Chitosan/Xanthan/Hydroxyapatite-graphene oxide porous scaffold associated with mesenchymal stem cells for dentin-pulp complex regeneration.

The aim of this paper was to synthesize and characterize polymeric scaffolds of Chitosan/Xanthan/Hydroxyapatite-Graphene Oxide nanocomposite associated with mesenchymal stem cells for regenerative dentistry application. The chitosan-xanthan gum (CX) complex was associated with Hydroxyapatite-Graphene Oxide (HA-GO) nanocomposite with different Graphene Oxides (GO) concentration (0.5 wt%; 1.0 wt%; 1.5 wt%). The scaffolds characterizations were performed by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and contact angle. The mechanical properties were assessed by compressive strength. The in vitro bioactivity and the in vitro cytotoxicity test (MTT test) were analyzed as well. The data was submitted to the Normality and Homogeneity tests. In vitro Indirect Cytotoxicity assay data was statistically analyzed by ANOVA two-way, followed by Tukey's test (α = 0.05). Compressive strength and contact angle data were statistically analyzed by one-way ANOVA, followed by Tukey's test (α = 0.05). XRD showed the presence of Hydroxyapatite (HA) peaks in the structures CXHA, CXHAGO 0.5%,1.0% and 1.5%. FT-IR showed amino and carboxylic bands characteristic of CX. Raman spectroscopy analysis evidenced a high quality of the GO. In the TGA it was observed the mass loss associated with the CX degradation by depolymerization. SEM analysis showed pores in the scaffolds, in addition to HA incorporated and adhered to the polymer. Contact angle test showed that scaffolds have a hydrophilic characteristic, with the CX group the highest contact angle and CXHA the lowest (p < 0.05). 1.0 wt% GO significantly increased the compressive strength compared to other compositions. In the bioactivity test, the apatite crystals precipitation on the scaffold surface was observed. MTT test showed high cell viability in CXHAGO 1.0% and CXHAGO 1.5% scaffold. CXHAGO scaffolds are promising for regenerative dentistry application because they have morphological characteristics, mechanical and biological properties favorable for the regeneration process.

Chitosan/Xanthan membrane containing hydroxyapatite/Graphene oxide nanocomposite for guided bone regeneration.

OBJECTIVE: To develop a chitosan-xanthan (CX) membrane associated with Hydroxyapatite (HA) and different concentrations of graphene oxide (GO). METHODOLOGY: The CX complex was associated with the hydroxyapatite-graphene oxide (HAGO) nanocomposite in different concentrations. The experimental groups were:1) CX; 2) Chitosan-Xanthan/Hydroxyapatite (CXHA); 3) Chitosan-Xanthan/Hydroxyapatite-Graphene Oxide 0.5% (CXHAGO 0.5%); 4) CXHAGO 1.0%; 5) CXHAGO 1.5%. The membranes characterizations were performed by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy, Scanning Electron Microscopy (SEM), Contact angle, Tensile Strength, in vitro Bioactivity and the in vitro Cell viability (MTT test). The data was submitted to the Normality and Homogeneity tests. In vitro Indirect Cytotoxicity assay data was statistically analyzed by two-way ANOVA and Tukey's test (α = 0.05). Tensile Strength and Contact Angle data were statistically analyzed by one-way ANOVA followed by Tukey's test (α = 0.05). RESULTS: XRD, FTIR and Raman spectroscopy confirmed the characteristic bands of the CX polymeric complex, the phosphate bands related to HA, and the presence of GO. SEM images demonstrated the non-porous and homogeneous surface of membranes. The contact angle test showed the hydrophilic characteristic of all membranes (p > 0.05). CX showed tensile strength significantly higher than other membranes. The apatite deposition was observed in all membranes after performing the bioactivity test. The cell viability of CXHAGO 1.0% and CXHAGO 1.5% was significantly higher than CX. CONCLUSION: The addition of HAGO reduced the mechanical strength of membranes, but improved its cell viability. It demonstrated the potential of CXHAGO membranes to be used in guided bone regeneration therapies.

Chondrogenesis of human amniotic fluid stem cells in Chitosan-Xanthan scaffold for cartilage tissue engineering.

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.

Towards wound dressings with improved properties: Effects of poly(dimethylsiloxane) on chitosan-alginate films loaded with thymol and beta-carotene.

