m). The irrigation was performed by drip lines with not-compensated emitters, spaced 0.3 m. The fertigation was realized using a fertilizer injector pump of electric action with injection of 0.25 h. The experimental design used completely randomized blocks with five treatments and four replications. The treatments consisted of injection in five distances, located at 10; 20; 30; 40; 50 m of the first drip line. Samples were collected in emitters located at the start, at 1/3, at 2/3 and at the end of the drip lines. The nutrient concentration was determined by flame spectrophotometry. The Christiansen's uniformity coefficients (CUC), of distribution (DUC), of statistical (SUC) and of emission (eUC) were estimated. The K2O concentration and distribution decreased linearly with the increase of the injection distance. In all treatments, the CUC, SUC and DUC were described as 'excellent'. The eUC was described as 'recommended' only at smaller injection distances.[PT] Objetivando fornecer subsídios para os produtores aperfeiçoarem o manejo da fertirrigação em pequenas áreas, avaliou-se, na Universitat Politècnica de València, Valência, Espanha (39° 29′ N, 0° 23′ W, 20 m), a uniformidade de distribuição do K2O via irrigação por gotejamento superficial em função da distância do ponto de injeção na linha principal. A irrigação foi efetuada por linhas laterais com emissores não compensantes, espaçados de 0,3 m. Na fertirrigação, foi utilizada uma bomba injetora de fertilizante de acionamento elétrico, com o tempo de injeção de 0,25 h. O delineamento experimental foi o de blocos casualizados, com cinco tratamentos e quatro repetições. Os tratamentos consistiram em cinco pontos de injeção na linha principal, situados a: 10; 20; 30; 40 e 50 m da primeira linha lateral. Foram coletadas amostras em emissores localizados no início, a 1/3, a 2/3 e no final das linhas laterais. A concentração do nutriente foi determinada por espectrofotometria de chama. Foram estimados os coeficientes de uniformidade de Christiansen (CUC), de distribuição (CUD), estatístico (CUE) e de emissão (CUe). A concentração e a distribuição de K2O diminuíram linearmente com o aumento da distância do ponto de injeção. Em todos os tratamentos, o CUC, CUE e CUD foram classificados como ‘excelente’. O CUe foi classificado como ‘recomendado’ apenas na menor distância de injeção.To the Coordination of Improvement of Higher Education Personnel (CAPES) for grant the Doctoral Sandwich Abroad scholarship and the Universitat Politècnica de València (UPV), for providing the experimental area and all equipment and supplies needed for the research.Do Bomfim, GV.; Manzano Juarez, J.; De Azevedo, BM.; Vasconcelos, DV.; Viana, TVDA. (2014). Potassium distribution in drip irrigation with fertigation for different injection distances in the main line. Engenharia Agrícola. 34(6):1151-1161. doi:10.1590/S0100-69162014000600011S1151116134

Application of Low-Frequency Alternating Current Electric Fields Via Interdigitated Electrodes: Effects on Cellular Viability, Cytoplasmic Calcium, and Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and β-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively

Age-Related Effects on the Potency of Human Adipose-Derived Stem Cells: Creation and Evaluation of Superlots and Implications for Musculoskeletal Tissue Engineering Applications

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed “superlots,” containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold

Sensitization of Transforming Growth Factor-β Signaling by Multiple Peptides Patterned on DNA Nanostructures

We report sensitization of a cellular signaling pathway by addition of functionalized DNA nanostructures. Signaling by transforming growth factor β (TGFβ) has been shown to be dependent on receptor clustering. By patterning a DNA nanostructure with closely spaced peptides that bind to TGFβ we observe increased sensitivity of NMuMG cells to TGFβ ligand. This is evidenced by translocation of secondary messenger proteins to the nucleus and stimulation of an inducible luciferase reporter at lower concentrations of TGFβ ligand. We believe this represents an important initial step towards realization of DNA as a self assembling and biologically compatible material for use in tissue engineering and drug delivery