This study aimed to evaluate the effect of poly(dimethylsiloxane) on the mechanical properties of chitosan-alginate (CA) polyelectrolyte complexes (PECs) with potential application as wound dressing biomaterials. For that purpose, different amounts of poly(dimethylsiloxane) were incorporated during the formulation of the PECs. Results showed that the highest tensile strength was observed when using 0.1 g of poly(dimethylsiloxane) per gram of PEC (CAS10). This formulation was also non-hemolytic, capable of inducing thrombus formation to potentially reduce bleeding, and additionally presented high stability when exposed to physiological fluids and/or conditions simulating patient bathing. To improve its wound healing capacity, this formulation was loaded with thymol and beta-carotene (anesthetic, anti-inflammatory and antioxidant compounds) by the supercritical carbon dioxide impregnation/deposition (SSI/D) method at 250 bar and 45 °C for 14 h and at two depressurization rates (5 and 10 bar/min). The PECs were also loaded by conventional impregnation in solution for comparison purposes. Higher bioactive loadings, of 1.8 ±â€¯0.2 and 1.3 ±â€¯0.03 µg per milligram of PEC for thymol and beta-carotene, respectively, were observed when using SSI/D and a higher depressurization rate (10 bar/min). These values do not correspond to the maximum loaded amount of each bioactive, which were strongly retained in the PEC structure due to favorable bioactive-polymer interactions, originating matrices that should present a more sustained release during in vivo applications.

Differentiation of dental pulp stem cells into chondrocytes upon culture on porous chitosan-xanthan scaffolds in the presence of kartogenin.

Adhesion, proliferation and differentiation of dental pulp stem cells (DPSCs) into chondrocytes were investigated in this work with the purpose of broadening the array of cell alternatives to the therapy of cartilage lesions related to tissue engineering approaches. A porous chitosan-xanthan (C-X) matrix was used as scaffold and kartogenin was used as a selective chondrogenic differentiation promoter. The scaffold was characterized regarding aspect and surface morphology, absorption and stability in culture medium, thickness, porosity, thermogravimetric behavior, X-ray diffraction, mechanical properties and indirect cytocompatibility. The behavior of DPSCs cultured on the scaffold was evaluated by scanning electron microscopy and cell differentiation, by histological analysis. A sufficiently stable amorphous scaffold with mean thickness of 0.89±0.01mm and high culture medium absorption capacity (13.20±1.88g/g) was obtained, and kartogenin concentrations as low as 100nmol/L were sufficient to efficiently induce DPSCs differentiation into chondrocytes, showing that the strategy proposed may be a straightforward and effective approach for tissue engineering aiming at the therapy of cartilage lesions.

Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds.

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Influence of a triazine derivative-based biocide on microbial biofilms of cutting fluids in contact with different substrates.

Although biofilms are often associated with hospital infection problems owing to their high resistance to antimicrobial agents, in recent years biofilms have also been studied in the industrial sector, mainly because they are a major cause of contamination outbreaks in facilities and products. The aim of this study was to investigate whether different materials commonly found in the metalworking industries have different biofilm formation characteristics when in contact with contaminated cutting fluid as well as to establish an optimal concentration of a triazine-based antimicrobial agent to protect the oil/water emulsion and also to delay or interrupt the development of biofilms. Biofilms grown on the surface of carbon steel, stainless steel, aluminum, polyvinyl chloride, and glass were analyzed in terms of cell growth and susceptibility to the tested biocide. The results showed that the type of material used had little influence on cell adhesion or on the microbicide concentration required to control and eradicate microorganisms suspended in the emulsion and in the biofilms.

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development.

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.

Behavior of wild-type and transfected S2 cells cultured in two different media.

An animal protein-free medium composed of IPL-41 containing 6 g L(-1) yeastolate ultrafiltrate, 10 g L(-1) glucose, 2 g L(-1) lactose, 5 g L(-1) glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2 × 10(7) cells mL(-1) in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL(-1) and 638 ng RVGP mL(-1), respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL(-1) and 322 ng HBsAg mL(-1) in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3 × 10(7) cells mL(-1) and produced up to 758 ng RVGP mL(-1). In general, glucose was consumed by cells, while lactate and ammonia were produced.

Behavior of wild-type and transfected S2 cells cultured in two different media

An animal protein-free medium composed of IPL-41 containing 6 g L−1 yeastolate ultrafiltrate, 10 g L−1 glucose, 2 g L−1 lactose, 5 g L−1 glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2 × 107 cells mL−1 in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL−1 and 638 ng RVGP mL−1, respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL−1 and 322 ng HBsAg mL−1 in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3 × 107 cells mL−1 and produced up to 758 ng RVGP mL−1. In general, glucose was consumed by cells, while lactate and ammonia were produced.

Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring.

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.

Influence of culture conditions on recombinant Drosophila melanogaster S2 cells producing rabies virus glycoprotein cultivated in serum-free medium.

The recombinant G glycoprotein from the surface of the rabies virus (RVGP) is a promising candidate as a rabies vaccine component and also for diagnostic purposes. In this study, RVGP production by transfected Drosophila melanogaster S2 cells cultivated in a serum-free medium (supplemented IPL-41 medium) was carried out. The effects of pH and pO(2) were evaluated in batch culture in parallel spinner flasks. The use of a pH equal to 6.3 and a pO(2) of 40% air saturation resulted in the highest RVGP content. These conditions were also used in fed-batch mode, yielding a RVGP content level of 98g/10(7) cells. The main nutrients consumed were glucose, glutamine, asparagine, serine and proline and the major metabolites produced were alanine and ammonia, according to the metabolism studies performed. Since RVGP is a transmembrane protein, two different methods for protein recovery were assessed and compared. Detergent-based cell disruption showed to be more effective than mechanical disruption with glass beads for glycoprotein recovery.

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells.

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L(-1) yeastolate ultrafiltrate, 10 g L(-1) glucose, 3.5 g L(-1) glutamine, 0.5 g L(-1) fructose, 2 g L(-1) lactose, 0.6 g L(-1) tyrosine, 1.48 g L(-1) methionine and 1% (v/v) lipid emulsion, reaching 19 x 10(6) cells mL(-1). Maximum specific growth rate and cell productivity were 0.025 h(-1) and 0.57 x 10(5) cells mL(-1) h(-1), respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L(-1), however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.

Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium.

Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 x 10(6) cells mL(-1)) was only obtained when Pluronic F68((R)) percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10(-7) cells.

Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium.

Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

Insect cell culture has become increasingly useful for the production of heterologous proteins as well as of baculovirus polyhedra, and several different culture media formulations can be employed for this purpose. The goal of this work was to assess the potential of lyophylized milk whey ultra filtration retentate (MWR) when associated to yeast extract (YE), glucose and Pluronic F68 (PF68) to partially replace fetal calf serum (FBS) in Spodoptera frugiperda Sf9 cells culture in Grace's medium, aiming AgMNPV baculovirus production. Batch cultivation results showed that the yeast extract and the milk whey concentrate effectively increased cell concentration to about half the level commonly verified for the Sf900II serum-free medium. A 4-fold increase in viable cell concentration was achieved when employing 5 percent (w/v) MWR, 8 g/L YE, 1 percent (v/v) of FBS and 2.7 g.l-1 of glucose, resulting in 1.6 x 10(7) polyhedra.ml-1 after infection with baculovirus.

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace's Medium Supplemented with Milk Whey Ultrafiltrate.

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace's medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace's medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace's medium with 2.7 g l(-1) glucose, 8 g l(-1) YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7x10(6) cells ml(-1)) when compared to Grace's containing 10% (v/v) FBS (9.5x10(5) cells ml(-1)). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6x10(7) PIBs ml(-1)) than in Grace's medium with 10% FBS (0.6x10(7) PIBs ml(-1)). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium.

Incorporation of antibiotics in liposomes designed for tuberculosis therapy by inhalation.

Liposomal encapsulation of tuberculostatic drugs can potentially increase their therapeutic index. The incorporation of isoniazid, pyrazinamide, rifampicin, ethionamide, and streptomycin in extruded distearoylphosphatidylcholine/cholesterol liposomes designed for administration through inhalation was evaluated. Ethionamide and rifampicin were incorporated during lipid film formation, whereas solutions of the remaining drugs were used to hydrate preformed lipid bilayers. Final drug to lipid ratios around 0.3 were achieved for isoniazid and pyrazinamide, and mean vesicle diameters varied from 286 to 329 nm. No expressive drug leakage or mean vesicle diameter changes occurred during 3 weeks. No significant incorporation was achieved for streptomycin, ethionamide, and rifampicin.